STING Senses Microbial Viability to Orchestrate Stress-Mediated Autophagy of the Endoplasmic Reticulum.

Constitutive cell-autonomous immunity in metazoans predates interferon-inducible immunity and comprises primordial innate defense. Phagocytes mobilize interferon-inducible responses upon engagement of well-characterized signaling pathways by pathogen-associated molecular patterns (PAMPs). The signals controlling deployment of constitutive cell-autonomous responses during infection have remained elusive. Vita-PAMPs denote microbial viability, signaling the danger of cellular exploitation by intracellular pathogens. We show that cyclic-di-adenosine monophosphate in live Gram-positive bacteria is a vita-PAMP, engaging the innate sensor stimulator of interferon genes (STING) to mediate endoplasmic reticulum (ER) stress. Subsequent inactivation of the mechanistic target of rapamycin mobilizes autophagy, which sequesters stressed ER membranes, resolves ER stress, and curtails phagocyte death. This vita-PAMP-induced ER-phagy additionally orchestrates an interferon response by localizing ER-resident STING to autophagosomes. Our findings identify stress-mediated ER-phagy as a cell-autonomous response mobilized by STING-dependent sensing of a specific vita-PAMP and elucidate how innate receptors engage multilayered homeostatic mechanisms to promote immunity and survival after infection.

Systematic Fe(II)-EDTA Method of Dose-Dependent Hydroxyl Radical Generation for Protein Oxidative Footprinting.

Correlating the structure and dynamics of proteins with biological function is critical to understanding normal and dysfunctional cellular mechanisms. We describe a quantitative method of hydroxyl radical generation via Fe(II)-ethylenediaminetetraacetic acid (EDTA)-catalyzed Fenton chemistry that provides ready access to protein oxidative footprinting using equipment commonly found in research and process control laboratories. Robust and reproducible dose-dependent oxidation of protein samples is observed and quantitated by mass spectrometry with as fine a single residue resolution. An oxidation analysis of lysozyme provides a readily accessible benchmark for our method. The efficacy of our oxidation method is demonstrated by mapping the interface of a RAS-monobody complex, the surface of the NIST mAb, and the interface between PRC2 complex components. These studies are executed using standard laboratory tools and a few pennies of reagents; the mass spectrometry analysis can be streamlined to map the protein structure with single amino acid residue resolution.

Fibrinogen alpha amyloidosis: insights from proteomics.

Introduction: Systemic amyloidosis is a diverse group of diseases that, although rare, pose a serious health issue and can lead to organ failure and death. Amyloid typing is essential in determining the causative protein and initiating proper treatment. Mass spectrometry-based proteomics is currently the most sensitive and accurate means of typing amyloid. Areas covered: Amyloidosis can be systemic or localized, acquired or hereditary, and can affect any organ or tissue. Diagnosis requires biopsy, histological analysis, and typing of the causative protein to determine treatment. The kidneys are the most commonly affected organ in systemic disease. Fibrinogen alpha chain amyloidosis (AFib) is the most prevalent form of hereditary renal amyloidosis. Select mutations in the fibrinogen Aα (FGA) gene lead to AFib. Expert commentary: Mass spectrometry is currently the most specific and sensitive method for amyloid typing. Identification of the mutated fibrinogen alpha chain can be difficult in the case of 'private' frameshift mutations, which dramatically change the sequences of the expressed fibrinogen alpha chain. A combination of expert pathologist review, mass spectrometry, and gene sequencing can allow for confident diagnosis and determination of the fibrinogen alpha chain mutated sequence.

Using Quantitative Spectrometry to Understand the Influence of Genetics and Nutritional Perturbations On the Virulence Potential of Staphylococcus aureus.

