Analysis of Signaling Endosome Composition and Dynamics Using SILAC in Embryonic Stem Cell-Derived Neurons

Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first quantitative proteomic analysis of signaling endosomes isolated from motor neurons and allows the assembly of a functional map of these axonal carriers involved in long-range neuronal signaling

The fidelity of synaptonemal complex assembly is regulated by a signaling mechanism that controls early meiotic progression

© 2014 Elsevier Inc.Proper chromosome segregation during meiosis requires the assembly of the synaptonemal complex (SC) between homologous chromosomes. However, the SC structure itself is indifferent to homology, andpoorly understood mechanisms that depend on conserved HORMA-domain proteins prevent ectopic SC assembly. Although HORMA-domain proteins are thought to regulate SC assembly as intrinsic components of meiotic chromosomes, here we uncover a key role for nuclear soluble HORMA-domain protein HTP-1 in the quality control of SC assembly. We show that a mutant form of HTP-1 impaired in chromosome loading provides functionality of an HTP-1-dependent checkpoint that delays exit from homology search-competent stages until all homolog pairs are linked by the SC. Bypassing of this regulatory mechanism results in premature meiotic progression and licensing of homology-independent SC assembly. These findings identify nuclear soluble HTP-1 as a regulator of early meiotic progression, suggesting parallels with the mode of action of Mad2 in the spindle assembly checkpoint

Cyclin D mediates tolerance of genome-doubling in cancers with functional p53

BACKGROUND: Aneuploidy and chromosomal instability (CIN) are common features of human malignancy that fuel genetic heterogeneity. Although tolerance to tetraploidization, an intermediate state that further exacerbates CIN, is frequently mediated by TP53 dysfunction, we find that some genome-doubled tumours retain wild-type TP53. We sought to understand how tetraploid cells with a functional p53/p21-axis tolerate genome-doubling events. METHOD: We performed quantitative proteomics in a diploid/tetraploid pair within a system of multiple independently derived TP53 wild-type tetraploid clones arising spontaneously from a diploid progenitor. We characterized adapted and acute tetraploidization in a variety of flow cytometry and biochemical assays and tested our findings against human tumours through bioinformatics analysis of the TCGA dataset. RESULTS: Cyclin D1 was found to be specifically overexpressed in early but not late passage tetraploid clones, and this overexpression was sufficient to promote tolerance to spontaneous and pharmacologically induced tetraploidy. We provide evidence that this role extends to D-type cyclins and their overexpression confers specific proliferative advantage to tetraploid cells. We demonstrate that tetraploid clones exhibit elevated levels of functional p53 and p21 but override the p53/p21 checkpoint by elevated expression of cyclin D1, via a stoichiometry-dependent and CDK activity-independent mechanism. Tetraploid cells do not exhibit increased sensitivity to abemaciclib, suggesting that cyclin D-overexpressing tumours might not be specifically amenable to treatment with CDK4/6 inhibitors. CONCLUSION: Our study suggests that D-type cyclin overexpression is an acute event, permissive for rapid adaptation to a genome-doubled state in TP53 wild-type tumours and that its overexpression is dispensable in later stages of tumour progression

Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia

Patients with autosomal dominant hypercholesterolemia (ADH) have a high risk of developing cardiovascular disease that can be effectively treated using statin drugs. Molecular diagnosis and family cascade screening is recommended for early identification of individuals at risk, but up to 40% of families have no mutation detected in known genes. This study combined linkage analysis and exome sequencing to identify a novel variant in exon 3 of APOB (Arg50Trp). Mass spectrometry established that low-density lipoprotein (LDL) containing Arg50Trp APOB accumulates in the circulation of affected individuals, suggesting defective hepatic uptake. Previously reported mutations in APOB causing ADH have been located in exon 26. This is the first report of a mutation outside this region causing this phenotype, therefore, more extensive screening of this large and highly polymorphic gene may be necessary in ADH families. This is now feasible due to the high capacity of recently available sequencing platforms

Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis

Microbiological testing of adults hospitalised with community-acquired pneumonia: An international study

