Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

Defining the KRAS- and ERK-dependent transcriptome in KRAS-mutant cancers.

How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.

Impact of preoperative haemoglobin A1c levels on postoperative outcomes in adults undergoing major noncardiac surgery: A systematic review.

AIMS: Diabetes is known to increase morbidity and mortality after major surgery. However, literature is conflicting on whether elevated preoperative haemoglobin A1c (HbA1c) levels are associated with worse outcomes following major noncardiac surgery. We aimed to investigate the effect of incremental preoperative HbA1c levels on postoperative outcomes in adults who had undergone major noncardiac surgery. METHODS: We systematically searched PubMed, EMBASE and the Cochrane Library databases for eligible studies published between January 2012 and July 2023. Randomised controlled trials and observational studies (cohort and case-control studies) which measured HbA1c within 6 months before surgery and compared outcomes between at least three incremental subgroups or analysed HbA1c as a continuous variable were included. The systematic review protocol was registered with PROSPERO (CRD42023391946). RESULTS: Twenty observational studies investigating outcomes across multiple surgical types were included. Higher preoperative HbA1c levels were associated with increased odds of overall postoperative complications, postoperative acute kidney injury, anastomotic leak, surgical site infections and increased length of stay. Each 1% increase in preoperative HbA1c was associated with increased odds of these complications. No association with reoperations and 30-day mortality was identified. The literature was highly variable with respect to composite major complications, perioperative cardiovascular events, hospital readmissions, postoperative pneumonia and systemic thromboembolism. CONCLUSIONS: Current evidence suggested that higher preoperative HbA1c levels were associated with increased odds of postoperative complications and extended length of stay in adults undergoing major noncardiac surgery. Further high-quality studies would be needed to quantify the risks posed and determine whether early intervention improves outcomes.

Discovery of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC 50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k inact /K I of 6.4 x 10 3 M -1 s -1 . LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic. Significance Statement: Chikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.

Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer.

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance.

The E3 ubiquitin ligase TRIM9 regulates synaptic function and actin dynamics in response to netrin-1.

During neuronal development, dynamic filopodia emerge from dendrites and mature into functional dendritic spines during synaptogenesis. Dendritic filopodia and spines respond to extracellular cues, influencing dendritic spine shape and size as well as synaptic function. Previously, the E3 ubiquitin ligase TRIM9 was shown to regulate filopodia in early stages of neuronal development, including netrin-1-dependent axon guidance and branching. Here, we demonstrate that TRIM9 also localizes to dendritic filopodia and spines of murine cortical and hippocampal neurons during synaptogenesis and is required for synaptic responses to netrin. In particular, TRIM9 is enriched in the postsynaptic density (PSD) within dendritic spines and loss of Trim9 alters the PSD proteome, including the actin cytoskeleton landscape. While netrin exposure induces accumulation of the Arp2/3 complex and filamentous actin in dendritic spine heads, this response is disrupted by genetic deletion of Trim9. In addition, we document changes in the synaptic receptors associated with loss of Trim9. These defects converge on a loss of netrin-dependent increases in neuronal firing rates, indicating TRIM9 is required downstream of synaptic netrin-1 signaling. We propose that TRIM9 regulates cytoskeletal dynamics in dendritic spines and is required for the proper response to synaptic stimuli.

Loss of the E3 ubiquitin ligase TRIM67 alters the post-synaptic density proteome.

The E3 ubiquitin ligase TRIM67 is enriched in the central nervous system and is required for proper neuronal development. Previously we demonstrated TRIM67 coordinates with the closely related E3 ubiquitin ligase TRIM9 to regulate cytoskeletal dynamics downstream of the netrin-1 during axon guidance and axon branching in early neuronal morphogenesis. Interestingly, loss of Trim67 impacts cognitive flexibility in a spatial learning and memory task. Despite this behavioral phenotype, it was previously uninvestigated if TRIM67 was involved in synapse formation or function. Here we demonstrate TRIM67 localizes to the post-synaptic density (PSD) within dendritic spines. Furthermore, we show that loss of Trim67 significantly changes the PSD proteome, including changes in the regulation of the actin and microtubule cytoskeletons. Collectively, our data propose a synaptic role for TRIM67.

