Differential Proteomic Expression Research Articles

Differential proteomic expression in non-functional pituitary neuroendocrine tumours and pituitary glands.

Pituitary neuroendocrine tumours (PitNETs) are common accounting for 10 to 25 % of all intracranial tumours. This project describes the feasibility of developing a novel membrane-based biomarker that could be used for fluorescent guided surgery. The aim was to catalogue the differential expression of membrane proteins between non-functional PitNETs and pituitary glands. Ten pituitary gland tissue specimens were obtained from the National Institute of Health (NIH) NeuroBio-Bank and twenty non-functional PitNETs were obtained from the Northwestern University Nervous System Tumour Bank. Mass spectrometry analysis using an Orbitrap Fusion Lumos Tribrid Mass Spectrometer linked to a Dionex Ultimate 3000 UPLC system was undertaken. Data Dependent Acquisition Mass Spectrometry and Data Independent Acquisition Mass Spectrometry was then completed. Pathway enrichment analysis was performed using clusterProfiler v4.6.0. Functional enrichment analysis was conducted using Gene Ontology terms and Reactome pathways. Differential expression analysis between the two groups revealed a total of 2110 significant differently expressed proteins (DEPs), with 1387 of these also having a Log2 fold change either greater than 1, or less than -1. Of the 2110 DEPs, 925 were upregulated in tumours compared to control, while 1185 were downregulated. We have demonstrated a proteomic comparison between non-functional PitNETs and normal pituitary glands. These results demonstrate differences consistent with contemporary literature but shows that NOTCH3 and PTPRJ are up-regulated in non-functional PitNETs compared to pituitary glands.

Longitudinal plasma proteomic analysis identifies biomarkers and combinational targets for anti-PD1-resistant cancer patients

The response rate of anti-PD1 therapy is limited, and the influence of anti-PD1 therapy on cancer patients is unclear. To address these challenges, we conducted a longitudinal analysis of plasma proteomic changes with anti-PD1 therapy in non-small cell lung cancer (NSCLC), alveolar soft part sarcoma (ASPS), and lymphoma patients. We included 339 plasma samples before and after anti-PD1 therapy from 193 patients with NSCLC, ASPS, or lymphoma. The plasma proteins were detected using data-independent acquisition-mass spectrometry and customable antibody microarrays. Differential proteomic characteristics in responders (R) and non-responders (NR) before and after anti-PD1 therapy were elucidated. A total of 1019 proteins were detected using our in-depth proteomics platform and distributed across 10–12 orders of abundance. By comparing the differential plasma proteome expression between R and NR groups, 50, 206, and 268 proteins were identified in NSCLC, ASPS, and lymphoma patients, respectively. Th17, IL-17, and JAK-STAT signal pathways were identified upregulated in NR group, while cellular senescence and transcriptional misregulation pathways were activated in R group. Longitudinal proteomics analysis revealed the IL-17 signaling pathway was downregulated after treatment. Consistently, many proteins were identified as potential combinatorial therapeutic targets (e.g., IL-17A and CD22). Five noninvasive biomarkers (FLT4, SFTPB, GNPTG, F5, and IL-17A) were further validated in an independent lymphoma cohort (n = 39), and another three noninvasive biomarkers (KIT, CCL3, and TNFSF1) were validated in NSCLC cohort (n = 76). Our results provide molecular insights into the anti-PD1 therapy in cancer patients and identify new therapeutic strategies for anti-PD1-resistant patients.

Serum proteomic profile of wild stump-tailed macaques (Macaca arctoides) infected with malaria parasites in Thailand.

The number of patients infected with simian malaria is gradually increasing in many countries of Southeast Asia and South America. The most important risk factor for a zoonotic spillover event of malarial infection is mostly influenced by the interaction between humans, monkeys, and vectors. In this study, we determine the protein expression profile of a wild stump-tailed macaque (Macaca arctoides) from a total of 32 blood samples collected from Prachuap Kiri Khan Province, Thailand. The malarial parasite was analyzed using nested polymerase chain reaction (PCR) assays by dividing the samples into three groups: non-infected, mono-infected, and multiple-infected. The identification and differential proteomic expression profiles were determined using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and bioinformatics tools. A total of 9,532 proteins (total proteins) were identified with the filter-based selection methods analysis, and a subset of 440 proteins were found to be different between each group. Within these proteins, the GhostKOALA functional enrichment analysis indicated that 142 important proteins were associated with either of the organismal system (28.87%), genetic information processing (23.24%), environmental information processing (16.20%), metabolism (13.38%), cellular processes (11.97%), or causing human disease (6.34%). Additionally, using interaction network analysis, nine potential reporter proteins were identified. Here, we report the first study on the protein profiles differentially expressed in the serum of wild stump-tailed macaques between non, mono, and multiple malarial infected living in a natural transmission environment. Our findings demonstrate that differentially expressed proteins implicated in host defense through lipid metabolism, involved with TGF pathway were suppressed, while those with the apoptosis pathway, such as cytokines and proinflammation signals were increased. Including the parasite's response via induced hemolysis and disruption of myeloid cells. A greater understanding of the fundamental processes involved in a malarial infection and host response can be crucial for developing diagnostic tools, medication development, and therapies to improve the health of those affected by the disease.

