Insufficient Clearance Research Articles

WS20.1 Structure–activity relationship for OligoG-induced normalization of the CF mucus phenotype

Objectives Cystic fibrosis (CF) is caused by nonfunctional ion transport via CFTR. In the lungs insufficient clearance of mucus leads to lung infections and tissue damage. We have previously shown that the gel-forming MUC2 mucin is packed in an orderly way that allows unpacking by calcium chelation and increased pH. Using an explant system of mouse ileum, we found that in contrast to normal mucus, the mucus is attached to the epithelium in CF mice and was normalized by apical application of 100 mM bicarbonate. OligoG CF-5/20 is derived from polymeric sodium alginate comprising mainly guluronate and some mannuronate, capable of chelating calcium. OligoG is in clinical trials as inhalation therapy in CF patients. Methods Explants from the small intestine of CF mice were mounted in the horizontal Ussing-type chamber. Alginate at different concentrations or composition was added to the apical buffer, pH 7.4. The attachment of the already formed mucus was assessed by comparing the total mucus thickness before and after aspiration. Results OligoG at 1.5% or higher normalized the mucus phenotype without increase in mucus thickness. The alginate fraction containing 12 guluronate units was most effective, whereas shorter and longer molecules were less efficient. Mannuronic polymers were inactive. Conclusion These results suggest that it is guluronic acid and its calcium chelating capacity that is the important and active component in OligoG. These observations indicate that OligoG could act by normalizing the mucus layers in both the intestine and potentially the lungs of cystic fibrosis patients at therapeutically relevant concentrations.

Small molecule TBTC as a new selective retinoid X receptor α agonist improves behavioral deficit in Alzheimer's disease model mice

Alzheimer's disease (AD) is a neurodegenerative disease, which is characterized by progressive cognitive impairments. The β-amyloid (Aβ)-induced neurodegeneration is determined as the main pathogenesis of AD, and either decrease of Aβ production or increase of Aβ clearance is beneficial in the treatment of AD, while Aβ clearance regulation seems to be more attractive as a promising therapeutic strategy against AD based on the fact that the insufficient clearance of Aβ is tightly associated with the late onset of AD that is represented as the majority of AD cases. Here, we report that the small molecular compound, methyl 2-amino-6-(tert-butyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (TBTC), as a selective agonist of retinoid X receptor α (RXRα) can effectively activate the heterodimerization of RXRα with either liver X receptor α (LXRα) or peroxisome proliferator activated receptor γ (PPARγ), stimulate the expressions of the genes of apoE, ABCA1 and ABCG1, and decrease Aβ content both in cells and animal models. In addition, administration of TBTC (30mg/kg/day) in the transgenic APP–PS1 mice could also reduce the formation of senile plaques and improve the daily living activity of the mice. Therefore, our findings have suggested that TBTC might hold the potential as a drug lead compound for the treatment of AD.

Clearance of amyloid β-protein and its role in the spreading of Alzheimer's disease pathology.

Amyloid β-protein (Aβ) containing amyloid plaques and abnormal phosphorylated τ-protein containing neurofibrillary tangles (NFTs) are hallmark lesions of Alzheimer's disease. Both Aβ plaques and NFTs show hierarchical patterns in which the areas of the brain are subsequently affected by Aβ plaques and NFTs, respectively (Braak and Braak, 1991; Thal et al., 2002). Aβ plaques start to develop in the neocortex (phase 1) and spread from there into allocortical regions (phase 2), diencephalon, basal forebrain and striatum (phase 3), midbrain and medulla oblongata (phase 4), and finally into the pons and the cerebellum (phase 5) (Thal et al., 2002). The first NFTs in the brain hemispheres are found in the transentorhinal cortex (stage I), then in the entorhinal cortex (stage II), the hippocampus (stage III), the temporal cortex (stage IV), further neocortical areas except the primary fields (stage V), and, finally, also in primary cortical areas, such as the primary visual cortex (stage VI) (Braak and Braak, 1991). Axonal connections between subsequently affected brain regions suggest that AD pathology spreads along neuronal pathways (Thal et al., 2002; Braak and Del Tredici, 2011). Insufficient clearance of Aβ has been considered to play an essential role in the pathogenesis of AD. Clearance mechanisms that contribute to Aβ elimination from brain are cellular enzymatic proteolysis in glial cells, neurons or in the extracellular space (Qiu et al., 1998; Yamaguchi et al., 1998; Iwata et al., 2000; Thal et al., 2000; Farris et al., 2003), transport through the blood-brain barrier (Shibata et al., 2000; Ito et al., 2007), and perivascular drainage (Weller et al., 2008) (Figure ​(Figure1A1A). Figure 1 Schematic representation of Aβ clearance and propagation. (A) Aβ clearance mechanisms: enzymatic clearance within neurons (N) and glial cells [here shown in the example is an astrocyte (AG)] or in the extracellular space (ECS) (Qiu et ... Here, I will discuss the potential impact of impaired Aβ clearance on propagation mechanisms for Aβ and τ.