The E3 ubiquitin ligase TRIM9 regulates synaptic function and actin dynamics.

During neuronal development, dynamic filopodia emerge from dendrites and mature into functional dendritic spines during synaptogenesis. Dendritic filopodia and spines respond to extracellular cues, influencing dendritic spine shape and size as well as synaptic function. Previously, the E3 ubiquitin ligase TRIM9 was shown to regulate filopodia in early stages of neuronal development, including netrin-1 dependent axon guidance and branching. Here we demonstrate TRIM9 also localizes to dendritic filopodia and spines of murine cortical and hippocampal neurons during synaptogenesis and is required for synaptic responses to netrin. In particular, TRIM9 is enriched in the post-synaptic density (PSD) within dendritic spines and loss of Trim9 alters the PSD proteome, including the actin cytoskeleton landscape. While netrin exposure induces accumulation of the Arp2/3 complex and filamentous actin in dendritic spine heads, this response is disrupted by genetic deletion of Trim9. In addition, we document changes in the synaptic receptors associated with loss of Trim9. These defects converge on a loss of netrin-dependent increases in neuronal firing rates, indicating TRIM9 is required downstream of synaptic netrin-1 signaling. We propose TRIM9 regulates cytoskeletal dynamics in dendritic spines and is required for the proper response to synaptic stimuli.

The impact of endoscopic treatment on health-related quality of life in patients with Barrett's neoplasia: a scoping review.

PURPOSE: The objective of this scoping review is to understand the extent, type of evidence, and overall findings in relation to the impact of endoscopic treatment (ET) on health-related quality of life (HR-QoL) in patients with Barrett's dysplasia and early oesophageal cancer. METHODS: A comprehensive search was conducted for literature between 2001 and 2022 in computerised databases (PubMed, Embase, Cochrane Library, and CINAHL Complete). Additionally, sources of unpublished literature were searched in Google Advanced Search. After title and abstract checking, full-text papers were retrieved. Data were extracted, synthesised, key information tabulated, and a narrative synthesis completed. RESULTS: Six studies were included in the final analysis. Twelve different survey tools were utilised across all studies. Study designs included three randomised controlled studies, two prospective observational studies, and a single retrospective observational study. The average age of study participants ranged from 60.3 to 71.0 years. Two studies evaluated HR-QoL as primary outcome measures, but most research evaluated HR-QoL as a secondary outcome. Health domains evaluated in the studies focussed on the biophysical and psychosocial aspects of quality of life. CONCLUSION: A small number of research studies have been conducted in this area. Due to the heterogeneity and small number of included studies, it was difficult to draw conclusions about the impact of specific ET types on HR-QoL. Overall, there were perceived psychological benefits while undergoing ET. Future research could target specific ET subtypes and measure HR-QoL at baseline and post-procedures in the short and long term.

Developing forebrain synapses are uniquely vulnerable to sleep loss.

Sleep is an essential behavior that supports lifelong brain health and cognition. Neuronal synapses are a major target for restorative sleep function and a locus of dysfunction in response to sleep deprivation (SD). Synapse density is highly dynamic during development, becoming stabilized with maturation to adulthood, suggesting sleep exerts distinct synaptic functions between development and adulthood. Importantly, problems with sleep are common in neurodevelopmental disorders including autism spectrum disorder (ASD). Moreover, early life sleep disruption in animal models causes long lasting changes in adult behavior. Different plasticity engaged during sleep necessarily implies that developing and adult synapses will show differential vulnerability to SD. To investigate distinct sleep functions and mechanisms of vulnerability to SD across development, we systematically examined the behavioral and molecular responses to acute SD between juvenile (P21-28), adolescent (P42-49) and adult (P70-100) mice of both sexes. Compared to adults, juveniles lack robust adaptations to SD, precipitating cognitive deficits in the novel object recognition test. Subcellular fractionation, combined with proteome and phosphoproteome analysis revealed the developing synapse is profoundly vulnerable to SD, whereas adults exhibit comparative resilience. SD in juveniles, and not older mice, aberrantly drives induction of synapse potentiation, synaptogenesis, and expression of peri-neuronal nets. Our analysis further reveals the developing synapse as a convergent node between vulnerability to SD and ASD genetic risk. Together, our systematic analysis supports a distinct developmental function of sleep and reveals how sleep disruption impacts key aspects of brain development, providing mechanistic insights for ASD susceptibility.

