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Objectives Chronic inflammation is independently associated with the incidence and progression of chronic disease. Exercise has been found to reduce chronic inflammation, however the role of exercise intensity (work rate) is unknown. This review aimed to determine the pooled effect of higher- compared to lower-intensity aerobic and resistance exercise on chronic inflammation in adults. Design Systematic review and meta-analysis. Methods Five electronic databases were searched. Intervention trials that assessed the effect of ≥2 different exercise intensities on peripheral markers of chronic inflammation [c-reactive protein (CRP), interleukin (IL)-6, tumour necrosis factor (TNF)-α and IL-10] in adults were included. Random-effect meta-analyses were conducted to calculate the mean difference in change scores between groups [effect size (ES)]. Sub-group analyses were performed to explore the influence of age, chronic disease, body mass index and intervention duration on inflammation heterogeneity. Results Of 3952 studies identified, 27 were included. There were no significant effects of exercise intensity on IL-6 (ES=-0.039, 95%CI=-0.353–0.275; p = 0.806), TNF-α (ES = 0.296, 95%CI=-0.184–0.777; p = 0.227) and IL-10 (ES = 0.007, 95%CI=-0.904–0.919; p = 0.987). A significant pooled ES was observed for higher- versus lower-intensity exercise on CRP concentrations, in studies of middle-aged adults (ES=-0.412, 95%CI=-0.821– -0.004, p = 0.048) or interventions >9 weeks in duration (ES=-0.520, 95%CI=-0.882–-0.159, p = 0.005). Conclusions Exercise intensity did not influence chronic inflammatory response. However, sub-analyses suggest that higher-intensity training may be more efficacious than lower-intensity for middle-aged adults, or when longer duration interventions are implemented (>9 weeks), in the most commonly-reported analyte (CRP).

OBJECTIVES HIV-exposed uninfected (HEU) infants exhibit altered vaccine responses and an increased mortality compared with HIV-unexposed infants. Here, vaccine responses in HEU and HIV-unexposed cord blood monocytes (CBMs) were assessed following Bacillus Calmette--Guerin (BCG) treatment. DESIGN Innate responses to in-vitro BCG treatment were assessed through transcriptional profiling using CBMs obtained from a Nigerian cohort of HIV-infected and uninfected women. METHODS HIV-unexposed (n = 9) and HEU (n = 10) infant CBMs were treated with BCG and transcriptionally profiled with the Nanostring nCounter platform. Differential expression and pathway enrichment analyses were performed, and transcripts were identified with enhanced or dampened BCG responses. RESULTS Following BCG stimulation, several pathways associated with inflammatory gene expression were upregulated irrespective of HIV exposure status. Both HIV-unexposed and HEU monocytes increased expression of several cytokines characteristic of innate BCG responses, including IL1β, TNFα, and IL-6. Using differential expression analysis, we identified genes significantly upregulated in HEU compared with HIV-unexposed monocytes including monocyte chemokine CCL7 and anti-inflammatory cytokine TNFAIP6. In contrast, genes significantly upregulated in HIV-unexposed compared with HEU monocytes include chemokine CCL3 and cytokine IL23A, both of which influence anti-mycobacterial T-cell responses. Finally, two genes, which regulate prostaglandin production, CSF2 and PTGS2, were also more significantly upregulated in the HIV-unexposed cord blood indicating that inflammatory mediators are suppressed in the HEU infants. CONCLUSION HEU monocytes exhibit altered induction of several key innate immune responses, providing mechanistic insights into dysregulated innate response pathways that can be therapeutically targeted to improve vaccine responses in HEU infants.

Helicobacter pylori infection is the main risk factor for the development of gastric cancer, the third leading cause of cancer death worldwide. H. pylori colonizes the human gastric mucosa and persists for decades. The inflammatory response is ineffective in clearing the infection, leading to disease progression that may result in gastric adenocarcinoma. We have shown that polyamines are regulators of the host response to H. pylori, and that spermine oxidase (SMOX), which metabolizes the polyamine spermine into spermidine plus H2O2, is associated with increased human gastric cancer risk. We now used a molecular approach to directly address the role of SMOX, and demonstrate that Smox-deficient mice exhibit significant reductions of gastric spermidine levels and H. pylori-induced inflammation. Proteomic analysis revealed that cancer was the most significantly altered functional pathway in Smox-/- gastric organoids. Moreover, there was also less DNA damage and β-catenin activation in H. pylori-infected Smox-/- mice or gastric organoids, compared to infected wild-type animals or gastroids. The link between SMOX and β-catenin activation was confirmed in human gastric organoids that were treated with a novel SMOX inhibitor. These findings indicate that SMOX promotes H. pylori-induced carcinogenesis by causing inflammation, DNA damage, and activation of β-catenin signaling.

We tested whether individual females of two species of aquatic turtles, Texas cooters (Pseudemys texana) and red-eared sliders (Trachemys scripta elegans), inhabiting headwaters of the San Marcos River, Hays County, Texas, selectively used the same nesting areas within and among years over a 20-year period. The study area was divided into four quadrants, and chi-square analyses confirmed that for within and among years, females of both species selected quadrants unequally. Our results thus confirm that females of both species at the study site exhibited nest area fidelity within and among years.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development. Statement of significance PDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors., Arginine methylation by PRMTs is dysregulated in cancer. Here, the authors use functional genomics screens and identify PRMT1 as a vulnerability in pancreatic ductal adenocarcinoma, and further show that PRMT1 regulates RNA metabolism and coordinates expression of genes in cell cycle progression, maintaining genomic stability and tumour growth.

