Proteomic Analysis of Serum Differentially Expressed Proteins Between Allergic Bronchopulmonary Aspergillosis and Asthma

Mycopathologia - Allergic bronchopulmonary aspergillosis (ABPA) constantly develops in asthmatics, which has not been fully investigated. This study aimed to investigate serum differentially...     

Effects of Bacillus cereus PAS38 on Immune-Related Differentially Expressed Genes of Spleen in Broilers

This study mainly explored the immunomodulatory mechanisms of the probiotic Bacillus cereus PAS38 (PB) on broiler spleen. A total of 120 avian white feathe     

Differentially Expressed Proteins Associated with Fusarium Head Blight Resistance in Wheat

Background

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, substantially reduces wheat grain yield and quality worldwide. Proteins play important roles in defense against the fungal infection. This study characterized differentially expressed proteins between near-isogenic lines (NILs) contrasting in alleles of Fhb1, a major FHB resistance gene in wheat, to identify proteins underlining FHB resistance of Fhb1.

Methods

The two-dimensional protein profiles were compared between the Fusarium-inoculated spikes of the two NILs collected 72 h after inoculation. The protein profiles of mock- and Fusarium-inoculated Fhb1⁺NIL were also compared to identify pathogen-responsive proteins.

Results

Eight proteins were either induced or upregulated in inoculated Fhb1⁺NIL when compared with mock-inoculated Fhb1⁺NIL; nine proteins were either induced or upregulated in the Fusarium-inoculated Fhb1⁺NIL when compared with Fusarium-inoculated Fhb1⁻NIL. Proteins that were differentially expressed in the Fhb1⁺NIL, not in the Fhb1⁻NIL, after Fusarium inoculation included wheat proteins for defending fungal penetration, photosynthesis, energy metabolism, and detoxification.

Conclusions

Coordinated expression of the identified proteins resulted in FHB resistance in Fhb1⁺NIL. The results provide insight into the pathway of Fhb1-mediated FHB resistance.     

