2-dimensional Gel Electrophoresis Research Articles

A molecular circuit linking the BCR to the NAD biosynthetic enzyme NAMPT is an actionable target in Richter syndrome

AbstractThis works defines, to the best of our knowledge, for the first time a molecular circuit connecting nicotinamide mononucleoside phosphoribosyl transferase (NAMPT) activity to the B-cell receptor (BCR) pathway. Using 4 distinct xenograft models derived from patients with Richter syndrome (RS-PDX), we show that BCR cross-linking results in transcriptional activation of the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme NAMPT, with increased protein expression, in turn, positively affecting global cellular NAD levels and sirtuins activity. NAMPT blockade, by using the novel OT-82 inhibitor in combination with either BTK or PI3K inhibitors (BTKi or PI3Ki), induces rapid and potent apoptotic responses in all 4 models, independently of their mutational profile and the expression of the other NAD biosynthetic enzymes, including nicotinate phosphoribosyltransferase. The connecting link in the circuit is represented by AKT that is both tyrosine- and serine-phosphorylated by PI3K and deacetylated by sirtuin 1 and 2 to obtain full kinase activation. Acetylation (ie, inhibition) of AKT after OT-82 administration was shown by 2-dimensional gel electrophoresis and immunoprecipitation. Consistently, pharmacological inhibition or silencing of sirtuin 1 and 2 impairs AKT activation and induces apoptosis of RS cells in combination with PI3Ki or BTKi. Lastly, treatment of RS-PDX mice with the combination of PI3Ki and OT-82 results in significant inhibition of tumor growth, with evidence of in vivo activation of apoptosis. Collectively, these data highlight a novel application for NAMPT inhibitors in combination with BTKi or PI3Ki in aggressive lymphomas.

2-DE-based and shotgun proteomics approach in the analysis of the seed proteome of a low phytic acid rice (Oryza sativa, ssp. japonica) mutant

A low phytic acid (lpa) rice mutant line, denoted as Os-lpa-XS110-2, was developed through gamma irradiation (Liu et al., 2007). Subsequently, this mutant line was cultivated alongside its parental wild-type counterpart, Xiushui 110, in four distinct field trials conducted in China. The primary objective of this research was to conduct a comparative proteomic analysis between the parental and mutant lines, aiming to detect and compare protein species with varying levels of relative abundance and their potential involvement in phytic acid biosynthesis. To elucidate the molecular underpinnings of this alteration, proteome analyses were conducted on both the parental and mutant lines, employing LC-MS/MS in conjunction with 2-dimensional gel electrophoresis. Only, 7 proteins consistently exhibited significant differential abundance across all trial locations, suggesting that the regulation of these proteins is less susceptible to environmental influences. In a broader context, among the 700 detected proteins, 56 displayed noteworthy up- or down-regulation across diverse geographical regions. Notably, storage and housekeeping proteins emerged as the most frequently identified protein categories, with globulin and glutelin demonstrating prominent differences in abundance. However, based on our current understanding, none of the identified proteins appear to have direct roles in phytic acid biosynthesis.

Traditional halal meat production without stunning versus commercial slaughter with electrical stunning of slow-growing broiler chicken: impact on meat quality and proteome changes

Impact of traditional halal meat production without stunning (NST) and commercial slaughter with electrical stunning (ST) of 100 slow-growing broiler chicken on blood plasma and different biochemical, enzymatic, hormonal, meat quality, and proteomic changes was evaluated. The results revealed lower (P < 0.05) postmortem pH values and higher redness (a*) scores for ST samples relative to NST group. Myofibrillar fragmentation index and bleeding efficiency (%) were lower (P < 0.05) in ST compared to NST samples. The ST group had higher (P < 0.05) creatinine, total protein, alanine aminotransferase (ALT), and triiodothyronine (T3) than NST group, however, no difference (P > 0.05) in blood glucose, lactate dehydrogenase (LDH), creatine kinase (CK), thyroxine (T4), cortisol, and aspartate aminotransferase (AST) was observed relative to NST samples. The 2-dimensional gel electrophoresis (2-DE) coupled to MALDI-TOF MS of meat samples has identified 14 differentially abundant proteins between 2 groups. Proteins demonstrating positive correlation with stress namely adenylate kinase isoenzyme-1, Rho guanine nucleotide exchange factor (NST), and apolipoprotein A-I (ST) were overabundant. From the current study, it is concluded that electrical stunning of broilers prior to slaughter or traditional halal slaughter without stunning does not adversely affect the meat quality.