Failure Analysis of an Inter-shaft Bearing of an Aero Gas Turbine Engine

Failure of an inter-shaft bearing in an aero gas turbine engine is investigated to determine its root cause. Teardown inspections followed by metallurgical investigations are carried out on the parts of the failed bearing. None of the components of the bearing has shown any discolouration, wear, or overheating marks. But severe flaking is observed on one side of the outer raceway. It indicates progressive fatigue due to overload. Insufficient clearance due to differential expansion of inner ring and contraction of outer ring due to heating and cooling, respectively, can also result in this type of phenomena. Maintaining oil discipline and using correct grade of oil at proper frequency can address these issues. A comprehensive engine-bearing prognostic approach may be followed utilizing available sensor information on-board the aircraft such as rotor speed, vibration, lube system information, and aircraft maneuvers to predict bearing life and incipient bearing failure.

Choroid plexus dysfunction impairs beta-amyloid clearance in a triple transgenic mouse model of Alzheimer's disease.

Compromised secretory function of choroid plexus (CP) and defective cerebrospinal fluid (CSF) production, along with accumulation of beta-amyloid (Aβ) peptides at the blood-CSF barrier (BCSFB), contribute to complications of Alzheimer’s disease (AD). The AD triple transgenic mouse model (3xTg-AD) at 16 month-old mimics critical hallmarks of the human disease: β-amyloid (Aβ) plaques and neurofibrillary tangles (NFT) with a temporal- and regional- specific profile. Currently, little is known about transport and metabolic responses by CP to the disrupted homeostasis of CNS Aβ in AD. This study analyzed the effects of highly-expressed AD-linked human transgenes (APP, PS1 and tau) on lateral ventricle CP function. Confocal imaging and immunohistochemistry revealed an increase only of Aβ42 isoform in epithelial cytosol and in stroma surrounding choroidal capillaries; this buildup may reflect insufficient clearance transport from CSF to blood. Still, there was increased expression, presumably compensatory, of the choroidal Aβ transporters: the low density lipoprotein receptor-related protein 1 (LRP1) and the receptor for advanced glycation end product (RAGE). A thickening of the epithelial basal membrane and greater collagen-IV deposition occurred around capillaries in CP, probably curtailing solute exchanges. Moreover, there was attenuated expression of epithelial aquaporin-1 and transthyretin (TTR) protein compared to Non-Tg mice. Collectively these findings indicate CP dysfunction hypothetically linked to increasing Aβ burden resulting in less efficient ion transport, concurrently with reduced production of CSF (less sink action on brain Aβ) and diminished secretion of TTR (less neuroprotection against cortical Aβ toxicity). The putative effects of a disabled CP-CSF system on CNS functions are discussed in the context of AD.

