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Cornified envelopes (CEs) of human epidermis ordinarily consist of transglutaminase-mediated cross-linked proteins and are essential for skin barrier function. However, in addition to enzyme-mediated isopeptide bonding, protein cross-linking could also arise from oxidative damage. Our group recently demonstrated abnormal incorporation of cellular proteins into CEs by pro-oxidants in woodsmoke. In this study, we focused on 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mesquite liquid smoke (MLS), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to further understand the mechanisms through which environmental pro-oxidants induce CE formation and alter the CE proteome. CEs induced by the ionophore X537A were used for comparison. Similar to X537A, DMNQ- and MLS-induced CE formation was associated with membrane permeabilization. However, since DMNQ is non-adduct forming, its CEs were similar in protein profile to those from X537A. By contrast, MLS, rich in reactive carbonyls that can form protein adducts, caused a dramatic change in the CE proteome. TCDD-CEs were found to contain many CE precursors, such as small proline-rich proteins and late cornified envelope proteins, encoded by the epidermal differentiation complex. Since expression of these proteins is mediated by the aryl hydrocarbon receptor (AhR), and its well-known downstream protein, CYP1A1, was exclusively present in the TCDD group, we suggest that TCDD alters the CE proteome through persistent AhR activation. This study demonstrates the potential of environmental pro-oxidants to alter the epidermal CE proteome and indicates that the cellular redox state has an important role in CE formation., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.

The interaction between the plant host, walnut ( Juglans regia ; Jr), and a deadly pathogen ( Xanthomonas arboricola pv. juglandis 417; Xaj) can lead to walnut bacterial blight (WB), which depletes walnut productivity by degrading the nut quality. Here, we dissect this pathosystem using tandem mass tag quantitative proteomics. Walnut hull tissues inoculated with Xaj were compared to mock-inoculated tissues, and 3972 proteins were identified, of which 3296 are from Jr and 676 from Xaj. Proteins with differential abundance include oxidoreductases, proteases, and enzymes involved in energy metabolism and amino acid interconversion pathways. Defense responses and plant hormone biosynthesis were also increased. Xaj proteins detected in infected tissues demonstrate its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Secreted proteases and extracellular secretion apparatus such as type IV pilus for twitching motility and type III secretion effectors indicate putative factors recognized by the host. Taken together, these results suggest intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Our results provide insights into molecular mechanisms and highlight potential molecular tools for early detection and disease control strategies.

Ibuprofen, an inhibitor of prostanoid biosynthesis, is a common pharmacological agent used for the management of pain, inflammation and fever. However, the chronic use of ibuprofen at high doses is associated with increased risk for cardiovascular, renal, gastrointestinal and liver injuries. The underlying mechanisms of ibuprofen-mediated effects on liver remain unclear. To determine the mechanisms and signaling pathways affected by ibuprofen (100 mg/kg/day for seven days), we performed proteomic profiling of male mice liver with quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. More than 300 proteins were significantly altered between the control and ibuprofen-treated groups. The data suggests that several major pathways including (1) energy metabolism, (2) protein degradation, (3) fatty acid metabolism and (4) antioxidant system are altered in livers from ibuprofen treated mice. Independent validation of protein changes in energy metabolism and the antioxidant system was carried out by Western blotting and showed sex-related differences. Proteasome and immunoproteasome activity/expression assays showed ibuprofen induced gender-specific proteasome and immunoproteasome dysfunction in liver. The study observed multifactorial gender-specific ibuprofen-mediated effects on mice liver and suggests that males and females are affected differently by ibuprofen.

