An exploration of mechanisms underlying Desemzia incerta colonization resistance to methicillin-resistant Staphylococcus aureus on the skin.

Colonization of human skin and nares by methicillin-resistant Staphylococcus aureus (MRSA) leads to the community spread of MRSA. This spread is exacerbated by the transfer of MRSA between humans and livestock, particularly swine. Here, we capitalized on the shared features between human and porcine skin, including shared MRSA colonization, to study novel bacterial mediators of MRSA colonization resistance. We focused on the poorly studied bacterial species Desemzia incerta, which we found to exert antimicrobial activity through a secreted product and exhibited colonization resistance against MRSA in an in vivo murine skin model. Using parallel genomic and biochemical investigation, we discovered that D. incerta secretes an antimicrobial protein. Sequential protein purification and proteomics analysis identified 24 candidate inhibitory proteins, including a promising peptidoglycan hydrolase candidate. Aided by transcriptional analysis of D. incerta and MRSA cocultures, we found that exposure to D. incerta leads to decreased MRSA biofilm production. These results emphasize the value of exploring microbial communities across a spectrum of hosts, which can lead to novel therapeutic agents as well as an increased understanding of microbial competition.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) causes a significant healthcare burden and can be spread to the human population via livestock transmission. Members of the skin microbiome can prevent MRSA colonization via a poorly understood phenomenon known as colonization resistance. Here, we studied the colonization resistance of S. aureus by bacterial inhibitors previously identified from a porcine skin model. We identify a pig skin commensal, Desemzia incerta, that reduced MRSA colonization in a murine model. We employ a combination of genomic, proteomic, and transcriptomic analyses to explore the mechanisms of inhibition between D. incerta and S. aureus. We identify 24 candidate antimicrobial proteins secreted by D. incerta that could be responsible for its antimicrobial activity. We also find that exposure to D. incerta leads to decreased S. aureus biofilm formation. These findings show that the livestock transmission of MRSA can be exploited to uncover novel mechanisms of MRSA colonization resistance.

Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons.

Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover αSyn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous αSyn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic αSyn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous αSyn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous αSyn, identifies mechanisms mediating the degradation of nonacetylated αSyn, and illustrates potential therapeutic pathways for decreasing αSyn levels in synucleinopathies.

CRISPR screen for protein inclusion formation uncovers a role for SRRD in the regulation of intermediate filament dynamics and aggresome assembly.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.

Stratification of neurogenic bladder risk in spina bifida using the urinary peptidome.

Neurogenic bladder poses a major morbidity in children with spina bifida (SB), and videourodynamic studies (VUDS) are used to stratify this risk. This small-scale pilot study utilized current mass-spectrometry-based proteomic approaches to identify peptides or proteins in urine that may differentiate children at high risk of developing renal complications from a neurogenic bladder. Twenty-two urine samples of which nine had high bladder pressure storage that put the upper urinary tract at risk, while 13 with a lower risk for renal compromise were analyzed. More than 1,900 peptides across all 22 samples were quantified, and 115 peptides differed significantly (P < 0.05) between the two groups. Using machine learning approaches five peptides that showed the greatest differences between these two clinical categories were used to build a classifier. We tested this classifier by blind analysis of an additional six urine samples and showed that it correctly assigned the unknown samples in their proper risk category. These promising results indicate that a urinary screening test based on peptides could be performed on a regular basis to stratify the neurogenic bladder into low or high-risk categories. Expanding this work to larger cohorts as well as across a broad spectrum of urodynamics outcomes may provide a useful diagnostic test for neurogenic bladder.NEW & NOTEWORTHY This approach could help risk stratify the neurogenic bladder in patients with spina bifida and could allow us to safely defer on up to 1/3 of urodynamic studies. These pilot data justify a larger trial before this approach becomes a clinical tool.

Quantitative label-free mass spectrometry reveals content and signaling differences between neonatal and adult platelets.