Alzheimer's disease is associated with low density of the long CR1 isoform

The long complement receptor type 1 (CR1) isoform, CR1*2 (S), has been identified as being associated with Alzheimer's disease (AD) risk. We aimed to analyze the phenotypic structural and expression aspects (length and density) of CR1 in erythrocytes of 135 Caucasian subjects (100 AD and 35 controls). CR1 length polymorphism was assessed at protein and gene levels using Western blot and high-resolution melting, respectively. CR1 sites on erythrocytes were enumerated by flow cytometry. CR1 gene analysis, spotting the rs6656401 and rs3818361 polymorphisms, was performed by pyrosequencing. The CR1 density was significantly lower in AD patients expressing the CR1*2 isoform compared with the controls (p = 0.001), demonstrating lower expression of CR1 in CR1*2 carriers. Our data suggested the existence of silent CR1 alleles. Finally, rs6656401 and rs3818361 were strongly associated with CR1 length polymorphism (p < 0.0001). These observations indicate that AD susceptibility is associated with the long CR1 isoform (CR1*2), albeit at a lower density, suggesting that AD results from insufficient clearance of plaque deposits rather than increased inflammation.

Acetylated histones contribute to the immunostimulatory potential of neutrophil extracellular traps in systemic lupus erythematosus.

In addition to disturbed apoptosis and insufficient clearance of apoptotic cells, there is recent evidence for a role of neutrophils in the aetiopathogenesis of systemic lupus erythematosus (SLE). In response to various stimuli, neutrophils can rapidly release DNA fibres decorated with citrullinated histones and anti-microbial peptides. These structures are referred to as neutrophil extracellular traps (NETs). In addition to apoptotic cell-derived microparticles, these NETs may comprise a further source of autoantigens, able to drive the autoimmune response in SLE. Our group recently identified specific histone modifications occurring during apoptosis that play an important role in the autoimmune response in SLE. In the current study, we evaluated the presence and immunostimulatory potential of these previously identified histone modifications in NETs. Compared to NETs from healthy donors, the histones present in NETs formed by SLE-derived neutrophils contain increased amounts of acetylated and methylated residues, which we previously observed to be associated with apoptosis and SLE. Treatment of neutrophils with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), prior to induction of NETosis, induced NETs containing hyperacetylated histones, endowed with an increased capacity to activate macrophages. This implies that specific histone modifications, in particular acetylation, might enhance the immunostimulatory potential of NETs in SLE.

Abstract 11636: Right Ventricle Lymphatic Vessel Insufficiency Contributes to Interstitial Fluid Stasis, Inflammation, Fibrosis, and Failure in Pulmonary Hypertension

Introduction: Pulmonary hypertension (PH) is characterized by cardiopulmonary remodeling. The ensuing right ventricular (RV) hypertrophy and failure contribute to significant mortality. Pericardial effusion worsens the prognosis for PH patients, yet no mechanistic data are available. Hypothesis: We hypothesized that lymphatic vessels in rat models of PH would be decreased and dysfunctional compared to controls. Methods: We used the Sugen 5416-hypoxia (Su-Hx) and monocrotaline (MCT) models of PH. To assess the degree of cardiac lymphatic vascularity, identify cell types, and to evaluate cell junction complexes in PH rats vs. controls, we used flow cytomtery, qPCR, immunofluorescence, and immunoblot. To assess myocardial edema, we used wet-to-dry weight ratios, echocardiography, and magnetic resonance imaging (MRI). In patients, we used echocardiography and magnetic resonance imaging to quantify RV edema and pericardial effusion. Results: We found that the presence of pericardial effusion by either MRI or echocardiography correlated with lower cardiac output in patients with PH (n =6). We found decreased lymphatics in RVs of rats with PH due to either Su-Hx or MCT, compared to controls (n= 5 per group). In the heart, AQP-1+/vWF-/KDR- lymphatic vessels co-expressed podoplanin and were distributed throughout the RV in controls and much less so in PH rats. Lymphatics appeared unchanged in size or number in the left ventricle of all rats. We found increases in myocardial fluid in RVs of PH rats vs. controls, which correlated positively with fibrosis and negatively to cardiac output. The presence of excess myocardial fluid coincided with collagen deposition but without increases in CD68, CD11b, CD11c, or CD163 macrophage infiltration. We observed disturbed connexin-43 and claudin-5 junctional complexes around alpha-actinin+ myocytes and TCF21+ cardiac fibroblasts in PH RVs compared to controls. Conclusions: In severe PH, RV function is likely impaired by insufficient lymphatic-mediated fluid clearance and the presence of dysfunctional cell junctions. A clearer understanding of how the cardiac lymphatic system impacts the pathobiology of PH may lead to novel therapy.

