The Effects of Social Distance and Front-of-Package Claims on Healthy Food Selection: Moderating Role of Perceived Importance of Eating Healthily.

Applying construal level theory, this study examined how social distance (thinking of self/children), front-of-package (FOP) claim type (nutrient/health/control), and perceived importance of eating healthily (low/high) impact consumer responses (attitudes/purchase intent) to healthier food products through an online experiment with 171 U.S. parents from low-to-mid socio-economic households. Participants were randomly assigned to view controlled images of healthier foods with packaging that bore different claim types for real and fictitious brands. Results revealed that when choosing for themselves, consumer attitudes were more positive when the healthier food package carried a nutrient (vs. health) claim, however, control claims received the most positive evaluations. When choosing for children, attitudes were more positive when the package carried a health (vs. nutrient/control) claim. Attitudes toward healthier foods were higher for consumers with high (vs. low) perceived importance of eating healthily when the package bore a nutrient claim, however, their attitudes did not significantly differ when the package bore a health/control claim. Purchase intent for healthier foods was higher for consumers with high (vs. low) perceived importance of healthy eating when shopping for self; whereas, when shopping for children, purchase intent did not significantly differ between consumers who varied in perceived importance of eating healthily.

Long-Term Cellulose Enrichment Selects for Highly Cellulolytic Consortia and Competition for Public Goods.

The complexity of microbial communities hinders our understanding of how microbial diversity and microbe-microbe interactions impact community functions. Here, using six independent communities originating from the refuse dumps of leaf-cutter ants and enriched using the plant polymer cellulose as the sole source of carbon, we examine how changes in bacterial diversity and interactions impact plant biomass decomposition. Over up to 60 serial transfers (∼8 months) using Whatman cellulose filter paper, cellulolytic ability increased and then stabilized in four enrichment lines and was variable in two lines. Bacterial community characterization using 16S rRNA gene amplicon sequencing showed community succession differed between the highly cellulolytic enrichment lines and those that had slower and more variable cellulose degradation rates. Metagenomic and metatranscriptomic analyses revealed that Cellvibrio and/or Cellulomonas dominated each enrichment line and produced the majority of cellulase enzymes, while diverse taxa were retained within these communities over the duration of transfers. Interestingly, the less cellulolytic communities had a higher diversity of organisms competing for the cellulose breakdown product cellobiose, suggesting that cheating slowed cellulose degradation. In addition, we found competitive exclusion as an important factor shaping all of the communities, with a negative correlation of Cellvibrio and Cellulomonas abundance within individual enrichment lines and the expression of genes associated with the production of secondary metabolites, toxins, and other antagonistic compounds. Our results provide insights into how microbial diversity and competition affect the stability and function of cellulose-degrading communities. IMPORTANCE Microbial communities are a key driver of the carbon cycle through the breakdown of complex polysaccharides in diverse environments including soil, marine systems, and the mammalian gut. However, due to the complexity of these communities, the species-species interactions that impact community structure and ultimately shape the rate of decomposition are difficult to define. Here, we performed serial enrichment on cellulose using communities inoculated from leaf-cutter ant refuse dumps, a cellulose-rich environment. By concurrently tracking cellulolytic ability and community composition and through metagenomic and metatranscriptomic sequencing, we analyzed the ecological dynamics of the enrichment lines. Our data suggest that antagonism is prevalent in these communities and that competition for soluble sugars may slow degradation and lead to community instability. Together, these results help reveal the relationships between competition and polysaccharide decomposition, with implications in diverse areas ranging from microbial community ecology to cellulosic biofuels production.

Validation of a Novel Multitarget Blood Test Shows High Sensitivity to Detect Early Stage Hepatocellular Carcinoma.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Although biannual ultrasound surveillance with or without α-fetoprotein (AFP) testing is recommended for at-risk patients, sensitivity for early stage HCC, for which potentially curative treatments exist, is suboptimal. We conducted studies to establish the multitarget HCC blood test (mt-HBT) algorithm and cut-off values and to validate test performance in patients with chronic liver disease. METHODS: Algorithm development and clinical validation studies were conducted with participants in an international, multicenter, case-control study. Study subjects had underlying cirrhosis or chronic hepatitis B virus; HCC cases were diagnosed per the American Association for the Study of Liver Diseases criteria and controls were matched for age and liver disease etiology. Whole blood and serum were shipped to a central laboratory and processed while blinded to case/control status. An algorithm was developed for the mt-HBT, which incorporates methylation biomarkers (HOXA1, TSPYL5, and B3GALT6), AFP, and sex. RESULTS: In algorithm development, with 136 HCC cases (60% early stage) and 404 controls, the mt-HBT showed 72% sensitivity for early stage HCC at 88% specificity. Test performance was validated in an independent cohort of 156 HCC cases (50% early stage) and 245 controls, showing 88% overall sensitivity, 82% early stage sensitivity, and 87% specificity. Early stage sensitivity in clinical validation was significantly higher than AFP at 20 ng/mL or greater (40%; P < .0001) and GALAD (gender, age, Lens culinaris agglutinin-reactive AFP, AFP, and des-γ-carboxy-prothrombin score) of -0.63 or greater (71%; P = .03), although AFP and GALAD at these cut-off values had higher specificities (100% and 93%, respectively). CONCLUSIONS: The mt-HBT may significantly improve early stage HCC detection for patients undergoing HCC surveillance, a critical step to increasing curative treatment opportunities and reducing mortality. ClinicalTrials.gov number NCT03628651.

