Heritable differences in abundance of bacterial rhizosphere taxa are correlated with fungal necrotrophic pathogen resistance.

Host-microbe interactions are increasingly recognized as important drivers of organismal health, growth, longevity and community-scale ecological processes. However, less is known about how genetic variation affects hosts' associated microbiomes and downstream phenotypes. We demonstrate that sunflower (Helianthus annuus) harbours substantial, heritable variation in microbial communities under field conditions. We show that microbial communities co-vary with heritable variation in resistance to root infection caused by the necrotrophic pathogen Sclerotinia sclerotiorum and that plants grown in autoclaved soil showed almost complete elimination of pathogen resistance. Association mapping suggests at least 59 genetic locations with effects on both microbial relative abundance and Sclerotinia resistance. Although the genetic architecture appears quantitative, we have elucidated previously unexplained genetic variation for resistance to this pathogen. We identify new targets for plant breeding and demonstrate the potential for heritable microbial associations to play important roles in defence in natural and human-altered environments.

RNA-protein analysis using a conditional CRISPR nuclease.

RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5' ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.

Extended-release naltrexone and harm reduction counseling for chronically homeless people with alcohol dependence.

BACKGROUND: Abstinence-based alcohol interventions are minimally desirable to and effective for chronically homeless individuals with alcohol dependence who have multimorbidity and high publicly funded service utilization and associated costs. Lower-barrier, patient-centered combined pharmacobehavioral interventions may more effectively treat this population. Harm reduction counseling involves a nonjudgmental, empathic style and patient-driven goal setting that requires neither abstinence nor use reduction. Extended-release naltrexone (XR-NTX), a monthly injectable formulation of an opioid receptor antagonist, reduces craving, is safe and effective for active drinkers, and may thereby support harm reduction goal setting. The aims of this 12-week, single-arm pilot were to initially document some aspects of feasibility, acceptability, and alcohol outcomes following XR-NTX administration and harm reduction counseling for chronically homeless individuals with alcohol dependence. METHODS: Participants were currently/formerly chronically homeless, alcohol-dependent individuals (N = 31) from 2 community-based agencies in the US Pacific Northwest. Measures included self-reported alcohol craving, quantity/frequency, problems, and biomarkers (ethyl glucuronide [EtG], liver transaminases). XR-NTX and harm reduction counseling were administered monthly over the 3-month treatment course. RESULTS: Of the 45 individuals approached, 43 were interested in participation. The first injection was received by 31 participants, and 24 complied with all study procedures. Participants reported the treatment was acceptable. Participants evinced decreases in alcohol craving (33%), typical (25%) and peak (34%) use, frequency (17%), problems (60%), and EtG from the baseline to the 12-week follow-up (Ps < .05). CONCLUSIONS: XR-NTX and harm reduction counseling are promising means of supporting reductions in alcohol use and alcohol-related harm among chronically homeless, alcohol-dependent individuals.

Survival analysis of freezing stress in the North American native perennial flax, Linum lewisii.

Abstract. The expansive range of Lewis flax (Linum lewisii), an herbaceous perennial, exposes the species to a diversity of climatic conditions. As interest in the domestication and adoption of perennial crop alternatives grows and interest in this species for natural area restoration continues, the assurance of a commercial plant variety's ability to endure the full range of possible climatic extremes is paramount. This study examines the freezing tolerance of a geographically representative sampling of 44 Lewis flax accessions at winter temperature extremes experienced in the northern Great Plains of the USA. Survival analysis models were adapted to include temperature exposure, in replacement of ordinal time typically used in such models, to produce statistics evaluating reactions to extreme temperatures that Lewis flax would encounter in our field environments. Our results revealed Lewis flax is more freezing tolerant than previously reported, and revealed four accessions with significantly superior genetic freezing tolerance than the released 'Maple Grove' cultivar. Furthermore, regrowth analyses indicate variation among accessions not associated with survival, which could lead to improving regrowth rate and survival simultaneously. These findings and their methodology expand the understanding of Lewis flax adaptation for winter hardiness and offer an efficient, new model that can be used to evaluate freezing tolerance at ordinal temperatures without requiring extensive prior physiological knowledge for a species.

Targeted metabolic labeling of yeast N-glycans with unnatural sugars.

Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridine-diphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1Delta strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.

A bifunctional role for group IIA secreted phospholipase A2 in human rheumatoid fibroblast-like synoviocyte arachidonic acid metabolism.