Cerebral ABC Transporter-common Mechanisms May Modulate Neurodegenerative Diseases and Depression in Elderly Subjects

In elderly subjects, depression and dementia often coincide but the actual reason is currently unknown. Does a causal link exist or is it just a reactive effect of the knowledge to suffer from dementia? The ABC transporter superfamily may represent a causal link between these mental disorders. Since the transporters ABCB1 and ABCC1 have been discovered as major β-amyloid-exporting molecules at the blood-brain barrier and ABCC1 was found to be directly activated by St. John's wort (SJW), depression and dementia certainly share an important pathophysiologic link. It was recognized that herbal anti-depressant formulations made from SJW are at least as effective for the treatment of unipolar depression in old age as classical pharmacotherapy, while having fewer side effects (Cochrane reports, 2008). SJW is known to activate various metabolizing and transport systems in the body, with cytochrome P450 enzymes and ABC transporters being most important. Does the treatment of depression in elderly subjects using pharmacological compounds or phytomedical extracts target a mechanism that also accounts for peptide storage in Alzheimer's disease and perhaps other proteopathies of the brain? In this review we summarize recent data that point to a common mechanism and present the first promising causal treatment results of demented elderly subjects with distinct SJW extracts. Insufficient trans-barrier clearance may indeed present a common problem in all the proteopathies of the brain where toxic peptides are deposited in a location-specific manner. Thus, activation of efflux molecules holds promise for future treatment of this large group of devastating disorders.

Aldehydic load and aldehyde dehydrogenase 2 profile during the progression of post-myocardial infarction cardiomyopathy: Benefits of Alda-1

Background/objectivesWe previously demonstrated that reducing cardiac aldehydic load by aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme responsible for metabolizing the major lipid peroxidation product, protects against acute ischemia/reperfusion injury and chronic heart failure. However, time-dependent changes in ALDH2 profile, aldehydic load and mitochondrial bioenergetics during progression of post-myocardial infarction (post-MI) cardiomyopathy are unknown and should be established to determine the optimal time window for drug treatment. MethodsHere we characterized cardiac ALDH2 activity and expression, lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE) adduct formation, glutathione pool and mitochondrial energy metabolism and H2O2 release during the 4weeks after permanent left anterior descending (LAD) coronary artery occlusion in rats. ResultsWe observed a sustained disruption of cardiac mitochondrial function during the progression of post-MI cardiomyopathy, characterized by >50% reduced mitochondrial respiratory control ratios and up to 2 fold increase in H2O2 release. Mitochondrial dysfunction was accompanied by accumulation of cardiac and circulating lipid peroxides and 4-HNE protein adducts and down-regulation of electron transport chain complexes I and V. Moreover, increased aldehydic load was associated with a 90% reduction in cardiac ALDH2 activity and increased glutathione pool. Further supporting an ALDH2 mechanism, sustained Alda-1 treatment (starting 24h after permanent LAD occlusion surgery) prevented aldehydic overload, mitochondrial dysfunction and improved ventricular function in post-MI cardiomyopathy rats. ConclusionTaken together, our findings demonstrate a disrupted mitochondrial metabolism along with an insufficient cardiac ALDH2-mediated aldehyde clearance during the progression of ventricular dysfunction, suggesting a potential therapeutic value of ALDH2 activators during the progression of post-myocardial infarction cardiomyopathy.