Staphylococcus aureus (Sa) is the leading cause of a variety of bacterial infections ranging from superficial skin infections to invasive and life threatening diseases such as septic bacteremia, necrotizing pneumonia, and endocarditis. The success of Sa as a human pathogen is contributed to its ability to adapt to different environments by changing expression, production, or secretion of virulence factors. Although Sa immune evasion is well-studied, the regulation of virulence factors under different nutrient and growth conditions is still not well understood. Here, we used label-free quantitative mass spectrometry to quantify and compare the Sa exoproteins (i.e. exoproteomes) of master regulator mutants or established reference strains. Different environmental conditions were addressed by growing the bacteria in rich or minimal media at different phases of growth. We observed clear differences in the composition of the exoproteomes depending on the genetic background or growth conditions. The relative abundance of cytotoxins determined in our study correlated well with differences in cytotoxicity measured by lysis of human neutrophils. Our findings demonstrate that label-free quantitative mass spectrometry is a versatile tool for predicting the virulence of bacterial strains and highlights the importance of the experimental design for in vitro studies. Furthermore, the results indicate that label-free proteomics can be used to cluster isolates into groups with similar virulence properties, highlighting the power of label-free quantitative mass spectrometry to distinguish Sa strains.

Phosphoproteomics of Fibroblast Growth Factor 1 (FGF1) Signaling in Chondrocytes: Identifying the Signature of Inhibitory Response.

Fibroblast growth factor (FGF) signaling is vital for many biological processes, beginning with development. The importance of FGF signaling for skeleton formation was first discovered by the analysis of genetic FGFR mutations which cause several bone morphogenetic disorders, including achondroplasia, the most common form of human dwarfism. The formation of the long bones is mediated through proliferation and differentiation of highly specialized cells - chondrocytes.Chondrocytes respond to FGF with growth inhibition, a unique response which differs from the proliferative response of the majority of cell types; however, its molecular determinants are still unclear. Quantitative phosphoproteomic analysis was utilized to catalogue the proteins whose phosphorylation status is changed upon FGF1 treatment. The generated dataset consists of 756 proteins. We could localize the divergence between proliferative (canonical) and inhibitory (chondrocyte specific) FGF transduction pathways immediately upstream of AKT kinase. Gene Ontology (GO) analysis of the FGF1 regulated peptides revealed that many of the identified phosphorylated proteins are assigned to negative regulation clusters, in accordance with the observed inhibitory growth response. This is the first time a comprehensive subset of proteins involved in FGF inhibitory response is defined. We were able to identify a number of targets and specifically discover glycogen synthase kinase3ß (GSK3ß) as a novel key mediator of FGF inhibitory response in chondrocytes.

Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence.

Mycobacterium tuberculosis (Mtb) encodes five type VII secretion systems (T7SS), designated ESX-1-ESX-5, that are critical for growth and pathogenesis. The best characterized is ESX-1, which profoundly impacts host cell interactions. In contrast, the ESX-3 T7SS is implicated in metal homeostasis, but efforts to define its function have been limited by an inability to recover deletion mutants. We overcame this impediment using medium supplemented with various iron complexes to recover mutants with deletions encompassing select genes within esx-3 or the entire operon. The esx-3 mutants were defective in uptake of siderophore-bound iron and dramatically accumulated cell-associated mycobactin siderophores. Proteomic analyses of culture filtrate revealed that secretion of EsxG and EsxH was codependent and that EsxG-EsxH also facilitated secretion of several members of the proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) protein families (named for conserved PE and PPE N-terminal motifs). Substrates that depended on EsxG-EsxH for secretion included PE5, encoded within the esx-3 locus, and the evolutionarily related PE15-PPE20 encoded outside the esx-3 locus. In vivo characterization of the mutants unexpectedly showed that the ESX-3 secretion system plays both iron-dependent and -independent roles in Mtb pathogenesis. PE5-PPE4 was found to be critical for the siderophore-mediated iron-acquisition functions of ESX-3. The importance of this iron-acquisition function was dependent upon host genotype, suggesting a role for ESX-3 secretion in counteracting host defense mechanisms that restrict iron availability. Further, we demonstrate that the ESX-3 T7SS secretes certain effectors that are important for iron uptake while additional secreted effectors modulate virulence in an iron-independent fashion.