Detection of Differentially Expressed Basal Cell Proteins by Mass Spectrometry

The ability of cells to modulate interactions with each other and the substrate is essential for epithelial tissue remodeling during processes such as wound healing and tumor progression. However, despite strides made in the field of proteomics, proteins involved in adhesion have been difficult to study. Here, we report a method for the enrichment and analysis of proteins associated with the basal surface of the cell and its underlying matrix. The enrichment involves deroofing the cells with 20 mm ammonium hydroxide and the removal of cytosolic and organellar proteins by stringent water wash. Proteomic profiling was achieved by LC-FTMS, which allowed comparison of differentially expressed or shared proteins under different cell states. First, we analyzed and compared the basal cell components of mouse keratinocytes lacking the cell-cell junction molecule plakoglobin with their control counterparts. Changes in the molecules involved in motility and invasion were detected in plakoglobin-deficient cells, including decreased detection of fibronectin, integrin β₄, and FAT tumor suppressor. Second, we assessed the differences in basal cell components between two human oral squamous cell carcinoma lines originating from different sites in the oral cavity (CAL33 and UM-SCC-1). The data show differences between the two lines in the type and abundance of proteins specific to cell adhesion, migration, and angiogenesis. Therefore, the method described here has the potential to serve as a platform to assess proteomic changes in basal cell components including extracellular and adhesion-specific proteins involved in wound healing, cancer, and chronic and acquired adhesion-related disorders.There is an urgent need for tools to comprehensively identify markers of normal and pathological processes at the molecular level. DNA microarrays have enabled researchers to follow gene expression changes with respect to many of these processes, including individual tumors in the case of cancer (1). Direct detection of proteins is typically required to validate changes at the gene product level; however, the changes in protein levels do not always reflect changes in gene expression because of post-translational modifications, differential compartmentalization, recycling, and degradation. Because it is ultimately the proteins that convey cellular phenotypes, it is necessary to develop methods for direct screening of proteins, and mass spectrometry shows promise for this purpose. However, the usefulness of mass spectrometry as an analytical tool to detect proteins in cells or tissue is limited to the extent to which the sample is sufficiently enriched for the specific fraction of interest. It is still challenging to identify molecules involved in specific normal or pathological processes because the relevant proteins are often difficult to isolate from the majority of cellular proteins that are not correlated to the process of interest. In this context, an ideal proteomics approach would require a minimal amount of starting material, be amenable to an efficient enrichment strategy, and would provide results quickly.It has been well established that molecules directly involved in cell-cell and cell-substrate adhesions are critical for processes such as epithelial to mesenchymal transition and wound healing. Their further role in regulation of tissue integrity, cell polarity, motility, and invasion is emphasized by a variety of disorders stemming from their inappropriate expression and mutations (2, 3). Selectins, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 have been established both as biomarkers (4) and predictive factors (5, 6) for the development of accelerated atherosclerosis and heart disease. In epithelial tissues, reduced expression of the cell-cell adhesion molecule E-cadherin correlates with epithelial to mesenchymal transition, tissue invasion, and metastasis and is a prognostic biomarker of poor clinical outcome in many cell types (7–9). Furthermore, up-regulating E-cadherin is considered as a treatment option in several types of cancer (10). Therefore, methods are also needed to not only identify adhesion molecules as disease markers but to also understand the pathology of underlying medical problems caused by impairment in adhesion molecule function (e.g. inability to heal chronic wounds (11)). However, the lack of knowledge about regulation and functional interactions of the specific adhesion-related proteins has so far thwarted the attempts at direct targeting of these molecules in basic and clinical research (12, 13). Therefore, a comprehensive understanding of how proteins that function in adhesive processes work together to maintain proper tissue form and function is critical.Some of the same barriers to effective application of mass spectrometry as an analytical tool (as discussed above) have impeded analysis of cell-cell and cell-matrix adhesion-dependent processes such as wound healing and cancer (14). The study of extracellular matrix (ECM)1 and adhesion-related proteins is further complicated by the difficulty in sample preparation because compared with cytosolic proteins basal cell proteins are often highly insoluble (e.g. transmembrane and plaque components) and difficult to isolate from intracellular proteins. One general strategy involves using ECM-specific enzymes to dislodge the cells at their points of attachment (15). The supernatant from the partial digest is collected for further proteomics analysis. However, most mass spectrometric analyses depend on detection of peptides with specific ionization and fragmentation properties that are most readily achieved using trypsin as the sole enzyme. The use of ECM-specific enzymes may result in a distribution of peptides that are not optimal for detection (i.e. the generation of non-tryptic termini). The other general approach to isolate components of the ECM involves using detergents to lyse cells on the surfaces to which they are attached and collect the remaining cell debris for analysis (15). Although progress has been made with respect to the creation of “mass spectrometry-friendly” detergents (16), the use of chemicals for the purpose of protein solubilization is generally not ideal. To overcome these problems, we adapted a fast, simple method of isolating extracellular, transmembrane, and associated proteins (from here on collectively referred to as “basal cell proteins”) from cells attached to a solid substrate. The method consists of “deroofing” the cells attached to glass coverslips by 20 mm NH₄OH solution followed by rapid water rinses to remove the bulk of the cell and its remaining debris (17). Our results show efficient removal of cytoplasm and organelles and detection of basal cell proteins by mass spectrometry, including those involved in cell-cell and cell-extracellular matrix interactions. These proteins were liberated from the surface with trypsin, and the subsequently generated peptides were detected and profiled for differences using LC-FTMS.The approach was first validated by comparing basal cell protein composition in mouse keratinocytes with or without a critical cell-cell junction protein called plakoglobin (PG). This desmosomal protein is required for cell-cell adhesion and maintenance of tissue integrity (18). Plakoglobin inhibits keratinocyte motility (19) and is down-regulated in several distinct tumor types, including bladder, breast, and cervical cancers (20–22). Moreover, we were able to dissect the molecular differences between an independent isolate of PG^−/− keratinocytes that behaved differently in motility assays from the rest of the PG-null cells, further emphasizing the potential for using the method to differentiate between cells with distinct adhesive and motile behaviors. The method was then evaluated in clinically relevant human tumor cell lines by extending the analysis to include two human oral squamous cancers of different origin. Because they lack precisely defined changes in cell adhesion molecules and phenotype, we compared the basal cell protein expression of UM-SCC-1 (23) and CAL33 (24) cell lines isolated from the roof of the mouth and tongue, respectively. These experiments revealed 40 proteins differentially expressed between the cell lines among over 100 detected. Moreover, the proteomic profile reveals a set of motility- and invasion-related genes unique to tongue-derived CAL33 cells. This could indicate the difference between oral cancers derived from different parts of the mouth, or it may indicate a potential difference in aggressiveness between these cell lines. These results show that our detection method is applicable for both detection and comparative studies in human cancer model systems.     