Duration of Gestation and Mode of Delivery Affect the Genes of Transepithelial Sodium Transport in Pulmonary Adaptation

Background: Respiratory distress due to inadequate lung liquid clearance is a significant problem in infants delivered late preterm or early term, especially by elective cesarean delivery (CD). Lung liquid clearance depends on epithelial ion transport and in animals is induced by glucocorticoids. Objectives: In newborn late preterm and term infants to study airway epithelial gene expressions of epithelial sodium channel (ENaC), and the serum and glucocorticoid-inducible kinase 1 (SGK1), and their association with cortisol, mode of delivery, and gestational age (GA). Methods: Infants were delivered at 35^0/7-41^6/7 weeks. Cortisol in umbilical cord plasma was analyzed with liquid chromatography-tandem mass spectrometry. ENaC and SGK1 mRNAs in airway epithelial cells obtained within 3 h and at 1 day postnatally were quantified with real-time PCR. Results: ENaC and SGK1 mRNAs were significantly lower in late preterm and early term infants than in those ≥39^0/7 weeks. Significant correlations existed between both ENaC and SGK1 and cord cortisol and GA. In term infants, SGK1 mRNA at 1.5 h was higher after vaginal delivery than elective CD. Conclusions: In late preterm and early term infants, low expression of ENaC and SGK1 may parallel insufficient lung liquid clearance predisposing to respiratory distress. Lower SGK1 expression after term CD could translate into insufficient sodium and lung liquid absorption. The findings demonstrate a central role for cortisol in regulation of ENaC and potentially perinatal sodium and lung liquid clearance.

Phagocytosis of Abeta by infiltrating myeloid cells in a mouse model of Alzheimer's disease

During Alzheimer's disease (AD) pathogenesis, Abeta peptides accumulate, build plaques in the brain and lead to neurodegeneration and thus, cognitive impairment. Plaque formation depends not solely on the amount of Abeta which is produced, but also on the effective removal of the peptide. The latter process is predominantly accomplished by microglia, the resident immune cells of the central nervous system (CNS). However, it has been shown that upon chronic exposure to Abeta microglia become inactivated, leading to insufficient clearance.

Lysosomes and α-synuclein form a dangerous duet leading to neuronal cell death.

Neurodegenerative diseases are (i) characterized by a selective neuronal vulnerability to degeneration in specific brain regions; and (ii) likely to be caused by disease-specific protein misfolding. Parkinson’s disease (PD) is characterized by the presence of intraneuronal proteinacious cytoplasmic inclusions, called Lewy Bodies (LB). α-Synuclein, an aggregation prone protein, has been identified as a major protein component of LB and the causative for autosomal dominant PD. Lysosomes are responsible for the clearance of long-lived proteins, such as α-synuclein, and for the removal of old or damaged organelles, such as mitochondria. Interestingly, PD-linked α-synuclein mutants and dopamine-modified wild-type α-synuclein block its own degradation, which result in insufficient clearance, leading to its aggregation and cell toxicity. Moreover, both lysosomes and lysosomal proteases have been found to be involved in the activation of certain cell death pathways. Interestingly, lysosomal alterations are observed in the brains of patients suffering from sporadic PD and also in toxic and genetic rodent models of PD-related neurodegeneration. All these events have unraveled a causal link between lysosomal impairment, α-synuclein accumulation, and neurotoxicity. In this review, we emphasize the pathophysiological mechanisms connecting α-synuclein and lysosomal dysfunction in neuronal cell death.

Fragility curves for isolated bridges in eastern Canada using experimental results