Investigation of the Host Kinome Response to Coronavirus Infection Reveals PI3K/mTOR Inhibitors as Betacoronavirus Antivirals.

Host kinases play essential roles in the host cell cycle, innate immune signaling, the stress response to viral infection, and inflammation. Previous work has demonstrated that coronaviruses specifically target kinase cascades to subvert host cell responses to infection and rely upon host kinase activity to phosphorylate viral proteins to enhance replication. Given the number of kinase inhibitors that are already FDA approved to treat cancers, fibrosis, and other human disease, they represent an attractive class of compounds to repurpose for host-targeted therapies against emerging coronavirus infections. To further understand the host kinome response to betacoronavirus infection, we employed multiplex inhibitory bead mass spectrometry (MIB-MS) following MERS-CoV and SARS-CoV-2 infection of human lung epithelial cell lines. Our MIB-MS analyses revealed activation of mTOR and MAPK signaling following MERS-CoV and SARS-CoV-2 infection, respectively. SARS-CoV-2 host kinome responses were further characterized using paired phosphoproteomics, which identified activation of MAPK, PI3K, and mTOR signaling. Through chemogenomic screening, we found that clinically relevant PI3K/mTOR inhibitors were able to inhibit coronavirus replication at nanomolar concentrations similar to direct-acting antivirals. This study lays the groundwork for identifying broad-acting, host-targeted therapies to reduce betacoronavirus replication that can be rapidly repurposed during future outbreaks and epidemics. The proteomics, phosphoproteomics, and MIB-MS datasets generated in this study are available in the Proteomics Identification Database (PRIDE) repository under project identifiers PXD040897 and PXD040901.

Tandem detergent-extraction and immunoprecipitation of proteinopathy: Scalable enrichment of ALS-associated TDP-43 aggregates.

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a highly conserved, ubiquitously expressed nucleic acid-binding protein that regulates DNA/RNA metabolism. Genetics and neuropathology studies have linked TDP-43 to several neuromuscular and neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Under pathological conditions, TDP-43 mislocalizes to the cytoplasm where it forms insoluble, hyper-phosphorylated aggregates during disease progression. Here, we optimized a scalable in vitro immuno-purification strategy referred to as tandem detergent-extraction and immunoprecipitation of proteinopathy (TDiP) to isolate TDP-43 aggregates that recapitulate those identified in postmortem ALS tissue. Moreover, we demonstrate that these purified aggregates can be utilized in biochemical, proteomics, and live-cell assays. This platform offers a rapid, accessible, and streamlined approach to study ALS disease mechanisms, while overcoming many limitations that have hampered TDP-43 disease modeling and therapeutic drug discovery efforts.

Metatranscriptomics analysis reveals a novel transcriptional and translational landscape during Middle East respiratory syndrome coronavirus infection.

Among all RNA viruses, coronavirus RNA transcription is the most complex and involves a process termed "discontinuous transcription" that results in the production of a set of 3'-nested, co-terminal genomic and subgenomic RNAs during infection. While the expression of the classic canonical set of subgenomic RNAs depends on the recognition of a 6- to 7-nt transcription regulatory core sequence (TRS), here, we use deep sequence and metagenomics analysis strategies and show that the coronavirus transcriptome is even more vast and more complex than previously appreciated and involves the production of leader-containing transcripts that have canonical and noncanonical leader-body junctions. Moreover, by ribosome protection and proteomics analyses, we show that both positive- and negative-sense transcripts are translationally active. The data support the hypothesis that the coronavirus proteome is much vaster than previously noted in the literature.

Quantitative proteomic profiling identifies global protein network dynamics in murine embryonic heart development.

