Diet-Microbiota Interactions Mediate Global Epigenetic Programming in Multiple Host Tissues.

Histone-modifying enzymes regulate transcription and are sensitive to availability of endogenous small-molecule metabolites, allowing chromatin to respond to changes in environment. The gut microbiota produces a myriad of metabolites that affect host physiology and susceptibility to disease; however, the underlying molecular events remain largely unknown. Here we demonstrate that microbial colonization regulates global histone acetylation and methylation in multiple host tissues in a diet-dependent manner: consumption of a "Western-type" diet prevents many of the microbiota-dependent chromatin changes that occur in a polysaccharide-rich diet. Finally, we demonstrate that supplementation of germ-free mice with short-chain fatty acids, major products of gut bacterial fermentation, is sufficient to recapitulate chromatin modification states and transcriptional responses associated with colonization. These findings have profound implications for understanding the complex functional interactions between diet, gut microbiota, and host health.

Influence of hybrid class and ensiling duration on deoxynivalenol accumulation and its derivative deoxynivalenol-3-glucoside while ensiling corn for silage.

In silage corn (Zea mays L.), Fusarium graminearum causes diseases and produces the mycotoxin deoxynivalenol (DON). The work presented here investigated DON accumulation and its fate during the ensiling of ground, whole-plant material obtained from dual-purpose (DP) and brown midrib (BMR) corn hybrids. Multi-year field trials arranged in a randomized complete block design were conducted in Wisconsin to evaluate BMR and DP corn hybrids in response to fungicide treatment. At harvest, the samples were chopped and vacuum sealed for a mini-silo time series assessment with silos opened following anaerobic fermentation for 0, 30, 60, 90 and 120 days. Repeated measures analysis of ensiled corn showed that hybrid (P < 0.01) and ensiling duration (P < 0.01) significantly impacted DON concentration through ensiling, while fungicide treatment had no significant effect (P > 0.05). Across hybrids and treatments, DON concentrations detected at harvest were the lowest with DON3G at harvest significantly (P < 0.01) and highly correlated (r = 0.74) with DON concentration 30-days post ensiling. These findings suggest that mycotoxin testing in corn should include not only DON but also for conjugates of DON that can be metabolized back to DON and increase the final DON concentration during ensiling.

Dietary adaptation to high starch involves increased relative abundance of sucrase-isomaltase and its mRNA in nestling house sparrows.

Dietary flexibility in digestive enzyme activity is widespread in vertebrates but mechanisms are poorly understood. When laboratory rats are switched to a higher carbohydrate diet, the activities of the apical intestinal α-glucosidases (AGs) increase within 6-12 h, mainly by rapid increase in enzyme transcription, followed by rapid translation and translocation to the intestine's apical, brush-border membrane (BBM). We performed the first unified study of the overall process in birds, relying on activity, proteomic, and transcriptomic data from the same animals. Our avian model was nestling house sparrows (Passer domesticus), which switch naturally from a low-starch insect diet to a higher starch seed diet and in whom the protein sucrase-isomaltase (SI) is responsible for all maltase and sucrase intestinal activities. Twenty-four hours after the switch to a high-starch diet, SI activity was increased but not at 12 h post diet switch. SI was the only hydrolase increased in the BBM, and its relative abundance and activity were positively correlated. Twenty-four hours after a reverse switch back to the lower starch diet, SI activity was decreased but not at 12 h post diet switch. Parallel changes in SI mRNA relative abundance were associated with the changes in SI activity in both diet-switch experiments, but our data also revealed an apparent diurnal rhythm in SI mRNA. This is the first demonstration that birds may rely on rapid increase in abundance of SI and its mRNA when adjusting to high-starch diet. Although the mechanisms underlying dietary induction of intestinal enzymes seem similar in nestling house sparrows and laboratory rodents, the time course for modulation in nestlings seemed half as fast compared with laboratory rodents. Before undertaking modulation, an opportunistic forager facing limited resources might rely on more extensive or prolonged environmental sampling, because the redesign of the intestine's hydrolytic capacity shortly after just one or a few meals of a new substrate might be a costly mistake.