筛选差异表达基因和蛋白质的方法进展

分离和鉴定差异表达基因和蛋白质不仅有助于发现基因和蛋白质的功能,更有助于揭示某些疾病的发生机理.目前筛选差异表达基因的方法主要有差异显示PCR方法(differential display RT-PCR,DDRT-PCR)、消减杂交法(subtractive hybridization,SH)、基因芯片技术(DNA chip technique)和基因表达的系统分析(serial analysis of gene expression,SAGE)等,其中消减杂交法中又先后建立了代表性差异分析技术(representational difference analysis,RDA)、抑制消减杂交法(suppression subtractive hybridization,SSH)和获得全长基因的消减杂交法(full-length-gene-obtainable subtractive hybridization).筛选差异表达蛋白质的方法主要有双向电泳技术(two-dimentional gel electrophoresis)和噬菌体全套抗体库技术(phage display antibody repertoire library technique).这些方法各有特点,各有利弊,研究者可根据自己的需要选择适合于自己的方法.     

五倍体草莓及其十倍体的叶片差异表达蛋白分析

以二倍体绿色草莓(Fragaria viridis Duch.)为父本、八倍体栽培草莓‘房香’(F.×ananassa‘Fusanoka’)为母本杂交所得的五倍体草莓(株系代号:FxLs-11-37,2n=5x=35)及其染色体加倍而成的十倍体(株系代号:A3,2n=10x=70)草莓为试材,观察记载其部分农艺性状、SPAD值以及净光合速率,利用双向凝胶电泳技术对草莓染色体加倍后叶片蛋白质进行了分析,获得了分辨率高、重复性好的电泳图谱。结果表明:(1)与五倍体FxLs-11-37相比,十倍体A3的株型明显矮化、植株冠径变大、叶长叶宽增大、叶片增厚以及叶色浓绿,其叶片的SPAD值与净光合速率显著高于FxLs-11-37。(2)通过PDQuest软件对图谱分析表明,两者在等电点4~7、分子量14.4~66.2kD范围内蛋白质斑点分布最多,可识别的总蛋白质斑点数超过700个,其中蛋白质表达差异水平在1.5倍以上的有18个,2.0倍以上的有4个。利用MALDI-TOF-MS/MS质谱技术鉴定了这4个差异蛋白质,分别是草莓主要过敏原a 1-E、核内不均一核糖核蛋白1、叶绿体硫辛酰基合酶1、NAD(P)H脱氢酶(醌)FQR1类似蛋白质。这些差异蛋白质主要与抗逆、mRNA转运、物质与能量代谢相关。荧光定量PCR对上述4个蛋白编码基因的转录表达水平检测表明,与蛋白质表达差异的趋势一致。该研究获得了草莓染色体加倍后差异表达的蛋白质,为深入研究提供了线索。     

Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development.     

Evaluation of Connectivity Among American Black Bear Populations in Georgia

Habitat fragmentation and loss contribute to isolation of wildlife populations and increased extinction risks for various species, including many large carnivores. We studied a small and isolated population of American black bears (Ursus americanus) that is of conservation concern in central Georgia, USA (i.e., central Georgia bear population [CGBP]). Our goal was to evaluate the potential for demographic and genetic interchange from neighboring bear populations to the CGBP. To evaluate resource selection and movement potential, we used 35,487 global positioning system locations collected every 20 minutes from 2012 to 2014 from 33 male bears in the CGBP. We then developed a step selection function model based on conditional logistic regression. Male bears chose steps that avoided crops, roads, and human developments and were closer to forests and woody wetlands than expected based on availability. We used a geographic information system to simulate 300 bear movement paths from nearby bear populations in northern Florida, northern Georgia, and southern Georgia to estimate the potential for immigration to the CGBP. Only 4 simulated movement paths from the nearby populations intersected the CGBP. The creation of a hypothetical 1-km-wide corridor between the southern Georgia population and the CGBP produced only minor improvements in interchange. Our findings suggest that demographic connectivity between the CGBP and surrounding bear populations may be limited, and coupled with previous works showing genetic isolation in the CGBP, that creation of corridors may have only marginal effects on restoring gene flow, at least in the near term. Management actions such as translocation and the establishment of stepping stone populations may be needed to increase the genetic diversity and demographic stability of bears in the CGBP. © 2021 The Wildlife Society.     

