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1.  Discovery of functional non-coding conserved regions in the α-synuclein gene locus 
F1000Research  2014;3:259.
Several single nucleotide polymorphisms (SNPs) and the Rep-1 microsatellite marker of the α-synuclein ( SNCA) gene have consistently been shown to be associated with Parkinson’s disease, but the functional relevance is unclear. Based on these findings we hypothesized that conserved cis-regulatory elements in the SNCA genomic region regulate expression of SNCA, and that SNPs in these regions could be functionally modulating the expression of SNCA, thus contributing to neuronal demise and predisposing to Parkinson’s disease.
In a pair-wise comparison of a 206kb genomic region encompassing the SNCA gene, we revealed 34 evolutionary conserved DNA sequences between human and mouse. All elements were cloned into reporter vectors and assessed for expression modulation in dual luciferase reporter assays.  We found that 12 out of 34 elements exhibited either an enhancement or reduction of the expression of the reporter gene. Three elements upstream of the SNCA gene displayed an approximately 1.5 fold (p<0.009) increase in expression. Of the intronic regions, three showed a 1.5 fold increase and two others indicated a 2 and 2.5 fold increase in expression (p<0.002). Three elements downstream of the SNCA gene showed 1.5 fold and 2.5 fold increase (p<0.0009). One element downstream of SNCA had a reduced expression of the reporter gene of 0.35 fold (p<0.0009) of normal activity.
Our results demonstrate that the SNCA gene contains cis-regulatory regions that might regulate the transcription and expression of SNCA. Further studies in disease-relevant tissue types will be important to understand the functional impact of regulatory regions and specific Parkinson’s disease-associated SNPs and its function in the disease process.
doi:10.12688/f1000research.3281.2
PMCID: PMC4275022  PMID: 25566351
2.  Subtalar extra-articular screw arthroereisis (SESA) for the treatment of flexible flatfoot in children 
Purpose
The aim of this study was to describe a subtalar extra-articular screw arthroereisis (SESA) technique for the correction of flexible flatfoot (FFF) in children and report the outcome.
Methods
From 1990 to 2012, data were collected on 485 patients who underwent SESA at the San Raffaele Hospital. The average age of the patient cohort was 11.5 ± 1.81 years (range 5.0–17.9 years; median 11.5 years). Inclusion criteria were FFF and marked flexible hindfoot valgus, and the exclusion criterion was rigid flatfoot. SESA was performed in 732 cases of FFF—bilaterally in 247 patients and monolaterally in 238 patients.
Results
The values of the pre- and post-SESA weight-bearing X-ray angles were 146° ± 7° and 129° ± 5°, respectively, for the Costa-Bartani angle, 43° ± 8° and 25° ± 6°, respectively, for the talar inclination angle and 11° ± 6° and 14° ± 5°, respectively, for calcaneal pitch (p <0.001). All data were analysed statistically with Student’s t test. Data on 398 patients were ultimately available for analysis. In 93.7 % of cases the results were good in terms of improved clinical aspects and X-ray measurement, absence of complications, normal foot function 3 months post-SESA and no requirement for further surgery. The complication rate was 6.3 % and included ankle joint effusion, painful contracture of peroneal muscles and fourth metatarsal bone stress fractures. A sample of 76 patients (121 feet) were evaluated after screw removal, which occurred on average 2.9 years after SESA. The angle measurements of this sample showed no statistically significant modification.
Conclusion
Based on our >20 years of experience, we believe that SESA is an optimal technique for the correction of FFF as it is simple and can be performed rapidly, and the corrective effect results from the screw’s mechanical and proprioceptive effect. The indication for surgery must be accurate. We suggest that the patient be at least 10 years of age in order that all of the foot’s growth potential can be utilized and to allow for spontaneous resolution and thereby avoid the possibility of over-treatment.
doi:10.1007/s11832-014-0619-7
PMCID: PMC4252272  PMID: 25413354
Flexible flatfoot; Arthroereisis; Calcaneo-stop; Minimally invasive surgery
3.  Seating 
Introduction
Patients with neuromuscular disorders are not able to adapt their sitting posture continuously. Seating devices, like seating orthoses, braces, seating shells, and custom-made cushions for wheelchairs, however may improve their quality of life by stabilizing their pelvis and trunk. Sitting should be regarded as a dynamic process regulated by motor reactions of trunk and pelvic muscles due to endogenic and exogenic influences.
Methods
Prerequisites for the indication of high-quality and cost effective seating devices are guidelines for planning and fitting which consider both pathomorphologic mechanisms and the patient’s personality. In order to avoid functional problems and pain caused by an insufficient seating device it is necessary to pay attention to the exact indication, time, and combination of technical options. Planning within a seating clinic needs teamwork. Primarily the goal of treatment is defined; it depends on the functional deficit, on the daily living activities of the patient, and on the social environmental factors. Secondly, fitting of the devices follows defined treatment guidelines.
Conclusion
By examination of the sensor and musculoskeletal system, it is possible to classify the patient’s sitting or seating ability for simplifying indication: three groups of ACTIVE sitters who are able to change position of trunk and pelvis actively are differentiated from three groups of PASSIVE sitters who have to be seated.
doi:10.1007/s11832-013-0513-8
PMCID: PMC3838517  PMID: 24432101
Neuromuscular spine deformities; Sitting; Seating; Technical aids for sitting and positioning; Seating devices; Seating shells; Wheelchairs
4.  Predictors of Response to Tiotropium Versus Salmeterol in Adults with Asthma1 
The Journal of allergy and clinical immunology  2013;132(5):10.1016/j.jaci.2013.08.003.