Phosphorylation-dependent interaction between antigenic peptides and MHC class I: a molecular basis for the presentation of transformed self.

Protein phosphorylation generates a source of phosphopeptides that are presented by major histocompatibility complex class I molecules and recognized by T cells. As deregulated phosphorylation is a hallmark of malignant transformation, the differential display of phosphopeptides on cancer cells provides an immunological signature of 'transformed self'. Here we demonstrate that phosphorylation can considerably increase peptide binding affinity for HLA-A2. To understand this, we solved crystal structures of four phosphopeptide-HLA-A2 complexes. These identified a novel peptide-binding motif centered on a solvent-exposed phosphate anchor. Our findings indicate that deregulated phosphorylation can create neoantigens by promoting binding to major histocompatibility complex molecules or by affecting the antigenic identity of presented epitopes. These results highlight the potential of phosphopeptides as novel targets for cancer immunotherapy.

Identification of Molecular Targets of Dietary Grape-Mediated Chemoprevention of Ultraviolet B Skin Carcinogenesis: A Comparative Quantitative Proteomics Analysis.

We recently showed that dietary grape powder (GP) imparts considerable protection against ultraviolet B (UVB)-mediated skin carcinogenesis in SKH-1 mice. To determine molecular mechanisms of this response, we employed tandem mass tag (TMT) quantitative global proteomics approach on skin tumors from mice exposed to 180 mJ/cm2 UVB twice per week and fed control or 5% GP diet. We found 2629 proteins modulated by GP feeding, with 34 identified using stringent cutoffs (false discovery rate (FDR) q-value ≤ 0.1, fold change ≥ 1.2, p-value ≤ 0.05, ≥ 3 unique peptides). Ingenuity Pathway Analysis helped identify seven proteins involved in protein ubiquitination, including the deubiquitinase UCHL5 and 6 subunits of the 20S proteasome (PSMA1,3,4,6 and PSMB4,7). A second data set without the FDR q-value identified 239 modulated proteins, seven of which are involved in protein ubiquitination. Further, 14 proteins involved in acute phase response signaling were modulated >1.5-fold, including acute phase proteins APCS, FGA, FGB, HP, HPX, and RBP1. Evaluation of upstream regulators found inhibition of ERK1/2 phosphorylation and NF-κB p65, and an increase in IκBα in GP-treated tumors. Overall, our data suggested that GP consumption may mitigate tumorigenesis by enhancing protein ubiquitination and degradation caused by oxidative stress, and manipulates an otherwise tumor-promoting anti-inflammatory environment.

Glial fibrillary acidic protein exhibits altered turnover kinetics in a mouse model of Alexander disease.

Mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP) lead to the rare and fatal disorder, Alexander disease (AxD). A prominent feature of the disease is aberrant accumulation of GFAP. It has been proposed that this accumulation occurs because of an increase in gene transcription coupled with impaired proteasomal degradation, yet this hypothesis remains untested. We therefore sought to directly investigate GFAP turnover in a mouse model of AxD that is heterozygous for a disease-causing point mutation (GfapR236H/+) (and thus expresses both wild-type and mutant protein). Stable isotope labeling by amino acids in cell culture, using primary cortical astrocytes, indicated that the in vitro half-lives of total GFAP in astrocytes from wild-type and mutant mice were similar at ∼3-4 days. Surprisingly, results obtained with stable isotope labeling of mammals revealed that, in vivo, the half-life of GFAP in mutant mice (15.4 ± 0.5 days) was much shorter than that in wild-type mice (27.5 ± 1.6 days). These unexpected in vivo data are most consistent with a model in which synthesis and degradation are both increased. Our work reveals that an AxD-causing mutation alters GFAP turnover kinetics in vivo and provides an essential foundation for future studies aimed at preventing or reducing the accumulation of GFAP. In particular, these data suggest that elimination of GFAP might be possible and occurs more quickly than previously surmised.

Comparative genomic and proteomic analysis of cytoskeletal changes in dexamethasone-treated trabecular meshwork cells.