Verticillium dahliae colonizes vascular tissue and causes vascular discoloration in susceptible hosts. Two well-defined races exist in V. dahliae populations from tomato and lettuce. In this study, proteins and metabolites obtained from stems of race 1-incompatible (Beefsteak) and -compatible (Early Pak) tomato cultivars were characterized. A total of 814 and 584 proteins in Beefsteak; and 456 and 637 proteins in Early Pak were identified in stem extracts of plants inoculated with races 1 and 2, respectively. A significant number of defense-related proteins were expressed in each tomato-V. dahliae interaction, as anticipated. However, phenylalanine ammonia-lyase (PAL), an important defense-associated enzyme of the phenylpropanoid pathway, in addition to remorin 1, NAD-dependent epimerase/dehydratase, and polyphenol oxidase were uniquely expressed in the incompatible interaction. Compared with the uninoculated control, significant overexpression of gene ontology terms associated with lignin biosynthesis, phenylpropanoid pathway and carbohydrate methylation were identified exclusively in the incompatible interaction. Phenolic compounds known to be involved in plant defense mechanisms were at higher levels in the incompatible relative to the compatible interactions. Based on our findings, PAL and enzymes involved defense-related secondary metabolism and the strengthening of cell walls is likely critical to confer resistance to race 1 of V. dahliae in tomato. SIGNIFICANCE: Verticillium dahliae, a soilborne fungal pathogen and a widely distributed fungal pathogen, colonizes vascular tissue and causes vascular discoloration in roots and stems, leaf wilting, and death of susceptible plant hosts. It causes billions of dollars in annual crop losses all over the world. The study focused on the proteomic and metabalomic of V. dahliae interactions (incompatible with Beefsteak and compatible with Early Pak tomato cultivars). Based on our findings, PAL and enzymes involved defense-related secondary metabolism and the strengthening of cell walls is likely critical to confer resistance to race 1 of V. dahliae in tomato., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Proteomic genotyping detects single amino acid polymorphisms to infer the genotype of corresponding non-synonymous SNPs. Like any DNA genotype, these inferences can be used to estimate random match probability. Fingermarks are a common source of biological evidence that is sample limited and a highly variable source of identifying DNA. Genetically variant peptides from fingermarks, that contain single amino acid polymorphisms, are an additional source of identifying genetic information. To discover these peptide biomarkers epidermal corneocytes from 9 subjects were isolated, processed, digested with trypsin and applied to mass spectrometry. The resulting proteomic and matching exome datasets were used to discover, characterize and validate 60 genetically variant peptides. An average of 28.8 ± 4.4 genetically variant peptides were detected from each subject resulting in a total of 264 SNP allele inferences with 260 true and 4 false positives, a false discovery rate of 1.5%. Random match probabilities were estimated using the genotype frequencies from the matching major populations in the 1000 Genomes Project. Estimates ranged up to a value of 1 in 1.7 × 10 8 , with a median probability of 1 in 2.4 × 10 6 . Furthermore, the proteomically-inferred genotypes are likely to be compatible with the STR-based random match probability estimates since the closest STR locus was 2.2 Mb from the nearest GVP-inferred SNP. This project represents a novel mode of genetic information that can be obtained from fingermarks and has the potential to complement other methods of human identification including analysis of ridge patterns or touch DNA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Defects in keratinocyte transglutaminase (TGM1), resulting in an improper protein scaffold for deposition of the lipid barrier, comprise a major source of autosomal recessive congenital ichthyosis. For that reason, the composition and formation of the cornified (cross-linked) protein envelope of the epidermis have been of considerable interest. Since the isopeptide cross-linked protein components are not individually isolable once incorporated, purified envelopes were analysed by mass spectrometry after trypsin digestion. Quantitative estimates of the identified components revealed some 170 proteins, each comprising at least 0.001% of the total, of which keratins were major constituents accounting for ≈74% of the total. Some prevalent non-keratin constituents such as keratinocyte proline-rich protein, loricrin and late envelope protein-7 were preferentially incorporated into envelopes. The results suggest a model where, as previously observed in hair shaft and nail plate, a diversity of cellular proteins are incorporated. They also help rationalize the minimal effect on epidermis of ablating genes for specific single envelope structural components. The quantitative profile of constituent proteins provides a foundation for future exploration of envelope perturbations that may occur in pathological conditions., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Callus samples from the ball and the arch of the foot, collected on tape circles, were compared by shotgun proteomic profiling. Pachyonychia congenita subjects were sampled who exhibited a mutation in KRT6A, KRT6B, KRT6C, KRT16 or KRT17, and the proteins were digested and analyzed by tandem mass spectrometry. In comparison with samples from unaffected control subjects, those from subjects with KRT6A or KRT16 mutations displayed the most differences in profile from normal, while those from subjects with KRT6C or KRT17 mutations showed few differences from normal. The profiles from subjects with KRT6B mutations were intermediate in protein profile differences. Degree of departure from the normal profile could be estimated by expression of numerous proteins in callus from the ball of the foot that were consistently different. By contrast, the protein profile from the arch of the foot was hardly affected. The results provide a foundation for noninvasive monitoring of the efficacy of treatments with quantitative assessment of departure from the normal phenotype., Significance: Pachyonychia congenita is an orphan disease in which the connection between the basic defect (keratin mutation) and debilitating symptoms (severe plantar pain) is poorly understood. Present work addresses the degree to which the protein profile is altered in the epidermis where the severe pain originates. The results indicate that the mutated keratins differ greatly in the degree to which they elicit perturbations in protein profile. In those cases with markedly altered protein levels, monitoring the callus profile may provide an objective measure of treatment efficacy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Detection of differentially abundant proteins in label-free quantitative shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments requires a series of computational steps that identify and quantify LC-MS features. It also requires statistical analyses that distinguish systematic changes in abundance between conditions from artifacts of biological and technical variation. The 2015 study of the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) aimed to evaluate the effects of the statistical analysis on the accuracy of the results. The study used LC-tandem mass spectra acquired from a controlled mixture, and made the data available to anonymous volunteer participants. The participants used methods of their choice to detect differentially abundant proteins, estimate the associated fold changes, and characterize the uncertainty of the results. The study found that multiple strategies (including the use of spectral counts versus peak intensities, and various software tools) could lead to accurate results, and that the performance was primarily determined by the analysts' expertise. This manuscript summarizes the outcome of the study, and provides representative examples of good computational and statistical practice. The data set generated as part of this study is publicly available.