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is an important regulator of cytokine-mediated inflammatory responses in both in vitro and in vivo models of rheumatoid arthritis (RA). However, treatment of RA patients with sPLA(2)-IIA inhibitors shows only transient benefit. Using an activity-impaired sPLA(2)-IIA mutant protein (H48Q), we show that up-regulation of TNF-dependent PGE(2) production and cyclooxygenase-2 (COX-2) induction by exogenous sPLA(2)-IIA in RA fibroblast-like synoviocytes (FLSs) is independent of its enzyme function. Selective cytosolic phospholipase A(2)-α (cPLA(2)-α) inhibitors abrogate TNF/sPLA(2)-IIA-mediated PGE(2) production without affecting COX-2 levels, indicating arachidonic acid (AA) flux to COX-2 occurs exclusively through TNF-mediated activation of cPLA(2)-α. Nonetheless, exogenous sPLA(2)-IIA, but not H48Q, stimulates both AA mobilization from FLSs and microparticle-derived AA release that is not used for COX-2-dependent PGE(2) production. sPLA(2)-IIA-mediated AA production is inhibited by pharmacological blockade of sPLA(2)-IIA but not cPLA(2)-α. Exogenous H48Q alone, like sPLA(2)-IIA, increases COX-2 protein levels without inducing PGE(2) production. Unlike TNF, sPLA(2)-IIA alone does not rapidly mobilize NF-κB or activate phosphorylation of p38 MAPK, two key regulators of COX-2 protein expression, but does activate the ERK1/2 pathway. Thus, sPLA(2)-IIA regulates AA flux through the cPLA(2)-α/COX-2 pathway in RA FLSs by up-regulating steady state levels of these biosynthetic enzymes through an indirect mechanism, rather than direct provision of substrate to the pathway. Inhibitors that have been optimized for their potency in enzyme activity inhibition alone may not adequately block the activity-independent function of sPLA(2)-IIA.

Membrane proteomics of phagosomes suggests a connection to autophagy.

Phagocytosis is the central process by which macrophage cells internalize and eliminate infectious microbes as well as apoptotic cells. During maturation, phagosomes containing engulfed particles fuse with various endosomal compartments through the action of regulatory molecules on the phagosomal membrane. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling, and autophagy. Many identified proteins were not previously known to reside in the phagosome. We characterized several proteins in LBC phagosomes that change in abundance on induction of autophagy, a process that has been previously implicated in the host defense against microbial pathogens. These observations suggest crosstalk between autophagy and phagocytosis that may be relevant to the innate immune response of macrophages.

Nasal and sinus endoscopy for medical management of resistant rhinosinusitis, including postsurgical patients.

Nasal endoscopy has been practiced by allergists since the early 1980s; however, allergists in general have not embraced endoscopic evaluation of patients with sinus disease, either before or after surgery. Allergists are in a unique position to render medical (as opposed to surgical) care of patients with sinusitis. There has been a growing realization that endoscopy is a valuable procedure for the evaluation and medical treatment of patients with difficult sinusitis. This has resulted in the need for a resource to allow allergists to understand the nature of endoscopic findings in patients with sinusitis, either preoperatively or postoperatively. This article introduces the findings at endoscopy that are common in patients with sinusitis, including those that may be seen after surgery. The findings include perforation of the septum, retained secretions, small surgical ostium caused by postoperative ostial stenosis, previous Caldwell Luc procedure, recirculation of mucus, hyperplastic nasal disease, synechiae, recurrent disease in previously unaffected sinuses, empty nose syndrome, frontal sinus disease, dental disease, and other, more complicated entities.

WITHDRAWN: Endoscopic evaluation of resistant rhinosinusitis (before and after surgery) is now available on the American Academy of Allergy, Asthma & Immunology Web site.

This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The Publisher apologizes for any inconvenience this may cause.

Second generation tetrahydroquinoline-based protein farnesyltransferase inhibitors as antimalarials.

Substituted tetrahydroquinolines (THQs) have been previously identified as inhibitors of mammalian protein farnesyltransferase (PFT). Previously we showed that blocking PFT in the malaria parasite led to cell death and that THQ-based inhibitors are the most potent among several structural classes of PFT inhibitors (PFTIs). We have prepared 266 THQ-based PFTIs and discovered several compounds that inhibit the malarial enzyme in the sub- to low-nanomolar range and that block the growth of the parasite (P. falciparum) in the low-nanomolar range. This body of structure-activity data can be rationalized in most cases by consideration of the X-ray structure of one of the THQs bound to mammalian PFT together with a homology structural model of the malarial enzyme. The results of this study provide the basis for selection of antimalarial PFTIs for further evaluation in preclinical drug discovery assays.

The first potent inhibitor of mammalian group X secreted phospholipase A2: elucidation of sites for enhanced binding.

Using the X-ray structure of human group X secreted phospholipase A(2) (hGX), we carried out structure-based design of indole-based inhibitors and prepared the compounds using a new synthetic route. The most potent compound inhibited hGX and the mouse orthologue with an IC(50) of 75 nM. This compound is the most potent hGX inhibitor reported to date and was also found to inhibit a subset of the other mouse and human sPLA(2)s.

Is rhinosinusitis a cause of asthma?