Changes in the actin cytoskeleton, especially the formation of cross-linked actin networks (CLANs) are thought to contribute to the increased intraocular pressure observed in primary open-angle and steroid-induced glaucoma. To better understand the effects of glucocorticoids, we employed a shotgun method to analyze global changes in the cytoskeleton and integrin signaling pathways following dexamethasone (DEX) treatment of human trabecular meshwork (HTM) cells. RNA and cell lysates were obtained from HTM cells incubated with or without DEX. Changes in protein expression were determined by mass spectrometry (MS) following differential centrifugation of cell lysates to enrich for low-abundance cytoskeletal and signaling proteins, proteolytic digestion, and a titanium dioxide column to enrich for phosphopeptides. Results were validated by Western blots. Changes in RNA levels were determined with gene arrays and RT-PCR. Overall, MS identified 318 cytoskeleton associated proteins. Five of these proteins (PDLIM1, FGFR1OP, leiomodin-1, ZO-2 and LRP16A) were only detected in DEX-treated cells by MS. However, only PDLIM1 showed a statistically significant increase at the RNA level. Other proteins with differences at both the RNA and protein levels included ß3 integrin, caveolin-1, Borg2, raftlin1, PI-3 kinase regulatory subunit α, transgelin, and filamin B. By immunofluorescence microscopy filamin B and PDLIM1 showed enhanced expression in human trabecular meshwork cells, but only PDLIM1 demonstrated significant localization within CLANs. Finally, MS showed that some of the cytoskeleton proteins (Borg2, leiomodin-1, LRP16A, raftlin1 and CKAP4) contained phosphorylated residues. This study suggests that DEX affects the expression of cytoskeleton proteins at the transcriptional and translational level and shows that a combined genomic and proteomic approach can be used for rapid analysis of proteins in the TM. It also shows that DEX altered the expression of components (PDLIM1 and ß3 integrins) involved in CLAN formation and provides new findings into the effects of glucocorticoids on the cytoskeleton.

Multi-omics analysis of green lineage osmotic stress pathways unveils crucial roles of different cellular compartments.

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway.

Proteomic analyses identify a diverse array of nuclear processes affected by small ubiquitin-like modifier conjugation in Arabidopsis.

The covalent attachment of SUMO (small ubiquitin-like modifier) to other intracellular proteins affects a broad range of nuclear processes in yeast and animals, including chromatin maintenance, transcription, and transport across the nuclear envelope, as well as protects proteins from ubiquitin addition. Substantial increases in SUMOylated proteins upon various stresses have also implicated this modification in the general stress response. To help understand the role(s) of SUMOylation in plants, we developed a stringent method to isolate SUMO-protein conjugates from Arabidopsis thaliana that exploits a tagged SUMO1 variant that faithfully replaces the wild-type protein. Following purification under denaturing conditions, SUMOylated proteins were identified by tandem mass spectrometry from both nonstressed plants and those exposed to heat and oxidative stress. The list of targets is enriched for factors that direct SUMOylation and for nuclear proteins involved in chromatin remodeling/repair, transcription, RNA metabolism, and protein trafficking. Targets of particular interest include histone H2B, components in the LEUNIG/TOPLESS corepressor complexes, and proteins that control histone acetylation and DNA methylation, which affect genome-wide transcription. SUMO attachment site(s) were identified in a subset of targets, including SUMO1 itself to confirm the assembly of poly-SUMO chains. SUMO1 also becomes conjugated with ubiquitin during heat stress, thus connecting these two posttranslational modifications in plants. Taken together, we propose that SUMOylation represents a rapid and global mechanism for reversibly manipulating plant chromosomal functions, especially during environmental stress.

In planta changes in protein phosphorylation induced by the plant hormone abscisic acid.

