Myeloperoxidase Promoter Polymorphism −463G Is Associated With More Severe Clinical Expression of Cystic Fibrosis Pulmonary Disease

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the −463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation

The status of the world's land and marine mammals: diversity, threat, and knowledge

Knowledge of mammalian diversity is still surprisingly disparate, both regionally and taxonomically. Here, we present a comprehensive assessment of the conservation status and distribution of the world's mammals. Data, compiled by 1700+ experts, cover all 5487 species, including marine mammals. Global macroecological patterns are very different for land and marine species but suggest common mechanisms driving diversity and endemism across systems. Compared with land species, threat levels are higher among marine mammals, driven by different processes (accidental mortality and pollution, rather than habitat loss), and are spatially distinct (peaking in northern oceans, rather than in Southeast Asia). Marine mammals are also disproportionately poorly known. These data are made freely available to support further scientific developments and conservation action

The bii4africa dataset of faunal and floral population intactness estimates across Africa’s major land uses

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species’ population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate ‘intactness scores’: the remaining proportion of an ‘intact’ reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region’s major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/ taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems

The bii4africa dataset of faunal and floral population intactness estimates across Africa’s major land uses

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species’ population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate ‘intactness scores’: the remaining proportion of an ‘intact’ reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region’s major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems

Thiocyanate Reduces Motor Impairment in the hMPO-A53T PD Mouse Model While Reducing MPO-Oxidation of Alpha Synuclein in Enlarged LYVE1/AQP4 Positive Periventricular Glymphatic Vessels

Parkinson’s disease (PD) is due to the oxidation of alpha synuclein (αSyn) contributing to motor impairment. We developed a transgenic mouse model of PD that overexpresses the mutated human αSyn gene (A53T) crossed to a mouse expressing the human MPO gene. This model exhibits increased oxidation and chlorination of αSyn leading to greater motor impairment. In the current study, the hMPO-A53T mice were treated with thiocyanate (SCN−) which is a favored substrate of MPO as compared to chlorine. We show that hMPO-A53T mice treated with SCN− have less chlorination in the brain and show an improvement in motor skills compared to the nontreated hMPO-A53T mice. Interestingly, in the hMPO-A53T mice we found a possible link between MPO-related disease and the glymphatic system which clears waste including αSyn from the brain. The untreated hMPO-A53T mice exhibited an increase in the size of periventricular glymphatic vessels expressing the glymphatic marker LYVE1 and aquaporin 4 (AQP4). These vessels also exhibited an increase in MPO and HOCl-modified epitopes in the glymphatic vessels correlating with loss of ependymal cells lining the ventricles. These findings suggest that MPO may significantly promote the impairment of the glymphatic waste removal system thus contributing to neurodegeneration in PD. Moreover, the inhibition of MPO chlorination/oxidation by SCN− may provide a potential therapeutic approach to this disease

Candidate receptors queried for a role in MPO-trafficking using shRNA knockdown and NH₄Cl in T47D-MPO cells.

<p>(A) T47D-MPO stable cells were grown for 48 hrs in media supplemented with either 10 mM NH4Cl, or 8 mM mannose + 8 mM mannose-6-phosphate (M6P) or both. The conditioned media and cell extracts were then assayed for: (i) hexosaminidase activity using a fluorogenic substrate; (ii) MPO content by ELISA to assess the treatments effects on the secretion; (iii) hexosaminidase activity and MPO concentration determined from the corresponding assays in i and ii were graphed as a ratio of that present in cell extracts divided by that present in the conditioned media (cells/media). Assay points are quadruplicate (i) and triplicate (II) measurements and plotted as mean ± SE. The data shown is representative of at least three independent experiments. (B) Immunolocalization of cation-dependent mannose-6-phosphate receptor (cdMPR) (red) with MPO, EEA1 and RCAS1 (green) in T47D-MPO cells. (C) shRNA knockdown of the cdMPR in the T47D-MPO stable cell line was carried out using lentiviral transduction as described in the “Materials and Methods”. (i) Membrane preparations derived from cells expressing either of two cdMPR shRNAs (990 & 029), a control shRNA (scramble) or the T47D-MPO parent cell line was analyzed by immunoblot with Mab-22d4 to quantify knockdown of cdMPR protein levels. Micrograms of membrane protein loaded into each lane are indicated and, as an additional loading control for membrane protein content, the blot was reprobed for E-cadheren (E-cad). (ii, iii) To examine any combined role of ciMPR and cdMPR in MPO trafficking, the cdMPR knockdown and control cell lines were grown for 44 h ± 10 mM NH₄Cl and assayed for hexosaminidase activity and MPO content as in panel A. (iv) To examine any combined role of ciMPR and cdMPR on the processing of MPO, the cdMPR knockdown and control cell extracts from ii and iii were immunoblotted for MPO under non-reducing conditions in order to detect any changes in the relative amounts of heterotetramer and proMPO. (v) Hexosaminidase activity in CHO cells treated with and without NH₄Cl. (D) Quantitative PCR to compare expression levels of VPS10p family members in cell lines that do (HL60, T47D and MCF7) and do not (MDA231 and BT549) efficiently form the MPO heterotetramer. Expression levels are expressed as the percent of GADPH mRNA. The experiments were repeated three times with the data from a representative experiment shown. (E) shRNA knockdown of the SorLA receptor and, as a control, GADPH was carried out using lentiviral transduction as described in the “Materials and Methods”. The selected cell lines were characterized as in panels A and C. (i) Results are summarized for each knockdown cell line as the ratio of hexosaminidase or MPO in cells/media as in panel A-iii and did not differ significantly from our observations of the parent cell line. (ii) Immunoblot analysis under non-reducing conditions of cell extracts from SorLA and GADPH shRNA-expressing cell lines to demonstrate knockdown of the respective proteins (two upper blots). Blots were reprobed with MPO antibodies to detect any changes in the relative amounts of heterotetramer and proMPO linked to decreased SorLA expression, with or without concomitant inhibition of ciMPR activity by treatment of cells with 10 mM NH₄Cl (bottom blot). E-cadheren immunoreactivity was used as a loading control.</p

Effect of cathepsin inhibitors on the processing, activity and endocytic trafficking in T47D-MPO cells.