Ficus deltoidea Leaf Alters Oxidative Stress, Protein Homeostasis and Ubiquitin-Proteasome Pathways in Fatty Acid-Induced Cell Line.

Ficus deltoidea (mistletoe fig) is a shrub well known among locals in Malaysia primarily for its treatment of toothaches, colds and wounds. The aim of this study is to determine the potential of leaves, sourced from three different varieties of F. deltoidea, to exhibit antioxidant activity, a reduction of lipid concentration, and protein expression in steatosis-induced liver cell lines. The leaves of three F. deltoidea varieties, namely Ficus deltoidea var. angustifolia, Ficus deltoidea var. trengganuensis and Ficus deltoidea var. kunstleri, were subjected to water extraction. The resulting crude extracts were fractionated using water and ethyl acetate. Palmitic acid was used to induce lipid accumulation (steatosis) in human liver (WRL68) cells, before all the samples were tested for their lipid-reducing activity. Several proteomic approaches were incorporated. The changes in protein expression were determined using 2-dimensional gel electrophoresis separation, whereas identification of our protein spots of interest was carried out via matrix-assisted laser desorption/ionization time-of-flight. Ficus deltoidea var. kunstleri alone demonstrated the ability to reduce lipids at the highest tested concentration (200 µg/mL) and was, therefore, used for subsequent experiments. Treatment with Ficus deltoidea var. kunstleri was found to restore redox status by increasing superoxide dismutase and glutathione peroxidase amounts and decreasing malondialdehyde formation. Six proteins were successfully identified; these were heat shock protein beta-1 (HSPB1), proteasome subunit alpha type 1 (PSMA1), glutathione S-transferase omega 1 (GSTO1), peroxiredoxin-1 (PRDX1), histone H2B (HIST1H2BD) and ubiquitin c-terminal hydrolase L3 (UCHL3). Through bioinformatics analysis, it was found that these proteins were significantly involved in specific pathways such as oxidative stress (PRDX1 and GSTO1), protein homeostasis (HSPB1) and degradation (UCHL3 and PSMA1). F. deltoidea pretreatment was shown to reduce lipid accumulation, thus improving the redox status and protein homeostasis. This suggests the role of F. deltoidea as a preventive mechanism in non-alcohol fatty liver disease.

Behavior of protein aggregates via electrostatic interactions or hydrogen bonds during dough formation

The aggregation behavior of gluten proteins via electrostatic interactions or hydrogen bonds during dough formation was investigated. Proteins were extracted with 50 mM acetic acid or 4 M urea to break electrostatic interactions or hydrogen bonds, respectively. The extracted proteins were analyzed by size exclusion high performance liquid chromatography and two dimensional fluorescence difference gel electrophoresis. Acetic acid- and urea-soluble protein aggregates increased with dough development, indicating that electrostatic interactions and hydrogen bonds weakened during dough mixing. On the other hand, the some monomeric proteins (mainly α-gliadins) in dough aggregated via electrostatic interactions rather than hydrogen bonds. A model is proposed for gluten formation during dough mixing based on non-covalent bonds.