Abscisic acid (ABA) is a hormone that controls seed dormancy and germination as well as the overall plant response to important environmental stresses such as drought. Recent studies have demonstrated that the ABA-bound receptor binds to and inhibits a class of protein phosphatases. To identify more broadly the phosphoproteins affected by this hormone in vivo, we used (14)N/(15)N metabolic labeling to perform a quantitative untargeted mass spectrometric analysis of the Arabidopsis thaliana phosphoproteome following ABA treatment. We found that 50 different phosphopeptides had their phosphorylation state significantly altered by ABA over a treatment period lasting 5-30 min. Among these changes were increases in phosphorylation of subfamily 2 SNF1-related kinases and ABA-responsive basic leucine zipper transcription factors implicated in ABA signaling by previous in vitro studies. Furthermore, four members of the aquaporin family showed decreased phosphorylation at a carboxy-terminal serine which is predicted to cause closure of the water-transporting aquaporin gate, consistent with ABA's role in ameliorating the effect of drought. Finally, more than 20 proteins not previously known to be involved with ABA were found to have significantly altered phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that an expanded model of ABA signaling, beyond simple phosphatase inhibition, may be necessary. This quantitative proteomics dataset provides a more comprehensive, albeit incomplete, view both of the protein targets whose biochemical activities are likely to be controlled by ABA and of the nature of the emerging phosphorylation and dephosphorylation cascades triggered by this hormone.

Assessment of Truck-Mounted Area-Wide S-methoprene Applications to Manage West Nile Virus Vector Species in the Suburbs of Chicago, IL, USA.

West Nile virus remains the leading cause of arboviral neuroinvasive disease in the United States, despite extensive efforts to control the mosquito vectors involved in transmission. In this study, we evaluated the effectiveness of Altosid SR-20 (active ingredient, S-methoprene 20%) larvicide applications using truck-mounted ultra-low volume (ULV) dispersal equipment to target Culex pipiens Linnaeus (Diptera: Culicidae) and Cx. restuans (Theobald)larvae. A combination of emergence bioassays, open-field measurements of deposited S-methoprene and spray distribution using gas chromatography-mass spectrometry, and assessments of adult Culex spp. populations in response to applications were conducted over the summer of 2020 within the North Shore Mosquito Abatement District (IL, USA). Open-field applications revealed that dispersed Altosid SR-20 using ULV equipment was effective (75% emergence inhibition in susceptible lab strain Cx. pipiens larvae) up to 53 m. In suburban neighborhood applications, we found that S-methoprene deposition and larval emergence inhibition (EI) in front yards did not differ significantly from backyards. An overall EI of 46% and 28% were observed for laboratory strain Cx. pipiens and wild Cx. restuans larvae respectively, and both had an EI significantly higher than the untreated control group. The EI of exposed wild Cx. pipiens larvae did not differ from the untreated controls, suggesting an increased tolerance to S-methoprene. No difference in abundance of gravid or host-seeking adult Culex spp. post-application was detected between treated and untreated sites. These results document the ability of area-wide application to distribute S-methoprene, but this strategy will need further modifications and evaluation for Culex spp. management.

Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution.

The unique biology of a neoplasm is reflected by its distinct molecular profile compared with normal tissue. To understand tumor development better, we have undertaken a quantitative proteomic search for abnormally expressed proteins in colonic tumors from Apc(Min/+) (Min) mice. By raising pairs of Min and wild-type mice on diets derived from natural-abundance or (15)N-labeled algae, we used metabolic labeling to compare protein levels in colonic tumor versus normal tissue. Because metabolic labeling allows internal control throughout sample preparation and analysis, technical error is minimized as compared with in vitro labeling. Several proteins displayed altered expression, and a subset was validated via stable isotopic dilution using synthetic peptide standards. We also compared gene and protein expression among tumor and nontumor tissue, revealing limited correlation. This divergence was especially pronounced for species showing biological change, highlighting the complementary perspectives provided by transcriptomics and proteomics. Our work demonstrates the power of metabolic labeling combined with stable isotopic dilution as an integrated strategy for the identification and validation of differentially expressed proteins using rodent models of human disease.

Chronic Jetlag Alters the Landscape of the Pancreatic Lipidome.