An evolutionarily distinct chaperone promotes 20S proteasome α-ring assembly in plants.

The core protease (CP) subcomplex of the 26S proteasome houses the proteolytic active sites and assumes a barrel shape comprised of four co-axially stacked heptameric rings formed by structurally related α- and ß-subunits. CP biogenesis typically begins with the assembly of the α-ring, which then provides a template for ß-subunit integration. In eukaryotes, α-ring assembly is partially mediated by two hetero-dimeric chaperones, termed Pba1-Pba2 (Add66) and Pba3-Pba4 (also known as Irc25-Poc4) in yeast. Pba1-Pba2 initially promotes orderly recruitment of the α-subunits through interactions between their C-terminal HbYX or HbF motifs and pockets at the α5-α6 and α6-α7 interfaces. Here, we identified PBAC5 as a fifth α-ring assembly chaperone in Arabidopsis that directly binds the Pba1 homolog PBAC1 to form a trimeric PBAC5-PBAC1-PBAC2 complex. PBAC5 harbors a HbYX motif that docks with a pocket between the α4 and α5 subunits during α-ring construction. Arabidopsis lacking PBAC5, PBAC1 and/or PBAC2 are hypersensitive to proteotoxic, salt and osmotic stresses, and display proteasome assembly defects. Remarkably, whereas PBAC5 is evolutionarily conserved among plants, sequence relatives are also dispersed within other kingdoms, including a scattered array of fungal, metazoan and oomycete species.

Bacteria Contribute to Plant Secondary Compound Degradation in a Generalist Herbivore System.

Herbivores must overcome a variety of plant defenses, including coping with plant secondary compounds (PSCs). To help detoxify these defensive chemicals, several insect herbivores are known to harbor gut microbiota with the metabolic capacity to degrade PSCs. Leaf-cutter ants are generalist herbivores, obtaining sustenance from specialized fungus gardens that act as external digestive systems and which degrade the diverse collection of plants foraged by the ants. There is in vitro evidence that certain PSCs harm Leucoagaricus gongylophorus, the fungal cultivar of leaf-cutter ants, suggesting a role for the Proteobacteria-dominant bacterial community present within fungus gardens. In this study, we investigated the ability of symbiotic bacteria present within fungus gardens of leaf-cutter ants to degrade PSCs. We cultured fungus garden bacteria, sequenced the genomes of 42 isolates, and identified genes involved in PSC degradation, including genes encoding cytochrome P450 enzymes and genes in geraniol, cumate, cinnamate, and α-pinene/limonene degradation pathways. Using metatranscriptomic analysis, we showed that some of these degradation genes are expressed in situ Most of the bacterial isolates grew unhindered in the presence of PSCs and, using gas chromatography-mass spectrometry (GC-MS), we determined that isolates from the genera Bacillus, Burkholderia, Enterobacter, Klebsiella, and Pseudomonas degrade α-pinene, ß-caryophyllene, or linalool. Using a headspace sampler, we show that subcolonies of fungus gardens reduced α-pinene and linalool over a 36-h period, while L. gongylophorus strains alone reduced only linalool. Overall, our results reveal that the bacterial communities in fungus gardens play a pivotal role in alleviating the effect of PSCs on the leaf-cutter ant system.IMPORTANCE Leaf-cutter ants are dominant neotropical herbivores capable of deriving energy from a wide range of plant substrates. The success of leaf-cutter ants is largely due to their external gut, composed of key microbial symbionts, specifically, the fungal mutualist L. gongylophorus and a consistent bacterial community. Both symbionts are known to have critical roles in extracting energy from plant material, yet comparatively little is known about their roles in the detoxification of plant secondary compounds. In this study, we assessed if the bacterial communities associated with leaf-cutter ant fungus gardens can degrade harmful plant chemicals. We identify plant secondary compound detoxification in leaf-cutter ant gardens as a process that depends on the degradative potential of both the bacterial community and L. gongylophorus Our findings suggest that the fungus garden and its associated microbial community influence the generalist foraging abilities of the ants, underscoring the importance of microbial symbionts in plant substrate suitability for herbivores.