We found that incubation of probiotic Lactobacillus casei BL23 in milk at 4 °C prior to ingestion increased its survival in the mammalian digestive tract. To investigate the specific molecular adaptations of L. casei to milk, we used tandem mass spectrometry to compare proteins produced by L. casei BL23 at 4 °C in milk to those in exponential and stationary phase cells in laboratory culture medium at either 37 or 4 °C. These comparisons revealed a core of expressed L. casei proteins as well as proteins produced in either a growth-phase or temperature-specific manner. In total, 205 L. casei proteins were uniquely expressed or detected in higher abundance specifically as a result of incubation in milk and included an over-representation of proteins for cell surface modification, fatty acid metabolism, amino acid transport and metabolism, and inorganic ion transport. Genes for DltD (d-alanine transfer protein), FabH (3-oxoacyl-ACP synthase), RecA (recombinase A), and Sod (superoxide dismutase) were targeted for inactivation. The competitive fitness of the mutants was altered in the mouse intestine compared with wild-type cells. These results show that the food matrix can have a profound influence on dietary (probiotic) bacteria and their functional significance in the mammalian gut.

Milk has been well established as the optimal nutrition source for infants, yet there is still much to be understood about its molecular composition. Therefore, our objective was to develop and compare comprehensive milk proteomes for human and rhesus macaques to highlight differences in neonatal nutrition. We developed a milk proteomics technique that overcomes previous technical barriers including pervasive post-translational modifications and limited sample volume. We identified 1606 and 518 proteins in human and macaque milk, respectively. During analysis of detected protein orthologs, we identified 88 differentially abundant proteins. Of these, 93% exhibited increased abundance in human milk relative to macaque and include lactoferrin, polymeric immunoglobulin receptor, alpha-1 antichymotrypsin, vitamin D-binding protein, and haptocorrin. Furthermore, proteins more abundant in human milk compared with macaque are associated with development of the gastrointestinal tract, the immune system, and the brain. Overall, our novel proteomics method reveals the first comprehensive macaque milk proteome and 524 newly identified human milk proteins. The differentially abundant proteins observed are consistent with the perspective that human infants, compared with nonhuman primates, are born at a slightly earlier stage of somatic development and require additional support through higher quantities of specific proteins to nurture human infant maturation.