OBJECTIVES: The innate biologic clock plays a significant role in lipid metabolism, including the peripheral clock in the pancreas. However, an evaluation of the downstream lipids in the pancreatic lipidome is lacking. We sought to understand the diurnal variations of lipids within the pancreatic lipidome. METHODS: At 4 weeks of age, C57Bl/6J mice were subjected to either normal lighting conditions or a chronic jetlag (CJ) condition known to mimic chronic shiftwork in humans. At 9 months, mice were serially killed at 4-hour intervals for 24 hours. The pancreas was removed and subjected to untargeted liquid chromatography-mass spectrometry to examine the pancreatic lipidome. RESULTS: A total of 4.7% of the pancreatic lipidome was rhythmically expressed, which increased to 12.9% after CJ. After CJ, there was a 4.58-hour shift in the timing of peak 24-hour lipid expression. Chronic jetlag also led to the enrichment of diacylglycerols and triglycerides, while promoting a decrease in lysophosphatidylcholines and 44-carbon acyl chain lipids. CONCLUSIONS: The pancreatic lipidome exhibits diurnal rhythmicity across a broad number of lipid classes. Chronic jetlag led to alterations in lipid composition that mirrored other metabolically active organs. Several of the reported changes may link altered sleep-wake cycles with known circadian disruption-induced pancreatic diseases.

Sex and genetic background define the metabolic, physiologic, and molecular response to protein restriction.

Low-protein diets promote metabolic health in humans and rodents. Despite evidence that sex and genetic background are key factors in the response to diet, most protein intake studies examine only a single strain and sex of mice. Using multiple strains and both sexes of mice, we find that improvements in metabolic health in response to reduced dietary protein strongly depend on sex and strain. While some phenotypes were conserved across strains and sexes, including increased glucose tolerance and energy expenditure, we observed high variability in adiposity, insulin sensitivity, and circulating hormones. Using a multi-omics approach, we identified mega-clusters of differentially expressed hepatic genes, metabolites, and lipids associated with each phenotype, providing molecular insight into the differential response to protein restriction. Our results highlight the importance of sex and genetic background in the response to dietary protein level, and the potential importance of a personalized medicine approach to dietary interventions.

A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS.

A simple method for the identification of brush-border membrane α-glucosidases is described. The proteins were first solubilized and separated in a gel under native, non-denaturing, conditions. The gel was then incubated in substrate solutions (maltose or sucrose), and the product (glucose) exposed in situ by the oxidation of o-dianisidine, which yields a brown-orange color. Nano-liquid chromatography coupled to mass spectrometry analyses of proteins (nano LC-MS/MS) present in the colored bands excised from the gels, was used to confirm the presence of the enzymes. The stain is inexpensive and the procedure permits testing several substrates in the same gel. Once enzymes are identified, their abundance, relative to that of other proteins in the brush border, can be semi-quantified using nano LC-MS/MS.

Co-culture with mouse embryonic fibroblasts improves maintenance of metabolic function of human small hepatocyte progenitor cells.

Derivation and culture of small hepatocyte progenitor cells (SHPCs) capable of proliferating in vitro has been described in rodents and recently in humans. These cells are capable of engrafting in injured livers, however, they display de-differentiated morphology and reduced xenobiotic metabolism activity in culture over passages. Here we report that SHPCs derived from adult primary human hepatocytes (PHHs) and cultured on mouse embryonic fibroblasts (MEFs) not only display differentiated morphology and exhibit gene expression profiles similar to adult PHHs, but importantly, they retain their phenotype over several passages. Further, unlike previous reports, where extensive manipulations of culture conditions are required to convert SHPCs to metabolically functional hepatocytes, SHPCs in our co-culture system maintain expression of xenobiotic metabolism-associated genes. We show that SHPCs in co-culture are able to perform xenobiotic metabolism at rates equal to their parent PHHs as evidenced by the metabolism of acetaminophen to all of its major metabolites. In summary, we present an improved co-culture system that allows generation of SHPCs from adult PHHs that maintain their differentiated phenotype over multiple passages. Our findings would be useful for expansion of limited PHHs for use in studies of drug metabolism and toxicity testing.

Effect of twice-daily oral administration of a chondroitin sulfate-containing supplement on urine chondroitin sulfate concentrations in dogs.