A film set for the elicitation of emotion in research: A comprehensive catalog derived from four decades of investigation.

Emotions are highly influential to many psychological processes. Indeed, research employing emotional stimuli is rapidly escalating across the field of psychology. However, challenges remain regarding discrete evocation of frequently co-elicited emotions such as amusement and happiness, or anger and disgust. Further, as much contemporary work in emotion employs college students, we sought to additionally evaluate the efficacy of film clips to discretely elicit these more challenging emotions in a young adult population using an online medium. The internet is an important tool for investigating responses to emotional stimuli, but validations of emotionally evocative film clips across laboratory and web-based settings are limited in the literature. An additional obstacle is identifying stimuli amidst the numerous film clip validation studies. During our investigation, we recognized the lack of a categorical database to facilitate rapid identification of useful film clips for individual researchers' unique investigations. Consequently, here we also sought to produce the first compilation of such stimuli into an accessible and comprehensive catalog. We based our catalog upon prior work as well as our own, and identified 24 articles and 295 film clips from four decades of research. We present information on the validation of these clips in addition to our own research validating six clips using online administration settings. The results of our search in the literature and our own study are presented in tables designed to facilitate and improve a selection of highly valid film stimuli for future research.

Effectiveness of Delayed Cord Clamping in Reducing Postdelivery Complications in Preterm Infants: A Systematic Review.

This systematic review evaluates the effectiveness of delayed cord clamping in preterm infants on reducing postdelivery complications of anemia, hemodynamic instability, and the development of intraventricular hemorrhages. Interventions included varying durations of delayed cord clamping with and without cord milking as compared with immediate cord clamping, shorter delays in cord clamping, and delayed cord clamping without cord milking. A comprehensive search of randomized controlled trials, observational, cohort, and before-after studies was conducted between 1946 and 2015 in the electronic databases of Ovid MEDLINE, Embase, and Google Scholar. Studies were critically appraised using the Critical Appraisal Skills Program guidelines. Twenty-seven studies were included in the review from 1997 to 2015 from varying countries. Outcome measures included hematocrit/hemoglobin levels, measured or calculated blood volumes levels, number and volume of blood transfusions, presence of hypotension and need for treatment, and development of intraventricular hemorrhage. Delayed cord clamping can lead to improved outcomes measures in preterm infants. This review supports the current recommendation to perform delayed cord clamping during preterm deliveries.

Community Health Asset Mapping Partnership Engages Hispanic/Latino Health Seekers and Providers.

BACKGROUND: The Hispanic/Latino population in Forsyth County, North Carolina, is growing quickly and experiencing significant disparities in access to care and health outcomes. Assessing community perceptions and utilization of health care resources in order to improve health equity among Hispanics/Latinos at both the county and state levels is critical. METHODS: Our community engagement process was guided by the Community Health Assets Mapping Partnerships (CHAMP) approach, which helps identify gaps in health care availability and areas for immediate action to improve access to and quality of health care. Specifically, we invited and encouraged the Hispanic/Latino population to participate in 4 different workshops conducted in Spanish or English. Participants were identified as either health care providers, defined as anyone who provides health care or a related service, or health care seekers, defined as anyone who utilizes such services. RESULTS: The most commonly cited challenges to access to care were cost of health care, documentation status, lack of public transportation, racism, lack of care, lack of respect, and education/language. These data were utilized to drive continued engagement with the Hispanic community, and action steps were outlined. LIMITATIONS: While participation in the workshops was acceptable, greater representation of health care seekers and community providers is needed. CONCLUSIONS: This process is fundamental to multilevel initiatives under way to develop trust and improve relationships between the Hispanic/Latino community and local health care entities in Forsyth County. Follow-through on recommended action steps will continue to further identify disparities, close gaps in care, and potentially impact local and state policies with regard to improving the health status of the Hispanic/Latino community.