Pancreatic ductal adenocarcinoma (PDAC) remains recalcitrant to all forms of cancer treatment and carries a five-year survival rate of only 8%1. Inhibition of oncogenic KRAS (hereafter KRAS*), the earliest lesion in disease development that is present in more than 90% of PDACs, and its signalling surrogates has yielded encouraging preclinical results with experimental agents2–4. However, KRAS*-independent disease recurrence following genetic extinction of Kras* in mouse models anticipates the need for co-extinction strategies5,6. Multiple oncogenic processes are initiated at the cell surface, where KRAS* physically and functionally interacts to direct signalling that is essential for malignant transformation and tumour maintenance. Insights into the complexity of the functional cell-surface-protein repertoire (surfaceome) have been technologically limited until recently and—in the case of PDAC—the genetic control of the function and composition of the PDAC surfaceome in the context of KRAS* signalling remains largely unknown. Here we develop an unbiased, functional target-discovery platform to query KRAS*-dependent changes of the PDAC surfaceome, which reveals syndecan 1 (SDC1, also known as CD138) as a protein that is upregulated at the cell surface by KRAS*. Localization of SDC1 at the cell surface—where it regulates macropinocytosis, an essential metabolic pathway that fuels PDAC cell growth—is essential for disease maintenance and progression. Thus, our study forges a mechanistic link between KRAS* signalling and a targetable molecule driving nutrient salvage pathways in PDAC and validates oncogene-driven surfaceome annotation as a strategy to identify cancer-specific vulnerabilities. In an inducible mouse model of pancreatic ductal adenocarcinoma, the signalling defect that underlies 90% of these tumours causes increased cell-surface expression of syndecan 1, leading to misregulation of macropinocytosis, and linking the defective signalling with nutrient-salvage pathways.

The classical pathway of complement (CP) can mediate C3 opsonization of Ags responsible for the costimulation and activation of cognate B lymphocytes. In this manner, the complement system acts as a bridge between the innate and adaptive immune systems critical for establishing a humoral response. However, aberrant complement activation is often observed in autoimmune diseases in which C3 deposition on self-antigens may serve to activate self-reactive B cell clones. In this study, we use BIVV009 (Sutimlimab), a clinical stage, humanized mAb that specifically inhibits the CP-specific serine protease C1s to evaluate the impact of upstream CP antagonism on activation and proliferation of normal and autoimmune human B cells. We report that BIVV009 significantly inhibited complement-mediated activation and proliferation of primary human B cells. Strikingly, CP antagonism suppressed human Ig–induced activation of B cells derived from patients with rheumatoid arthritis. These results suggest that clinical use of CP inhibitors in autoimmune patients may not only block complement-mediated tissue damage, but may also prevent the long-term activation of autoimmune B cells and the production of autoantibodies that contribute to the underlying pathologic condition of these diseases.

Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+ H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans.

Natural Killer (NK) cells are among the first effectors to directly contact influenza and influenza-infected cells and their activation affects not only their intrinsic functions, but also subsequent CD8+ T cell responses. We utilized a NK cell depletion model to interrogate the contribution of NK cells to the development of anti-influenza CD8+ T cell memory. NK cell ablation increased the number of influenza-specific memory CD8+ T cells in the respiratory tract and lung-draining lymph node. Interestingly, animals depleted of NK cells during primary influenza infection were protected as well as their NK-intact counterparts despite significantly fewer reactivated CD8+ T cells infiltrating the respiratory tract after lethal, heterosubtypic challenge. Instead, protection in NK-deficient animals seems to be conferred by rapid reactivation of an enlarged pool of lung tissue-resident (TRM) memory cells within two days post challenge. Further interrogation of how NK cell ablation enhances respiratory TRM indicated that TRM development is independent of global and NK cell derived IFN-γ. These data suggest that reduction in NK cell activation after vaccination with live, non-lethal influenza virus increases compartmentalized, broadly protective memory CD8+ T cell generation and decreases the risk of CD8+ T cell-mediated pathology following subsequent influenza infections.

Background Urothelial cancer patients treated with immune checkpoint inhibitor (ICI) therapy have varied response and survival.1 Clinical and immunogenomic biomarkers could help predict ICI response and survival to inform decisions about patient selection for ICI treatment. Methods The association of clinical metadata and immunogenomic signatures with response and survival was analyzed in a set of 347 urothelial cancer patients treated with the PD-L1 inhibitor atezolizumab as part of the IMVigor210 study.1 Data were divided into a discovery set (2/3 of patients) and validation set (1/3 of patients). We analyzed as potential predictors 70 total variables, of which 16 were clinical metadata and 54 were immunogenomic signatures. Categorical variables were converted to dummy variables (89 total variables: 35 clinical, 54 immunogenomic). Using the discovery set, elastic net regression with Monte Carlo cross-validation was used to build optimal models for response (logistic regression) and survival (Cox proportional-hazards). Model performance was evaluated using the validation set. Results In the optimal model of response, 17 variables (10 clinical, 7 immunogenomic) were selected as informative predictors, including Baseline Eastern Cooperative Oncology Group (ECOG) Score = 0, Neoantigen Burden, Lymph Node Metastases, and Tumor Mutation Burden (figure 1). The final model predicted patient response with good performance (Area Under Curve = 0.828, pAUC = 2.38e-3; True Negative Rate = 91.7%, True Positive Rate = 87.5%, pconfusion matrix = 0.0252). In the optimal model of survival, 32 variables (17 clinical, 15 immunogenomic) were selected as informative predictors, including baseline ECOG Score = 0, IC Level 2+, Race = Asian, and Consensus Tumor Subtype = Neuroendocrine (figure 2). The final model predicted patient survival with good performance (c-indexmodel = 0.652, pc-index = 0.0290). Conclusions Models incorporating clinical metadata and immunogenomic signatures can predict response and survival for urothelial cancer patients treated with atezolizumab. Among predictors in those models, baseline performance status is the greatest and most positive predictor of response and survival. Reference Mariathasan S, Turley S, Nickles D, et al. TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells. Nature 2018;554:544–548.