OBJECTIVE: To quantify the magnitude and duration of changes in urine chondroitin sulfate concentration (uCS) as a result of oral administration of a chondroitin sulfate-containing supplement in dogs. ANIMALS: 8 healthy privately owned dogs. PROCEDURES: A urine sample was collected from each dog via cystocentesis on day 1; free-catch midstream urine samples were collected once daily on days 2 through 5. Pretreatment uCS was established from those samples. Each dog then received a chondroitin sulfate-containing supplement (20 to 30 mg/kg, PO, q 12 h) for 8 days (on days 7 through 14). Urine samples were collected on days 8 through 12 and day 15. For each sample, uCS was quantified by liquid chromatography-tandem mass spectrometry. Variable urine concentration was accounted for by dividing the uCS by urine creatinine concentration (uCrea) to determine the uCS:uCrea ratio. Pretreatment uCS:uCrea ratios were compared with treatment uCS:uCrea ratios to calculate the fold change in uCS after supplement administration. RESULTS: Among the study dogs, oral administration of the chondroitin sulfate-containing supplement resulted in a 1.9-fold increase in the median uCS:uCrea ratio. Data obtained on days 8 through 12 and day 15 indicated that the daily increase in uCS remained consistent and was not additive. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that oral administration of supplemental chondroitin sulfate to dogs modestly increased uCS within 24 hours; however, subsequent supplement administration did not have an additive effect. A potential therapeutic benefit of persistently increased uCS in preventing recurrent urinary tract infections in dogs warrants investigation.

Bacteria: A novel source for potent mosquito feeding-deterrents.

Antibiotic and insecticidal bioactivities of the extracellular secondary metabolites produced by entomopathogenic bacteria belonging to genus Xenorhabdus have been identified; however, their novel applications such as mosquito feeding-deterrence have not been reported. Here, we show that a mixture of compounds isolated from Xenorhabdus budapestensis in vitro cultures exhibits potent feeding-deterrent activity against three deadly mosquito vectors: Aedes aegypti, Anopheles gambiae, and Culex pipiens. We demonstrate that the deterrent active fraction isolated from replicate bacterial cultures is highly enriched in two compounds consistent with the previously described fabclavines, strongly suggesting that these are the molecular species responsible for feeding-deterrence. The mosquito feeding-deterrent activity in the putative fabclavine-rich fraction is comparable to or better than that of N,N-diethyl-3-methylbenzamide (also known as DEET) or picaridin in side-by-side assays. These findings lay the groundwork for research into biologically derived, peptide-based, low-molecular weight compounds isolated from bacteria for exploitation as mosquito repellents and feeding-deterrents.

Aronia Berry Supplementation Mitigates Inflammation in T Cell Transfer-Induced Colitis by Decreasing Oxidative Stress.

Oxidative stress is involved in the pathogenesis and progression of inflammatory bowel disease. Consumption of aronia berry inhibits T cell transfer colitis, but the antioxidant mechanisms pertinent to immune function are unclear. We hypothesized that aronia berry consumption could inhibit inflammation by modulating the antioxidant function of immunocytes and gastrointestinal tissues. Colitis was induced in recombinase activating gene-1 deficient (Rag1-/-) mice injected with syngeneic CD4+CD62L+ naïve T cells. Concurrent with transfer, mice consumed either 4.5% w/w aronia berry-supplemented or a control diet for five weeks. Aronia berry inhibited intestinal inflammation evidenced by lower colon weight/length ratios, 2-deoxy-2-[18F]fluoro-d-glucose (FDG) uptake, mRNA expressions of tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) in the colon. Aronia berry also suppressed systemic inflammation evidenced by lower FDG uptake in the spleen, liver, and lung. Colitis induced increased colon malondialdehyde (MDA), decreased colon glutathione peroxidase (GPx) activity, reduced glutathione (rGSH) level, and suppressed expression of antioxidant enzymes in the colon and mesenteric lymph node (MLN). Aronia berry upregulated expression of antioxidant enzymes, prevented colitis-associated depletion of rGSH, and maintained GPx activity. Moreover, aronia berry modulated mitochondria-specific antioxidant activity and decreased splenic mitochondrial H2O2 production in colitic mice. Thus, aronia berry consumption inhibits oxidative stress in the colon during T cell transfer colitis because of its multifaceted antioxidant function in both the cytosol and mitochondria of immunocytes.

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: identification of proteins binding the delta-endotoxin, Cry1Ac of Bacillus thuringiensis.

Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) delta-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.