Background
Tiotropium has activity as an asthma controller. However, predictors of a positive response to tiotropium have not been described.
Objective
To describe individual and differential response of patients with asthma to salmeterol and tiotropium, when added to an ICS, as well as predictors of a positive clinical response.
Methods
Data from the double-blind, three-way crossover NHLBI Asthma Clinical Research Network’s TALC trial (ClinicalTrials.gov number, NCT00565266) were analyzed for individual and differential treatment responses to salmeterol and tiotropium, and predictors of a positive response to the endpoints FEV1, morning peak expiratory flow (AM PEF), and asthma control days (ACDs).
Results
While approximately equal numbers of patients showed a differential response to salmeterol and tiotropium in terms of AM PEF (90 and 78, respectively), and ACDs (49 and 53, respectively), more showed a differential response to tiotropium for FEV1 (104) than salmeterol (62). An acute response to a short-acting bronchodilator, especially albuterol, predicted a positive clinical response to tiotropium for FEV1 (OR 4.08 [CI 2.00–8.31], P < 0.001) and AM PEF (OR 2.12 [CI 1.12–4.01], P = 0.021), as did a decreased FEV1/FVC ratio (FEV1 response increased 0.39% of baseline for every 1% decrease in the FEV1/FVC ratio). Higher cholinergic tone was also a predictor, while ethnicity, gender, atopy, IgE Level, sputum eosinophils, FENO, asthma duration, and BMI were not.
Conclusion
While these results need confirmation, predictors of a positive clinical response to tiotropium include a positive response to albuterol and airway obstruction, factors which could help identify appropriate patients for this therapy.
doi:10.1016/j.jaci.2013.08.003
PMCID: PMC3826080  PMID: 24084072
asthma; tiotropium; salmeterol; responder analysis; predictor of response
5.  Discovery of functional non-coding conserved regions in the α-synuclein gene locus 
F1000Research  2014;3:259.
Several single nucleotide polymorphisms (SNPs) and the Rep-1 microsatellite marker of the α-synuclein ( SNCA) gene have consistently been shown to be associated with Parkinson’s disease, but the functional relevance is unclear. Based on these findings we hypothesized that conserved cis-regulatory elements in the SNCA genomic region regulate expression of SNCA, and that SNPs in these regions could be functionally modulating the expression of SNCA, thus contributing to neuronal demise and predisposing to Parkinson’s disease.
In a pair-wise comparison of a 206kb genomic region encompassing the SNCA gene, we revealed 32 evolutionary conserved DNA sequences between human and mouse. All elements were cloned into reporter vectors and assessed for expression modulation in dual luciferase reporter assays.  We found that 11 out of 32 elements exhibited either an enhancement or reduction of the expression of the reporter gene. Three elements upstream of the SNCA gene displayed an approximately 1.5 fold (p<0.009) increase in expression. Of the intronic regions, three showed a 1.5 fold increase and two others indicated a 2 and 2.5 fold increase in expression (p<0.002). Two elements downstream of the SNCA gene showed 1.5 fold and 2.5 fold increase (p<0.0009). One element downstream of SNCA had a reduced expression of the reporter gene of 0.35 fold (p<0.0009) of normal activity.
Our results demonstrate that the SNCA gene contains cis-regulatory regions that might regulate the transcription and expression of SNCA. Further studies in disease-relevant tissue types will be important to understand the functional impact of regulatory regions and specific Parkinson’s disease-associated SNPs and its function in the disease process.
doi:10.12688/f1000research.3281.1
PMCID: PMC4275022  PMID: 25566351
6.  Dysregulated expression of lipid storage and membrane dynamics factors in Tia1 knockout mouse nervous tissue 
Neurogenetics  2014;15(2):135-144.
During cell stress, the transcription and translation of immediate early genes are prioritized, while most other messenger RNAs (mRNAs) are stored away in stress granules or degraded in processing bodies (P-bodies). TIA-1 is an mRNA-binding protein that needs to translocate from the nucleus to seed the formation of stress granules in the cytoplasm. Because other stress granule components such as TDP-43, FUS, ATXN2, SMN, MAPT, HNRNPA2B1, and HNRNPA1 are crucial for the motor neuron diseases amyotrophic lateral sclerosis (ALS)/spinal muscular atrophy (SMA) and for the frontotemporal dementia (FTD), here we studied mouse nervous tissue to identify mRNAs with selective dependence on Tia1 deletion. Transcriptome profiling with oligonucleotide microarrays in comparison of spinal cord and cerebellum, together with independent validation in quantitative reverse transcriptase PCR and immunoblots demonstrated several strong and consistent dysregulations. In agreement with previously reported TIA1 knock down effects, cell cycle and apoptosis regulators were affected markedly with expression changes up to +2-fold, exhibiting increased levels for Cdkn1a, Ccnf, and Tprkb vs. decreased levels for Bid and Inca1 transcripts. Novel and surprisingly strong expression alterations were detected for fat storage and membrane trafficking factors, with prominent +3-fold upregulations of Plin4, Wdfy1, Tbc1d24, and Pnpla2 vs. a −2.4-fold downregulation of Cntn4 transcript, encoding an axonal membrane adhesion factor with established haploinsufficiency. In comparison, subtle effects on the RNA processing machinery included up to 1.2-fold upregulations of Dcp1b and Tial1. The effect on lipid dynamics factors is noteworthy, since also the gene deletion of Tardbp (encoding TDP-43) and Atxn2 led to fat metabolism phenotypes in mouse. In conclusion, genetic ablation of the stress granule nucleator TIA-1 has a novel major effect on mRNAs encoding lipid homeostasis factors in the brain, similar to the fasting effect.
