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BackgroundThere continues to be a great need for better biomarkers and host-directed treatment targets for community-acquired pneumonia (CAP). Alterations in phospholipid metabolism may constitute a source of small molecule biomarkers for acute infections including CAP. Evidence from animal models of pulmonary infections and sepsis suggests that inhibiting acid sphingomyelinase (which releases ceramides from sphingomyelins) may reduce end-organ damage.MethodsWe measured concentrations of 105 phospholipids, 40 acylcarnitines, and 4 ceramides, as well as acid sphingomyelinase activity, in plasma from patients with CAP (n=29, sampled on admission and 4 subsequent time points), chronic obstructive pulmonary disease exacerbation with infection (COPD, n=13) as a clinically important disease control, and 33 age- and sex-matched controls.ResultsPhospholipid concentrations were greatly decreased in CAP and normalized along clinical improvement. Greatest changes were seen in phosphatidylcholines, followed by lysophosphatidylcholines, sphingomyelins and ceramides (three of which were upregulated), and were least in acylcarnitines. Changes in COPD were less pronounced, but also differed qualitatively, e.g. by increases in selected sphingomyelins. We identified highly accurate biomarkers for CAP (AUC <= 0.97) and COPD (AUC <= 0.93) vs. Controls, and moderately accurate biomarkers for CAP vs. COPD (AUC <= 0.83), all of which were phospholipids. Phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins were also markedly decreased in S. aureus-infected human A549 and differentiated THP1 cells. Correlations with C-reactive protein and procalcitonin were predominantly negative but only of mild-to-moderate extent, suggesting that these markers reflect more than merely inflammation. Consistent with the increased ceramide concentrations, increased acid sphingomyelinase activity accurately distinguished CAP (fold change=2.8, AUC=0.94) and COPD (1.75, 0.88) from Controls and normalized with clinical resolution.ConclusionsThe results underscore the high potential of plasma phospholipids as biomarkers for CAP, begin to reveal differences in lipid dysregulation between CAP and infection-associated COPD exacerbation, and suggest that the decreases in plasma concentrations are at least partially determined by changes in host target cells. Furthermore, they provide validation in clinical blood samples of acid sphingomyelinase as a potential treatment target to improve clinical outcome of CAP.

Methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage is a major risk factor for infection, namely among populations in the community with inherent prompting factors, such as the homeless. In Portugal, there are no data on S. aureus/MRSA nasal carriage among the homeless community. A total of 84 homeless individuals living in Lisbon (34 with no permanent address and 50 living in shelter) were nasally screened for S. aureus/ MRSA. All isolates were characterized to determine antimicrobial susceptibility and clonal type. A total of 43 (51.2%) S. aureus carriers were identified, including a single individual colonized with MRSA (1.2%). S. aureus carriage rate was higher among individuals with no permanent address (58.8% versus 46%), younger (45.7 +/- 12.7 versus 52.5 +/- 10.8 years), and with diagnosis of asthma (9% versus 0%). The single MRSA belonged to the EMRSA-15 clone (PFGE D, ST15-SCCmec IVh, and spa type t790). Almost half of the methicillin-susceptible S. aureus (MSSA) isolates (41.9%, n = 18) belonged to two major clones, ST398-t1451 (n = 13) and ST30-t399/t11980/t12808 associated with PFGE I (n = 5). A high proportion of isolates showed non-susceptibility to mupirocin (64%), erythromycin (45%), and fusidic acid (20%) and induced resistance to clindamycin (39%). None of the isolates harboured PVL. Our results suggest that the homeless population of Lisbon does not constitute a reservoir of MRSA in the community, but harbour the highly transmissible ST398-t1451 MSSA lineage.