Identification of Differentially Expressed Proteins in Murine Embryonic and Postnatal Cortical Neural Progenitors

Background

The central nervous system (CNS) develops from a heterogeneous pool of neural stem and progenitor cells (NSPC), the underlying differences among which are poorly understood. The study of NSPC would be greatly facilitated by the identification of additional proteins that mediate their function and that would distinguish amongst different progenitor populations.

Methodology/Principal Findings

To identify membrane and membrane-associated proteins expressed by NSPC, we used a proteomics approach to profile NSPC cultured as neurospheres (NS) isolated from the murine cortex during a period of neurogenesis (embryonic day 11.5, E11.5), as compared to NSPC isolated at a peak of gliogenesis (postnatal day 1, P0) and to differentiated E11.5 NS. 54 proteins were identified with high expression in E11.5 NS, including the TrkC receptor, several heterotrimeric G proteins, and the Neogenin receptor. 24 proteins were identified with similar expression in E11.5 and P0 NS over differentiated E11.5 NS, and 13 proteins were identified with high expression specifically in P0 NS compared to E11.5 NS. To illustrate the potential relevance of these identified proteins to neural stem cell biology, the function of Neogenin was further studied. Using Fluorescence Activated Cell Sorting (FACS) analysis, expression of Neogenin was associated with a self-renewing population present in both E11.5 and adult subventricular zone (SVZ) NS but not in P0 NS. E11.5 NS expressed a putative Neogenin ligand, RGMa, and underwent apoptosis when exposed to a ligand-blocking antibody.

Conclusions/Significance

There are fundamental differences between the continuously self-renewing and more limited progenitors of the developing cortex. We identified a subset of differentially expressed proteins that serve not only as a set of functionally important proteins, but as a useful set of markers for the subsequent analysis of NSPC. Neogenin is associated with the continuously self-renewing and neurogenic cells present in E11.5 cortical and adult SVZ NS, and the Neogenin/RGMa receptor/ligand pair may regulate cell survival during development.     

A Statistical Model to Identify Differentially Expressed Proteins in 2D PAGE Gels

Two dimensional polyacrylamide gel electrophoresis (2D PAGE) is used to identify differentially expressed proteins and may be applied to biomarker discovery. A limitation of this approach is the inability to detect a protein when its concentration falls below the limit of detection. Consequently, differential expression of proteins may be missed when the level of a protein in the cases or controls is below the limit of detection for 2D PAGE. Standard statistical techniques have difficulty dealing with undetected proteins. To address this issue, we propose a mixture model that takes into account both detected and non-detected proteins. Non-detected proteins are classified either as (a) proteins that are not expressed in at least one replicate, or (b) proteins that are expressed but are below the limit of detection. We obtain maximum likelihood estimates of the parameters of the mixture model, including the group-specific probability of expression and mean expression intensities. Differentially expressed proteins can be detected by using a Likelihood Ratio Test (LRT). Our simulation results, using data generated from biological experiments, show that the likelihood model has higher statistical power than standard statistical approaches to detect differentially expressed proteins. An R package, Slider (Statistical Likelihood model for Identifying Differential Expression in R), is freely available at http://www.cebl.auckland.ac.nz/slider.php.     

Induction of pseudopregnancy in the American Black Bear (Ursus americanus)

The aim of this study was to determine whether American Black Bears (Ursus americanus) can experience a pseudopregnancy of the same duration as pregnancy. To do this, we treated three nonmated, captive female bears with human chorionic gonadotropin (hCG) during one breeding season, and saline during another. Progesterone concentrations were measured in monthly blood samples to determine whether pseudopregnancy had occurred. Elevated progesterone concentrations were observed in two out of three bears treated with hCG. We conclude that 1) Elevated progesterone concentrations can be induced in black bears by injection of 35 U/kg hCG during the mating season. 2) Bears can experience a pseudopregnancy, identical in length to pregnancy, in which progesterone profiles are indistinguishable from those of pregnancy.     