Electronic supplementary material
The online version of this article (doi:10.1007/s10048-014-0397-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s10048-014-0397-x
PMCID: PMC3994287  PMID: 24659297
TIA-1; Transcriptome; Cell cycle; Lipid trafficking; RNA processing machinery; Motor neuron disease; Frontotemporal dementia; Cerebellar ataxia
7.  The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography 
Virology  2011;421(2):141-148.
The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T=16 icosahedron attached to a contractile tail via a head–tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid.
doi:10.1016/j.virol.2011.08.028
PMCID: PMC3939024  PMID: 22018785
Bacteriophage; Myoviridae; DNA ejection; Contractile tail; Tail contraction; Bacillus anthracis; Cryo-electron tomography; Phage infection; Tail sheath; Base plate
8.  Platelet Glycoproteins and Fibrinogen in Recovery from Idiopathic Sudden Hearing Loss 
PLoS ONE  2014;9(1):e86898.
Background
The pathomechanism and location of idiopathic sudden sensorineural hearing loss (ISSHL) is unclear. In a previous case-control study, we found elevated fibrinogen concentrations and a higher prevalence of T allele carriers of the glycoprotein (Gp) Ia C807T polymorphism in ISSHL patients.
Methodology
127 patients with ISSHL (mean age 53.3 years, 48.8% females), who underwent a standard therapy with high dose steroids, pentoxifyllin and sterofundine over 8 days were included. We examined the influence of GpIa genotype and fibrinogen (BclI-, A312-, HaeIII-) genotype and fibrinogen plasma levels on hearing recovery after 8 weeks (change from baseline: 0 dB  =  no recovery, >0 to 10 dB = moderate recovery, >10 dB = good recovery). In a subsample of 59 patients with ISSHL, we further studied the association of platelet glycoprotein GpIa, Ib and IIIa densities on hearing recovery as well as the possible effect-modification of platelet glycoproteins on hearing recovery by plasma fibrinogen.
Results
In univariate analysis, neither the GpIa genotype nor fibrinogen genotype (all p>0.1) but lower fibrinogen levels (p = 0.029), less vertigo (p = 0.002) and lower GpIIIa receptor density (p = 0.037, n = 59) were associated with hearing recovery. In multivariate analysis, fibrinogen significantly modified the effect of GPIa receptor density on good hearing recovery (effect-modification on multiplicative scale OR = 0.45 (95% confidence interval (0.21–0.94)), p = 0.03). GPIb receptor density below the mean was associated with a 2-fold increase in good hearing recovery both in patients with fibrinogen levels above (p = 0.04) as well as in patients with fibrinogen levels below the mean (p = 0.06). There was no indication for an effect-modification (p = 0.97).
Conclusions
The findings suggest a vascular/rheological origin of ISSHL with unique features of thrombosis in the inner ear artery that may include complex interrelationships among platelet glycoproteins and plasma fibrinogen.
doi:10.1371/journal.pone.0086898
PMCID: PMC3900659  PMID: 24466284
9.  Sensory functions of motile cilia and implication for bronchiectasis 
Cilia are specialized organelles that extend from the surface of cells into the local environment. Airway epithelial cell cilia are motile to provide mucociliary clearance for host defense. On other cells, solitary cilia are specialized to detect chemical or mechanosensory signals. Sensory proteins in motile cilia have recently been identified that detect shear stress, osmotic force, fluid flow, bitter taste and sex hormones. The relationship of sensory function in human motile cilia to disease is now being revealed. One example is polycystin-1 and polycystin-2. As a complex, these proteins function as a flow sensor in cilia and are mutated in autosomal dominant polycystic kidney disease (ADPKD). The polycystins are also expressed in motile cilia of the airways, potentially operating as sensors in the lung. Computed tomography studies from patients with ADPKD revealed radiographic evidence for bronchiectasis, suggesting that polycystin-1 and -2 are important in lung function. The expression of this complex and sensory channel TRPV4, and bitter taste and sex hormones receptors in motile cilia indicate that the cell is wired to interpret environmental cues to regulate cilia beat frequency and other functions. Defective signaling of sensory proteins may result in a ciliopathy that includes lung disease.
PMCID: PMC3841983  PMID: 22202111
cilia; sensory; bronchiectasis; ADPKD; signaling
10.  Management of dabigatran-induced bleeding: expert statement 
Wiener Klinische Wochenschrift  2013;125(21-22):721-729.
Summary
The interdisciplinary group of experts has compiled a clinical guidance for manifest dabigatran-induced haemorrhage and envisaged invasive interventions on patients under dabigatran. It recommends an escalation of treatment measures as summarized in a pocket guide (see electronic supplementary material online and insert in the print issue).
