Impact of inflammation,emphysema, and smoking cessation on V/Q in mouse models of lung obstruction

Background

Chronic obstructive pulmonary disease (COPD) is known to greatly affect ventilation (V) and perfusion (Q) of the lung through pathologies such as inflammation and emphysema. However, there is little direct evidence regarding how these pathologies contribute to the V/Q mismatch observed in COPD and models thereof. Also, little is known regarding how smoking cessation affects V/Q relationships after inflammation and airspace enlargement have become established. To this end, we have quantified V/Q on a per-voxel basis using single photon emission computed tomography (SPECT) in mouse models of COPD and lung obstruction.

Methods

Three distinct murine models were used to investigate the impact of different pathologies on V/Q, as measured by SPECT. Lipopolysaccharide (LPS) was used to produce neutrophilic inflammation, porcine pancreatic elastase (PPE) was used to produce emphysema, and long-term cigarette smoke (CS) exposure and cessation were used to investigate the combination of these pathologies.

Results

CS exposure resulted in an increase in mononuclear cells and neutrophils, an increase in airspace enlargement, and an increase in V/Q mismatching. The inflammation produced by LPS was more robust and predominantly neutrophilic, compared to that of cigarette smoke; nevertheless, inflammation alone caused V/Q mismatching similar to that seen with long-term CS exposure. The emphysematous lesions caused by PPE administration were also capable of causing V/Q mismatch in the absence of inflammation. Following CS cessation, inflammatory cell levels returned to those of controls and, similarly, V/Q measures returned to normal despite evidence of persistent mild airspace enlargement.

Conclusions

Both robust inflammation and extensive airspace enlargement, on their own, were capable of producing V/Q mismatch. As CS cessation resulted in a return of V/Q mismatching and inflammatory cell counts to control levels, lung inflammation is likely a major contributor to V/Q mismatch observed in the cigarette smoke exposure model as well as in COPD patients. This return of V/Q mismatching to control values also took place in the presence of mild airspace enlargement, indicating that emphysematous lesions must be of a larger volume before affecting the lung significantly. Early smoking cessation is therefore critical before emphysema has an irreversible impact on gas exchange.     

Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.Cilia and flagella are prominent organelles of many eukaryotic cells. The names “cilia” and “flagella” are often related to historical reasons but they correspond to the same entity: a cylindrical organelle surrounded by a membrane and composed of an axoneme, a set of nine doublet microtubules originating from the basal body. Motile cilia usually contain a central pair of single microtubules and various substructures involved in the generation or the control of flagellar or ciliary beating, such as dynein arms, radial spokes, or central pair projections. This structural organization is remarkably well conserved across evolution, being encountered from protists to mammals (1). The conservation is also found at the molecular level as observed by comparative genomics between species with or without cilia and flagella (2, 3). Nevertheless, proteomic analysis revealed that in addition to the common core, many components unique to each group of eukaryotes are also present (4–8).The cilium represents a separate compartment from the cell body and does not contain any ribosomes or vesicles of any kind. The base of cilia and flagella contains projections that link each microtubule triplet of the basal body to the flagellum membrane (9). This region has been proposed to act as a barrier restricting entry of cytoplasmic proteins and ensuring retention of flagellum matrix elements (10). The transition zone is found in-between this area and the axoneme and contains several complexes of proteins (many of whom are mutated in the case of ciliopathies, genetic diseases affecting cilia function and/or formation) that contribute to the definition of the ciliary compartment (11, 12). Recent data showed that dextrans of low molecular weight are free to diffuse in the ciliary compartment as well as in the nucleus, whereas molecules of higher size (30 kDa or above) could not access these organelles. This led to the finding that a structure equivalent to the nucleopore complex is localized at the basal body area and could control access to the ciliary compartment (13). Finally, a septin barrier appears to be present close to the basis of the cilium and could control the trafficking of specific ciliary membrane proteins (14). The existence of a specific compartment comprising a large number of skeletal, matrix, and membrane proteins raises the issue of its internal organization. Key questions include the distribution of proteins, the mechanisms involved in specific distribution and the turnover during the life of the organelle.We selected to address these basic phenomena in the protist Trypanosoma brucei, well known as the etiological agent of sleeping sickness in Africa, but that is also an amenable model for cilia studies (15). It possesses a single flagellum that contains a typical 9 + 2 axoneme emerging from a depression of the cell surface called the flagellar pocket. This structure can be related to the ciliary pocket found at the base of different types of cilia in mammalian cells (16, 17). The axoneme is flanked by a lattice-like structure called the paraflagellar rod (PFR)¹ that is present as soon as the flagellum emerges from the pocket and runs to its distal end (18). The PFR contains at least 30 different proteins (19) and has been proposed to contribute to cell motility because its ablation results in cell paralysis in T. brucei (20) and in the related parasite Leishmania mexicana (21). The flagellum is attached to the cell body for most of its length, with the PFR lying close to the cell body side where a specific cytoskeletal structure termed the flagellum attachment zone (FAZ) is found (22). It is made of a unique filament composed of trypanosome-specific proteins (23, 24) and of four specialized microtubules flanked by the smooth endoplasmic reticulum (25). The flagellum plays key cellular functions as it drives cell motility (4, 26, 27), controls cell morphogenesis (28) and is responsible for parasite attachment during invasion of the salivary glands in the tsetse fly (29). Moreover, it could perform sensory functions and contribute to detection of the environment during the parasite life cycle (30). Recent data revealed the essential role of flagellum beating during fly invasion (31) but surprisingly reduction of forward motility did not affect infectivity in a mouse model (32).Purification of intact flagella from trypanosomes is a challenging task because of the adhesion to the cell body. Detergent and high-salt treatment have been used to efficiently purify the skeletal fraction of the flagellum that contains the axoneme, the PFR, and the basal body but that also includes the kinetoplast (mitochondrial genome), the FAZ, and the flagellar pocket collar (4, 33, 34). However, membrane and matrix components are totally lost during this procedure. For example, none of the intraflagellar transport (IFT) proteins that normally traffic in the flagellum matrix along peripheral microtubules (35) could be detected in samples purified by this procedure (4). We therefore decided to purify intact flagella by using a mutant strain called FLA1^RNAi where expression of an mRNA encoding a protein essential for flagellum attachment to the cell body (36) can be conditionally knocked-down by RNAi (37). FLA1^RNAi cells exhibit detached flagella from the main cell body, with the exception of the anchoring point at the basal body (37). By mechanical shearing, we found out that flagella could be severed from the cell body while preserving their membrane and their matrix elements. After purification, flagellar fractions were exhaustively characterized at the level of light and electron microscopy and their content was determined by mass spectrometry that confirmed the presence of the majority of known flagellar markers and revealed novel flagellar components. Three previously characterized proteins (the arginine kinase and two 14-3-3 proteins) and 10 hypothetical proteins were investigated in detail. Out of these 13 candidate proteins, 10 turned out to be associated to the flagellum whereas the others could not be detected experimentally. The novel ones were termed FLAM, for Flagellum Members. Remarkably, these proteins showed very specific location patterns within the flagellum including the membrane, the distal tip of the axoneme or the first proximal half of the axoneme, and displayed unexpected variations in their turnover rate. Overall, we revealed the existence of multiple subdomains within the flagellum with very specific dynamics, further demonstrating the highly sophisticated organization of the organelle.     