BACKGROUND: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. OBJECTIVES: This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. METHODS: We isolated resting umbilical cord blood-derived platelets from healthy full-term neonates (n = 8) and resting blood platelets from healthy adults (n = 6) and compared protein and phosphoprotein contents using data-independent acquisition mass spectrometry. RESULTS: We identified 4770 platelet proteins with high confidence across all samples. Adult and neonatal platelets were clustered separately by principal component analysis. Adult platelets were significantly enriched in immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched in ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched in phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched in phosphorylated proteins involved in insulin growth factor signaling. CONCLUSION: Using label-free data-independent acquisition mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation between neonatal and adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

c-Myc uses Cul4b to preserve genome integrity and promote antiviral CD8+ T cell immunity.

During infection, virus-specific CD8+ T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral load. This rapid clonal expansion can put T cells at significant risk for replication-induced DNA damage. Here, we find that c-Myc links CD8+ T cell expansion to DNA damage response pathways though the E3 ubiquitin ligase, Cullin 4b (Cul4b). Following activation, c-Myc increases the levels of Cul4b and other members of the Cullin RING Ligase 4 (CRL4) complex. Despite expressing c-Myc at high levels, Cul4b-deficient CD8+ T cells do not expand and clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) in vivo. Cul4b-deficient CD8+ T cells accrue DNA damage and succumb to proliferative catastrophe early after antigen encounter. Mechanistically, Cul4b knockout induces an accumulation of p21 and Cyclin E2, resulting in replication stress. Our data show that c-Myc supports cell proliferation by maintaining genome stability via Cul4b, thereby directly coupling these two interdependent pathways. These data clarify how CD8+ T cells use c-Myc and Cul4b to sustain their potential for extraordinary population expansion, longevity and antiviral responses.

An exploration of mechanisms underlying Desemzia incerta colonization resistance to methicillin-resistant Staphylococcus aureus on the skin.

Colonization of human skin and nares by methicillin-resistant Staphylococcus aureus (MRSA) leads to community spread of MRSA. This spread is exacerbated by transfer of MRSA between humans and livestock, particularly swine. Here we capitalized on the shared features between human and porcine skin, including shared MRSA colonization, to study novel bacterial mediators of MRSA colonization resistance. We focused on the poorly studied bacterial species Desemzia incerta, which we found to exert antimicrobial activity through a secreted product and exhibited colonization resistance against MRSA in an in vivo murine skin model. Using parallel genomic and biochemical investigation, we discovered that D. incerta secretes an antimicrobial protein. Sequential protein purification and proteomics analysis identified 24 candidate inhibitory proteins, including a promising peptidoglycan hydrolase candidate. Aided by transcriptional analysis of D. incerta and MRSA cocultures, we found that exposure to D. incerta leads to decreased MRSA biofilm production. These results emphasize the value in exploring microbial communities across a spectrum of hosts, which can lead to novel therapeutic agents as well as increased understanding of microbial competition.

Neutral sphingomyelinase blockade enhances hematopoietic stem cell fitness through an integrated stress response.

Hematopoietic stem and progenitor cell (HSPC) transplantation serves as a curative therapy for many benign and malignant hematopoietic disorders and as a platform for gene therapy. However, growing needs for ex vivo manipulation of HSPC-graft products are limited by barriers in maintaining critical self-renewal and quiescence properties. The role of sphingolipid metabolism in safeguarding these essential cellular properties has been recently recognized, but not yet widely explored. Here, we demonstrate that pharmacologic and genetic inhibition of neutral sphingomyelinase 2 (nSMase-2) leads to sustained improvements in long-term competitive transplantation efficiency after ex vivo culture. Mechanistically, nSMase-2 blockade activates a canonical integrated stress response (ISR) and promotes metabolic quiescence in human and murine HSPCs. These adaptations result in part from disruption in sphingolipid metabolism that impairs the release of nSMase-2-dependent extracellular vesicles (EVs). The aggregate findings link EV trafficking and the ISR as a regulatory dyad guarding HSPC homeostasis and long-term fitness. Translationally, transient nSMase-2 inhibition enables ex vivo graft manipulation with enhanced HSPC potency.

Phosphoproteomics reveals content and signaling differences between neonatal and adult platelets.