In polarized MDCK cells, disruption of the tyrosine-based YXXΦ basolateral trafficking motif (Y156A) in the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG), results in its apical mistrafficking and transformation in vivo. However, the mechanisms underlying these dramatic effects are unknown. Using a doxycycline-inducible system in 3D Matrigel cultures, we now show that induction of Y156A EREG in fully formed MDCK cysts results in direct and complete delivery of mutant EREG to the apical cell surface. Within 3 days of induction, ectopic lumens were detected in mutant, but not wild-type, EREG-expressing cysts. Of note, these structures resembled histological features found in subcutaneous xenografts of mutant EREG-expressing MDCK cells. These ectopic lumens formed de novo rather than budding from the central lumen and depended on metalloprotease-mediated cleavage of EREG and subsequent EGFR activity. Moreover, the most frequent EREG mutation in human cancer (R147stop) resulted in its apical mistrafficking in engineered MDCK cells. Thus, induction of EREG apical mistrafficking is sufficient to disrupt selective aspects of polarity of a preformed polarized epithelium. This article has an associated First Person interview with the first author of the paper.

Cancers are highly complex ecosystems composed of molecularly distinct sub-populations of tumor cells, each exhibiting a unique spectrum of genetic features and phenotypes, and embedded within a complex organ context. To substantially improve clinical outcomes, there is a need to comprehensively define inter- and intra-tumor phenotypic diversity, as well as to understand the genetic dependencies that underlie discrete molecular subpopulations. To this end, we integrated CRISPR-based co-dependency annotations with a tissue-specific co-expression network developed from patient-derived models to establish CoDEX, a framework to quantitatively associate gene-cluster patterns with genetic vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). Using CoDEX, we defined multiple prominent anticorrelated gene-cluster signatures and specific pathway dependencies, both across genetically distinct PDAC models and intratumorally at the single-cell level. Of these, one differential signature recapitulated the characteristics of classical and basal-like PDAC molecular subtypes on a continuous scale. Anchoring genetic dependencies identified through functional genomics within the gene-cluster signature defined fundamental vulnerabilities associated with transcriptomic signatures of PDAC subtypes. Subtype-associated dependencies were validated by feature-barcoded CRISPR knockout of prioritized basal-like-associated genetic vulnerabilities (SMAD4, ILK, and ZEB1) followed by scRNAseq in multiple PDAC models. Silencing of these genes resulted in a significant and directional clonal shift toward the classical-like signature of more indolent tumors. These results validate CoDEX as a novel, quantitative approach to identify specific genetic dependencies within defined molecular contexts that may guide clinical positioning of targeted therapeutics.

Background & Aims Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. Methods To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. Results Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. Conclusions Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

Altered serotonin (5-HT) signaling is associated with multiple brain disorders, including major depressive disorder (MDD), obsessive-compulsive disorder (OCD), and autism spectrum disorder (ASD). The presynaptic, high-affinity 5-HT transporter (SERT) tightly regulates 5-HT clearance after release from serotonergic neurons in the brain and enteric nervous systems, among other sites. Accumulating evidence suggests that SERT is dynamically regulated in distinct activity states as a result of environmental and intracellular stimuli, with regulation perturbed by disease-associated coding variants. Our lab identified a rare, hypermorphic SERT coding substitution, Gly56Ala, in subjects with ASD, finding that the Ala56 variant stabilizes a high-affinity outward-facing conformation (SERT∗) that leads to elevated 5-HT uptake in vitro and in vivo. Hyperactive SERT Ala56 appears to preclude further activity enhancements by p38α mitogen-activated protein kinase (MAPK) and can be normalized by pharmacological p38α MAPK inhibition, consistent with SERT Ala56 mimicking, constitutively, a high-activity conformation entered into transiently by p38α MAPK activation. We hypothesize that changes in SERT-interacting proteins (SIPs) support the shift of SERT into the SERT∗ state which may be captured by comparing the composition of SERT Ala56 protein complexes with those of wildtype (WT) SERT, defining specific interactions through comparisons of protein complexes recovered using preparations from SERT–/– (knockout; KO) mice. Using quantitative proteomic-based approaches, we identify a total of 459 SIPs, that demonstrate both SERT specificity and sensitivity to the Gly56Ala substitution, with a striking bias being a loss of SIP interactions with SERT Ala56 compared to WT SERT. Among this group are previously validated SIPs, such as flotillin-1 (FLOT1) and protein phosphatase 2A (PP2A), whose functions are believed to contribute to SERT microdomain localization and regulation. Interestingly, our studies nominate a number of novel SIPs implicated in ASD, including fragile X mental retardation 1 protein (FMR1) and SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), of potential relevance to long-standing evidence of serotonergic contributions to ASD. Further investigation of these SIPs, and the broader networks they engage, may afford a greater understanding of ASD as well as other brain and peripheral disorders associated with perturbed 5-HT signaling.

The North Atlantic subpolar gyre (SPG) connects tropical and high latitude waters, playing a leading role in deep‐water formation, propagation of Atlantic water into the Arctic, and as habitat for many ecosystems. Instrumental records spanning recent decades document significant decadal variability in SPG circulation, with associated hydrographic and ecological changes. Emerging longer‐term records provide circumstantial evidence that the North Atlantic also experienced centennial trends during the 20th century. Here, we use marine sediment records to show that there has been a long‐term change in SPG circulation during the industrial era, largely during the 20th century. Moreover, we show that the shift and late 20th century SPG configuration were unprecedented in the last 10,000 years. Recent SPG dynamics resulted in an expansion of subtropical ecosystems into new habitats and likely also altered the transport of heat to high latitudes.