Defining the mechanisms that govern heart development is essential for identifying the etiology of congenital heart disease. Here, quantitative proteomics was used to measure temporal changes in the proteome at critical stages of murine embryonic heart development. Global temporal profiles of the over 7,300 proteins uncovered signature cardiac protein interaction networks that linked protein dynamics with molecular pathways. Using this integrated dataset, we identified and demonstrated a functional role for the mevalonate pathway in regulating the cell cycle of embryonic cardiomyocytes. Overall, our proteomic datasets are a resource for studying events that regulate embryonic heart development and contribute to congenital heart disease.

Multi-omics analyses reveal ClpP activators disrupt essential mitochondrial pathways in triple-negative breast cancer.

ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified ∼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying ∼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation.

Discovery of a Potent and Selective Targeted NSD2 Degrader for the Reduction of H3K36me2.

Nuclear receptor-binding SET domain-containing 2 (NSD2) plays important roles in gene regulation, largely through its ability to dimethylate lysine 36 of histone 3 (H3K36me2). Despite aberrant activity of NSD2 reported in numerous cancers, efforts to selectively inhibit the catalytic activity of this protein with small molecules have been unsuccessful to date. Here, we report the development of UNC8153, a novel NSD2-targeted degrader that potently and selectively reduces the cellular levels of both NSD2 protein and the H3K36me2 chromatin mark. UNC8153 contains a simple warhead that confers proteasome-dependent degradation of NSD2 through a novel mechanism. Importantly, UNC8153-mediated reduction of H3K36me2 through the degradation of NSD2 results in the downregulation of pathological phenotypes in multiple myeloma cells including mild antiproliferative effects in MM1.S cells containing an activating point mutation and antiadhesive effects in KMS11 cells harboring the t(4;14) translocation that upregulates NSD2 expression.

Metagenomics combined with activity-based proteomics point to gut bacterial enzymes that reactivate mycophenolate.

Mycophenolate mofetil (MMF) is an important immunosuppressant prodrug prescribed to prevent organ transplant rejection and to treat autoimmune diseases. MMF usage, however, is limited by severe gastrointestinal toxicity that is observed in approximately 45% of MMF recipients. The active form of the drug, mycophenolic acid (MPA), undergoes extensive enterohepatic recirculation by bacterial ß-glucuronidase (GUS) enzymes, which reactivate MPA from mycophenolate glucuronide (MPAG) within the gastrointestinal tract. GUS enzymes demonstrate distinct substrate preferences based on their structural features, and gut microbial GUS enzymes that reactivate MPA have not been identified. Here, we compare the fecal microbiomes of transplant recipients receiving MMF to healthy individuals using shotgun metagenomic sequencing. We find that neither microbial composition nor the presence of specific structural classes of GUS genes are sufficient to explain the differences in MPA reactivation measured between fecal samples from the two cohorts. We next employed a GUS-specific activity-based chemical probe and targeted metaproteomics to identify and quantify the GUS proteins present in the human fecal samples. The identification of specific GUS enzymes was improved by using the metagenomics data collected from the fecal samples. We found that the presence of GUS enzymes that bind the flavin mononucleotide (FMN) is significantly correlated with efficient MPA reactivation. Furthermore, structural analysis identified motifs unique to these FMN-binding GUS enzymes that provide molecular support for their ability to process this drug glucuronide. These results indicate that FMN-binding GUS enzymes may be responsible for reactivation of MPA and could be a driving force behind MPA-induced GI toxicity.

Antagonism of the mu-delta opioid receptor heterodimer enhances opioid antinociception by activating Src and calcium/calmodulin-dependent protein kinase II signaling.