Comparative protein analysis of two maize genotypes with contrasting tolerance to low temperature

BackgroundLow temperature (LT) stress is one of the major environmental stress factors affecting the growth and yield of maize (Zea mays L.). Hence, it is important to unravel the molecular mechanisms behind LT stress tolerance to improve molecular breeding in LT tolerant genotypes. In the present study, two maize genotypes viz. Gurez local from Kashmir Himalaya and tropical grown GM6, were dissected for their LT stress response in terms of accumulation of differentially regulated proteins (DRPs). Leaf proteome analysis at three-leaf stage of maize seedlings subjected to LT stress of 6 °C for a total of 12 h duration was performed using two dimensional gel electrophoresis (2D-PAGE) followed by subsequent identification of the proteins involved.ResultsAfter MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) and bioinformatics analysis, 19 proteins were successfully identified in Gurez local, while as 10 proteins were found to get successful identification in GM6. The interesting observations from the present investigation is the identification of three novel proteins viz. threonine dehydratase biosynthetic chloroplastic, thylakoidal processing peptidase 1 chloroplastic, and nodulin-like protein, whose role in abiotic stress tolerance, in general, and LT stress, in particular, has not been reported so far. It is important to highlight here that most of LT responsive proteins including the three novel proteins were identified from Gurez local only, owing to its exceptional LT tolerance. From the protein profiles, obtained in both genotypes immediately after LT stress perception, it was inferred that stress responsive protein accumulation and their expression fashion help the Gurez local in seedling establishment and withstand unfavorable conditions as compared to GM6. This was inferred from the findings of pathway enrichment analysis like regulation of seed growth, timing of floral transition, lipid glycosylation, and aspartate family amino acid catabolic processes, besides other key stress defense mechanisms. However, in GM6, metabolic pathways enriched were found to be involved in more general processes including cell cycle DNA replication and regulation of phenylpropanoid metabolism. Furthermore, majority of the qRT-PCR results of the selected proteins demonstrated positive correlation between protein levels and transcript abundance, thereby strengthening our findings.ConclusionsIn conclusion, our findings reported majority of the identified proteins in Gurez local exhibiting up-regulated pattern under LT stress as compared to GM6. Furthermore, three novel proteins induced by LT stress were found in Gurez local, requiring further functional validation. Therefore, our results offer more insights for elucidating the molecular networks mediating LT stress tolerance in maize.

Proteomic analysis of hepatic effects of okadaic acid in HepaRG human liver cells.

The marine biotoxin okadaic acid (OA) is produced by dinoflagellates and enters the human food chain by accumulating in the fatty tissue of filter-feeding shellfish. Consumption of highly contaminated shellfish can lead to diarrheic shellfish poisoning. However, apart from the acute effects in the intestine, OA can also provoke toxic effects in the liver, as it is able to pass the intestinal barrier into the blood stream. However, molecular details of OA-induced hepatotoxicity are still insufficiently characterized, and especially at the proteomic level data are scarce. In this study, we used human HepaRG liver cells and exposed them to non-cytotoxic OA concentrations for 24 hours. Global changes in protein expression were analyzed using 2-dimensional gel electrophoresis in combination with mass-spectrometric protein identification. The results constitute the first proteomic analysis of OA effects in human liver cells and indicate, amongst others, that OA affects the energy homeostasis, induces oxidative stress, and induces cytoskeletal changes.

Elevated plasma α1-antichymotrypsin is a biomarker candidate for malaria patients

Advancements in the field of proteomics have provided opportunities to develop diagnostic and therapeutic strategies against various diseases. About half of the world's population remains at risk of malaria. Caused by protozoan parasites of the genus Plasmodium, malaria is one of the oldest and largest risk factors responsible for the global burden of infectious diseases with an estimated 3.2 billion persons at risk of infection. For epidemiological surveillance and appropriate treatment of individuals infected with Plasmodium spp., timely detection is critical. In this study, we used combinations of depletion of abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, LC-MS/MS and western blot analysis on the plasma of healthy donors (100 individuals) and vivax and falciparum malaria patients (100 vivax malaria patients and 8 falciparum malaria patients). These analyses revealed that α1-antichymotrypsin (AACT) protein levels were elevated in vivax malaria patient plasma samples (mean fold-change ± standard error: 2.83 ± 0.11, based on band intensities), but not in plasma from patients with other mosquito-borne infectious diseases. The results of AACT immunoblot analyses showed that AACT protein was significantly elevated in vivax and falciparum malaria patient plasma samples (≥ 2-fold) compared to healthy control donor plasma samples, which has not been previously reported. [BMB Reports 2022; 55(11): 571-576].