Glioblastoma survival is associated with distinct proteomic alteration signatures post chemoirradiation in a large-scale proteomic panel.

Glioblastomas (GBM) are rapidly progressive, nearly uniformly fatal brain tumors. Proteomic analysis represents an opportunity for noninvasive GBM classification and biological understanding of treatment response. We analyzed differential proteomic expression pre vs. post completion of concurrent chemoirradiation (CRT) in patient serum samples to explore proteomic alterations and classify GBM by integrating clinical and proteomic parameters. 82 patients with GBM were clinically annotated and serum samples obtained pre- and post-CRT. Serum samples were then screened using the aptamer-based SOMAScan® proteomic assay. Significant traits from uni- and multivariate Cox models for overall survival (OS) were designated independent prognostic factors and principal component analysis (PCA) was carried out. Differential expression of protein signals was calculated using paired t-tests, with KOBAS used to identify associated KEGG pathways. GSEA pre-ranked analysis was employed on the overall list of differentially expressed proteins (DEPs) against the MSigDB Hallmark, GO Biological Process, and Reactome databases with weighted gene correlation network analysis (WGCNA) and Enrichr used to validate pathway hits internally. 3 clinical clusters of patients with differential survival were identified. 389 significantly DEPs pre vs. post-treatment were identified, including 284 upregulated and 105 downregulated, representing several pathways relevant to cancer metabolism and progression. The lowest survival group (median OS 13.2 months) was associated with DEPs affiliated with proliferative pathways and exhibiting distinct oppositional response including with respect to radiation therapy related pathways, as compared to better-performing groups (intermediate, median OS 22.4 months; highest, median OS 28.7 months). Opposite signaling patterns across multiple analyses in several pathways (notably fatty acid metabolism, NOTCH, TNFα via NF-κB, Myc target V1 signaling, UV response, unfolded protein response, peroxisome, and interferon response) were distinct between clinical survival groups and supported by WGCNA. 23 proteins were statistically signficant for OS with 5 (NETO2, CST7, SEMA6D, CBLN4, NPS) supported by KM. Distinct proteomic alterations with hallmarks of cancer, including progression, resistance, stemness, and invasion, were identified in serum samples obtained from GBM patients pre vs. post CRT and corresponded with clinical survival. The proteome can potentially be employed for glioma classification and biological interrogation of cancer pathways.

Differential proteomic expression profiles in vulvar lichen planus as compared to normal vulvar tissue, vulvar lichen sclerosus, or oral lichen planus: An exploratory study.

Vulvar lichen planus (VLP) is a chronic inflammatory disease which adversely affects patients' quality of life. The pathogenesis of VLP is unknown although Th1 immune response has been implicated. We aimed to discover specific tissue-based protein biomarkers in VLP compared to normal vulvar tissue (NVT), vulvar lichen sclerosus (VLS) and oral lichen planus (OLP). We used laser capture microdissection-liquid chromatography- tandem mass spectrometry to assess protein expression in fixed lesional mucosal specimens from patients with VLP (n = 5). We then compared proteomic profiles against those of NVT (n = 4), VLS (n = 5), OLP (n = 6) and normal oral mucosa (n = 5), previously published by our group. IL16, PTPRC, PTPRCAP, TAP1 and ITGB2 and were significantly overexpressed in VLP compared to NVT. Ingenuity pathway analysis identified antigen presentation and integrin signalling pathways. Proteins overexpressed in both VLP versus NVT and OLP versus NOM included IL16, PTPRC, PTPRCAP, TAP1, HLA-DPB1, HLA-B and HLA-DRA. This proteomic analysis revealed several overexpressed proteins in VLP that relate to Th1 autoimmunity, including IL16. Overlapping pathways, including those involving IFNγ and Th1 signalling, were observed between VLP, VLS, and OLP.