ABSTRACT: The opioid receptors are important regulators of pain, reward, and addiction. Limited evidence suggests the mu and delta opioid receptors form a heterodimer (MDOR), which may act as a negative feedback brake on opioid-induced analgesia. However, evidence for the MDOR in vivo is indirect and limited, and there are few selective tools available. We recently published the first MDOR-selective antagonist, D24M, allowing us to test the role of the MDOR in mice. We thus cotreated CD-1 mice with D24M and opioids in tail flick, paw incision, and chemotherapy-induced peripheral neuropathy pain models. D24M treatment enhanced oxymorphone antinociception in all models by 54.7% to 628%. This enhancement could not be replicated with the mu and delta selective antagonists CTAP, naltrindole, and naloxonazine, and D24M had a mild transient effect in the rotarod test, suggesting this increase is selective to the MDOR. However, D24M had no effect on morphine or buprenorphine, suggesting that only specific opioids interact with the MDOR. To find a mechanism, we performed phosphoproteomic analysis on brainstems of mice. We found that the kinases Src and CaMKII were repressed by oxymorphone, which was restored by D24M. We were able to confirm the role of Src and CaMKII in D24M-enhanced antinociception using small molecule inhibitors (KN93 and Src-I1). Together, these results provide direct in vivo evidence that the MDOR acts as an opioid negative feedback brake, which occurs through the repression of Src and CaMKII signal transduction. These results further suggest that MDOR antagonism could be a means to improve clinical opioid therapy.

Myeloid-associated differentiation marker is a novel SP-A-associated transmembrane protein whose expression on airway epithelial cells correlates with asthma severity.

Surfactant protein A (SP-A) is well-known for its protective role in pulmonary immunity. Previous studies from our group have shown that SP-A mediates eosinophil activities, including degranulation and apoptosis. In order to identify potential binding partners on eosinophils for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. We identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Blocking MYADM on mouse and human eosinophils ex vivo prevented SP-A from inducing apoptosis; blocking MYADM in vivo led to increased persistence of eosinophilia and airway hyper-responsiveness in an ovalbumin (OVA) allergy model and increased airways resistance and mucus production in a house dust mite (HDM) asthma model. Examination of a subset of participants in the Severe Asthma Research Program (SARP) cohort revealed a significant association between epithelial expression of MYADM in asthma patients and parameters of airway inflammation, including: peripheral blood eosinophilia, exhaled nitric oxide (FeNO) and the number of exacerbations in the past 12 months. Taken together, our studies provide the first evidence of MYADM as a novel SP-A-associated protein that is necessary for SP-A to induce eosinophil apoptosis and we bring to light the potential importance of this previously unrecognized transmembrane protein in patients with asthma.

Mode of action evaluation for reduced reproduction in Daphnia pulex exposed to the insensitive munition, 1-methyl-3-nitro-1-nitroguanidine (MeNQ).

The US Department of Defense (DOD) is developing insensitive munitions (IMs) that are resistant to unintended detonation to protect warfighters. To enable material life-cycle analysis for the IM, 1-methyl-3-nitro-1-nitroguanidine (MeNQ), ecotoxicological impacts assessment was required. A previous investigation of MeNQ exposures in Daphnia pulex revealed concentration-responsive decreases in reproduction relative to controls (0 mg/L) across a 174, 346, 709, 1385, and 2286 mg/L exposure range. The present study used those exposures to conduct global transcriptomic expression analyses to establish hypothetical mode(s) of action underlying inhibited reproduction. The number of significantly affected transcripts and the magnitude of fold-change differences relative to controls tended to increase with increasing MeNQ concentration where hierarchical clustering analysis identified separation among the "low" (174 and 346 mg/L) and "high" (709, 1385, and 2286 mg/L) exposures. Vitellogenin is critical to Daphnia reproductive processes and MeNQ exposures significantly decreased transcriptional expression for vitellogenin-1 precursor at the lowest exposure level (174 mg/L) with benchmark dose (BMD) levels closely tracking concentrations that caused inhibited reproduction. Additionally, juvenile hormone-inducible protein, chorion peroxidase, and high choriolytic enzyme transcriptional expression were impacted by MeNQ exposure having potential implications for egg production / maturation and overall fecundity. In concert with these effects on specific genes involved in Daphnia reproductive physiology, MeNQ exposures caused significant enrichment of several canonical-pathways responsible for metabolism of cellular energy substrates where BMD levels for transcriptional expression were observed at ≤100 mg/L. These observations imply possible effects on whole-organism energy budgets that may also incur indirect costs on reproduction.