SUMMARY Innate responses of myeloid cells defend against pathogenic bacteria via inducible effectors. Deoxyhypusine synthase (DHPS) catalyzes the transfer of the N-moiety of spermidine to the lysine-50 residue of eukaryotic translation initiation factor 5A (EIF5A) to form the amino acid hypusine. Hypusinated EIF5A (EIF5AHyp) transports specific mRNAs to ribosomes for translation. We show that DHPS is induced in macrophages by two gastrointestinal pathogens, Helicobacter pylori and Citrobacter rodentium, resulting in enhanced hypusination of EIF5A. EIF5AHyp was also increased in gastric macrophages from patients with H. pylori gastritis. Furthermore, we identify the bacteria-induced immune effectors regulated by hypusination. This set of proteins includes essential constituents of antimicrobial response and autophagy. Mice with myeloid cell-specific deletion of Dhps exhibit reduced EIF5AHyp in macrophages and increased bacterial burden and inflammation. Thus, regulation of translation through hypusination is a critical hallmark of the defense of eukaryotic hosts against pathogenic bacteria., Graphical Abstract, In Brief Gobert et al. demonstrate that hypusination, a specific mechanism regulating translation, is induced in macrophages by bacteria. Hypusination is required for the translation of inducible antimicrobial effectors. Mice that specifically lack hypusination in macrophages are highly susceptible to Helicobacter pylori and Citrobacter rodentium, two pathogens of the gastrointestinal tract.

Various injuries to the neural tissues can cause irreversible damage to multiple functions of the nervous system ranging from motor control to cognitive function. The limited treatment options available for patients have led to extensive interest in studying the mechanisms of neuronal regeneration and recovery from injury. Since many neurons are terminally differentiated, by increasing cell survival following injury it may be possible to minimize the impact of these injuries and provide translational potential for treatment of neuronal diseases. While several cell types are known to survive injury through plasma membrane repair mechanisms, there has been little investigation of membrane repair in neurons and even fewer efforts to target membrane repair as a therapy in neurons. Studies from our laboratory group and others demonstrated that mitsugumin 53 (MG53), a muscle-enriched tripartite motif (TRIM) family protein also known as TRIM72, is an essential component of the cell membrane repair machinery in skeletal muscle. Interestingly, recombinant human MG53 (rhMG53) can be applied exogenously to increase membrane repair capacity both in vitro and in vivo. Increasing the membrane repair capacity of neurons could potentially minimize the death of these cells and affect the progression of various neuronal diseases. In this study we assess the therapeutic potential of rhMG53 to increase membrane repair in cultured neurons and in an in vivo mouse model of neurotrauma. We found that a robust repair response exists in various neuronal cells and that rhMG53 can increase neuronal membrane repair both in vitro and in vivo. These findings provide direct evidence of conserved membrane repair responses in neurons and that these repair mechanisms can be targeted as a potential therapeutic approach for neuronal injury.

We report the case of a 57-year-old woman with familial adenomatous polyposis (FAP) who presented with bilateral ovarian microcystic stromal tumors (MCSTs) and a cribriform-morular variant of papillary thyroid carcinoma, as well as concurrent noninvasive endometrial adenocarcinoma. The ovarian masses were confined to the ovaries. The tumor cells were positive for nuclear expression of β-catenin. The papillary thyroid carcinoma revealed the cribriform morular architecture associated with FAP, and immunohistochemistry also showed aberrant nuclear β-catenin. The endometrial adenocarcinoma, in contrast, showed an immunophenotype of negative nuclear β-catenin. Whole exome sequencing of blood was performed, and analysis revealed a rarely reported variant in the adenomatous polyposis coli (APC) gene, c.475dupT(pTyr159Leufs*9). The concurrence of these various neoplasms within one patient could provide insights into the pathogenesis of MCST, which has only recently been described, and underscores the importance of recognizing this rare entity in the setting of FAP.

Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.

Low concentrations and complex speciation can present major challenges for measuring Fe in natural waters easily and accurately. This study describes an optimized ferrozine method for measuring total dissolved Fe in the nanomolar range in seawater samples containing various concentrations of organic matter, as well as the commercially available Fe ligands ethylenediaminetetraacetic acid (EDTA) and desferrioxamine B (DFB), which are widely used in phytoplankton cultures. The method involves sample acidification to liberate Fe from strong complexes and/or colloids, followed by reduction of Fe(III) to Fe(II) using sulfite , and finally the measurement of Fe(II)-ferrozine complexes using long optical path spectrophotometry . The performance of each step was improved in such a way as to achieve the most efficient dissociation rate of Fe from the given ligands in the least possible time, regardless of their type or concentration. Storage of samples at pH 1 for 15 days enabled full recovery of Fe in the presence of 50 μmol/L DFB, while the duration required for full Fe recovery in the presence of the same concentration of EDTA was only about 1 h. Addition of ferrozine after a brief (about 15 min) reduction step with subsequent incubation for 24 h resulted in stable color development in the samples over time. The approach was successfully applied to determine total dissolved Fe in samples from coastal waters containing 10.5 μmol/L of particulate organic carbon (POC), and also in samples containing 2 μmol/L EDTA and 800 μmol/L of POC collected from laboratory cultures of the marine cyanobacterium, Trichodesmium erythraeum.