Targeted Proteomic Analysis of Small GTPases in Radioresistant Breast Cancer Cells.

Radiation therapy benefits more than 50% of all cancer patients and cures 40% of them, where ionizing radiation (IR) deposits energy to cells and tissues, thereby eliciting DNA damage and resulting in cell death. Small GTPases are a superfamily of proteins that play critical roles in cell signaling. Several small GTPases, including RAC1, RHOB, and RALA, were previously shown to modulate radioresistance in cancer cells. However, there is no systematic proteomic study on small GTPases that regulate radioresistance in cancer cells. Herein, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled (SIL) peptides, to identify differentially expressed small GTPase proteins in two pairs of breast cancer cell lines, MDA-MB-231 and MCF7, and their corresponding radioresistant cell lines. We identified 7 commonly altered small GTPase proteins with over 1.5-fold changes in the two pairs of cell lines. We also discovered ARFRP1 as a novel regulator of radioresistance, where its downregulation promotes radioresistance in breast cancer cells. Together, this represents the first comprehensive investigation about the differential expression of the small GTPase proteome associated with the development of radioresistance in breast cancer cells. Our work also uncovered ARFRP1 as a new target for enhancing radiation sensitivity in breast cancer.

Using a Relative Quantitative Proteomic Method to Identify Differentially Abundant Proteins in Brucella melitensis Biovar 3 and Brucella melitensis M5-90.

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.

Comparative Proteomic Analysis of Drug Trichosanthin Addition to BeWo Cell Line.

Trichosanthin (TCS) is a traditional Chinese herbal medicine used to treat some gynecological diseases. Its effective component has diverse biological functions, including antineoplastic activity. The human trophoblast cell line BeWo was chosen as an experimental model for in vitro testing of a drug screen for anticancer properties of TCS. The MTT method was used in this study to get a primary screen result. The result showed that 100 mM had the best IC50 value. Proteomics analysis was then performed for further investigation of the drug effect of TCS on the BeWo cell line. In this differential proteomic expression analysis, the total proteins extracted from the BeWo cell line and their protein expression level after the drug treatment were compared by 2DE. Then, 24 unique three-fold differentially expressed proteins (DEPs) were successfully identified by MALDI-TOF/TOF MS. Label-free proteomics was run as a complemental method for the same experimental procedure. There are two proteins that were identified in both the 2DE and label-free methods. Among those identified proteins, bioinformatics analysis showed the importance of pathway and signal transduction and gives us the potential possibility for the disease treatment hypothesis.

Effects of long-term moderate-small intensity aerobic exercise on differential expression of proteome in left ventricular muscle of rats

Objective: To investigate the effects of long-term moderate-small intensity aerobic exercise on the differential expression of proteome in left ventricular muscle of rats, and to screen the target proteins sensitive to moderate-small intensity aerobic exercise stimulation. This study will enrich the basic theory of exercise and fitness and provide new ideas and experimental basis for the rehabilitation treatment of chronic cardiovascular disease. Methods: Twenty male SD rats were randomly divided into exercise group and control group (n=10). The treadmill training model of long-term moderate-small intensity aerobic exercise was established, and the whole protein samples of left ventricular muscle were extracted and separated by two-dimensional gel electrophoresis (2-DE). The two-dimensional gel electrophoresis map was analyzed by Bio PD quest image analysis software. The protein spots with differential expression more than 5 times or down-regulated over 80% after exercise were identified by tandem time-of-flight mass spectrometry (ULGRAFL-FLEX-TOF/TOF). Results: Compared with the group C, the heart weight index of the group E was increased by 32.0%, and the difference was significant (P＜0.05). Compared with the group C, there were 71 protein spots expression were up-regulated≥2 times or down-regulated≥50% in the group E. 4 protein spots expression were up-regulated≥5 times or down-regulated≥80% were identified by mass spectrometry, 3 proteins and 1 unknown protein were identified. Conclusion: After long-term moderate-small intensity aerobic exercise, the rats heart had a good adaptive change, and the proteome of left ventricular muscle changed significantly. Long-term moderate-small intensity aerobic exercise can effective enhance the ability of myocardial antioxidation.