In the last years, the ionic conductance behavior of solid-state nanochannels (SSN) has been extensively studied with both basic and applied purposes. In particular, the interactions between confined groups and dissolved species have been widely used for the design of biosensors and smart devices. Being the species confined to the small volume of the SSN, the ionic equilibrium usually differs from that in the solution bulk and nanoconfinement effects appear. In this work, we study the binding equilibrium between surface-confined amine groups and phosphate anions taking place within SSN by measuring the changes in the iontronic transmembrane current response of single nanochannels at different phosphate concentrations. Phosphate binding is higher compared with other divalent anions and takes place even in electrostatically hindered conditions, which reinforces the idea of chemical specificity of the amine-phosphate interaction. The sensitivity of the iontronic response of asymmetric SSN to changes in the surface charge allowed the interpretation of the experimental results in terms of a simple binding model, which reveals that the nanoconfinement effects are responsible for a one order of magnitude increase in the effective constants for the anion binding to the surface amine groups in the nanochannel walls. Furthermore, polyphosphates show a more pronounced binding tendency toward amine moieties, which allows the detection and quantification of ATP in the micromolar range from the analysis of the iontronic response.

Chronic Intestinal Pseudo-Obstruction (CIPO) is a rare gastrointestinal disorder, which affects the smooth muscle contractions of the gastrointestinal tract. Dominant mutations in the smooth muscle actin gene, ACTG2, accounts for 44%-50% of CIPO patients. Other recessive or X-linked genes, including MYLK, LMOD1, RAD21, MYH11, MYL9, and FLNA were reported in single cases. In this study, we used Whole-Exome Sequencing (WES) to study 23 independent CIPO families including one extended family with 13 affected members. A dominantly inherited rare mutation, c.5819delC (p.Pro1940HisfsTer91), in the smooth muscle myosin gene, MYH11, was found in the extended family, shared by 7 affected family members but not by 3 unaffected family members with available DNA, suggesting a high probability of genetic linkage. Gene burden analysis indicates that additional genes, COL4A1, FBLN1 and HK2, may be associated with the disease. This study expanded our understanding of CIPO etiology and provided additional genetic evidence to physicians and genetic counselors for CIPO diagnosis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Microsporidia are eukaryotic parasites that infect essentially all animal species, including many of agricultural importance(1-3), and are significant opportunistic parasites of humane. They are characterized by having a specialized infection apparatus, an obligate intracellular lifestyles(5), rudimentary mitochondria and the smallest known eukaryotic genomess(5-7). Extreme genome compaction led to minimal gene sizes affecting even conserved ancient complexes such as the ribosomes(8-10). In the present study, the cryo-electron microscopy structure of the ribosome from the microsporidium Vairimorpha necatrix is presented, which illustrates how genome compaction has resulted in the smallest known eukaryotic cytoplasmic ribosome. Selection pressure led to the loss of two ribosomal proteins and removal of essentially all eukaryote-specific ribosomal RNA (rRNA) expansion segments, reducing the rRNA to a functionally conserved core. The structure highlights how one microsporidia-specific and several repurposed existing ribosomal proteins compensate for the extensive rRNA reduction. The microsporidian ribosome is kept in an inactive state by two previously uncharacterized dormancy factors that specifically target the functionally important E-site, P-site and polypeptide exit tunnel. The present study illustrates the distinct effects of evolutionary pressure on RNA and proteincoding genes, provides a mechanism for ribosome inhibition and can serve as a structural basis for the development of inhibitors against microsporidian parasites.

Eukaryotic chromosomes contain compartments of various functions, which are marked by and enriched with specific histone modifications. However, the molecular mechanisms by which these histone marks function in chromosome compartmentalization are poorly understood. Constitutive heterochromatin is a largely silent chromosome compartment characterized in part by H3K9me2 and 3. Here, we show that heterochromatin protein 1 (HP1), an H3K9me2 and 3 "reader," interacts with SUV39H1, an H3K9me2 and 3 "writer," and with TRIM28, an abundant HP1 scaffolding protein, to form complexes with increased multivalent engagement of H3K9me2 and 3-modified chromatin. H3K9me2 and 3-marked nucleosomal arrays and associated complexes undergo phase separation to form macromolecule-enriched liquid droplets. The droplets are reminiscent of heterochromatin as they are highly dense chromatin-containing structures that are resistant to DNase and exclude the general transcription factor TFIIB. Our data suggest a general mechanism by which histone marks regulate chromosome compartmentalization by promoting phase separation.