Stress Proteins in Mammalian Hibernation

Many organisms whose body temperatures (T_b) vary when they areexposed to a wide range of environmental temperatures exhibitdifferential expression of stress (heat shock) proteins, presumablyto minimize protein damage during thermal stress. In contrast,we know relatively little about natural variation in stressproteins in homeotherms (i.e., birds and mammals), perhaps dueto the relatively constant T_b that is the hallmark of this vertebrategroup. The significant changes in T_b and metabolism characteristicof mammalian hibernators suggest they could provide insightinto the ecological relevance of stress proteins in mammals.Here we examined differential expression of stress proteinsin several tissues of active and torpid 13-lined ground squirrels.There were few significant differences in expression of inducibleHsp70 protein in liver, kidney, heart, intestine, and skeletalmuscle between active and torpid squirrels, and neither fastingin active squirrels nor torpor bout length in hibernators significantlyaltered Hsp70 abundance in these tissues. However, Hsp70 proteinwas lower in brown adipose tissue (BAT) of torpid compared withactive squirrels. In contrast, abundance of GRP75, the mitochondria]form of Hsp70, was greater in liver, skeletal muscle and intestineof torpid compared with active squirrels, with the greatestchange in intestine. Because GRP75 has been shown to be inducedby non-thermal stressors including glucose deprivation and oxidativestress, these results suggest that the ecological significanceof stress proteins for hibernators may be more closely associatedwith the metabolic demands of heterothermy rather than thermalstress per se.     

MS-EmpiRe Utilizes Peptide-level Noise Distributions for Ultra-sensitive Detection of Differentially Expressed Proteins

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Highlights

• •New software to determine differentially expressed proteins in quantitative MS experiments.
• •Applicable to any modern quantitative MS setup and study type, including clinical setups.
• •Ultra-sensitive at strict FDR control, up to 1000 additional proteins in a single comparison.
• •Easy to use, fast processing and readily available package, including user friendly manual.

     

Identification of Streptomyces coelicolor Proteins That Are Differentially Expressed in the Presence of Plant Material

Streptomyces coelicolor and Lemna minor were used as a model to study the modulation of bacterial gene expression during plant-streptomycete interactions. S. coelicolor was grown in minimal medium with and without L. minor fronds. Bacterial proteomes were analyzed by two-dimensional gel electrophoresis, and a comparison of the two culture conditions resulted in identification of 31 proteins that were induced or repressed by the presence of plant material. One-half of these proteins were identified by peptide mass fingerprinting by using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The induced proteins were involved in energetic metabolism (glycolysis, pentose phosphate pathway, oxidative phosphorylation), protein synthesis, degradation of amino acids, alkenes, or cellulose, tellurite resistance, and growth under general physiological or oxidative stress conditions. The repressed proteins were proteins synthesized under starvation stress conditions. These results suggest that root exudates provide additional carbon sources to the bacteria and that physiological adaptations are required for efficient bacterial growth in the presence of plants.     

Screening for Differentially Expressed Proteins Relevant to the Differential Diagnosis of Sarcoidosis and Tuberculosis

Background

In this study, we sought to identify differentially expressed proteins in the serum of patients with sarcoidosis or tuberculosis and to evaluate these proteins as markers for the differential diagnosis of sarcoidosis and sputum-negative tuberculosis.

Methods

Using protein microarrays, we identified 3 proteins exhibiting differential expression between patients with sarcoidosis and tuberculosis. Elevated expression of these proteins was verified using the enzyme-linked immunosorbent assay (ELISA) and was further confirmed by immunohistochemistry. Receiver operating characteristic (ROC) curve, logistic regression analysis, parallel, and serial tests were used to evaluate the diagnostic efficacy of the proteins.

Results

Intercellular Adhesion Molecule 1(ICAM-1) and leptin were screened for differentially expressed proteins relevant to sarcoidosis and tuberculosis. Using ROC curves, we found that ICAM-1 (cutoff value: 57740 pg/mL) had an area under the curve (AUC), sensitivity, and specificity of 0.718, 62.3%, and 79.5% respectively, while leptin (cutoff value: 1193.186 pg/mL) had an AUC, sensitivity, and specificity of 0.763, 88.3%, and 65.8%, respectively. Logistic regression analysis revealed that the AUC, sensitivity, and specificity of combined leptin and ICAM-1 were 0.787, 89.6%, and 65.8%, respectively, while those of combined leptin, ICAM-1, and body mass index (BMI) were 0.837, 90.9%, and 64.4%, respectively, which had the greatest diagnostic value. Parallel and serial tests indicated that the BMI-leptin parallel with the ICAM-1 serial was the best diagnostic method, achieving a sensitivity and specificity of 86.5% and 73.1%, respectively. Thus, our results identified elevated expression of ICAM-1 and leptin in serum and granulomas of sarcoidosis patients.

Conclusions

ICAM-1 and leptin were found to be potential markers for the diagnosis of sarcoidosis and differential diagnosis of sarcoidosis and sputum-negative tuberculosis.     