Radiation-Induced Intestinal Normal Tissue Toxicity: Implications for Altered Proteome Profile

Radiation-induced toxicity to healthy/normal intestinal tissues, especially during radiotherapy, limits the radiation dose necessary to effectively eradicate tumors of the abdomen and pelvis. Although the pathogenesis of intestinal radiation toxicity is highly complex, understanding post-irradiation alterations in protein profiles can provide crucial insights that make radiotherapy safer and more efficient and allow for increasing the radiation dose during cancer treatment. Recent preclinical and clinical studies have advanced our current understanding of the molecular changes associated with radiation-induced intestinal damage by assessing changes in protein expression with mass spectrometry-based approaches and 2-dimensional difference gel electrophoresis. Studies by various groups have demonstrated that proteins that are involved in the inflammatory response, the apoptotic pathway, reactive oxygen species scavenging, and cell proliferation can be targeted to develop effective radiation countermeasures. Moreover, altered protein profiles serve as a crucial biomarkers for intestinal radiation damage. In this review, we present alterations in protein signatures following intestinal radiation damage as detected by proteomics approaches in preclinical and clinical models with the aim of providing a better understanding of how to accomplish intestinal protection against radiation damage.

Gene Expression Analyses of Mutant Flammulina velutipes (Enokitake Mushroom) with Clogging Phenomenon

Regulation of proper gene expression is important for cellular and organismal survival, maintenance, and growth. Abnormal gene expression, even for a single critical gene, can thwart cellular integrity and normal physiology to cause diseases, aging, and death. Therefore, gene expression profiling serves as a powerful tool to understand the pathology of diseases and to cure them. In this study, the difference in gene expression in Flammulina velutipes was compared between the wild type (WT) mushroom and the mutant one with clogging phenomenon. Differentially expressed transcripts were screened to identify the candidate genes responsible for the mutant phenotype using the DNA microarray analysis. A total of 88 genes including 60 upregulated and 28 downregulated genes were validated using the real-time quantitative PCR analysis. In addition, proteomic differences between the WT and mutant mushroom were analyzed using two–dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Interestingly, the genes identified by these genomic and proteomic analyses were involved in stress response, translation, and energy/sugar metabolism, including HSP70, elongation factor 2, and pyruvate kinase. Together, our data suggest that the aberrant expression of these genes attributes to the mutant clogging phenotype. We propose that these genes can be targeted to foster normal growth in F. velutipes.

Agronomic and Proteomic Assessment of Salt Stress Responses in &lt;i&gt;Pennisetum glaucum&lt;/i&gt; (Pearl Millet) Genotypes

Salinity is one of the environmental stresses that alter protein expression. To evaluate that, two ICRISAT Pennisetum glaucum (pearl millet) genotypes (IP 19586 and IP 22269) were subjected to 0.3 dSm-1 and 10 dSm-1 salinities. The agronomic parameters and ion content were assessed to find the tolerance level of the genotype to salinity. Differential expression of proteins on salt stress in both genotypes were analyzed by 2-dimensional gel electrophoresis. MALDI–MS/MS-TOF-TOF analysis helped in ascertain the amino acid sequences and the MASCOT search suggested the biological process of the predicted proteins. IP 22269 exclude the accumulation of toxic level of sodium in leaf. Based on physiological responses, IP 22269 was proposed to be salt-sensitive, and IP 19586 salt-tolerant genotypes. 67 proteins were responsive either less or more abundant to salinity. As per findings, the proteins responsive to salt stress are granule-bound starch synthase 1, alpha-amylase, Meiotic recombination protein SPO11, flavonoid 3'- monooxygenase, ethylene receptor 4, protein transcription initiation factor Y 11g, and auxin-responsive protein IAA16. Further, based on the STRING analysis these proteins form a protein-protein interaction network with 171 proteins. This research elucidates few salt stress tolerance traits and network between the salt stress-responsive mechanisms in pearl millet.