Currently toxicity data for iron (Fe) in seawater are limited; furthermore, these data are of poor quality as a result of the importance of Fe solubility in test solutions being overlooked. The present study characterized the solubility and lability of Fe(III) in seawater and then examined the effects of Fe(III) on the fertilization success and larval survival of the tropical marine scleractinian corals Acropora spathulata and Platygyra daedalea. We present the first assessment of the effects of Fe on the early life stages of scleractinian corals. Concentrations of both soluble and labile forms of Fe were very low, with dissolved Fe concentrations ≤0.195 mg/L in bioassay test solutions and chemical determinations revealing labile Fe concentrations ≤1.21 mg/L. For fertilization experiments, the median effect concentration (EC50) value for total Fe was 25 mg/L for the most sensitive species, P. daedalea, whereas the EC50 values for A. spathulata ranged between 40 and 66 mg/L. The median lethal concentration value for P. daedalea larval survival was 47 mg/L Fe after 72-h exposure. We provide Fe toxicity data for tropical marine keystone species that could be used to help generate more reliable guideline values for Fe in marine waters. Environ Toxicol Chem 2018;37:1104-1114. © 2017 SETAC.

Introduction Severe asthma is a chronic lung disease characterized by inflammation, airway hyperresponsiveness (AHR) and airway remodelling with hyperplasia of airway smooth muscle cells (aSMC). Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 controls bronchoconstriction. The objective of this study was to assess the role of Rac1 in severe asthma associated airway remodelling. Methods We have studied the level of Rac1 activation by immunofluorescence in bronchial biopsies from severe asthmatic patients and control patients. In order to identify the role of Rac1 in aSMC remodelling, mice with specific deletion of Rac1 in SMC were sensitized to house dust mite to mimic the characteristics of severe allergic asthma in humans. Results Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from severe asthmatic patients compared to control subjects. The basal proliferation rate of aSMC from severe asthmatics subjects is increased compared to control subjects. We have also observed that bFGF and PDGF-BB, growth factors described to be implicated in airway remodelling, stimulate the proliferation of aSMC from severe asthmatic subjects by 33% and 37% respectively. Moreover, the inhibition of Rac1 by EHT1864 decreased this proliferation by more than 50%. By a biochemical approach we have demonstrated that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the STAT3 signalling pathway. In vivo, we have demonstrated that specific deletion of Rac1 in SMC prevents aSMC hyperplasia and hypertrophy. Conclusion This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation and airway remodelling. Inhibition of Rac1 dependant signalling pathway may represent an interesting target for the treatment of severe asthma.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable disease characterized by poor survival, dense desmoplastic stroma and activating mutations in KRAS ( Citation Format: Sanjana Srinivasan, Johnathon Rose, Wantong Yao, Sahil Seth, Michael Peoples, Annette Machado, Chieh-Yuan Li, I Lin Ho, Jaewon J. Lee, Paola A. Guerrera, Eiru Kim, Mustafa Syed, Joseph Daniele, Angela K. Deem, Michael Kim, Christopher A. Bristow, Eugene Koay, Giannicola Genovese, Andrea Viale, Timothy P. Heffernan, Anirban Maitra, Traver Hart, Alessandro Carugo, Giulio Draetta. Diversity across the pancreatic ductal adenocarcinoma disease spectrum revealed by network-anchored functional genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2136.

Background Molecular signaling events associated with the necroinflammatory changes in nonalcoholic steatohepatitis (NASH) are not well understood. Aims To understand the molecular basis of NASH, we evaluated reversible phosphorylation events in hepatic tissue derived from Class III obese subjects by phosphoproteomic means with the aim of highlighting key regulatory pathways that distinguish NASH from non-alcoholic fatty liver disease (also known as simple steatosis; SS). Materials & Methods Class III obese subjects undergoing bariatric surgery underwent liver biopsy (eight normal patients, eight with simple steatosis, and eight NASH patients). Our strategy was unbiased, comparing global differences in liver protein reversible phosphorylation events across the 24 subjects. Results Of the 3078 phosphorylation sites assigned (2465 phosphoserine, 445 phosphothreonine, 165 phosphotyrosine), 53 were altered by a factor of 2 among cohorts, and of those, 12 were significantly increased or decreased by ANOVA (P < 0.05). Discussion Statistical analyses of canonical signaling pathways identified carbohydrate metabolism and RNA post-transcriptional modification among the most over-represented networks. Conclusion Collectively, these results raise the possibility of abnormalities in carbohydrate metabolism as an important trigger for the development of NASH, in parallel with already established abnormalities in lipid metabolism.

Exercise improves whole-body metabolic health through adaptations to various tissues, including adipose tissue, but the effects of exercise training on the lipidome of white adipose tissue (WAT) and brown adipose tissue (BAT) are unknown. Here, we utilize MS/MSALL shotgun lipidomics to determine the molecular signatures of exercise-induced adaptations to subcutaneous WAT (scWAT) and BAT. Three weeks of exercise training decrease specific molecular species of phosphatidic acid (PA), phosphatidylcholines (PC), phosphatidylethanolamines (PE), and phosphatidylserines (PS) in scWAT and increase specific molecular species of PC and PE in BAT. Exercise also decreases most triacylglycerols (TAGs) in scWAT and BAT. In summary, exercise-induced changes to the scWAT and BAT lipidome are highly specific to certain molecular lipid species, indicating that changes in tissue lipid content reflect selective remodeling in scWAT and BAT of both phospholipids and glycerol lipids in response to exercise training, thus providing a comprehensive resource for future studies of lipid metabolism pathways.