Electronic supplementary material
The online version of this article (doi: 10.1007/s00508-013-0430-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00508-013-0430-3
PMCID: PMC3838590  PMID: 24217941
Thrombin time; Ccarin clotting time; Patient blood management; Haemodialysis; Prothrombincomplex concentrate; Recombinant factor VIIa; Thrombinzeit; Ecarin clotting time; Patient Blood Management; Hämodialyse; Prothrombinkomplexkonzentrat; rekombinanter Faktor VIIa
11.  Comparison of Physician-, Biomarker-, and Symptom-Based Strategies for Adjustment of Inhaled Corticosteroid Therapy in Adults With Asthma 
Context
No consensus exists for adjusting inhaled corticosteroid therapy in patients with asthma. Approaches include adjustment at outpatient visits guided by physician assessment of asthma control (symptoms, rescue therapy, pulmonary function), based on exhaled nitric oxide, or on a day-to-day basis guided by symptoms.
Objective
To determine if adjustment of inhaled corticosteroid therapy based on exhaled nitric oxide or day-to-day symptoms is superior to guideline-informed, physician assessment–based adjustment in preventing treatment failure in adults with mild to moderate asthma.
Design, Setting, and Participants
A randomized, parallel, 3-group, placebo-controlled, multiply-blinded trial of 342 adults with mild to moderate asthma controlled by low-dose inhaled corticosteroid therapy (n=114 assigned to physician assessment–based adjustment [101 completed], n=115 to biomarker-based [exhaled nitric oxide] adjustment [92 completed], and n=113 to symptom-based adjustment [97 completed]), the Best Adjustment Strategy for Asthma in the Long Term (BASALT) trial was conducted by the Asthma Clinical Research Network at 10 academic medical centers in the United States for 9 months between June 2007 and July 2010.
Interventions
For physician assessment–based adjustment and biomarker-based (exhaled nitric oxide) adjustment, the dose of inhaled corticosteroids was adjusted every 6 weeks; for symptom-based adjustment, inhaled corticosteroids were taken with each albuterol rescue use.
Main Outcome Measure
The primary outcome was time to treatment failure.
Results
There were no significant differences in time to treatment failure. The 9-month Kaplan-Meier failure rates were 22% (97.5% CI, 14%-33%; 24 events) for physician assessment–based adjustment, 20% (97.5% CI, 13%-30%; 21 events) for biomarker-based adjustment, and 15% (97.5% CI, 9%-25%; 16 events) for symptom-based adjustment. The hazard ratio for physician assessment–based adjustment vs biomarker-based adjustment was 1.2 (97.5% CI, 0.6-2.3). The hazard ratio for physician assessment–based adjustment vs symptom-based adjustment was 1.6 (97.5% CI, 0.8-3.3).
Conclusion
Among adults with mild to moderate persistent asthma controlled with low-dose inhaled corticosteroid therapy, the use of either biomarker-based or symptom-based adjustment of inhaled corticosteroids was not superior to physician assessment–based adjustment of inhaled corticosteroids in time to treatment failure.
Trial Registration
clinicaltrials.gov Identifier: NCT00495157
doi:10.1001/2012.jama.10893
PMCID: PMC3697088  PMID: 22968888
12.  Neonatal progeria: increased ratio of progerin to lamin A leads to progeria of the newborn 
Hutchinson–Gilford progeria syndrome (HGPS) is an important model disease for premature ageing. Affected children appear healthy at birth, but develop the first symptoms during their first year of life. They die at an average age of 13 years, mostly because of myocardial infarction or stroke. Classical progeria is caused by the heterozygous point mutation c.1824C>T in the LMNA gene, which activates a cryptic splice site. The affected protein cannot be processed correctly to mature lamin A, but is modified into a farnesylated protein truncated by 50 amino acids (progerin). Three more variations in LMNA result in the same mutant protein, but different grades of disease severity. We describe a patient with the heterozygous LMNA mutation c.1821G>A, leading to neonatal progeria with death in the first year of life. Intracellular lamin A was downregulated in the patient's fibroblasts and the ratio of progerin to lamin A was increased when compared with HGPS. It is suggestive that the ratio of farnesylated protein to mature lamin A determines the disease severity in progeria.
doi:10.1038/ejhg.2012.36
PMCID: PMC3421121  PMID: 22419169
neonatal progeria; HGPS; LMNA; progerin
13.  1,25-Dihydroxyvitamin D3 Suppresses TLR8 Expression and TLR8-Mediated Inflammatory Responses in Monocytes In Vitro and Experimental Autoimmune Encephalomyelitis In Vivo 
PLoS ONE  2013;8(3):e58808.
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) suppresses autoimmunity and inflammation; however, the mechanism of its action has not been fully understood. We sought in this study to determine whether the anti-immune/anti-inflammatory action of 1,25(OH)2D3 is in part mediated through an interplay between 1,25(OH)2D3 and toll-like receptor (TLR)7/8 signaling. 1,25(OH)2D3 treatment prior to and/or following experimental autoimmune encephalomyelitis (EAE) induction effectively reduced inflammatory cytokine expression in the spinal cord and ameliorated EAE. These effects were accompanied with a reduction in expression of several TLRs with the most profound effect observed for TLR8. The expression of TLR8 adaptor protein MyD88 was also significantly reduced by 1,25(OH)2D3. To determine the molecular mechanism by which 1,25(OH)2D3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we evaluated whether 1,25(OH)2D3 can directly inhibit TLR8 signaling and the resulting inflammatory responses in human THP-1 monocytes. 1,25(OH)2D3 treatment not only significantly reduced TLR8 expression but also the expression or activity of MyD88, IRF-4, IRF-7 and NF-kB in monocytes challenged with TLR8 ligands. TLR8 promoter-luciferase reporter assays indicated that 1,25(OH)2D3 decreases TLR8 mRNA level in part via inhibiting TLR8 gene transcription activity. As a result of inhibition on TLR8 signaling cascade at various stages, 1,25(OH)2D3 significantly diminished the TLR8 target gene expression (TNF-α and IL-1β). In summary, our novel findings suggest that TLR8 is a new target of 1,25(OH)2D3 and may mediate the anti-inflammatory action of 1,25(OH)2D3. Our findings also point to a destructive role of TLR8 in EAE and shed lights on pathogenesis of multiple sclerosis.