The galanin receptor agonist, galnon, attenuates cocaine-induced reinstatement and dopamine overflow in the frontal cortex.

Relapse represents one of the most significant problems in the long-term treatment of drug addiction. Cocaine blocks plasma membrane monoamine transporters and increases dopamine (DA) overflow in the brain, and DA is critical for the motivational and primary reinforcing effects of the drug as well as cocaine-primed reinstatement of cocaine seeking in rats, a model of relapse. Thus, modulators of the DA system may be effective for the treatment of cocaine dependence. The endogenous neuropeptide galanin inhibits DA transmission, and both galanin and the synthetic galanin receptor agonist, galnon, interfere with some rewarding properties of cocaine. The purpose of this study was to further assess the effects of galnon on cocaine-induced behaviors and neurochemistry in rats. We found that galnon attenuated cocaine-induced motor activity, reinstatement and DA overflow in the frontal cortex at a dose that did not reduce baseline motor activity, stable self-administration of cocaine, baseline extracellular DA levels or cocaine-induced DA overflow in the nucleus accumbens (NAc). Similar to cocaine, galnon had no effect on stable food self-administration but reduced food-primed reinstatement. These results indicate that galnon can diminish cocaine-induced hyperactivity and relapse-like behavior, possibly in part by modulating DA transmission in the frontal cortex.

Intestinal cell kinase (ICK) promotes activation of mTOR complex 1 (mTORC1) through phosphorylation of Raptor Thr-908.

Intestinal cell kinase (ICK), named after its cloning origin, the intestine, is actually a ubiquitously expressed and highly conserved serine/threonine protein kinase. Recently we reported that ICK supports cell proliferation and G(1) cell cycle progression. ICK deficiency significantly disrupted the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling events. However, the biological substrates that mediate the downstream signaling effects of ICK in proliferation and the molecular mechanisms by which ICK interacts with mTORC1 are not well defined. Our prior studies also provided biochemical evidence that ICK interacts with the mTOR/Raptor complex in cells and phosphorylates Raptor in vitro. In this report, we investigated whether and how ICK targets Raptor to regulate the activity of mTORC1. Using the ICK substrate consensus sequence [R-P-X-S/T-P/A/T/S], we identified a putative phosphorylation site, RPGT908T, for ICK in human Raptor. By mass spectrometry and a phospho-specific antibody, we showed that Raptor Thr-908 is a novel in vivo phosphorylation site. ICK is able to phosphorylate Raptor Thr-908 both in vitro and in vivo and when Raptor exists in protein complexes with or without mTOR. Although expression of the Raptor T908A mutant did not affect the mTORC1 integrity, it markedly impaired the mTORC1 activation by insulin or by overexpression of the small GTP-binding protein RheB under nutrient starvation. Our findings demonstrate an important role for ICK in modulating the activity of mTORC1 through phosphorylation of Raptor Thr-908 and thus implicate a potential signaling mechanism by which ICK regulates cell proliferation and division.

The core functions and forms paradigm throughout EPIS: designing and implementing an evidence-based practice with function fidelity.

There are numerous frameworks for implementing evidence-based practices (EBPs) in novel settings to achieve "fidelity." However, identifying appropriate referents for fidelity poses a challenge. The Core Functions and Forms paradigm offers a model that can inform adaptation decisions throughout all phases of the Exploration, Preparation, Implementation, Sustainment (EPIS) framework. We applied the Core Functions-Forms paradigm throughout the Exploration and Preparation phases of EPIS in the design of two EBPs targeting family protective factors among Latinos in San Diego, as well as describe plans for its use in Implementation and Sustainment. We employed a distinct approach for each intervention element to contrast adaptation decisions that prioritize adherence to either form or function fidelity. We describe our application of the functions-forms paradigm within the EPIS framework, focusing on the Preparation phase. We also provide functions-forms matrices that map out the relationship between individual intervention components (forms) and the essential processes (functions) by which components are theorized to exert their impact. This case study of how the core functions-forms framework can be mapped onto EPIS can support a conceptual shift from prioritizing form fidelity to also focusing on function fidelity. This might allow interventionists to target appropriate fidelity referents when adapting an EBP, rather than defaulting to maintaining fidelity to forms as described in the protocol. We see great promise for using this framework for guiding actions throughout all EPIS phases and informing future applications of this paradigm to foster more robust fidelity to function.