Rapidly proliferating cells require an increased rate of metabolism to allow for the production of nucleic acids, amino acids, and lipids. Pyruvate kinase catalyzes the final step in the glycolysis pathway, and different isoforms display vastly different catalytic efficiencies. The M2 isoform of pyruvate kinase (PKM2) is strongly expressed in cancer cells and contributes to aerobic glycolysis in what is commonly termed the Warburg effect. Here, we show that PKM2 is covalently modified by the lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE modify multiple sites on PKM2 in vitro, including Cys424 and His439, which play a role in protein-protein interactions and fructose 1,6-bis-phosphate binding, respectively. Modification of these sites results in a dose-dependent decrease in enzymatic activity. In addition, high concentrations of the electrophile, most notably in the case of ONE, result in substantial protein-protein cross-linking in vitro and in cells. Exposure of RKO cells to electrophiles results in modification of monomeric PKM2 in a dose-dependent manner. There is a concomitant decrease in PKM2 activity in cells upon ONE exposure, but not HNE exposure. Together, our data suggest that modification of PKM2 by certain electrophiles results in kinase inactivation.

Volume 4, no. 4, e00353-19, 2019, [https://doi.org/10.1128/mSphere.00353-19][1]. This article was published on 7 August 2019 with the fifth author’s name misspelled. The byline has been updated in the current version, posted on 30 August 2019. [1]: /lookup/doi/10.1128/mSphere.00353-19

Trypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei., Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We previously showed that downregulation of T. brucei mitochondrial inner membrane protein translocase 50 (TbTim50), an aspartate-based protein phosphatase and a component of the translocase of the mitochondrial inner membrane (TIM), increased the tolerance of procyclic cells to oxidative stress. Using comparative proteomics analysis and further validating the proteomics results by immunoblotting, here we discovered that TbTim50 downregulation caused an approximately 5-fold increase in the levels of PIP39, which is also an aspartate-based protein phosphatase and is primarily localized in glycosomes. A moderate upregulation of a number of glycosomal enzymes was also noticed due to TbTim50 knockdown. We found that the rate of mitochondrial ATP production by oxidative phosphorylation decreased and that substrate-level phosphorylation increased due to depletion of TbTim50. These results were correlated with relative increases in the levels of trypanosome alternative oxidase and hexokinase and a reduced-growth phenotype in low-glucose medium. The levels and activity of the mitochondrial superoxide dismutase and glutaredoxin levels were increased due to TbTim50 knockdown. Furthermore, we show that TbTim50 downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased. Knocking down both TbTim50 and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. These results suggest that TbTim50 and PIP39, two protein phosphatases in mitochondria and glycosomes, respectively, cross talk via the AMPK pathway to maintain cellular homeostasis in the procyclic form of T. brucei. IMPORTANCE Trypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.

SUMMARY The chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors., Graphical Abstract, In Brief Pharmacological inhibition of the WIN site of WDR5 is a promising anti-cancer strategy. Guarnaccia et al. use quantitative proteomics to characterize how inhibiting the WIN site alters the WDR5 interactome. This resource expands understanding of WDR5 and the action of WIN site inhibitors.

Neurofibromatosis type 1 (NF1) is a common neurogenetic disorder, in which affected individuals develop tumors of the nervous system. Children with NF1 are particularly prone to brain tumors (gliomas) involving the optic pathway that can result in impaired vision. Since tumor formation and expansion requires a cooperative tumor microenvironment, it is important to identify the cellular and acellular components associated with glioma development and growth. In this study, we used 3-D matrix assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) to measure the distributions of multiple molecular species throughout optic nerve tissue in mice with and without glioma, and to explore their spatial relationships within the 3-D volume of the optic nerve and chiasm. 3-D IMS studies often involve extensive workflows due to the high volume of sections required to generate high quality 3-D images. Herein, we present a workflow for 3-D data acquisition and volume reconstruction using mouse optic nerve tissue. The resulting 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions. Biological significance The current work addresses a number of challenges in 3-D MALDI IMS, driven by the small size of the mouse optic nerve and the need to maintain consistency across multiple 2-D IMS experiments. The 3-D IMS data yield both molecular similarities and differences between glioma-bearing and wild-type (WT) tissues, including protein distributions localizing to different anatomical subregions, which could then be targeted for identification and related back to the biology observed in gliomas of the optic nerve.

MALDI imaging mass spectrometry (IMS) has been applied to whole animal tissue sections of Pacific White Shrimp, Litopenaeus vannamei, in an effort to identify and spatially localize proteins in specific organ systems. Frozen shrimp were sectioned along the ventral-dorsal axis and methods were optimized for matrix application. In addition, tissue microextraction and homogenization was conducted followed by top-down LC-MS/MS analysis of intact proteins and searches of shrimp EST databases to identify imaged proteins. IMS images revealed organ system specific protein signals that highlighted the hepatopancreas, heart, nervous system, musculature, and cuticle. Top-down proteomics identification of abdominal muscle proteins revealed the sequence of the most abundant muscle protein that has no sequence homology to known proteins. Additional identifications of abdominal muscle proteins included titin, troponin-I, ubiquitin, as well as intact and multiple truncated forms of flightin; a protein known to function in high frequency contraction of insect wing muscles. The combined use of imaging mass spectrometry and top-down proteomics allowed for identification of novel proteins from the sparsely populated shrimp protein databases.

The primary interactions between incoming viral RNA genomes and host proteins are crucial to infection and immunity. Until now, the ability to study these events was lacking. We developed viral cross-linking and solid-phase purification (VIR-CLASP) to characterize the earliest interactions between viral RNA and cellular proteins. We investigated the infection of human cells using Chikungunya virus (CHIKV) and influenza A virus and identified hundreds of direct RNA-protein interactions. Here, we explore the biological impact of three protein classes that bind CHIKV RNA within minutes of infection. We find CHIKV RNA binds and hijacks the lipid-modifying enzyme fatty acid synthase (FASN) for pro-viral activity. We show that CHIKV genomes are N6-methyladenosine modified, and YTHDF1 binds and suppresses CHIKV replication. Finally, we find that the innate immune DNA sensor IFI16 associates with CHIKV RNA, reducing viral replication and maturation. Our findings have direct applicability to the investigation of potentially all RNA viruses.