Background and Objective: Recent clinical studies have shown that transfusions of adult platelets increase morbidity and mortality in preterm infants. Neonatal platelets are hyporesponsive to agonist stimulation, and emerging evidence suggests developmental differences in platelet immune functions. This study was designed to compare the proteome and phosphoproteome of resting adult and neonatal platelets. Methods: We isolated resting umbilical cord blood-derived platelets from healthy full term neonates (n=9) and resting blood platelets from healthy adults (n=7), and compared protein and phosphoprotein contents using data independent acquisition mass spectrometry. Results: We identified 4745 platelet proteins with high confidence across all samples. Adult and neonatal platelets clustered separately by principal component analysis. Adult platelets were significantly enriched for immunomodulatory proteins, including ß2 microglobulin and CXCL12, whereas neonatal platelets were enriched for ribosomal components and proteins involved in metabolic activities. Adult platelets were enriched for phosphorylated GTPase regulatory enzymes and proteins participating in trafficking, which may help prime them for activation and degranulation. Neonatal platelets were enriched for phosphorylated proteins involved in insulin growth factor signaling. Conclusions: Using state-of-the-art mass spectrometry, our findings expanded the known neonatal platelet proteome and identified important differences in protein content and phosphorylation compared with adult platelets. These developmental differences suggested enhanced immune functions for adult platelets and presence of a molecular machinery related to platelet activation. These findings are important to understanding mechanisms underlying key platelet functions as well as the harmful effects of adult platelet transfusions given to preterm infants.

Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells.

Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.

The Ubiquitin Ligase Itch Skews Light Zone Selection in Germinal Centers.

Ig diversification occurs in peripheral lymphoid organs after establishment of central tolerance during B cell development. In germinal centers (GCs), somatic hypermutation of Ig genes occurs in dark zones, followed by selection of mutated clones in light zones (LZs). This generates high-affinity Ig receptors to pathogens but can also produce autoreactive Ig receptors, which are removed by selection mechanisms that are incompletely understood. The ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop potentially fatal autoimmune diseases; yet, how Itch regulates GC B cells is not well understood. By studying Itch-deficient mice, we have recently shown that Itch directly limits the magnitude of GC responses. Proteomic profiling of GC B cells uncovered that Itch-deficient cells exhibit high mTORC1 and Myc activity, hallmarks of positive selection. Bone marrow chimera and adoptive transfer experiments revealed that B cell Itch restricts noncycling LZ cells. These results support, to our knowledge, a novel role for Itch in skewing selection of GC B cells to restrict LZ accumulation and shape GC-derived humoral immunity. Determining how B cells integrate cues within GCs to navigate through LZs and dark zones will aid in understanding how autoreactive clones emerge from GCs in people with autoimmune disease.

Proteomics identifies novel biomarkers of synovial joint disease in a canine model of mucopolysaccharidosis I.

Mucopolysaccharidosis I is a lysosomal storage disorder characterized by deficient alpha-L-iduronidase activity, leading to abnormal accumulation of glycosaminoglycans in cells and tissues. Synovial joint disease is prevalent and significantly reduces patient quality of life. There is a critical need for improved understanding of joint disease pathophysiology in MPS I, including specific biomarkers to predict and monitor joint disease progression, and response to treatment. The objective of this study was to leverage the naturally-occurring MPS I canine model and undertake an unbiased proteomic screen to identify systemic biomarkers predictive of local joint disease in MPS I. Synovial fluid and serum samples were collected from MPS I and healthy dogs at 12 months-of-age, and protein abundance characterized using liquid chromatography tandem mass spectrometry. Stifle joints were evaluated postmortem using magnetic resonance imaging (MRI) and histology. Proteomics identified 40 proteins for which abundance was significantly correlated between serum and synovial fluid, including markers of inflammatory joint disease and lysosomal dysfunction. Elevated expression of three biomarker candidates, matrix metalloproteinase 19, inter-alpha-trypsin inhibitor heavy-chain 3 and alpha-1-microglobulin, was confirmed in MPS I cartilage, and serum abundance of these molecules was found to correlate with MRI and histological degenerative grades. The candidate biomarkers identified have the potential to improve patient care by facilitating minimally-invasive, specific assessment of joint disease progression and response to therapeutic intervention.