Habitat selection of the Corsican Nuthatch (Sitta whiteheadi) after a fire

The Corsican Nuthatch Sitta whiteheadi is a bird endemic to Corsica Island and has a very small population. Its habitat, Corsican pine Pinus nigra laricio forest, is currently restricted to less than 16,000 ha and is threatened by forest fires. In this article, we aim (1) to evaluate the effects of a large wildfire on a Nuthatch population, and (2) to identify the habitat features that influence the presence/absence of the Nuthatch after fire, so as to promote appropriate forestry practices after fire. The study has been conducted on a study plot of 300 ha which is part of a larger area severely burnt in August 2003. Habitat characteristics have been investigated on 39 plots of 1,225 m² occupied by the bird, and 22 randomly chosen plots without the Nuthatch. We observed a decrease of 37.5% in Nuthatch abundance the first spring after the fire, but the impact showed great local variation as a function of fire severity. Logistic modelling showed that the presence of Nuthatch mainly depended on the degree of crown alteration: the Nuthatch tended to be present when at least one pine had less than 2.5 m of crown burned. We have no evidence of any direct fire-induced mortality, but several effects of fire can explain this population decrease, namely, the reduction of canopy volume, the decrease of the amount of pine seeds, and the reduction of nest-site availability. These results permit us to propose a simple criterion that can help in choosing the plots to be cut where salvage logging is necessary.     

Effects of myostatin deletion in aging mice

Inhibitors of myostatin, a negative regulator of skeletal muscle mass, are being developed to mitigate aging-related muscle loss. Knock-out (KO) mouse studies suggest myostatin also affects adiposity, glucose handling and cardiac growth. However, the cardiac consequences of inhibiting myostatin remain unclear. Myostatin inhibition can potentiate cardiac growth in specific settings ( Morissette et al., 2006) , a concern because of cardiac hypertrophy is associated with adverse clinical outcomes. Therefore, we examined the systemic and cardiac effects of myostatin deletion in aged mice (27–30 months old). Heart mass increased comparably in both wild-type (WT) and KO mice. Aged KO mice maintained twice as much quadriceps mass as aged WT; however, both groups lost the same percentage (36%) of adult muscle mass. Dual-energy X-ray absorptiometry revealed increased bone density, mineral content, and area in aged KO vs. aged WT mice. Serum insulin and glucose levels were lower in KO mice. Echocardiography showed preserved cardiac function with better fractional shortening (58.1% vs. 49.4%, P  = 0.002) and smaller left ventricular diastolic diameters (3.41 vs. 2.71, P  = 0.012) in KO vs. WT mice. Phospholamban phosphorylation was increased 3.3-fold in KO hearts ( P  < 0.05), without changes in total phospholamban, sarco(endo)plasmic reticulum calcium ATPase 2a or calsequestrin. Aged KO hearts showed less fibrosis by Masson's Trichrome staining. Thus, myostatin deletion does not affect aging-related increases in cardiac mass and appears beneficial for bone density, insulin sensitivity and heart function in senescent mice. These results suggest that clinical interventions designed to inhibit skeletal muscle mass loss with aging could have beneficial effects on other organ systems as well.     

Impaired Lysosomal Trimming of N-Linked Oligosaccharides Leads to Hyperglycosylation of Native Lysosomal Proteins in Mice with α-Mannosidosis