Proteomics of human milk has been used to identify the comprehensive cargo of proteins involved in immune and cellular function. Very little is known about the effects of gestational diabetes mellitus (GDM) on lactation and breast milk components. The objective of the current study was to examine the effect of GDM on the expression of proteins in the whey fraction of human colostrum. Colostrum was collected from women who were diagnosed with (n = 6) or without (n = 12) GDM at weeks 24-28 in pregnancy. Colostral whey was analyzed for protein abundances using high-resolution, high-mass accuracy liquid chromatography tandem mass spectrometry. A total of 601 proteins were identified, of which 260 were quantified using label free spectral counting. Orthogonal partial least-squares discriminant analysis identified 27 proteins that best predict GDM. The power law global error model corrected for multiple testing was used to confirm that 10 of the 27 proteins were also statistically significantly different between women with versus without GDM. The identified changes in protein expression suggest that diabetes mellitus during pregnancy has consequences on human colostral proteins involved in immunity and nutrition.

Aims: Mitochondrial dysfunction is an important part of the decline in cardiac function in heart failure. We hypothesized for hypothesized that there would be specific abnormalities in mitochondrial function and proteome with the progression of ischemic heart failure (HF)., Main Methods: We used a high left anterior descending artery (LAD) ligation in 3-4month old male rats to generate HF. Rats were studied 9weeks post-ligation., Key Findings: Electron microscopy of left ventricle samples showed mitochondrial changes including decreased size, increased number, abnormal distribution, and cristae loss. Mitochondria in ischemic HF exhibited decreased total ATP, impaired mitochondrial respiration, as well as reduced complex I activity. Analysis of LV mitochondrial proteins by mass spectrometry was performed, and 31 differentially expressed proteins (p<0.05) of more than 500 total proteins were identified. Of these proteins, 15 were up-regulated and 16 were down-regulated in the failing heart. A set of complex I proteins was significantly decreased, consistent with the impairment of complex I activity. There were distinct changes in mitochondrial function and proteome in ischemic HF. Although there were similarities, the distinction between the reported proteomic changed with TAC pressure overload induced HF and ischemic HF in the current study suggested different pathological mechanisms., Significance: Specific changes in mitochondrial protein expression, which correlate with changes in mitochondrial function, have been identified in ischemic HF for the first time., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mutant laboratory mice with distinctive hair phenotypes are useful for identifying genes responsible for hair diseases. The work presented here demonstrates that shotgun proteomic profiling can distinguish hair shafts from different inbred mouse strains. For this purpose, analyzing the total hair shaft provided better discrimination than analyzing the isolated solubilized and particulate (cross-linked) fractions. Over 100 proteins exhibited significant differences among the 11 strains and 5 mutant stocks across the wide spectrum of strains surveyed. Effects on the profile of single gene mutations causing hair shaft defects were profound. Since the hair shaft provides a discrete sampling of the species proteome, with constituents serving important functions in epidermal appendages and throughout the body, this work provides a foundation for non-invasive diagnosis of genetic diseases of hair and perhaps other tissues.

Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.