Developing a meta-understanding of 'human aspects' of providing palliative care.

Objectives: Our intention was to develop a meta-understanding of the 'human aspects' of providing palliative care. Integral to developing this meta-understanding was recognising the individuality of people, their varied involvements, situations, understandings, and responses, and the difficulty in stepping back to get a whole view of this while being in the midst of providing palliative care. We intended for this meta-understanding to inform reflections and sense-making conversations related to people's changing situations and diverse needs. Methods: Using collaborative inquiry, this qualitative research was undertaken 'with' clinicians rather than 'on' them. Our team (n = 7) was composed of palliative care clinicians and researchers from a co-located rural health service and university. We explored our personal perceptions and experiences through a series of 12 meetings over 8 months. In addition, through five focus groups, we acccessed perceptions and experiences of 13 purposively sampled participants with a range of roles as carers and/or healthcare providers. Data were dialogically and iteratively interpreted. Findings: Our meta-understanding of 'human aspects' of providing palliative care, represented diagrammatically in a model, is composed of ATTRIBUTES OF HUMANITY and ACTIONS OF CARING. ATTRIBUTES OF HUMANITY are death's inevitability, suffering's variability, compassion's dynamic nature, and hope's precariousness. ACTIONS OF CARING include recognising and responding, aligning expectations, valuing relationships, and using resources wisely. The meta-understanding is a framework to keep multiple complex concepts 'in view' as they interrelate with each other. Significance of findings: Our meta-understanding, highlighting 'human aspects' of providing palliative care, has scope to embrace complexity, uncertainty, and the interrelatedness of people in the midst of resourcing, requiring, and engaging in palliative care. Questions are posed for this purpose. The non-linear diagrammatic representation of ATTRIBUTES OF HUMANITY and ACTIONS OF CARING facilitates multiple ways of engaging and revisiting palliative care situations or navigating changes within and across them.

The cell envelope of Staphylococcus aureus selectively controls the sorting of virulence factors.

Staphylococcus aureus bi-component pore-forming leukocidins are secreted toxins that directly target and lyse immune cells. Intriguingly, one of the leukocidins, Leukocidin AB (LukAB), is found associated with the bacterial cell envelope in addition to secreted into the extracellular milieu. Here, we report that retention of LukAB on the bacterial cells provides S. aureus with a pre-synthesized active toxin that kills immune cells. On the bacteria, LukAB is distributed as discrete foci in two distinct compartments: membrane-proximal and surface-exposed. Through genetic screens, we show that a membrane lipid, lysyl-phosphatidylglycerol (LPG), and lipoteichoic acid (LTA) contribute to LukAB deposition and release. Furthermore, by studying non-covalently surface-bound proteins we discovered that the sorting of additional exoproteins, such as IsaB, Hel, ScaH, and Geh, are also controlled by LPG and LTA. Collectively, our study reveals a multistep secretion system that controls exoprotein storage and protein translocation across the S. aureus cell wall.

Proteomic analysis shows that the main constituent of subepidermal localised cutaneous amyloidosis is not galectin-7.