α-Mannosidosis is caused by the genetic defect of the lysosomal α-d-mannosidase (LAMAN), which is involved in the breakdown of free α-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans, and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a model of mice with α-mannosidosis that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by two-dimensional difference gel electrophoresis, deglycosylation assays, and mass spectrometry. The analysis of cathepsin B-derived oligosaccharides revealed a hypermannosylation of glycoproteins in mice with α-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human α-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is involved not only in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparagine-linked oligosaccharides on native lysosomal proteins.The lysosomal α-d-mannosidase (LAMAN; EC 3.2.1.24) belongs to the group of at least seven lysosomal exoglycosidases which sequentially degrade oligosaccharides derived from glycoproteins (2, 31). These glycoproteins enter the lysosomal compartment by either endocytic pathways (extracellular and plasma membrane proteins) or autophagic processes (intracellular proteins). In addition, free oligosaccharides originating from lipid-linked oligosaccharides in the endoplasmic reticulum and from glycoproteins by the endoplasmic reticulum-associated protein degradation (ERAD) pathway are transported into the lysosome, where these oligosaccharides are subsequently degraded (9, 45). Inside the lysosome, the degradation of the glycoproteins is described as a bidirectional process in which on the one hand the polypeptide is hydrolyzed by a cohort of lysosomal endo- and exoproteases with partially overlapping specificities like cathepsins and other peptidases (like DPP II and TPP-I [19, 40, 52). On the other hand, the sugar moiety is stepwise hydrolyzed into its monosaccharides by exoglycosidases. The precise order of the bidirectional breakdown of glycoproteins is unclear, although assumptions can be made based on the analysis of the storage products of the different glycoproteinoses (31). Therefore, it is assumed that an efficient degradation of the oligosaccharide chain is highly dependent on the cleavage of the protein-oligosaccharide linkage by the glycosylasparaginase (2, 31). In contrast, the proteolysis of the polypeptide backbone is mainly unaffected by intact oligosaccharide structures on the glycoproteins (1).LAMAN has a broad substrate specificity, cleaving nonreducing terminal α1,2-, α1,3-, and α1,6-mannosyl linkages found in complex-type, hybrid-type, and high-mannose-type asparagine-linked glycans (30, 60). Additionally, a second lysosomal mannosidase (MAN2B2) specific for the core α1,6 branch was characterized and found to be dependent on the prior enzymatic activity of lysosomal glycosylasparaginase or chitobiase, releasing Man3GlcNAc2 and Man3GlcNAc oligosaccharides, respectively (21, 36). The cooperation of this novel core-specific α1,6-mannosidase with chitobiase is also reflected by their similar tissue-specific expression patterns in humans and rodents and their simultaneous absence in cattle and cats (2, 14).LAMAN deficiency results in the rare lysosomal storage disorder (LSD) α-mannosidosis, which is clinically characterized by progressive mental retardation, dysostosis multiplex, impaired hearing, immune defects, and mild hepatosplenomegaly. However, the onset of symptoms varies greatly and the clinical severity of α-mannosidosis patients ranges from mildly affected to severely affected, lacking a genotype-phenotype correlation (29). Patients also show elevated serum and urine oligosaccharide levels and an enlargement of the lysosomal compartment which is considered to be caused by the accumulation of undegraded oligosaccharides. The major lysosomal storage product is the trisaccharide Man2GlcNAc, although oligosaccharides with up to eight mannosyl residues were detected in the urine and serum of patients, indicating their lysosomal accumulation as well (4, 33). From these findings, one can draw the conclusion that beside metabolic intermediates of the glycoprotein degradation, a considerable number of oligosaccharides originate from dolichol-linked oligosaccharides or from glycoproteins that failed quality control in the endoplasmic reticulum and thus are degraded by the proteasome, leaving behind highly mannosylated glycans (23, 32, 41). It is assumed that 70% of the stored oligosaccharides derive from complex- and hybrid-type glycans, 10% derive from high-mannose-type glycans, and 20% derive from biosynthetic intermediates, e.g., lipid-linked oligosaccharides (61).Naturally occurring animal models for α-mannosidosis have been described for cats (8, 55), cattle (6, 24), and guinea pigs (12). The animal models have been the subjects of various studies dealing with neuropathological, behavioral, and therapeutic aspects of α-mannosidosis (3, 13, 38). It was shown with guinea pigs and cats that enzyme replacement therapy (ERT) and bone marrow transplantation, respectively, provided a benefit concerning clinical manifestations and remarkable success in the central nervous system of cats after bone marrow transplantation (13, 56).Aside from the naturally occurring models, a mouse model for α-mannosidosis was generated in which the LAMAN gene was disrupted by gene targeting. This mouse model phenotypically resembled a mild variant of the human disease (46). We exploited this mouse model to develop an ERT approach as already clinically established for other LSDs like Gaucher disease, Hunter disease, or Pompe disease. For this purpose, LAMAN preparations from different species were proven to be efficacious for visceral organs, and most remarkably, we demonstrated that high-dose administration of recombinant human LAMAN (rhLAMAN) affected the central neural storage (39). Very recently, Blanz et al. confirmed the influence of high-dosage ERT on the peripheral as well as the central nervous system in the same mouse model and showed clearance of storage material in hippocampal neurons in particular (5). Here, we report on structural alterations of lysosomal proteins in mice with α-mannosidosis due to hyperglycosylation and the reversibility by ERT.     