doi:10.1371/journal.pone.0058808
PMCID: PMC3597563  PMID: 23516559
14.  ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice 
PLoS Genetics  2012;8(8):e1002920.
Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissue-specific effects, which may be partially alleviated by the induction of FBXW8.
Author Summary
Frequent age-associated neurodegenerative disorders like Alzheimer's, Parkinson's, and Lou Gehrig's disease are being elucidated molecularly by studying rare heritable variants. Various hereditary neurodegenerative disorders are caused by polyglutamine expansions in different proteins. In spite of this common pathogenesis and the pathological aggregation of most affected proteins, investigators were puzzled that the pattern of affected neuron population varies and that molecular mechanisms seem different between such disorders. The polyglutamine expansions in the Ataxin-2 (ATXN2) protein are exceptional in view of the lack of aggregate clumps in nuclei of affected Purkinje neurons and well documented alterations of RNA processing in the resulting disorders SCA2 and ALS. Here, as a faithful disease model and to overcome the unavailability of autopsied patient brain tissues, we generated and characterized an ATXN2-CAG42-knock-in mouse mutant. Our data show that the unspecific, chronically present mutation leads to progressive insolubility and to reduced soluble levels of the disease protein and of an interactor protein, which modulates RNA processing. Compensatory efforts are particularly weak in vulnerable tissue. They appear to include the increased degradation of the toxic disease protein by FBXW8. Thus the link between protein and RNA pathology becomes clear, and crucial molecular targets for preventive therapy are identified.
doi:10.1371/journal.pgen.1002920
PMCID: PMC3431311  PMID: 22956915
15.  De novo MECP2 duplication in two females with random X-inactivation and moderate mental retardation 
Xq28 duplications including MECP2 are a well-known cause of severe mental retardation in males with seizures, muscular hypotonia, progressive spasticity, poor speech and recurrent infections that often lead to early death. Female carriers usually show a normal intellectual performance due to skewed X-inactivation (XCI). We report on two female patients with a de novo MECP2 duplication associated with moderate mental retardation. In both patients, the de novo duplication occurred on the paternal allele, and both patients show a random XCI, which can be assumed as the triggering factor for the phenotype. Furthermore, we describe the phenotype that might be restricted to unspecific mild-to -moderate mental retardation with neurological features in early adulthood.
doi:10.1038/ejhg.2010.226
PMCID: PMC3083613  PMID: 21326285
Xq28 duplication; MECP2; X-inactivation; mental retardation in females
16.  PITX2 Is Involved in Stress Response in Cultured Human Trabecular Meshwork Cells through Regulation of SLC13A3 
The development of glaucoma in patients with Axenfeld-Rieger syndrome may be a distinct consequence of PITX2 mutation separate from the congenital structural defects that manifest in the anterior segment. The authors investigated a target gene of PITX2 regulation that may account for late-onset glaucoma due to oxidative stress.
Purpose.
Mutations of the PITX2 gene cause Axenfeld-Rieger syndrome (ARS) and glaucoma. In this study, the authors investigated genes directly regulated by the PITX2 transcription factor to gain insight into the mechanisms underlying these disorders.
Methods.
RNA from nonpigmented ciliary epithelium cells transfected with hormone-inducible PITX2 and activated by mifepristone was subjected to microarray analyses. Data were analyzed using dCHIP algorithms to detect significant differences in expression. Genes with significantly altered expression in multiple microarray experiments in the presence of activated PITX2 were subjected to in silico and biochemical analyses to validate them as direct regulatory targets. One target gene was further characterized by studying the effect of its knockdown in a cell model of oxidative stress, and its expression in zebrafish embryos was analyzed by in situ hybridization.
Results.
Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) was identified as 1 of 47 potential PITX2 target genes in ocular cells. PITX2 directly regulates SLC13A3 expression, as demonstrated by luciferase reporter and chromatin immunoprecipitation assays. Reduction of PITX2 or SLC13A3 levels by small interfering RNA (siRNA)-mediated knockdown augmented the death of transformed human trabecular meshwork cells exposed to hydrogen peroxide. Zebrafish slc13a3 is expressed in anterior ocular regions in a pattern similar to that of pitx2.
Conclusions.
The results indicate that SLC13A3 is a direct downstream target of PITX2 transcriptional regulation and that levels of PITX2 and SLC13A3 modulate responses to oxidative stress in ocular cells.
doi:10.1167/iovs.10-6967
PMCID: PMC3183983  PMID: 21873665
17.  A pilot study to assess feasibility of the water method to aid colonoscope insertion in community settings in the United States 
Background
The water method decreases patient discomfort and sedation requirement. Applicability in non-veteran community settings in the United States (U.S.) has not been reported.