A deafness-associated mitochondrial DNA mutation caused pleiotropic effects on DNA replication and tRNA metabolism.

In this report, we investigated the molecular mechanism underlying a deafness-associated m.5783C > T mutation that affects the canonical C50-G63 base-pairing of TΨC stem of tRNACys and immediately adjacent to 5′ end of light-strand origin of mitochondrial DNA (mtDNA) replication (OriL). Two dimensional agarose gel electrophoresis revealed marked decreases in the replication intermediates including ascending arm of Y-fork arcs spanning OriL in the mutant cybrids bearing m.5783C > T mutation. mtDNA replication alterations were further evidenced by decreased levels of PolγA, Twinkle and SSBP1, newly synthesized mtDNA and mtDNA contents in the mutant cybrids. The m.5783C > T mutation altered tRNACys structure and function, including decreased melting temperature, conformational changes, instability and deficient aminoacylation of mutated tRNACys. The m.5783C > T mutation impaired the 5′ end processing efficiency of tRNACys precursors and reduced the levels of tRNACys and downstream tRNATyr. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced effects in the polypeptides harboring higher numbers of cysteine and tyrosine codons. These alterations led to deficient oxidative phosphorylation including instability and reduced activities of the respiratory chain enzyme complexes I, III, IV and intact supercomplexes overall. Our findings highlight the impact of mitochondrial dysfunction on deafness arising from defects in mitochondrial DNA replication and tRNA metabolism.

Cancer and Associated Therapies Impact the Skeletal Muscle Proteome.

Introduction: Both cancer and cancer associated therapies (CAT; including chemotherapy or concurrent chemoradiation) disrupt cellular metabolism throughout the body, including the regulation of skeletal muscle mass and function. Adjunct testosterone therapy during standard of care chemotherapy and chemoradiation modulates CAT-induced dysregulation of skeletal muscle metabolism and protects lean body mass during CAT. However, the extent to which the skeletal muscle proteome is altered under these therapeutic conditions is unknown. Objective: We probed the skeletal muscle proteome of cancer patients as an ancillary analysis following a randomized, double-blind, placebo-controlled phase II trial investigating the effect of adjunct testosterone on body composition in men and women with advanced cancers undergoing CAT. Methods: Men and women diagnosed with late stage (≥IIB) or recurrent head and neck or cervical cancer who were scheduled to receive standard of care CAT were administered an adjunct 7 weeks treatment of weekly intramuscular injections of either 100 mg testosterone (CAT+T, n = 7; 2M/5F) or placebo/saline (CAT+P, n = 6; 4M/2F). Biopsies were performed on the vastus lateralis before (PRE) and after (POST) the 7 weeks treatment. Extracted proteins were separated with 2-dimensional gel electrophoresis (2DE), and subjected to analyses of total protein abundance, phosphorylation and S-nitrosylation. Proteoforms showing significant 1.5 fold differences (t-test p ≤ 0.05) between PRE and POST timepoints were identified by mass spectroscopy (MS), and lists of altered proteins were subjected to Gene Set Enrichment Analysis (GSEA) to identify affected pathways. Results: A total of 756 distinct protein spots were identified. Of those spots, 102 were found to be altered in terms of abundance, phosphorylation, or S-nitrosylation, and identified by mass spectroscopy analysis to represent 58 unique proteins. Among the biological processes and pathways identified, CAT+P predominantly impacted metabolic processes, cell assembly, oxygen transport, and apoptotic signaling, while CAT+T impacted transcription regulation, muscle differentiation, muscle development, and contraction. Conclusion: Cancer and CAT significantly altered the skeletal muscle proteome in a manner suggestive of loss of structural integrity, reduced contractile function, and disrupted metabolism. Proteomic analysis suggests that the addition of adjunct testosterone minimized the structural and contractile influence of cancer and its associated therapies.