Follicular dendritic cells (FDCs), a rare and enigmatic stromal cell type in the B cell follicles of secondary lymphoid organs, store and present antigen to B cells. While essential for germinal center (GC) responses, their exact role during GC B cell selection remains unknown. FDCs upregulate the inhibitory IgG Fc receptor Fc gamma RIIB during GC formation. We show that the stromal deficiency of Fc gamma RIIB does not affect GC B cell frequencies compared to wildtype mice. However, in the absence of Fc gamma RIIB on FDCs, GCs show aberrant B cell selection during autoreactive and selective foreign antigen responses. These GCs are more diverse as measured by the AidCre(ERT2) -confetti system and show the persistence of IgM(+) clones with decreased numbers of IgH mutations. Our results show that FDCs can modulate GC B cell diversity by the upregulation of Fc gamma RIIB. Permissive clonal selection and subsequent increased GC diversity may affect epitope spreading during autoimmunity and foreign responses.

The angiotensin II (AngII) type 1 receptor (AT1R) is a member of the G protein- coupled receptor (GPCR) family and binds beta-arrestins (beta-arrs), which regulate AT1R signaling and trafficking. These processes can be biased by different ligands or mutations in the AGTR1 gene. As for many GPCRs, the exact details for AT1R-beta-arr interactions driven by AngII or beta-arr-biased ligands remain largely unknown. Here, we used the amber-suppression technology to site-specifically introduce the unnatural amino acid (UAA) p-azido-L-phenylalanine (azF) into the intracellular loops (ICLs) and the C-tail of AT1R. Our goal was to generate competent photoreactive receptors that can be cross-linked to beta-arrs in cells. We performed UV-mediated photolysis of 25 different azF-labeled AT1Rs to cross-link beta-arr1 to AngII-bound receptors, enabling us to map important contact sites in the C-tail and in the ICL2 and ICL3 of the receptor. The extent of AT1R-beta-arr1 cross-linking among azF-labeled receptors differed, revealing variability in beta-arr's contact mode with the different AT1R domains. Moreover, the signature of ligated AT1R-beta-arr complexes from a subset of azF-labeled receptors also differed between AngII and beta-arr-biased ligand stimulation of receptors and between azF-labeled AT1R bearing and that lacking a bias signaling mutation. These observations further implied distinct interaction modalities of the AT1R-beta-arr1 complex in biased signaling conditions. Our findings demonstrate that this photocross-linking approach is useful for understanding GPCR-beta-arr complexes in different activation states and could be extended to study other protein-protein interactions in cells.

Inflammasomes are multiprotein complexes formed in response to pathogens. NLRP1 and CARD8 are related proteins that form inflammasomes, but the pathogen-associated signal(s) and the molecular mechanisms controlling their activation have not been established. Inhibitors of the serine dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both NLRP1 and CARD8. Interestingly, DPP9 binds directly to NLRP1 and CARDS, and this interaction may contribute to the inhibition of NLRP1. Here, we use activity-based probes, reconstituted inflammasome assays, and mass spectrometry-based proteomics to further investigate the DPP9-CARD8 interaction. We show that the. DPP9-CARD8 interaction, unlike the DPP9-NLRP1 interaction, is not disrupted by DPP9 inhibitors or CARD8 mutations that block autoproteolysis. Moreover, wild-type, but not catalytically inactive mutant, DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. Together, this work reveals that DPP9's catalytic activity and not its binding to CARD8 restrains the CARDS inflammasome and thus suggests the binding interaction likely serves some other biological purpose.