Aquatic macrophytes play a key role in providing habitat, refuge and food for a range of biota in shallow lakes. However, many shallow lakes have experienced declines in macrophyte vegetation in recent decades, principally due to eutrophication. As changes in macrophyte composition and abundance can affect overall ecological structure and function of a lake, an assessment of the timing and nature of such changes is crucial to our understanding of the wider lake ecosystem. In the typical absence of historical plant records, the macro-remains of macrophytes preserved in lake sediments can be used to assess long-term changes in aquatic vegetation. We generated recent (150-200 years) plant macrofossil records for six English lakes subject to conservation protection to define past macrophyte communities, assess trajectories of ecological change and consider the implications of our findings for conservation targets and strategies. The data for all six lakes reveal a diverse submerged macrophyte community, with charophytes as a key component, in the early part of the sedimentary records. The stratigraphies indicate considerable change to the aquatic vegetation over the last two centuries with a general shift towards species more typically associated with eutrophic conditions. A common feature is the decline in abundance of low-growing charophytes and an increase in tall canopy-forming angiosperms such as fine-leaved Potamogeton species, Zannichellia palustris and Callitriche species. We hypothesise, based on findings from long-term datasets and palaeoecological records from enriched shallow lakes where plants are now absent, that the observed shifts provide a warning to managers that the lakes are on a pathway to complete macrophyte loss such that nutrient load reduction is urgently needed. It is the sound understanding of present-day plant ecology that affords such reliable interpretation of the fossil data which, in turn, provide valuable context for current conservation decisions.

Chronic rejection is the major cause of long-term heart allograft failure, characterized by tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signaling, regulating both T cell activation and migration of primed T cells to non-lymphoid antigen-rich tissue. We investigated the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model in which HY-mismatched male hearts were transplanted into female recipients. We show that suppression of p110δ activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment by impairing the localization of antigen-specific T cells to the grafts, while not inducing specific T cell tolerance. p110δ pharmacologic inactivation is effective when initiated after transplantation. Targeting p110δ activity might be a viable strategy for the treatment of heart chronic rejection in humans.

Diseases are characterized by distinct changes in tissue molecular distribution. Molecular analysis of intact tissues traditionally requires preexisting knowledge of, and reagents for, the targets of interest. Conversely, label-free discovery of disease-associated tissue analytes requires destructive processing for downstream identification platforms. Tissue-based analyses therefore sacrifice discovery to gain spatial distribution of known targets or sacrifice tissue architecture for discovery of unknown targets. To overcome these obstacles, we developed a multimodality imaging platform for discovery-based molecular histology. We apply this platform to a model of disseminated infection triggered by the pathogen Staphylococcus aureus, leading to the discovery of infection-associated alterations in the distribution and abundance of proteins and elements in tissue in mice. These data provide an unbiased, three-dimensional analysis of how disease affects the molecular architecture of complex tissues, enable culture-free diagnosis of infection through imaging-based detection of bacterial and host analytes, and reveal molecular heterogeneity at the host-pathogen interface.

The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators.

Summary: Multi-indicator palaeoecological studies have become increasingly popular over the last decade as the need for a more complete understanding of lake ecological histories has increased. However, the true potential of the full biological record for assessing the potential drivers of observed ecological shifts in lake sediment records has rarely been demonstrated. Here, we examine the remains of a range of food-web components including algae (diatoms), macrophytes (plant macrofossils), zooplankton (chitinous and ephippial Cladocera remains), invertebrates (including chironomids, bryozoans, Mollusca) and fish (fish scales and fish leech egg cocoons) in multiple sediment cores from Groby Pool, an enriched English shallow lake, to assess whole-ecosystem response to eutrophication over the last two centuries. We focus on three striking changes in the palaeorecord, namely the post-1900 increase in Daphnia spp., the post-1840 decline in Cristatella mucedo and the post-1940 increase in Cocconeis placentula, and utilise the multi-indicator palaeoecological data to evaluate possible explanations for these patterns. Principal curves analysis revealed marked and broadly simultaneous changes in the plant macrofossils, cladocerans, diatoms and chironomids (as well as in other animal remains such as bryozoans and Mollusca), indicating an early period of enrichment most likely associated with land-use change in the late 18th century, followed by a more recent eutrophication phase coincident with the discharge of sewage effluent to the lake from 1935. Ecological change, resulting from eutrophication, was shown to have progressed slowly and steadily and to have occurred at all trophic levels with a shift from a relatively diverse 'mesotrophic' macrophyte assemblage, dominance by benthic diatoms and plant-associated Chydoridae and chironomids towards a relatively species-poor, 'eutrophic' macrophyte community with dominance by planktonic algae (e.g. Cyclostephanoid diatom taxa), planktonic Cladocera (Bosmina, Daphnia) and a chironomid fauna dominated by mud-associated taxa. The inferred shift in the macrophyte community from charophyte to fine-leaved pondweed and Callitriche truncata suggests a reduction in the seasonal duration of plant dominance. The multi-indicator analysis indicates that a combination of increased phytoplankton biomass and low zooplanktivorous fish predation is likely to explain the recent increases in Daphnia spp., while loss of plant habitat and increased competition for food appear to be the most likely causes of the observed decline in C. mucedo, and resistance to increased grazing pressure from invertebrates is the most probable driver of the C. placentula increase. Our study illustrates the potential of using the full array of fossil groups preserved in lake sediment cores to infer ecosystem dynamics over centennial timescales and to contribute to our understanding of the mechanisms that may link biological assemblages to a range of forcing factors. Further, this paper provides methodological guidance by demonstrating the ability of amalgamated records from three cores to reveal a strong sequence of events and coherent patterns.

Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue–residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid–derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of α1β1 integrin consistent with the modification of collagen IV by hypochlorous (HOCl) and hypobromous acids. Noncollagenous (NC1) hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W28 and W192 are significantly elevated compared with nondiabetic controls. Molecular dynamics simulations predicted a more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/refolding. Our results suggest that hypohalous acid–derived modifications of renal ECM, and specifically collagen IV networks, contribute to functional protein damage in diabetes.

Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity in vitro, the in vivo functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, αBa and αBb. We observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in αBa than αBb mutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.

X-linked inhibitor of apoptosis (XIAP) plays an important role in preventing apoptotic cell death. XIAP has been shown to participate in signaling pathways, including Wnt signaling. XIAP regulates Wnt signaling by promoting the monoubiquitylation of the co-repressor Groucho/TLE family proteins, decreasing its affinity for the TCF/Lef family of transcription factors and allowing assembly of transcriptionally active β-catenin–TCF/Lef complexes. We now demonstrate that XIAP is phosphorylated by GSK3 at threonine 180, and that an alanine mutant (XIAP(T180A)) exhibits decreased Wnt activity compared to wild-type XIAP in cultured human cells and in Xenopus embryos. Although XIAP(T180A) ubiquitylates TLE3 at wild-type levels in vitro, it exhibits a reduced capacity to ubiquitylate and bind TLE3 in human cells. XIAP(T180A) binds Smac (also known as DIABLO) and inhibits Fas-induced apoptosis to a similar degree to wild-type XIAP. Our studies uncover a new mechanism by which XIAP is specifically directed towards a Wnt signaling function versus its anti-apoptotic function. These findings have implications for development of anti-XIAP therapeutics for human cancers.

A luminol chemiluminescence method was employed to study the oxidation kinetics of Fe(II) in both the absence and the presence of organic exudates released by the marine cyanobacterium Trichodesmium erythraeum. The apparent Fe(II) oxidation rate constant was studied for batch cultures grown with varying Fe' concentrations of 0.05–0.29 and 1.44–2.03 nmol L−1 at pH ranges from 8.1–8.6, corresponding to the change in pH in the cultures during the entire growth cycle. Fe(II) oxidation was accelerated when cells were growing exponentially and gradually decreased towards the stationary phase, consistent with the presence of organic exudates. The best fit of the kinetic model to the data also demonstrated clear differences in apparent Fe(II) oxidation rate constants during different growth phases. However, no significant difference was observed in oxidation rate constants between the two Fe' treatments. These findings suggest that Trichodesmium releases organic compounds into the extracellular environment that influence Fe redox chemistry, potentially affecting Fe bioavailability, and that the nature of the Fe(II) complexes formed is not influenced by Fe limitation of the organism's growth.

Aldehyde dehydrogenase-1A1 (ALDH1A1), CD133, CD44, and CD24 have been reported as cancer stem cell markers in ovarian cancers. The goal of our study was to assess the prognostic significance of these markers in patients with advanced serous ovarian cancer. Formalin-fixed, paraffin-embedded tissues from 347 ovarian cancers were used to construct a microarray. Immunohistochemical studies for ALDH1A1, CD133, CD44, and CD24 were performed and scored semiquantitatively by 2 pathologists based on intensity and percent of positive immunoreactive cells. Immunohistochemistry was compared to clinical parameters and survival. Of the 347 cases, early stage disease, nonserous tumors, cases with incomplete therapy, and cores with no tumor were excluded. Immunohistochemistry was interpretable in 124 of the 136 stage III and IV ovarian serous carcinoma. ALDH1A1, CD24, and CD44 were variably detected in both tumor and stromal cells, and immunoreactivity in tumor was stronger than in stromal cells. CD133 immunoreactivity was not quantified due to nonspecific staining in tumor and stroma. Statistical analyses using χ2 and Student t test revealed that ALDH1A1-positive (n=53) carcinoma were 3 times more likely to demonstrate platinum refractoriness than ALDH1A1-negative (n=71) tumors (17% vs. 6%, respectively; p=.04); however, neither progression free nor overall survival was influenced by ALDH1A1 status in this cohort. The expression of CD44 and CD24 had no clinicopathological associations in the present study. Our study supports that ALDH1A1 expression is associated with poor response to platinum-based therapy in patients with high-grade ovarian serous carcinoma. Further study of this relationship is needed to understand how this could impact clinical care.

Signaling changes that occur in the striatum following the loss of dopamine neurons in the Parkinson disease (PD) are poorly understood. While increases in the activity of kinases and decreases in the activity of phosphatases have been observed, the specific consequences of these changes are less well understood. Phosphatases, such as protein phosphatase 1 (PP1), are highly promiscuous and obtain substrate selectivity via targeting proteins. Spinophilin is the major PP1-targeting protein enriched in the postsynaptic density of striatal dendritic spines. Spinophilin association with PP1 is increased concurrent with decreases in PP1 activity in an animal model of PD. Using proteomic-based approaches, we observed dopamine depletion-induced decreases in spinophilin binding to multiple protein classes in the striatum. Specifically, there was a decrease in the association of spinophilin with neurofilament medium (NF-M) in dopamine-depleted striatum. Using a heterologous cell line, we determined that spinophilin binding to NF-M required overexpression of the catalytic subunit of protein kinase A and was decreased by cyclin-dependent protein kinase 5. Functionally, we demonstrate that spinophilin can decrease NF-M phosphorylation. Our data determine mechanisms that regulate, and putative consequences of, pathological changes in the association of spinophilin with NF-M that are observed in animal models of PD.