There is a great deal of evidence of an association between rhinosinusitis and asthma. However, it is less clear whether rhinosinusitis is a direct trigger for asthma or the two conditions are simply manifestations of a common underlying process. Evidence for a role for rhinosinusitis as a trigger for asthma includes many examples of improvement in asthma once concomitant rhinosinusitis is treated medically or surgically. Possible mechanisms for this relationship include naso-pharyngo-bronchial reflexes, postnasal drip, abnormal breathing, and the local production of inflammatory mediators that trigger pulmonary inflammation via the bone marrow. On the other hand, evidence exists that rhinosinusitis and asthma are manifestations of a common process. For example, there are similarities between the histopathological changes in the epithelium in chronic rhinosinusitis and asthma. The bone marrow may provide the link between the upper and lower airways in creating a common disease. A second possible mechanism for a common disease is response to staphylococcal enterotoxins. Although evidence exists to suggest that rhinosinusitis either triggers asthma or represents a local manifestation of a shared disorder, the key to reconciling this apparent controversy is to consider that rhinosinusitis is not just a single, uniform disease. Current evidence suggests that rhinosinusitis with neither polyps nor eosinophilic inflammation acts as a direct trigger for asthma, whereas rhinosinusitis with both polyps and eosinophilic inflammation shares underlying mechanisms with asthma. Clearly, however, there is considerable overlap between the different, complex mechanisms that link rhinosinusitis to asthma.

Rhinosinusitis and pediatric asthma.

Rhinosinusitis is a common disease in patients of all age groups. Rhinosinusitis arises from a variety of infectious and inflammatory mechanisms. There is ample evidence that rhinosinusitis can directly influence asthma. There is also growing evidence that rhinosinusitis may be associated with asthma as different manifestations of the same disorder. A great deal of future research is required to fully elucidate the different mechanisms whereby rhinosinusitis influences or associates with asthma, but it is clear that rhinosinusitis needs to be considered in patients with severe or refractory asthma.

Harm reduction with pharmacotherapy for homeless people with alcohol dependence: protocol for a randomized controlled trial.

BACKGROUND: Interventions requiring abstinence from alcohol are neither preferred by nor shown to be highly effective with many homeless individuals with alcohol dependence. It is therefore important to develop lower-threshold, patient-centered interventions for this multimorbid and high-utilizing population. Harm-reduction counseling requires neither abstinence nor use reduction and pairs a compassionate style with patient-driven goal-setting. Extended-release naltrexone (XR-NTX), a monthly injectable formulation of an opioid receptor antagonist, reduces craving and may support achievement of harm-reduction goals. Together, harm-reduction counseling and XR-NTX may support alcohol harm reduction and quality-of-life improvement. AIMS: Study aims include testing: a) the relative efficacy of XR-NTX and harm-reduction counseling compared to a community-based, supportive-services-as-usual control, b) theory-based mediators of treatment effects, and c) treatment effects on publicly funded service costs. METHODS: This RCT involves four arms: a) XR-NTX+harm-reduction counseling, b) placebo+harm-reduction counseling, c) harm-reduction counseling only, and d) community-based, supportive-services-as-usual control conditions. Participants are currently/formerly homeless, alcohol dependent individuals (N=300). Outcomes include alcohol variables (i.e., craving, quantity/frequency, problems and biomarkers), health-related quality of life, and publicly funded service utilization and associated costs. Mediators include 10-point motivation rulers and the Penn Alcohol Craving Scale. XR-NTX and harm-reduction counseling are administered every 4weeks over the 12-week treatment course. Follow-up assessments are conducted at weeks 24 and 36. DISCUSSION: If found efficacious, XR-NTX and harm-reduction counseling will be well-positioned to support reductions in alcohol-related harm, decreases in costs associated with publicly funded service utilization, and increases in quality of life among homeless, alcohol-dependent individuals.

A chemical glycoproteomics platform reveals O-GlcNAcylation of mitochondrial voltage-dependent anion channel 2.

Protein modification by O-linked ß-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling modality, but identifying signal-specific O-GlcNAcylation events remains a significant experimental challenge. Here, we describe a method for visualizing and analyzing organelle- and stimulus-specific O-GlcNAcylated proteins and use it to identify the mitochondrial voltage-dependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2(-/-) cells resist the mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation, demonstrating a functional connection between O-GlcNAc signaling and mitochondrial physiology through VDAC2. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts.

Isotopic signature transfer and mass pattern prediction (IsoStamp): an enabling technique for chemically-directed proteomics.

Directed proteomics applies mass spectrometry analysis to a subset of information-rich proteins. Here we describe a method for targeting select proteins by chemical modification with a tag that imparts a distinct isotopic signature detectable in a full-scan mass spectrum. Termed isotopic signature transfer and mass pattern prediction (IsoStamp), the technique exploits the perturbing effects of a dibrominated chemical tag on a peptide's mass envelope, which can be detected with high sensitivity and fidelity using a computational method. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. To identify a tagged-peptide's sequence, we performed an inclusion list-driven shotgun proteomics experiment where peptides bearing a recoded mass envelope were targeted for fragmentation, allowing for direct site mapping. Using this approach, femtomole quantities of several targeted peptides were identified in total mammalian cell lysate, while traditional data-dependent methods were unable to identify as many peptides. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). IsoStamp has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for posttranslational modification.