Aims
Our aim is to perform a pilot study to establish feasibility of use the water method at 2 community sites. We tested the hypothesis that compared with air insufflation patients examined with the water method would require less sedation without adverse impact on outcomes.
Methods
Two performance improvement projects were carried out. Consecutive patients who consented to respond to a questionnaire after colonoscopy were enrolled. Project 1: The design was single-blinded (patient only); quasi-randomized - odd days (water), even days (air). Colonoscopy was performed by a staff attending. Project 2: A supervised trainee performed the reported procedures. In both, patient demographics (age, gender and body mass index), amount of sedation required during colonoscopy and procedure-related variables were recorded. The patients completed a questionnaire that enquired about discomfort during colonoscopy and willingness to repeat the procedure within 24 hours after the procedure.
Results
Project 1: Significantly lower doses of fentanyl and midazolam were used and a higher adenoma detection rate (ADR) was demonstrated in the water group. Project 2: 100% cecal intubation rate was achieved by the supervised trainee.
Conclusion
This is the first pilot report in the U.S. documenting feasibility of the water method as the principal modality to aid colonoscope insertion in both male and female community patients. In a head-to-head comparison, significant reduction of sedation requirement is confirmed as hypothesized. No adverse impact on outcomes was noted.
doi:10.4161/jig.20130
PMCID: PMC3350906  PMID: 22586546
water method; colonoscope insertion; feasibility
18.  Yeast two-hybrid analysis of a human trabecular meshwork cDNA library identified EFEMP2 as a novel PITX2 interacting protein 
Molecular Vision  2012;18:2182-2189.
Purpose
Mutations in the homeobox transcription factor paired-like homeodomain transcription factor 2 (PITX2) cause Axenfeld–Reiger syndrome (ARS), which is associated with anterior segment dysgenesis (ASD) and glaucoma. To understand ARS pathogenesis, it is essential to know the normal functions of PITX2 and the proteins with which PITX2 interacts in the eye. Therefore, we used a unique cDNA library that we created from human trabecular meshwork (TM) primary cells to discover PITX2-interacting proteins (PIPs).
Methods
A human TM cDNA library was created from primary cells in the ProQuest Two-Hybrid prey vector: pEXP-AD502. Human PITX2A and PITX2C isoforms were used independently as “bait” to identify novel PIPs. A total of 1.25×106 clones were screened by yeast two-hybrid (Y2H) analyses. PIPs obtained from each Y2H experiment were confirmed by yeast retransformation and mammalian co-immunoprecipitation assays.
Results
EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) was identified by both PITX2A and PITX2C isoforms as a novel PIP from Y2H analyses. EFEMP2 is 443 amino acids long with six epidermal growth factor (EGF)-like modules and one fibulin-like module. The PITX2-interaction domain in EFEMP2 lies between the second EGF-like module and the COOH-terminal fibulin-like module. Co-immunoprecipitation assays in COS-7 cells confirmed the interaction between PITX2 and EFEMP2.
Conclusions
We discovered EFEMP2 as a novel PITX2-interacting protein. Further, our cDNA library made from human TM primary cells is a unique and effective resource to identify novel interacting proteins for glaucoma and ASD candidates. This resource could be used both for discovery and validation of interactomes identified from in silico analysis.
PMCID: PMC3425575  PMID: 22919265
19.  A trial of clarithromycin for the treatment of suboptimally controlled asthma 
Background
Polymerase chain reaction (PCR) studies have demonstrated evidence of M. pneumoniae and C. pneumoniae in the lower airways of patients with asthma.
Objective
To test the hypothesis that clarithromycin would improve asthma control in individuals with mild-to-moderate persistent asthma that was not well-controlled despite treatment with low-dose inhaled corticosteroids (ICS).
Methods
Adults with an Asthma Control Questionnaire (ACQ) score ≥1.5 after a 4 week period of treatment with fluticasone propionate were entered into a PCR-stratified randomized trial to evaluate the effect of 16 weeks of either clarithromycin or placebo, added to fluticasone, on asthma control in individuals with or without lower airway PCR evidence of M. pneumoniae or C. pneumoniae.
Results
92 participants were randomized. Twelve (13%) subjects demonstrated PCR evidence of M. pneumoniae or C. pneumoniae in endobronchial biopsies; 80 were PCR negative for both organisms. In PCR positive participants, clarithromycin yielded a 0.4±0.4 unit improvement in the ACQ score, with a 0.1±0.3 unit improvement in those allocated to placebo. This between-group difference of 0.3±0.5 (p=0.6) was neither clinically nor statistically significant. In PCR negative participants, a non-significant between-group difference of 0.2±0.2 units (p=0.3) was observed. Clarithromycin did not improve lung function or airway inflammation but did improve airway hyperresponsiveness, increasing the methacholine PC20 by 1.2±0.5 doubling doses (p=0.02) in the study population.