Proteomic, biochemical, and anatomical influences of nanographene oxide on soybean (Glycine max)

Nano-graphene oxide (NGO) is an engineered nanostructure that is used in various fields including biology, chemistry, medicine, and environmental protection. This kind of highly used nanomaterial (NM) is being released and accumulated gradually in nature and can have some adverse influences on living organisms including plants. Soybean as a cultivated plant with a high importance in food industry, but sensitive to stresses, was chosen in the present study to be examined in terms of proteomic, biochemical, and anatomical properties under the NGO stress. Accordingly, a 2-dimensional gel electrophoresis (2-DE) approach was adopted for proteomic analysis of the NGO treated soybean roots, where significant changes were observed in the abundance of 48 proteins. MALDI TOF/TOF analysis revealed the upregulation of the proteins involved in the redox regulation in plants. Furthermore, anatomical examination of soybean roots under light microscopy showed that the NGO could enter into the root epidermis through the apoplastic pathway and accumulated in some parts of the root. With increasing NGO concentration, the diameter of the vascular apertures increased and then decreased at higher concentrations. To evaluate the toxicity of NGO, some of the growth parameters including fresh and dry weight, and height of the shoots, as well as some stress-related biochemical properties such as H2O2 production, antioxidant enzymes activity, and phenolics and flavonoids contents were measured. The results indicated that NGO could cause an oxidative stress, which can be considered a toxic effect evoking antioxidative and detoxification mechanisms in soybean.

Horse meat tenderization in relation to post-mortem evolution of the myofibrillar sub-proteome

The ageing process after animal slaughter enhances tenderness and influences the value of meat. Horse meat is becoming more popular but lacks standardized ageing practices that should be supported by a better understanding of post-mortem muscle biochemistry. Steaks from Longissimus Thoracis et Lumborum (LTL) of eight Hispano-Bretón horses were aged for 0, 7, 14 and 21 days and myofibrillar proteins were resolved by one dimensional gel electrophoresis (1-DE). Ten protein bands were found to change (p ≤ 0.05) among ageing periods. Most changes were observed between days 0 and 14, suggesting that tenderization occurred primary during the first two weeks. Liquid isoelectric focusing (OFFGEL) technology was applied to better resolve myofibrillar sub-proteome and evidenced fourteen protein bands that changed (p ≤ 0.05) between 0 and 21 days. Three of them were protein fragments coming from troponins T and I and from creatine kinase. Identified molecules could be further studied as potential markers for horse meat tenderness.

Severe heat stress modulated nuclear factor erythroid 2-related factor 2 and macrophage migration inhibitory factor pathway in rat liver.

Heat stress impairs physiology and overall functionality of the body at tissue and organ level in animals. Liver being a vital organ performs more than hundreds regulatory functions of the body. Present study investigates the modulation of molecular pathways that are responsible for liver damage triggered by heat stress. Male Sprague dawley rats were exposed to heat stress (45°C) in heat simulation chamber till core temperature reaches 40°C and 42°C in 25 and 42min respectively. For in-depth evaluation of liver functions during severe heat stress, hepatic transcriptome and proteome were analysed by microarray and two dimensional gel electrophoresis respectively. Results revealed major alterations in redox status, inflammation, mitochondrial dysfunction and proteostasis related pathways. Data of molecular pathway analysis demonstrate that nuclear factor erythroid 2-related factor 2 (NRF-2) mediated oxidative stress response and macrophage migration inhibitory factor (MIF) regulated inflammatory pathways were upregulated in severe heat stressed liver. Expression levels of downstream molecules of above pathways such as heat shock protein 90AB 1, peroxiredoxin 5, Jun N-terminal kinases 1/2, heme-oxygenase 1, apolipoprotein 1 and interleukin 10 were examined and result suggested the upregulation of these genes modulates the NRF-2 and MIF regulated pathways in heat stressed liver. Irregularity in molecular signalling networks lead to mitochondrial dysfunction indicated by upregulation of ATP synthase β and peroxiredoxin 1 along with decreased levels of glucose-6-phosphate dehydrogenase and enhanced activity of cytochrome c in liver mitochondria. Thus, current study demonstrated heat induced alterations in key liver functions were regulated by NRF-2 and MIF pathways.