One option to mitigate the seismic risk of highway bridges in Quebec is to replace typical elastomeric bearings used there with natural-rubber seismic-isolator devices. To support this alternative, this paper assesses the seismic vulnerability of typical bridge classes retrofitted with seismic-isolator devices through the development of fragility curves. Retrofitted-bridge fragility curves provide a powerful tool to evaluate the impact of a retrofit measure on the performance of different bridge classes. The analytical fragility approach uses nonlinear time-history analysis with 3-D detailed models for typical configurations of highway bridges. Experimental results of square bearings with different sizes and shape factors are used to account for uncertainties related to the mechanical properties of seismic-isolators. Critical load tests are conducted on slender seismic isolation bearings and a finite-element model is calibrated with the test results to define the seismic-isolator limit states. The fragility curves of different key components of the bridge system are compared and the results reveal that seismic isolation is effective significantly in reducing the probability of damage to columns and foundations. Due to insufficient clearance between the superstructure and abutment wing walls, however, the probability of damage in wing walls is increased and the fragility of this component controls the bridge-system fragility for all bridge classes evaluated. Concrete-girder bridges are found to be more vulnerable than steel-girder bridges due to the larger superstructure mass involved in the seismic response. The results from this work can be used to evaluate and select bridge retrofits, and can form the basis for cost-benefit retrofit studies.

WS15.2 The secreted mucus proteome in ileum of a cystic fibrosis mouse model

Objectives: Cystic fibrosis (CF) is caused by mutations in the gene encoding the CFTR, an ion channel transporting chloride and bicarbonate ions. Patients with CF have stagnant mucus, resulting in insufficient mucus clearance, bacterial infections and lung damage. The main constituent of mucus is secreted, gel forming mucins, stored in goblet cells. Mucus is densely packed in goblet cell granulae, the packing being tightly controlled by the N-terminal part of the mucin and requires calcium ions, as we have shown for the intestinal MUC2 mucin. Properties of mucus expansion in the ileum would presumably be general for all gel forming, secreted mucins, such as MUC5AC in the airways. To increase mucociliary clearance, the usefulness of hypertonic saline (HS) has been investigated in patients with CF at least since 1994, but the mechanism of action remains undetermined. Methods: Using an explant system, we have previously shown that the mucus of the small intestine is easily removable in the WT mouse ileum, whereas it is attached to the epithelium in mice without a functional CFTR channel (CftrDF508). This phenotype could be reverted to a non-attached phenotype by apical solutions containing above 100mM bicarbonate. Results: In the CF mouse ileum, hypertonic saline between 1.75 and 5% detached the mucus from the epithelium. Between 2 and 5% the mucus also expanded over 1 hour. At 2%, HS did not make CftrDF508 ileal mucus penetrable to beads the size of bacteria. Conclusion: HS, a common CF therapy, was effective in detaching the mucus, indicating a mechanism of action. Further research is required to identify the molecular identity of the mucus attachment in CF.

THU0055 Elevated Cfdna is Associated with Active Lupus Nephritis and May Mainly Derive from Netosis in Systemic Lupus Erythematosus

BackgroundAbnormal formation and insufficient clearance of neutrophil extracellular traps (NETs) has been involved in lupus nephritis (LN). The residual NETs is one potential source of circulating cell-free DNA (cfDNA). However,...

A pathogenic role for SHP-1 activity in macrophages (HUM1P.321)

Abstract Microbial infections elicit inflammatory responses that eradicate infection and remove damaged cells. Regulation of these events is critical as excessive inflammation may cause substantial bystander tissue destruction in the process of pathogen clearance while a deficient response may be insufficient to clear infections and damaged cells. Defective clearance of dying cells can trigger devastating chronic inflammatory diseases, and we report an unanticipated role for SHP-1 in facilitating this process. SHP-1 restricts innate immunity to pathogens, and therefore mice lacking SHP-1 suffer macrophage-mediated destruction of myelin in the CNS following infection with Theiler’s murine encephalomyelitis virus (TMEV). To the contrary, we have discovered that although resistant to CNS disease, TMEV-infected wild type animals develop a severe debilitating skeletal muscle disease not seen in SHP-1-/- mice. We show that SHP-1-mediated restriction of macrophage responses in WT mice corresponds with insufficient clearance of damaged tissue and increased muscle dysfunction. As SHP-1 controls macrophage phagocytic activity, our data suggests that increased macrophage activity in SHP-1-/- mice restricts muscle disease through rapid clearance of infected and damaged myofibers. These findings demonstrate a unique role of SHP-1 in exacerbating rather than resolving disease pathology by inhibiting clearance of cellular debris. These data suggest novel therapies for chronic inflammatory diseases.