Increasing evidence suggests that proteins present in the angiosperm sieve tube system play an important role in the long distance signaling system of plants. To identify the nature of these putatively non-cell-autonomous proteins, we adopted a large scale proteomics approach to analyze pumpkin phloem exudates. Phloem proteins were fractionated by fast protein liquid chromatography using both anion and cation exchange columns and then either in-solution or in-gel digested following further separation by SDS-PAGE. A total of 345 LC-MS/MS data sets were analyzed using a combination of Mascot and X!Tandem against the NCBI non-redundant green plant database and an extensive Cucurbit maxima expressed sequence tag database. In this analysis, 1,209 different consensi were obtained of which 1,121 could be annotated from GenBank and BLAST search analyses against three plant species, Arabidopsis thaliana, rice (Oryza sativa), and poplar (Populus trichocarpa). Gene ontology (GO) enrichment analyses identified sets of phloem proteins that function in RNA binding, mRNA translation, ubiquitin-mediated proteolysis, and macromolecular and vesicle trafficking. Our findings indicate that protein synthesis and turnover, processes that were thought to be absent in enucleate sieve elements, likely occur within the angiosperm phloem translocation stream. In addition, our GO analysis identified a set of phloem proteins that are associated with the GO term "embryonic development ending in seed dormancy"; this finding raises the intriguing question as to whether the phloem may exert some level of control over seed development. The universal significance of the phloem proteome was highlighted by conservation of the phloem proteome in species as diverse as monocots (rice), eudicots (Arabidopsis and pumpkin), and trees (poplar). These results are discussed from the perspective of the role played by the phloem proteome as an integral component of the whole plant communication system.

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.

Proteomic analysis of a Triton X-100 insoluble, 30,000 x g pellet from purified pea chloroplasts resulted in the identification of 179 nonredundant proteins. This chloroplast fraction was mostly depleted of chloroplast membranes since only 23% and 9% of the identified proteins were also observed in envelope and thylakoid membranes, respectively. One of the most abundant proteins in this fraction was sulfite reductase, a dual function protein previously shown to act as a plastid DNA condensing protein. Approximately 35 other proteins known (or predicted) to be associated with high-density protein-nucleic acid particles (nucleoids) were also identified including a family of DNA gyrases, as well as proteins involved in plastid transcription and translation. Although nucleoids appeared to be the predominant component of 30k x g Triton-insoluble chloroplast preparations, multi-enzyme protein complexes were also present including each subunit to the pyruvate dehydrogenase and acetyl-CoA carboxylase multi-enzyme complexes, as well as a proposed assembly of the first three enzymes of the Calvin cycle. Approximately 18% of the proteins identified were annonated as unknown or hypothetical proteins and another 20% contained "putative" or "like" in the identifier tag. This is the first proteomic characterization of a membrane-depleted, high-density fraction from plastids and demonstrates the utility of this simple procedure to isolate intact macromolecular structures from purified organelles for analysis of protein-protein and protein-nucleic acid interactions.

Alcohol-associated liver disease (ALD) is a prevalent medical problem with limited effective treatment strategies. Although many biological processes contributing to ALD have been elucidated, a complete understanding of the underlying mechanisms is still lacking. The current study employed a proteomic approach to identify hepatic changes resulting from ethanol (EtOH) consumption and the genetic ablation of the formyl peptide receptor 2 (FPR2), a G-protein coupled receptor known to regulate multiple signaling pathways and biological processes, in a mouse model of ALD. Since previous research from our team demonstrated a notable reduction in hepatic FPR2 protein levels in patients with alcohol-associated hepatitis (AH), the proteomic changes in the livers of Fpr2-/- EtOH mice were compared to those observed in patients with AH in order to identify common hepatic proteomic alterations. Several pathways linked to exacerbated ALD in Fpr2-/- EtOH mice, as well as hepatic protein changes resembling those found in patients suffering from AH, were identified. These alterations included decreased levels of coagulation factors F2 and F9, as well as reduced hepatic levels of glutamate-cysteine ligase catalytic subunit (GCLC) and total glutathione in Fpr2-/- EtOH compared to WT EtOH mice. In conclusion, the data suggest that FPR2 may play a regulatory role in hepatic blood coagulation and the antioxidant system, both in a pre-clinical model of ALD and in human AH, however further experiments are required to validate these findings.