Abstract LB220: Cellular Differentiation and Growth Control in the Human Colonic epithelium: Comparing Clinical Trial Outcomes to Responses in Human Colonoid Culture

Abstract Introduction: As part of our effort to understand the role of trace elements in colon polyp chemoprevention, we have conducted both colonoid culture (ex vivo) and human subject (in vivo) studies using Aquamin®— a calcium-, magnesium-, trace elements-rich, multi-mineral product derived from red marine algae as an intervention. Calcium alone was used as a comparator. Methods: Thirty subjects at-risk for colorectal cancer were enrolled and randomized to receive Aquamin® or calcium alone (each providing 800-mg calcium per day) or placebo. Colon biopsies were obtained before and after 90 days of intervention. We employed histologically normal colonic tissue from the same subjects (at baseline), to propagate in colonoid culture. Once established and expanded, we used the same interventions for a period of two weeks and compared the response (at 1.5mM) to the outcome of the studies conducted in human colonic biopsies after 90 days of intervention. Specifically, colonoid tissue and biopsies were subjected to analysis for markers of growth and differentiation by quantitative immunohistochemistry and tandem mass tag mass spectrometry-based proteomics. Results: The normal tissue colonoids demonstrated a high degree of differentiation as indicated by gross and microscopic appearance, and cytokeratin 20 (CK20) expression without either intervention. Only modest increases were seen in the CK20 expression with either calcium alone or Aquamin®. Similarly, CK20 expression was higher in baseline biopsies and there was no increase in the expression with calcium but a 5% increase seen with Aquamin® after 90 days of intervention. On proteomic screen, CK20 expression was increased by 40% in colonoid tissue in response to both interventions at 1.5mM, while CK20 expression increased by 10% and 21% with Calcium and Aquamin® in colon biopsies. When the colonoids were assessed for Ki67 (growth marker) expression, it was decreased by 17.2% and 18.4% with calcium alone and Aquamin® respectively as compared to the control. Analyzing colon biopsies after 90 days of intervention revealed that Aquamin® reduced the level of Ki67 expression by 20%, while no change was seen with calcium alone. Differential proteomic expression of proliferating cell nuclear antigen was decreased by 32% and 39% by calcium and Aquamin® at 1.5mM in colonoid tissue, whereas Ki67 protein was decreased by 4% and 22% with calcium and Aquamin®, respectively in colon biopsies. Conclusion: Colon tissue maintained in colonoid culture, thus, provides a good surrogate for human tissue collected in a clinical trial. It can be used to assess personalized responses or can provide a quick snapshot of the response in a relatively short time. Differential proteomic expression appears to be more sensitive than quantitative immunohistochemistry. A combination of calcium and additional trace elements proved to be more effective than calcium alone in altering differentiation and growth markers in either setting. Citation Format: Muhammad N. Aslam, Shannon McClintock, Mohamed Ali H Jawad-Makki, Karsten Knuver, Daniyal M. Nadeem, Haris Ahmad, Durga Attili, Danielle (Kim) Turgeon, James Varani. Cellular Differentiation and Growth Control in the Human Colonic epithelium: Comparing Clinical Trial Outcomes to Responses in Human Colonoid Culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB220.

Comparative proteomics and variations in extracellular matrix of Candida tropicalis biofilm in response to citral.

Candida tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. The formation of biofilm by C. tropicalis can cause dreadful and persistent infections which are difficult to treat due to acquired resistance. Presently, available anti-Candida drugs exhibit a high frequency of resistance, low specificity and toxicity at a higher dosage. In addition, the discovery of natural or synthetic anti-Candida drugs is slow paced and often does not pass clinical trials. Citral, a monoterpene aldehyde, has shown effective antimicrobial activities against various microorganisms. However, only few studies have elaborated the action of citral against the biofilm of C. tropicalis. In the present work, the aim was to study the fungicidal effect, differential expression of proteome and changes in extracellular matrix in response to the sub-lethal concentration (16µg/mL) of citral. The administration of citral on C. tropicalis biofilm leads to a fungicidal effect. Furthermore, the differential expression of proteome has revealed twenty-five proteins in C. tropicalis biofilm, which were differentially expressed in the presence of citral. Among these, amino acid biosynthesis (Met6p, Gln1p, Pha2p); nucleotide biosynthesis (Xpt1p); carbohydrate metabolism (Eno1p, Fba1p, Gpm1p); sterol biosynthesis (Mvd1p/Erg19p, Hem13p); energy metabolism (Dnm1p, Coa1p, Ndk1p, Atp2p, Atp4p, Hts1p); oxidative stress (Hda2p, Gre22p, Tsa1p, Pst2p, Sod2p) and biofilm-specific (Adh1p, Ape1p, Gsp1p) proteins were identified. The overexpression of oxidative stress-related proteins indicates the response of biofilm cell to combating oxidative stress during citral treatment. Moreover, the upregulation of Adh1p is of particular interest because it subsidizes the biofilm inhibition through ethanol production as a cellular response. The augmented expression of Mvd1p/Erg19p signifies the effect of citral on ergosterol biosynthesis. The presence of citral has also shown an increment in hexosamine and ergosterol component in extracellular matrix of C. tropicalis biofilm. Hence, it is indicated that the cellular response towards citral acts through multifactorial processes. This study will further help in the interpretation of the effect of citral on C. tropicalis biofilm and development of novel antifungal agents against these potential protein targets.