Arginine 469 is a pivotal residue for the Hsc70-GlcNAc-binding property

The members of the 70 kDa-heat shock proteins (HSP70) family play numerous fundamental functions in the cell such as promoting the assembly of multimeric complexes or helping the correct folding of nascent proteins to take place. In numerous previous studies we demonstrated that Hsp70 and its constitutive isoform Hsc70 are endowed of a GlcNAc-binding activity. The molecular modeling of the substrate binding domain of Hsc70 and in silico docking experiments using Ser/Thr-O-GlcNAc motifs allowed to define the potential carbohydrate-recognition region and to point out the crucial position of Arg469 as an amino-acid directly interacting with the sugar moiety. We cloned a flagged Hsc70 in a pCMV.SPORT6 vector and we showed that the mutation R469A decreased the GlcNAc-binding property of the chaperone of around 70%. This is the first work reporting the localization of the GlcNAc-binding domain of a member of the HSP70 family.     

Metabolomics to unveil and understand phenotypic diversity between pathogen populations

Leishmaniasis is a debilitating disease caused by the parasite Leishmania. There is extensive clinical polymorphism, including variable responsiveness to treatment. We study Leishmania donovani parasites isolated from visceral leishmaniasis patients in Nepal that responded differently to antimonial treatment due to differing intrinsic drug sensitivity of the parasites. Here, we present a proof-of-principle study in which we applied a metabolomics pipeline specifically developed for L. donovani to characterize the global metabolic differences between antimonial-sensitive and antimonial-resistant L. donovani isolates. Clones of drug-sensitive and drug-resistant parasite isolates from clinical samples were cultured in vitro and harvested for metabolomics analysis. The relative abundance of 340 metabolites was determined by ZIC-HILIC chromatography coupled to LTQ-Orbitrap mass spectrometry. Our measurements cover approximately 20% of the predicted core metabolome of Leishmania and additionally detected a large number of lipids. Drug-sensitive and drug-resistant parasites showed distinct metabolic profiles, and unsupervised clustering and principal component analysis clearly distinguished the two phenotypes. For 100 metabolites, the detected intensity differed more than three-fold between the 2 phenotypes. Many of these were in specific areas of lipid metabolism, suggesting that the membrane composition of the drug-resistant parasites is extensively modified. Untargeted metabolomics has been applied on clinical Leishmania isolates to uncover major metabolic differences between drug-sensitive and drug-resistant isolates. The identified major differences provide novel insights into the mechanisms involved in resistance to antimonial drugs, and facilitate investigations using targeted approaches to unravel the key changes mediating drug resistance.     

Fifty‐seven years of composition change in the eastern boreal forest of Canada

Question: In the boreal forest of eastern Canada, how does forest vegetation change in the sustained absence of fire? Location: Eastern boreal forest in Quebec's North Shore region, Canada (49°30′–50°00′N; 67°30′–68°35′W). Methods: Aerial photos from three different periods (1930, 1965 and 1987) were used to characterize changes in vegetation composition in 23 scenes of 200 ha. Time since fire, presence of secondary disturbances and data on soil and topographic variables were obtained. Ordination and clustering techniques were used to define compositional trajectories of change over the 57‐yr period. These trajectories were further grouped into pathways based on compositional changes, time since fire and preferential deposit‐drainage types. Results: Among the 26 compositional trajectories, three successional pathways were distinguished. Two start post‐fire succession with a dominance of intolerant hardwood. In one of these, this is followed by an increase in Abies balsamea, while in the second the importance of Picea mariana increases with time. In the third pathway P. mariana is an important component from the outset. In this pathway, we observed modest fluctuation in the relative dominance of P. mariana and A. balsamea and variation in stand structure. Conclusion: The boreal forest vegetation of Eastern Canada is diverse and dynamic even in the absence of fire, notably under the influence of partial disturbances. Such disturbances can be associated with changes in composition or stand structure. The development of management strategies aimed at maintaining stand diversity by emulating a broader variety of partial and secondary disturbances should be encouraged.