Conclusion
Adding clarithromycin to fluticasone in adults with mild-to-moderate persistent asthma that was suboptimally-controlled by low-dose ICS alone did not further improve asthma control. Although there was an improvement in airway hyperresponsiveness with clarithromycin, this benefit was not accompanied by improvements in other secondary outcomes.
doi:10.1016/j.jaci.2010.07.024
PMCID: PMC2950827  PMID: 20920764
asthma; infection; antibiotic
20.  A combination of transcriptome and methylation analyses reveals embryologically-relevant candidate genes in MRKH patients 
Background
The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is present in at least 1 out of 4,500 female live births and is the second most common cause for primary amenorrhea. It is characterized by vaginal and uterine aplasia in an XX individual with normal secondary characteristics. It has long been considered a sporadic anomaly, but familial clustering occurs. Several candidate genes have been studied although no single factor has yet been identified. Cases of discordant monozygotic twins suggest that the involvement of epigenetic factors is more likely.
Methods
Differences in gene expression and methylation patterns of uterine tissue between eight MRKH patients and eight controls were identified using whole-genome microarray analyses. Results obtained by expression and methylation arrays were confirmed by qRT-PCR and pyrosequencing.
Results
We delineated 293 differentially expressed and 194 differentially methylated genes of which nine overlap in both groups. These nine genes are mainly embryologically relevant for the development of the female genital tract.
Conclusion
Our study used, for the first time, a combined whole-genome expression and methylation approach to reveal the etiology of the MRKH syndrome. The findings suggest that either deficient estrogen receptors or the ectopic expression of certain HOXA genes might lead to abnormal development of the female reproductive tract. In utero exposure to endocrine disruptors or abnormally high maternal hormone levels might cause ectopic expression or anterior transformation of HOXA genes. It is, however, also possible that different factors influence the anti-Mullerian hormone promoter activity during embryological development causing regression of the Müllerian ducts. Thus, our data stimulate new research directions to decipher the pathogenic basis of MRKH syndrome.
doi:10.1186/1750-1172-6-32
PMCID: PMC3123171  PMID: 21619687
21.  AZITHROMYCIN IS ASSOCIATED WITH INCREASED SURVIVAL IN LUNG TRANSPLANT RECIPIENTS WITH BRONCHIOLITIS OBLITERANS SYNDROME 
Background
Previous studies have suggested that azithromycin improves lung function in lung transplant recipients with bronchiolitis obliterans syndrome (BOS). However, these studies did not include a non-treated BOS control cohort or perform survival analysis. This study was undertaken to estimate the effect of azithromycin treatment on survival in lung transplant recipients with BOS.
Methods
We conducted a retrospective cohort study of consecutive lung transplant recipients who developed BOS between 1999 and 2007. An association between azithromycin treatment and death was assessed using univariate and multivariable time-dependent Cox regression analysis.
Results
Of the 178 recipients that developed BOS in our study, 78 developed BOS after 2003 and were treated with azithromycin. The azithromycin treated and untreated cohorts had similar baseline characteristics. Univariate analysis demonstrated that azithromycin treatment was associated with a survival advantage and this beneficial treatment effect was more pronounced when treatment was initiated during BOS stage 1. Multivariable analysis demonstrated azithromycin treatment during BOS stage 1 (adjusted hazard ratio=0.23, p=0.01) and absolute FEV1 value at the time of BOS stage 1 (adjusted hazard ratio=0.52, p=0.003) were both associated with a decreased risk for death.
Conclusion
In lung transplant recipients with BOS stage 1, azithromycin treatment initiated before BOS stage 2 was independently associated with a significant reduction in the risk of death. This finding supports the need for a randomized controlled trial to confirm the impact of azithromycin on survival in lung transplant recipients.
doi:10.1016/j.healun.2009.12.003
PMCID: PMC2854291  PMID: 20133163
22.  Tiotropium Bromide Step-Up Therapy for Adults with Uncontrolled Asthma 
The New England journal of medicine  2010;363(18):1715-1726.
BACKGROUND
Long-acting beta-agonist (LABA) therapy improves symptoms in patients whose asthma is poorly controlled by an inhaled glucocorticoid alone. Alternative treatments for adults with uncontrolled asthma are needed.
METHODS
In a three-way, double-blind, triple-dummy crossover trial involving 210 patients with asthma, we evaluated the addition of tiotropium bromide (a long-acting anticholinergic agent approved for the treatment of chronic obstructive pulmonary disease but not asthma) to an inhaled glucocorticoid, as compared with a doubling of the dose of the inhaled glucocorticoid (primary superiority comparison) or the addition of the LABA salmeterol (secondary noninferiority comparison).
RESULTS
The use of tiotropium resulted in a superior primary outcome, as compared with a doubling of the dose of an inhaled glucocorticoid, as assessed by measuring the morning peak expiratory flow (PEF), with a mean difference of 25.8 liters per minute (P<0.001) and superiority in most secondary outcomes, including evening PEF, with a difference of 35.3 liters per minute (P<0.001); the proportion of asthma-control days, with a difference of 0.079 (P = 0.01); the forced expiratory volume in 1 second (FEV1) before bronchodilation, with a difference of 0.10 liters (P = 0.004); and daily symptom scores, with a difference of −0.11 points (P<0.001). The addition of tiotropium was also noninferior to the addition of salmeterol for all assessed outcomes and increased the prebronchodilator FEV1 more than did salmeterol, with a difference of 0.11 liters (P = 0.003).
CONCLUSIONS
When added to an inhaled glucocorticoid, tiotropium improved symptoms and lung function in patients with inadequately controlled asthma. Its effects appeared to be equivalent to those with the addition of salmeterol. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT00565266.)
doi:10.1056/NEJMoa1008770
PMCID: PMC3011177  PMID: 20979471
23.  Detection of respiratory viruses and the associated chemokine responses in serious acute respiratory illness 
Thorax  2010;65(7):639-644.