A Pilot Proteomic Study of Vestibular Fluid From Patients With Vulvodynia.

ObjectiveMany women are affected by vulvodynia, but medical therapies to date have proven ineffective. We performed a pilot study using gel-based proteomics to develop a map of proteins present in vaginal/vestibular secretions and identify proteins that could be considered for future evaluation as potential therapeutic targets.Materials and MethodsWe collected vestibular fluid from 4 controls and 4 patients with vulvodynia by placing a cotton swab in the vestibule and extracting the absorbed proteins. The proteins underwent 2-dimensional difference gel electrophoresis and mass spectrometry to develop a protein map. Immunohistochemistry was used to validate proteomic findings.ResultsA map was constructed of 32 of the more abundant proteins in vestibular fluid and their levels compared in control subjects and vulvodynia patients. Among these were annexin A1, interleukin 1 receptor antagonist, protein S100 A9, and a number of antiproteases and proteases. Many of these proteins differed by at least 50% between groups, but only annexin A1, one of the protease inhibitors, and immunoglobulin G κ chain were significantly different. The results with annexin A1 were validated by similar findings with immunohistochemistry.ConclusionsThe findings of this pilot study demonstrate a set of vestibule mucosa proteins that differ significantly—either increasing or decreasing—in vulvodynia patients compared with controls, and several others that exhibited greater than 1.5-fold change but did not reach statistical significance. This study constitutes a proof-of-principle that an open, unbiased proteomic approach can identify molecular participants in vulvodynia, some of which had not been identified to date by hypothesis-driven studies.

Green tea powder inclusion promoted hatchability through increased yolk antioxidant activity

Dietary supplementation of green tea powder (GTP) changes egg quality of hens, however, whether these changes affect incubation is still unknown. This study was to compare the proteomic difference of incubated eggs from hens with GTP supplemented or not. Huainan partridge chickens (1,080) at 35 wk of age were allocated into 2 groups, one group fed basal diet (CG) and one group fed basal diet plus 1% GTP (EG). After 4 wk feeding, artificially fertilized eggs were collected for yolk cholesterol determination and incubation. During incubation, 6 embryos from each group were randomly selected in each day for yolk protein extraction and quantification. Yolk cholesterol content was significantly lower, while the hatchability was significantly higher in EG than that of the CG group (P < 0.05). Yolk protein concentration at embryonic days (ED) of 0, 2, 6, and 13 showed significant changes and were selected for proteomic analysis by 2-dimensional gel electrophoresis combined with liquid chromatography-tandem mass spectrometry. Fifty-one differentially expressed (DE) protein spots were identified among different incubation stages between CG and EG group which were mainly classified into vitellogenin, immunoglobulin, and ovoinhibitor, and occupied 45.1, 23.5, and 15.7%, respectively, to the total DE proteins. Ovotransferrin, participated in extracellular sequestering of iron ion process, was significantly lower in EG group than that of the CG group (P < 0.05). Ig light chain precursor (Immunoglobulin) exhibited higher expression at ED6 in EG group as compared with that of the CG group, and was participated in immune response related processes. Ovoinhibitor, mainly involved in protease binding activity, showed lower abundance at ED13 in EG group as compared with that of the CG group. Vitellogenin-3, showed lower expression in EG group as compared with that of the CG group, was mainly participated in lipid transportation and localization according to GO enrichment. Chickens fed diet with GTP provided eggs more antioxidant ability that increased hatchability, indicated that GTP could be considered as additive in breeding layer.