鼠精子发生时期差异表达基因的克隆(英文)

为了分离鼠精子发生时期表达的基因,本文采用mRNA差异显示法,以鼠的粗线期卵母细胞为对照,检测了出生后60天和16天鼠的睾丸。得到12个有差异的片段(Fig.1&Table 1)。克隆测序结果表明,其中5个与已知基因非常吻合,另外6个与一些未知功能的cDNA、ESTs有较高的同源性,只有1个与已知序列没有同源性。Northern杂交分析显示sp1和sp8主要在成年鼠睾丸表达(Fig.4B)。采用5RACE对sp1的cDNA进行了全长分析,该基因编码一个推测是高度磷酸化蛋白的541个氨基酸(Fig.2),其中包括一个核定位信号,无论在核苷酸水平上,还是在氨基酸水平上均没有明显的同源性,仅在2个蛋白区有少量同源氨基酸(Fig.3)。该基因在20-60天龄鼠的睾丸均有表达,并且具有很高的组织特异性只在睾丸里表达(Fig.4A)。因而,这个基因有可能参与减数分裂及其以后的整个过程。可以认为这是一个新基因。我们把它命名为peat (predominantly expressed in adult testis)。     

Proteomic Analysis of Differentially Expressed Proteins in Resistant Soybean Leaves after Phakopsora pachyrhizi Infection

Soybean rust caused by Phakopsora pachyrhizi is a destructive foliar disease in nearly all soybean‐producing countries. Understanding the host responses at the molecular level is certainly essential for effective control of the disease. To identify proteins involved in the resistance to soybean rust, differential proteomic analysis was conducted in soybean leaves of a resistant genotype after P. pachyrhizi infection. A total of 41 protein spots exhibiting a fold change >1.5 between the non‐inoculated and P. pachyrhizi‐inoculated soybean leaves at 12 and 24 h postinoculation (hpi) were unambiguously identified and functionally grouped into seven categories. Twenty proteins were up‐regulated and four proteins were down‐regulated at 12 hpi, whereas 18 proteins were up‐regulated and eight proteins were down‐regulated at 24 hpi. Generally, proteins involved in photosynthesis were down‐regulated, whereas proteins associated with disease and defense response, protein folding and assembly, carbohydrate metabolism and energy production were up‐regulated. Results are discussed in terms of the functional implications of the proteins identified, with special emphasis on their putative roles in defense. Abundance changes of these proteins, together with their putative functions reveal a comprehensive picture of the host response in rust‐resistant soybean leaves and provide a useful platform for better understanding of the molecular basis of soybean rust resistance.     

R-Spondins Are Expressed by the Intestinal Stroma and are Differentially Regulated during Citrobacter rodentium- and DSS-Induced Colitis in Mice

The R-spondin family of proteins has recently been described as secreted enhancers of β-catenin activation through the canonical Wnt signaling pathway. We previously reported that Rspo2 is a major determinant of susceptibility to Citrobacter rodentium-mediated colitis in mice and recent genome-wide association studies have revealed RSPO3 as a candidate Crohn’s disease-specific inflammatory bowel disease susceptibility gene in humans. However, there is little information on the endogenous expression and cellular source of R-spondins in the colon at steady state and during intestinal inflammation. RNA sequencing and qRT-PCR were used to assess the expression of R-spondins at steady state and in two mouse models of colonic inflammation. The cellular source of R-spondins was assessed in specific colonic cell populations isolated by cell sorting. Data mining from publicly available datasets was used to assess the expression of R-spondins in the human colon. At steady state, colonic expression of R-spondins was found to be exclusive to non-epithelial CD45^- lamina propria cells, and Rspo3/RSPO3 was the most highly expressed R-spondin in both mouse and human colon. R-spondin expression was found to be highly dynamic and differentially regulated during C. rodentium infection and dextran sodium sulfate (DSS) colitis, with notably high levels of Rspo3 expression during DSS colitis, and high levels of Rspo2 expression during C. rodentium infection, specifically in susceptible mice. Our data are consistent with the hypothesis that in the colon, R-spondins are expressed by subepithelial stromal cells, and that Rspo3/RSPO3 is the family member most implicated in colonic homeostasis. The differential regulation of the R-spondins in different models of intestinal inflammation indicate they respond to specific pathogenic and inflammatory signals that differ in the two models and provides further evidence that this family of proteins plays a key role in linking intestinal inflammation and homeostasis.