<p>(A) T47D-MPO cells were grown with or without 8 μM ALLN for 44 h. MPO was partially purified from the resulting cell extracts on SP-sepharose and analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody to detect all MPO species (left panel) or antibodies specific for epitopes within the light chain of MPO (right panel). (B) T47D-MPO cells were grown in the presence of the indicated concentrations of e64d for 44 h and the resulting cell extracts analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody. (C) Samples analyzed in panel A were assayed for peroxidase activity and total MPO content and the relative specific activity (Active MPO/Total MPO) determined as described in “Methods and Materials”. (D) T47D-MPO cells were grown on glass coverslips in the presence of 8 μM ALLN for 72 h. The treated cells were fixed, permeabolized and double-labeled with antibodies against MPO (red) and Lamp1 (green). Mounted coverslips were imaged with a 63x oil objective on a Zeiss LSM 710 confocal microscope.</p

Subcellular localization of the C316A, C319S and R569W MPO mutants stably expressed in T47D cells.

<p>Cells grown on glass coverslips were fixed, permeabilized and double-labeled with polyclonal MPO antibodies and the indicated organelle marker antibody. Mounted coverslips were imaged with a 63X objective on a Zeiss LSM 710 confocal microscope. (A) T47D-C316A cells labeled with antibodies against MPO and Lamp1 and shows that the C316A mutant accumulates normally within lysosomes. (B) T47D-C319S cells labeled with antibodies against MPO (all panels) and Lamp1 (top panel), the ER marker KDEL (second panel), the trans-Golgi marker RCAS1 (third panel) and the cis-Golgi marker GM130 (fourth panel). Distinct localizations of the C319S mutant to both the ER (arrow) and Golgi (arrowhead) are indicated on the second panel. (C) T47D-R569W cells labeled with antibodies against MPO and the ER marker protein disulfide isomerase (PDI).</p

Characterization of N-Glycosylation and Mannose-6-Phosphate modification of wildtype and mutant MPO from recombinant cell lines.

<p>(A) Cell extracts from the indicated endogenous (HL60) or recombinant MPO-expressing cell lines were treated with the glycosidases EndoH or PNGaseF as indicated and analyzed for alterations in relative mobility by immunoblot (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149391#sec002" target="_blank">Materials and Methods</a>). The four left-hand panels were probed with polyclonal multi-epitope MPO antibody and the far right panel with anti-peptide antibody specific for the “pro” domain of MPO. The results in the right-hand panel aid in the visualization of the change in glycosylation pattern observed with the C319S MPO mutant as the pro-epitope is unaffected by glycosylation pattern whereas recognition by the multi-epitope polyclonal antibody is negatively affected by the increased complex oligosaccharide content of this mutant. *Non-specific band in HT29 extracts. (B) Secreted MPO was partially purified on SP-sepharose from the conditioned media of MPO-expressing cell lines, treated with the glycosidases EndoH or PNGaseF as indicated and analyzed by immunoblot as in panel A. Of the two secreted MPO species the full-length proMPO was predominant in MDA468, BT549 and T47D-C316A cells whereas proMPO with the pro domain proteolytically removed (“cleaved” proMPO) was the predominant form observed in T47D, T47D-C319S and T47D-C316AC319S cells. (C) Eight hundred ng of MPO partially purified on SP-sepharose from native (HL60) and recombinant cells extracts or 500 ng of pure neutrophil MPO was immunoprecipitated with the Mab-16E3 MPO antibody. Immunoprecipitates were analyzed by immunoblot under reducing conditions with multi-epitope polyclonal MPO antibody to detect all MPO species (upper blots) or with a single chain variable domain (ScFv) specific for the mannose-6-phosphate (M6P) modification (lower blots). (D) Eight hundred ng of secreted MPO partially purified on SP-sepharose from the indicated cell lines was immunoprecipitated with Mab-16E3 MPO antibody. Immunoprecipitates were analyzed by immunoblot as in panel C. (C & D) The specificity of the ScFv for M6P recognition is demonstrated by the loss of signal when immunoprecipitated MPO is treated with calf intestinal phosphatase (CIP). Faint bands on the bottom of ScFv blots are background recognition of the immunoprecipitating antibody that migrates just below the MPO heavy chain on SDS-PAGE. (E) Untransfected T47D, MCF7, BT547, MDA231 and MDA468 cells, were fixed, permeabilized and stained for total cellular M6P levels using the soluble form of bovine cation-independent mannose-6-phosphate receptor (sciMPR). Binding of the sciMPR was detected with Mab-86f7, which binds to bovine, but not human ciMPR. Lysosomes are stained with antibodies against Lamp2. Competitive inhibition of sciMPR binding with 5 mM free M6P in T47D cells was included as a specificity control.</p