Background
A specific diagnosis of a lower respiratory viral infection is often difficult despite frequent clinical suspicion. This low diagnostic yield may be improved by use of sensitive detection methods and biomarkers.
Methods
We investigated the prevalence, clinical predictors and inflammatory mediator profile of respiratory viral infection in serious acute respiratory illness. Sequential bronchoalveolar lavage (BAL) fluids from all patients hospitalized with acute respiratory illness over 12 months (n=283) were tested for the presence of 17 respiratory viruses by multiplex PCR assay and for newly-discovered respiratory viruses (bocavirus, WU and KI polyomaviruses) by single-target PCR. BAL samples also underwent conventional testing (direct immunoflorescence and viral culture) for respiratory virus at the clinician’s discretion. 27 inflammatory mediators were measured in subset of the patients (n=64) using a multiplex immunoassay.
Results
We detected 39 respiratory viruses in 37 (13.1% of total) patients by molecular testing, including rhinovirus (n=13), influenza virus (n=8), respiratory syncytial virus (n=6), human metapneumovirus (n=3), coronavirus NL63 (n=2), parainfluenza virus (n=2), adenovirus (n=1), and newly-discovered viruses (n=4). Molecular methods were 3.8-fold more sensitive than conventional methods. Clinical characteristics alone were insufficient to separate patients with and without respiratory virus. The presence of respiratory virus was associated with increased levels of interferon-γ-inducible protein 10 (IP -10)(p<0.001) and eotaxin-1 (p=0.017) in BAL.
Conclusions
Respiratory viruses can be found in patients with serious acute respiratory illness by use of PCR assays more frequently than previously appreciated. IP-10 may be a useful biomarker for respiratory viral infection.
doi:10.1136/thx.2009.132480
PMCID: PMC3018337  PMID: 20627924
Respiratory virus infection; Bronchoalveolar Lavage; IP-10 (CXCL10); Biological Markers
24.  Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia induce distinct host responses 
Critical care medicine  2010;38(1):223-241.
Objective
Pathogens that cause pneumonia may be treated in a targeted fashion by antibiotics, but if this therapy fails, treatment involves only non-specific supportive measures, independent of the inciting infection. The purpose of this study was to determine whether host response is similar following disparate infections with similar mortalities.
Design
Prospective, randomized controlled study.
Setting
Animal laboratory in a university medical center.
Interventions
Pneumonia was induced in FVB/N mice by either Streptococcus pneumoniae or two different concentrations of Pseudomonas aeruginosa. Plasma and bronchoalveolar lavage fluid from septic animals was assayed by a microarray immunoassay measuring 18 inflammatory mediators at multiple timepoints.
Measurements and Main Results
The host response was dependent upon the causative organism as well as kinetics of mortality, but the pro- and anti- inflammatory response was independent of inoculum concentration or degree of bacteremia. Pneumonia caused by different concentrations of the same bacteria, Pseudomonas aeruginosa, also yielded distinct inflammatory responses; however, inflammatory mediator expression did not directly track the severity of infection. For all infections, the host response was compartmentalized, with markedly different concentrations of inflammatory mediators in the systemic circulation and the lungs. Hierarchical clustering analysis resulted in the identification of 5 distinct clusters of the host response to bacterial infection. Principal components analysis correlated pulmonary MIP-2 and IL-10 with progression of infection while elevated plasma TNFsr2 and MCP-1 were indicative of fulminant disease with >90% mortality within 48 hours.
Conclusions
Septic mice have distinct local and systemic responses to Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia. Targeting specific host inflammatory responses induced by distinct bacterial infections could represent a potential therapeutic approach in the treatment of sepsis.
doi:10.1097/CCM.0b013e3181b4a76b
PMCID: PMC2796712  PMID: 19770740
Sepsis; Pneumonia; Streptococcus pneumoniae; Pseudomonas aeruginosa; host response; cytokine
25.  Entry of Burkholderia organisms into respiratory epithelium: CFTR, microfilament and microtubule dependence 
Background
The pathogenesis of infection with Burkholderia cepacia complex (Bcc) organisms may be linked to its capacity to invade respiratory epithelium.
Methods
An antibiotic exclusion assay was used to study B. dolosa AU4459 and B. cenocepacia J2315 invasion into wild-type (WT) and CFTR-deficient respiratory epithelial cells. Inhibitors were used to evaluate Bcc invasion dependency on host microtubule (mt) and microfilament (mf) systems.
Results
B. dolosa entered WT-CFTR cells with 5-fold greater efficiency than CFTR deficient cells (25% vs 5%, respectively). Invasion dropped to <0.5% after either mf or mt inhibition. B. cenocepacia entered WT (0.05%) and CFTR-deficient cells (0.07%) with similarly low efficiencies, which significantly decreased with either mf or mt inhibition (0.008% and 0.002%, respectively).
Conclusion
B. dolosa and B. cenocepacia enter respiratory epithelial cells in a mf and mt dependent fashion. Mutated CFTR leads to less internalization of B. dolosa, but not B. cenocepacia.
doi:10.1016/j.jcf.2009.10.002
PMCID: PMC2818354  PMID: 19914879
Burkholderia cenocepacia; Burkholderia dolosa; invasion; respiratory epithelium; LC3; LCFSN; CFTR; microfilaments; microtubules

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