Cornified envelopes (CEs) of human epidermis ordinarily consist of transglutaminase-mediated cross-linked proteins and are essential for skin barrier function. However, in addition to enzyme-mediated isopeptide bonding, protein cross-linking could also arise from oxidative damage. Our group recently demonstrated abnormal incorporation of cellular proteins into CEs by pro-oxidants in woodsmoke. In this study, we focused on 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mesquite liquid smoke (MLS), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to further understand the mechanisms through which environmental pro-oxidants induce CE formation and alter the CE proteome. CEs induced by the ionophore X537A were used for comparison. Similar to X537A, DMNQ- and MLS-induced CE formation was associated with membrane permeabilization. However, since DMNQ is non-adduct forming, its CEs were similar in protein profile to those from X537A. By contrast, MLS, rich in reactive carbonyls that can form protein adducts, caused a dramatic change in the CE proteome. TCDD-CEs were found to contain many CE precursors, such as small proline-rich proteins and late cornified envelope proteins, encoded by the epidermal differentiation complex. Since expression of these proteins is mediated by the aryl hydrocarbon receptor (AhR), and its well-known downstream protein, CYP1A1, was exclusively present in the TCDD group, we suggest that TCDD alters the CE proteome through persistent AhR activation. This study demonstrates the potential of environmental pro-oxidants to alter the epidermal CE proteome and indicates that the cellular redox state has an important role in CE formation.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

The interaction between the plant host, walnut (Juglans regia; Jr), and a deadly pathogen (Xanthomonas arboricola pv. juglandis 417; Xaj) can lead to walnut bacterial blight (WB), which depletes walnut productivity by degrading the nut quality. Here, we dissect this pathosystem using tandem mass tag quantitative proteomics. Walnut hull tissues inoculated with Xaj were compared to mock-inoculated tissues, and 3972 proteins were identified, of which 3296 are from Jr and 676 from Xaj. Proteins with differential abundance include oxidoreductases, proteases, and enzymes involved in energy metabolism and amino acid interconversion pathways. Defense responses and plant hormone biosynthesis were also increased. Xaj proteins detected in infected tissues demonstrate its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Secreted proteases and extracellular secretion apparatus such as type IV pilus for twitching motility and type III secretion effectors indicate putative factors recognized by the host. Taken together, these results suggest intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Our results provide insights into molecular mechanisms and highlight potential molecular tools for early detection and disease control strategies.

Soybean oil consumption is increasing worldwide and parallels a rise in obesity. Rich in unsaturated fats, especially linoleic acid, soybean oil is assumed to be healthy, and yet it induces obesity, diabetes, insulin resistance, and fatty liver in mice. Here, we show that the genetically modified soybean oil Plenish, which came on the U.S. market in 2014 and is low in linoleic acid, induces less obesity than conventional soybean oil in C57BL/6 male mice. Proteomic analysis of the liver reveals global differences in hepatic proteins when comparing diets rich in the two soybean oils, coconut oil, and a low-fat diet. Metabolomic analysis of the liver and plasma shows a positive correlation between obesity and hepatic C18 oxylipin metabolites of omega-6 (ω6) and omega-3 (ω3) fatty acids (linoleic and α-linolenic acid, respectively) in the cytochrome P450/soluble epoxide hydrolase pathway. While Plenish induced less insulin resistance than conventional soybean oil, it resulted in hepatomegaly and liver dysfunction as did olive oil, which has a similar fatty acid composition. These results implicate a new class of compounds in diet-induced obesity-C18 epoxide and diol oxylipins.

Callus samples from the ball and the arch of the foot, collected on tape circles, were compared by shotgun proteomic profiling. Pachyonychia congenita subjects were sampled who exhibited a mutation in KRT6A, KRT6B, KRT6C, KRT16 or KRT17, and the proteins were digested and analyzed by tandem mass spectrometry. In comparison with samples from unaffected control subjects, those from subjects with KRT6A or KRT16 mutations displayed the most differences in profile from normal, while those from subjects with KRT6C or KRT17 mutations showed few differences from normal. The profiles from subjects with KRT6B mutations were intermediate in protein profile differences. Degree of departure from the normal profile could be estimated by expression of numerous proteins in callus from the ball of the foot that were consistently different. By contrast, the protein profile from the arch of the foot was hardly affected. The results provide a foundation for noninvasive monitoring of the efficacy of treatments with quantitative assessment of departure from the normal phenotype.SignificancePachyonychia congenita is an orphan disease in which the connection between the basic defect (keratin mutation) and debilitating symptoms (severe plantar pain) is poorly understood. Present work addresses the degree to which the protein profile is altered in the epidermis where the severe pain originates. The results indicate that the mutated keratins differ greatly in the degree to which they elicit perturbations in protein profile. In those cases with markedly altered protein levels, monitoring the callus profile may provide an objective measure of treatment efficacy.