FBXW7ß isoform drives transcriptional activation of the proinflammatory TNF cluster in human pro-B cells.

Noncanonical exon usage plays many important roles in cellular phenotypes, but its contribution to human B-cell development remains sketchily understood. To fill this gap, we collected various B-cell fractions from bone marrow (BM) and tonsil donors, performed RNA sequencing, and examined transcript variants. We identified 150 genes that harbor local splicing variations in all pairwise comparisons. One of them encodes FBXW7, an E3 ubiquitin ligase implicated as a driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript used an alternative first exon to produce the poorly characterized FBXW7ß isoform, previously thought to be restricted to neural tissues. The FBXW7ß transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the BM. When overexpressed in a heterologous cell system, this transcript yielded the expected protein product, as judged by anti-FLAG immunoblotting and mass spectrometry. Furthermore, in REH B-ALL cells, FBXW7ß mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7ß, we used gain- and loss-of-function approaches and identified an FBXW7-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7ß expression. This signature contained several members of the tumor necrosis factor superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7ß expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis, and responses to anticancer therapies.

Differentiating children with sepsis with and without acute respiratory distress syndrome using proteomics.

Both sepsis and acute respiratory distress syndrome (ARDS) rely on imprecise clinical definitions leading to heterogeneity, which has contributed to negative trials. Because circulating protein/DNA complexes have been implicated in sepsis and ARDS, we aimed to develop a proteomic signature of DNA-bound proteins to discriminate between children with sepsis with and without ARDS. We performed a prospective case-control study in 12 children with sepsis with ARDS matched to 12 children with sepsis without ARDS on age, severity of illness score, and source of infection. We performed co-immunoprecipitation and downstream proteomics in plasma collected ≤ 24 h of intensive care unit admission. Expression profiles were generated, and a random forest classifier was used on differentially expressed proteins to develop a signature which discriminated ARDS. The classifier was tested in six independent blinded samples. Neutrophil and nucleosome proteins were over-represented in ARDS, including two S100A proteins, superoxide dismutase (SOD), and three histones. Random forest produced a 10-protein signature that accurately discriminated between children with sepsis with and without ARDS. This classifier perfectly assigned six independent blinded samples as having ARDS or not. We validated higher expression of the most informative discriminating protein, galectin-3-binding protein, in children with ARDS. Our methodology has applicability to isolation of DNA-bound proteins from plasma. Our results support the premise of a molecular definition of ARDS, and give preliminary insight into why some children with sepsis, but not others, develop ARDS.

Multimodality assessment of heart failure with preserved ejection fraction skeletal muscle reveals differences in the machinery of energy fuel metabolism.

AIMS: Skeletal muscle (SkM) abnormalities may impact exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF). We sought to quantify differences in SkM oxidative phosphorylation capacity (OxPhos), fibre composition, and the SkM proteome between HFpEF, hypertensive (HTN), and healthy participants. METHODS AND RESULTS: Fifty-nine subjects (20 healthy, 19 HTN, and 20 HFpEF) performed a maximal-effort cardiopulmonary exercise test to define peak oxygen consumption (VO2, peak ), ventilatory threshold (VT), and VO2 efficiency (ratio of total work performed to O2 consumed). SkM OxPhos was assessed using Creatine Chemical-Exchange Saturation Transfer (CrCEST, n = 51), which quantifies unphosphorylated Cr, before and after plantar flexion exercise. The half-time of Cr recovery (t1/2, Cr ) was taken as a metric of in vivo SkM OxPhos. In a subset of subjects (healthy = 13, HTN = 9, and HFpEF = 12), percutaneous biopsy of the vastus lateralis was performed for myofibre typing, mitochondrial morphology, and proteomic and phosphoproteomic analysis. HFpEF subjects demonstrated lower VO2,peak , VT, and VO2 efficiency than either control group (all P < 0.05). The t1/2, Cr was significantly longer in HFpEF (P = 0.005), indicative of impaired SkM OxPhos, and correlated with cycle ergometry exercise parameters. HFpEF SkM contained fewer Type I myofibres (P = 0.003). Proteomic analyses demonstrated (a) reduced levels of proteins related to OxPhos that correlated with exercise capacity and (b) reduced ERK signalling in HFpEF. CONCLUSIONS: Heart failure with preserved ejection fraction patients demonstrate impaired functional capacity and SkM OxPhos. Reductions in the proportions of Type I myofibres, proteins required for OxPhos, and altered phosphorylation signalling in the SkM may contribute to exercise intolerance in HFpEF.