The pathogenesis and diagnosis of systemic lupus erythematosus: still not resolved

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with various clinical manifestations affecting different tissues. A characteristic feature of SLE is the presence of autoantibodies against double-stranded (ds)DNA, histones and nucleosomes, and other chromatin components. SLE is a prototype type III hypersensitivity reaction. Local deposition of anti-nuclear antibodies in complex with released chromatin induces serious inflammatory conditions by activation of the complement system. The severe renal manifestation, lupus nephritis, is classified based on histological findings in renal biopsies. Apoptotic debris, including chromatin, is present in the extracellular matrix and circulation of patients with SLE. This may be due to an aberrant process of apoptosis and/or insufficient clearance of apoptotic cells/chromatin. The non-cleared apoptotic debris may lead to activation of both the innate and adaptive immune systems. In addition, an aberrant presentation of peptides by antigen-presenting cells, disturbed selection processes for lymphocytes, and deregulated lymphocyte responses may be involved in the development of autoimmunity. In the present review, we briefly will summarize current knowledge on the pathogenesis of SLE. We will also critically discuss and challenge central issues that need to be addressed in order to fully understand the pathogenic mechanisms involved in the development of SLE and in order to have an improved diagnosis for SLE. Disappointingly, in our opinion, there are still more questions than answers for the pathogenesis, diagnosis, and treatment of SLE.

Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia

Using a whole-exome sequencing strategy, we identified recessive CCNO (encoding cyclin O) mutations in 16 individuals suffering from chronic destructive lung disease due to insufficient airway clearance. Respiratory epithelial cells showed a marked reduction in the number of multiple motile cilia (MMC) covering the cell surface. The few residual cilia that correctly expressed axonemal motor proteins were motile and did not exhibit obvious beating defects. Careful subcellular analyses as well as in vitro ciliogenesis experiments in CCNO-mutant cells showed defective mother centriole generation and placement. Morpholino-based knockdown of the Xenopus ortholog of CCNO also resulted in reduced MMC and centriole numbers in embryonic epidermal cells. CCNO is expressed in the apical cytoplasm of multiciliated cells and acts downstream of multicilin, which governs the generation of multiciliated cells. To our knowledge, CCNO is the first reported gene linking an inherited human disease to reduced MMC generation due to a defect in centriole amplification and migration.

Insights into the molecular interactions between aminopeptidase and amyloid beta peptide using molecular modeling techniques.

Amyloid beta (Aβ) peptides play a central role in the pathogenesis of Alzheimer's disease. The accumulation of Aβ peptides in AD brain was caused due to overproduction or insufficient clearance and defects in the proteolytic degradation of Aβ peptides. Hence, Aβ peptide degradation could be a promising therapeutic approach in AD treatment. Recent experimental report suggests that aminopeptidase from Streptomyces griseus KK565 (SGAK) can degrade Aβ peptides but the interactive residues are yet to be known in detail at the atomic level. Hence, we developed the three-dimensional model of aminopeptidase (SGAK) using SWISS-MODEL, Geno3D and MODELLER. Model built by MODELLER was used for further studies. Molecular docking was performed between aminopeptidase (SGAK) with wild-type and mutated Aβ peptides. The docked complex of aminopeptidase (SGAK) and wild-type Aβ peptide (1IYT.pdb) shows more stability than the other complexes. Molecular docking and MD simulation results revealed that the residues His93, Asp105, Glu139, Glu140, Asp168 and His255 are involved in the hydrogen bonding with Aβ peptide and zinc ions. The interactions between carboxyl oxygen atoms of Glu139 of aminopeptidase (SGAK) with water molecule suggest that the Glu139 may be involved in the nucleophilic attack on Ala2-Glu3 peptide bond of Aβ peptide. Hence, amino acid Glu139 of aminopeptidase (SGAK) might play an important role to degrade Aβ peptides, a causative agent of Alzheimer's disease.