Extracellular matrix plays a role in differentiation and phenotype development of its resident cells. Although cardiac extracellular matrix from the contractile tissues has been studied and utilized in tissue engineering, extracellular matrix properties of the pacemaking sinoatrial node are largely unknown. In this study, the biomechanical properties and biochemical composition and distribution of extracellular matrix in the sinoatrial node were investigated relative to the left ventricle. Extracellular matrix of the sinoatrial node was found to be overall stiffer than that of the left ventricle and highly heterogeneous with interstitial regions composed of predominantly fibrillar collagens and rich in elastin. The extracellular matrix protein distribution suggests that resident pacemaking cardiomyocytes are enclosed in fibrillar collagens that can withstand greater tensile strength while the surrounding elastin-rich regions may undergo deformation to reduce the mechanical strain in these cells. Moreover, basement membrane-associated adhesion proteins that are ligands for integrins were of low abundance in the sinoatrial node, which may decrease force transduction in the pacemaking cardiomyocytes. In contrast to extracellular matrix of the left ventricle, extracellular matrix of the sinoatrial node may reduce mechanical strain and force transduction in pacemaking cardiomyocytes. These findings provide the criteria for a suitable matrix scaffold for engineering biopacemakers.

BackgroundFruit flies and mammals protect themselves against infection by mounting immune and metabolic responses that must be balanced against the metabolic needs of the pathogens. In this context, p38 mitogen-activated protein kinase (MAPK)-dependent signaling is critical to regulating both innate immunity and metabolism during infection. Accordingly, we asked to what extent the Asian malaria mosquito Anopheles stephensi utilizes p38 MAPK signaling during infection with the human malaria parasite Plasmodium falciparum.MethodsA. stephensi p38 MAPK (AsP38 MAPK) was identified and patterns of signaling in vitro and in vivo (midgut) were analyzed using phospho-specific antibodies and small molecule inhibitors. Functional effects of AsP38 MAPK inhibition were assessed using P. falciparum infection, quantitative real-time PCR, assays for reactive oxygen species and survivorship under oxidative stress, proteomics, and biochemical analyses.ResultsThe genome of A. stephensi encodes a single p38 MAPK that is activated in the midgut in response to parasite infection. Inhibition of AsP38 MAPK signaling significantly reduced P. falciparum sporogonic development. This phenotype was associated with AsP38 MAPK regulation of mitochondrial physiology and stress responses in the midgut epithelium, a tissue critical for parasite development. Specifically, inhibition of AsP38 MAPK resulted in reduction in mosquito protein synthesis machinery, a shift in glucose metabolism, reduced mitochondrial metabolism, enhanced production of mitochondrial reactive oxygen species, induction of an array of anti-parasite effector genes, and decreased resistance to oxidative stress-mediated damage. Hence, P. falciparum-induced activation of AsP38 MAPK in the midgut facilitates parasite infection through a combination of reduced anti-parasite immune defenses and enhanced host protein synthesis and bioenergetics to minimize the impact of infection on the host and to maximize parasite survival, and ultimately, transmission.ConclusionsThese observations suggest that, as in mammals, innate immunity and mitochondrial responses are integrated in mosquitoes and that AsP38 MAPK-dependent signaling facilitates mosquito survival during parasite infection, a fact that may attest to the relatively longer evolutionary relationship of these parasites with their invertebrate compared to their vertebrate hosts. On a practical level, improved understanding of the balances and trade-offs between resistance and metabolism could be leveraged to generate fit, resistant mosquitoes for malaria control.