Lichen or macular localised cutaneous amyloidoses have long been described as keratinic amyloidoses and believed to be due to the deposition of cytokeratin peptides originating from epidermis in the dermal papillae. However, recently it was suggested that galectin-7 is the causative protein for this type of amyloidosis. This was based on the detection of galectin-7 in a biopsy from a patient diagnosed with Bowen's disease and localised cutaneous amyloidosis. In this study we report mass spectrometry-based proteomic analysis of the protein composition of localised cutaneous amyloid deposits from seven patients using laser microdissection and show that basal keratins are the main constituents of the amyloid deposits. Galectin-7 was not present in the dermal amyloid deposits and was only present in the overlying Congo red negative epidermis.

Localized Peritumoral AL Amyloidosis Associated With Mantle Cell Lymphoma With Plasmacytic Differentiation.

Immunoglobulin light chain (AL) amyloidosis is characterized by the deposition of amyloid fibers derived from pathologic immunoglobulin light chains. Although systemic plasma cell neoplasms are the most common cause of AL amyloidosis, a subset of cases is caused by B-cell lymphoproliferative disorders such as lymphoplasmacytic lymphoma or extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue. Recently, SOX11-negative IGH hypermutated mantle cell lymphoma (MCL) is recognized to show frequent plasmacytic differentiation and indolent clinical course. Here, we report 3 cases of peritumoral AL amyloidosis associated with SOX11-negative MCL. All 3 cases showed cyclin D1 expression by immunohistochemistry and CCND1 translocation as detected by fluorescence in situ hybridization analysis. Peritumoral AL amyloidosis was observed at the biopsy sites in the gastrointestinal tract, a supraclavicular lymph node, and a cervical lymph node, and all presented with marked plasmacytic differentiation of lymphoma cells. None of the cases showed evidence of bone marrow involvement by morphology and immunophenotyping. None of the patients had distant organ involvement with systemic amyloidosis. All 3 patients had an indolent clinical course and are alive with disease at the time of the last follow-up (range: 48 to 74 mo). Our findings show that MCL with plasmacytic differentiation can cause amyloid deposition and CCND1 abnormalities should be performed in all cases of extramedullary AL amyloidosis. Recognition of indolent MCL as a cause of peritumoral AL amyloidosis may have important clinical management implications.

Minimal Residual Disease in Myeloma: Application for Clinical Care and New Drug Registration.

The development of novel agents has transformed the treatment paradigm for multiple myeloma, with minimal residual disease (MRD) negativity now achievable across the entire disease spectrum. Bone marrow-based technologies to assess MRD, including approaches using next-generation flow and next-generation sequencing, have provided real-time clinical tools for the sensitive detection and monitoring of MRD in patients with multiple myeloma. Complementary liquid biopsy-based assays are now quickly progressing with some, such as mass spectrometry methods, being very close to clinical use, while others utilizing nucleic acid-based technologies are still developing and will prove important to further our understanding of the biology of MRD. On the regulatory front, multiple retrospective individual patient and clinical trial level meta-analyses have already shown and will continue to assess the potential of MRD as a surrogate for patient outcome. Given all this progress, it is not surprising that a number of clinicians are now considering using MRD to inform real-world clinical care of patients across the spectrum from smoldering myeloma to relapsed refractory multiple myeloma, with each disease setting presenting key challenges and questions that will need to be addressed through clinical trials. The pace of advances in targeted and immune therapies in multiple myeloma is unprecedented, and novel MRD-driven biomarker strategies are essential to accelerate innovative clinical trials leading to regulatory approval of novel treatments and continued improvement in patient outcomes.

Immunosuppressive networks in the tumour environment and their effect in dendritic cells.

The failure of the immune system to provide protection against tumour cells is an important immunological problem. It is now evident that inadequate function of the host immune system is one of the main mechanisms by which tumours escape from immune control, as well as an important factor that limits the success of cancer immunotherapy. In recent years, it has become increasingly clear that defects in dendritic cells have a crucial role in non-responsiveness to tumours. This article focuses on the functional consequences and recently described mechanisms of the dendritic-cell defects in cancer.