Transcript and Protein Profiling Provides Insights Into the Molecular Mechanisms of Harvesting-Induced Latex Production in Rubber Tree.

Natural rubber, an important industrial raw material with wide applications, is harvested in the form of latex (cytoplasm of rubber-producing laticifers) from Hevea brasiliensis (para rubber tree) by the way of tapping. Conspicuous stimulation on latex production is observed for the first few tappings conducted on virgin (untapped before) or resting (tapped before but no tapping for a period) rubber trees. To understand the underlying mechanisms, an integrative analysis of the latex transcriptome and proteome was conducted on virgin or resting Hevea trees for the first five tappings. A total of 505 non-redundant differentially expressed (DE) transcript-derived fragments (TDFs) were identified by silver-staining cDNA-AFLP, with 217 exhibiting patterns of upregulated, 180 downregulated and 108 irregularly-regulated. Meanwhile, 117 two dimensional gel electrophoresis DE-protein spots were isolated and subjected to mass spectrometry analysis, with 89 and 57 being successfully identified by MALDI-TOF and MALDI-TOF/TOF, respectively. About 72.5% DE-TDFs and 76.1% DE-proteins were functionally annotated and categorized. Noteworthily, most of the DE-TDFs implicated in sugar transport and metabolism as well as rubber biosynthesis were upregulated by the tapping treatment. The importance of sugar metabolism in harvesting-induced latex production was reinforced by the identification of abundant relevant DE-protein spots. About 83.8% of the randomly selected DE-TDFs were validated for expression patterns by semi-quantitative RT-PCR, and an 89.7% consistency for the 29 latex regeneration-related DE-TDFs examined by quantitative RT-PCR analysis. In brief, our results reveal extensive physiological and molecular changes in Hevea laticifers incurred by the tapping treatment, and the vast number of DE genes and proteins identified here contribute to unraveling the gene regulatory network of tapping-stimulated latex production.

Proteomic identification of arginine-methylated proteins in colon cancer cells and comparison of messenger RNA expression between colorectal cancer and adjacent normal tissues.

PurposeIdentification of type I protein arginine methyltransferase (PRMT) substrates and their functional significance during tumorigenesis is becoming more important. The present study aimed to identify target substrates for type I PRMT using 2-dimensional (2D) gel electrophoresis (GE) and 2D Western blotting (WB).MethodsUsing immunoblot analysis, we compared the expression of type I PRMTs and endogenous levels of arginine methylation between the primary colorectal cancer (CRC) and adjacent noncancerous tissues paired from the same patient. To identify arginine-methylated proteins in HCT116 cells, we carried out 2D-GE and 2D-WB with a type I PRMT product-specific antibody (anti-dimethyl-arginine antibody, asymmetric [ASYM24]). Arginine-methylated protein spots were identified by mass spectrometry, and messenger RNA (mRNA) levels corresponding to the identified proteins were analyzed using National Center for Biotechnology Information (NCBI) microarray datasets between the primary CRC and noncancerous tissues.ResultsType I PRMTs and methylarginine-containing proteins were highly maintained in CRC tissues compared to noncancerous tissues. We matched 142 spots using spot analysis software between a Coomassie blue (CBB)-stained 2D gel and 2D-WB, and we successfully identified 7 proteins that reacted with the ASYM24 antibody: CACYBP, GLOD4, MAPRE1, CCT7, TKT, CK8, and HSPA8. Among these proteins, the levels of 4 mRNAs including MAPRE1, CCT7, TKT, and HSPA8 in CRC tissues showed a statistically significant increase compared to noncancerous tissues from patients using the NCBI microarray datasets.ConclusionOur results indicate that the method shown here is useful in identifying arginine-methylated proteins, and significance of arginine modification in the proteins identified here should be further identified during CRC development.