Mitochondrial morphology, bioenergetics and proteomic responses in fatty acid oxidation disorders.

Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondrial deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

Plasma Nucleosomes Are Associated With Mortality in Pediatric Acute Respiratory Distress Syndrome.

OBJECTIVES: Circulating nucleosomes and their component histones have been implicated as pathogenic in sepsis and acute respiratory distress syndrome in adults. However, their role in pediatric acute respiratory distress syndrome is unknown. DESIGN: We performed a prospective cohort study in children with acute respiratory distress syndrome, with plasma collection within 24 hours of acute respiratory distress syndrome onset. We associated nucleosome levels with severity of acute respiratory distress syndrome and with nonpulmonary organ failures and tested for association of nucleosomes with PICU mortality and ventilator-free days at 28 days in univariate and multivariable analyses. We also performed proteomics of DNA-bound plasma proteins in a matched case-control study of septic children with and without acute respiratory distress syndrome in order to identify specific histone proteins elevated in acute respiratory distress syndrome. SETTING: Large academic tertiary-care PICU. PATIENTS: Intubated children meeting Berlin criteria for acute respiratory distress syndrome. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We enrolled 333 children with acute respiratory distress syndrome, with 69 nonsurvivors (21%). Plasma nucleosomes were correlated with acute respiratory distress syndrome severity and with the number of nonpulmonary organ failures at acute respiratory distress syndrome onset. Nucleosomes were higher (p < 0.001) in nonsurvivors (0.40 [interquartile range, 0.20-0.71] arbitrary units) relative to survivors (0.10 [interquartile range, 0.04-0.25] arbitrary units). Nucleosomes were associated with PICU mortality in multivariable analysis (adjusted odds ratio 1.84 per 1 sd increase; 95% CI, 1.38-2.45; p < 0.001). Nucleosomes were also associated with a lower probability of being extubated alive by day 28 after multivariable adjustment (adjusted subdistribution hazard ratio, 0.74; 95% CI, 0.63-0.88; p = 0.001). Proteomic analysis demonstrated higher levels of the core nucleosome histones H2A, H2B, H3, and H4 in septic children with acute respiratory distress syndrome, relative to septic children without acute respiratory distress syndrome. CONCLUSIONS: Plasma nucleosomes are associated with acute respiratory distress syndrome severity, nonpulmonary organ failures, and worse outcomes in pediatric acute respiratory distress syndrome.

The E3 ubiquitin ligase Cul4b promotes CD4+ T cell expansion by aiding the repair of damaged DNA.

The capacity for T cells to become activated and clonally expand during pathogen invasion is pivotal for protective immunity. Our understanding of how T cell receptor (TCR) signaling prepares cells for this rapid expansion remains limited. Here we provide evidence that the E3 ubiquitin ligase Cullin-4b (Cul4b) regulates this process. The abundance of total and neddylated Cul4b increased following TCR stimulation. Disruption of Cul4b resulted in impaired proliferation and survival of activated T cells. Additionally, Cul4b-deficient CD4+ T cells accumulated DNA damage. In T cells, Cul4b preferentially associated with the substrate receptor DCAF1, and Cul4b and DCAF1 were found to interact with proteins that promote the sensing or repair of damaged DNA. While Cul4b-deficient CD4+ T cells showed evidence of DNA damage sensing, downstream phosphorylation of SMC1A did not occur. These findings reveal an essential role for Cul4b in promoting the repair of damaged DNA to allow survival and expansion of activated T cells.

Neutrophils promote clearance of nuclear debris following acid-induced lung injury.

Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared with wild type. Size analysis of DNA debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation, they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intratracheal administration of DNase I. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.

ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein.

Modulating cytoplasmic Ca2+ concentration ([Ca2+]i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.