This study aimed to describe real-life microbiological testing of adults hospitalised with community-acquired pneumonia (CAP) and to assess concordance with the 2007 Infectious Diseases Society of America (IDSA)/American Thoracic Society (ATS) and 2011 European Respiratory Society (ERS) CAP guidelines. This was a cohort study based on the Global Initiative for Methicillin-resistant Staphylococcus aureus Pneumonia (GLIMP) database, which contains point-prevalence data on adults hospitalised with CAP across 54 countries during 2015. In total, 3702 patients were included. Testing was performed in 3217 patients, and included blood culture (71.1%), sputum culture (61.8%), Legionella urinary antigen test (30.1%), pneumococcal urinary antigen test (30.0%), viral testing (14.9%), acute-phase serology (8.8%), bronchoalveolar lavage culture (8.4%) and pleural fluid culture (3.2%). A pathogen was detected in 1173 (36.5%) patients. Testing attitudes varied significantly according to geography and disease severity. Testing was concordant with IDSA/ATS and ERS guidelines in 16.7% and 23.9% of patients, respectively. IDSA/ATS concordance was higher in Europe than in North America (21.5% versus 9.8%; p<0.01), while ERS concordance was higher in North America than in Europe (33.5% versus 19.5%; p<0.01). Testing practices of adults hospitalised with CAP varied significantly by geography and disease severity. There was a wide discordance between real-life testing practices and IDSA/ATS/ERS guideline recommendations

Prevalence and etiology of community-acquired pneumonia in immunocompromised patients

Background. The correct management of immunocompromised patients with pneumonia is debated. We evaluated the prevalence, risk factors, and characteristics of immunocompromised patients coming from the community with pneumonia. Methods. We conducted a secondary analysis of an international, multicenter study enrolling adult patients coming from the community with pneumonia and hospitalized in 222 hospitals in 54 countries worldwide. Risk factors for immunocompromise included AIDS, aplastic anemia, asplenia, hematological cancer, chemotherapy, neutropenia, biological drug use, lung transplantation, chronic steroid use, and solid tumor. Results. At least 1 risk factor for immunocompromise was recorded in 18% of the 3702 patients enrolled. The prevalences of risk factors significantly differed across continents and countries, with chronic steroid use (45%), hematological cancer (25%), and chemotherapy (22%) the most common. Among immunocompromised patients, community-acquired pneumonia (CAP) pathogens were the most frequently identified, and prevalences did not differ from those in immunocompetent patients. Risk factors for immunocompromise were independently associated with neither Pseudomonas aeruginosa nor non\u2013community-acquired bacteria. Specific risk factors were independently associated with fungal infections (odds ratio for AIDS and hematological cancer, 15.10 and 4.65, respectively; both P = .001), mycobacterial infections (AIDS; P = .006), and viral infections other than influenza (hematological cancer, 5.49; P < .001). Conclusions. Our findings could be considered by clinicians in prescribing empiric antibiotic therapy for CAP in immunocompromised patients. Patients with AIDS and hematological cancer admitted with CAP may have higher prevalences of fungi, mycobacteria, and noninfluenza viruses

Atypical pathogens in hospitalized patients with community-acquired pneumonia: A worldwide perspective

Background: Empirical antibiotic coverage for atypical pathogens in community-acquired pneumonia (CAP) has long been debated, mainly because of a lack of epidemiological data. We aimed to assess both testing for atypical pathogens and their prevalence in hospitalized patients with CAP worldwide, especially in relation with disease severity. Methods: A secondary analysis of the GLIMP database, an international, multicentre, point-prevalence study of adult patients admitted for CAP in 222 hospitals across 6 continents in 2015, was performed. The study evaluated frequency of testing for atypical pathogens, including L. pneumophila, M. pneumoniae, C. pneumoniae, and their prevalence. Risk factors for testing and prevalence for atypical pathogens were assessed through univariate analysis. Results: Among 3702 CAP patients 1250 (33.8%) underwent at least one test for atypical pathogens. Testing varies greatly among countries and its frequency was higher in Europe than elsewhere (46.0% vs. 12.7%, respectively, p &lt; 0.0001). Detection of L. pneumophila urinary antigen was the most common test performed worldwide (32.0%). Patients with severe CAP were less likely to be tested for both atypical pathogens considered together (30.5% vs. 35.0%, p = 0.009) and specifically for legionellosis (28.3% vs. 33.5%, p = 0.003) than the rest of the population. Similarly, L. pneumophila testing was lower in ICU patients. At least one atypical pathogen was isolated in 62 patients (4.7%), including M. pneumoniae (26/251 patients, 10.3%), L. pneumophila (30/1186 patients, 2.5%), and C. pneumoniae (8/228 patients, 3.5%). Patients with CAP due to atypical pathogens were significantly younger, showed less cardiovascular, renal, and metabolic comorbidities in comparison to adult patients hospitalized due to non-atypical pathogen CAP. Conclusions: Testing for atypical pathogens in patients admitted for CAP in poorly standardized in real life and does not mirror atypical prevalence in different settings. Further evidence on the impact of atypical pathogens, expecially in the low-income countries, is needed to guidelines implementation