The antimicrobial potential of Streptomyces from insect microbiomes.

Antimicrobial resistance is a global health crisis and few novel antimicrobials have been discovered in recent decades. Natural products, particularly from Streptomyces, are the source of most antimicrobials, yet discovery campaigns focusing on Streptomyces from the soil largely rediscover known compounds. Investigation of understudied and symbiotic sources has seen some success, yet no studies have systematically explored microbiomes for antimicrobials. Here we assess the distinct evolutionary lineages of Streptomyces from insect microbiomes as a source of new antimicrobials through large-scale isolations, bioactivity assays, genomics, metabolomics, and in vivo infection models. Insect-associated Streptomyces inhibit antimicrobial-resistant pathogens more than soil Streptomyces. Genomics and metabolomics reveal their diverse biosynthetic capabilities. Further, we describe cyphomycin, a new molecule active against multidrug resistant fungal pathogens. The evolutionary trajectories of Streptomyces from the insect microbiome influence their biosynthetic potential and ability to inhibit resistant pathogens, supporting the promise of this source in augmenting future antimicrobial discovery.

Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability.

Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.

Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis.

Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. Here we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuing proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes.

Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases.

BACKGROUND: Understanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families. RESULTS: Comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site. CONCLUSIONS: The structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein-protein interactions. Together, these data indicate that AA10 LPMOs are under selection to change their function, which may optimize cellulolytic activity. This work provides a phylogenetic basis for identifying and classifying additional cellulolytic or chitinolytic LPMOs.

Cellulolytic Streptomyces strains associated with herbivorous insects share a phylogenetically linked capacity to degrade lignocellulose.

Actinobacteria in the genus Streptomyces are critical players in microbial communities that decompose complex carbohydrates in the soil, and these bacteria have recently been implicated in the deconstruction of plant polysaccharides for some herbivorous insects. Despite the importance of Streptomyces to carbon cycling, the extent of their plant biomass-degrading ability remains largely unknown. In this study, we compared four strains of Streptomyces isolated from insect herbivores that attack pine trees: DpondAA-B6 (SDPB6) from the mountain pine beetle, SPB74 from the southern pine beetle, and SirexAA-E (SACTE) and SirexAA-G from the woodwasp, Sirex noctilio. Biochemical analysis of secreted enzymes demonstrated that only two of these strains, SACTE and SDPB6, were efficient at degrading plant biomass. Genomic analyses indicated that SACTE and SDPB6 are closely related and that they share similar compositions of carbohydrate-active enzymes. Genome-wide proteomic and transcriptomic analyses revealed that the major exocellulases (GH6 and GH48), lytic polysaccharide monooxygenases (AA10), and mannanases (GH5) were conserved and secreted by both organisms, while the secreted endocellulases (GH5 and GH9 versus GH9 and GH12) were from diverged enzyme families. Together, these data identify two phylogenetically related insect-associated Streptomyces strains with high biomass-degrading activity and characterize key enzymatic similarities and differences used by these organisms to deconstruct plant biomass.

Biochemical properties and atomic resolution structure of a proteolytically processed ß-mannanase from cellulolytic Streptomyces sp. SirexAA-E.

ß-Mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only ß-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.

Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the ß6 (PBF1) and ß7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

Aerobic deconstruction of cellulosic biomass by an insect-associated Streptomyces.

Streptomyces are best known for producing antimicrobial secondary metabolites, but they are also recognized for their contributions to biomass utilization. Despite their importance to carbon cycling in terrestrial ecosystems, our understanding of the cellulolytic ability of Streptomyces is currently limited to a few soil-isolates. Here, we demonstrate the biomass-deconstructing capability of Streptomyces sp. SirexAA-E (ActE), an aerobic bacterium associated with the invasive pine-boring woodwasp Sirex noctilio. When grown on plant biomass, ActE secretes a suite of enzymes including endo- and exo-cellulases, CBM33 polysaccharide-monooxygenases, and hemicellulases. Genome-wide transcriptomic and proteomic analyses, and biochemical assays have revealed the key enzymes used to deconstruct crystalline cellulose, other pure polysaccharides, and biomass. The mixture of enzymes obtained from growth on biomass has biomass-degrading activity comparable to a cellulolytic enzyme cocktail from the fungus Trichoderma reesei, and thus provides a compelling example of high cellulolytic capacity in an aerobic bacterium.

The RPT2 subunit of the 26S proteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis.

The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly.

Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes.

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.