Host Response of Atlantic Salmon (Salmo salar) Re-Inoculated with Paramoeba perurans.

In aquaculture, recurrence rates of amoebic gill disease (AGD) caused by the ectoparasite Paramoeba perurans are high and no prophylactic strategies exist for disease prevention. In this study, Atlantic salmon (Salmo salar) were initially inoculated with P. perurans and following the development of amoebic gill disease were treated with freshwater immersion on day 21 and day 35 post inoculation. Fish were re-inoculated following a negative qPCR analysis for the presence of P. perurans. The gill host immune response was investigated at 7, 14, and 18 days post re-inoculation. Differential proteome expression of immune related proteins was assessed by comparison of each time point against naïve controls. In the gill, some proteins of the innate immune system were expressed in response to gill re-colonization by P. perurans, while no features of adaptive immunity were found to be differentially expressed. Many of the proteins identified are novel in the context of AGD and their expression profiles suggest that their roles in the response to disease development and progression in single or multiple infections warrant further investigation.

Platelet proteome changes in dogs with congestive heart failure

BackgroundPlatelets play a central role in the development of cardiovascular diseases and changes in their proteins are involved in the pathophysiology of heart diseases in humans. There is lack of knowledge about the possible role of platelets in congestive heart failure (CHF) in dogs. Thus, this study aimed to investigate the changes in global platelet proteomes in dogs with CHF, to clarify the possible role of platelets in the physiopathology of this disease. Healthy-dogs (n = 10) and dogs with acute CHF due to myxomatous mitral valve disease (MMVD, n = 10) were used. Acute CHF was defined based on the clinical (increased respiratory rate or difficulty breathing) and radiographic findings of pulmonary edema. Dogs Blood samples were collected into tubes with acid-citrate-dextrose, and platelet-pellets were obtained by centrifuge and washing steps. Platelet-proteomes were identified using LC-MS based label-free differential proteome expression analysis method and matched according to protein database for Canis lupus familiaris.ResultsTotally 104 different proteins were identified in the platelets of the dogs being 4 out of them were significantly up-regulated and 6 down-regulated in acute CHF dogs. Guanine-nucleotide-binding protein, apolipoproteins (A-II and C-III) and clusterin levels increased, but CXC-motif-chemokine-10, cytochrome-C-oxidase-subunit-2, cathepsin-D, serine/threonine-protein-phosphatase-PP1-gamma-catalytic-subunit, creatine-kinase-B-type and myotrophin levels decreased in acute CHF dogs. These proteins are associated with several molecular functions, biological processes, signaling systems and immune-inflammatory responses.ConclusionThis study describes by first time the changes in the protein composition in platelets of dogs with acute CHF due to MMVD. Our findings provide a resource for increase the knowledge about the proteome of canine platelets and their roles in CHF caused by MMVD and could be a tool for further investigations about the prevention and treatment of this disease.

The impact of differential expression of the pectoral muscle proteome in two groups of Japanese quail with different growth rates on‏ meat quality

ABSTRACT 1. In this study, the proteomics method was applied to genetically evaluate the performance and carcase characteristics of Japanese quail and their molecular mechanisms, and to further determine and analyse the meat quality and muscle proteins. 2. Ten Japanese quail were selected from among 400 quail from the seventh generation of a population. Various traits were recorded and multivariate analysis was applied using Wombat software. 3. Differential expression of pectoral muscle proteins was performed, whereby nine spots were selected (P < 0.05) for determination. All proteins from the quail group with the highest breeding value showed significantly greater relative intensity, except for serum albumin. 4. The results showed that an increase in growth rate can cause disturbances in most organs and their metabolism, although the increase in the expression of some proteins indicated that the bird’s body tends to adapt to special conditions. 5. The allocation of genetic resources is likely to maintain the balance between most organs, which does not overlap. However, most evidence shows that meat quality has been reduced, but to a limited extent, by selection for growth.

Differential Proteomic Expression of Equine Cardiac and Lamellar Tissue During Insulin-Induced Laminitis.

Endocrinopathic laminitis is pathologically similar to the multi-organ dysfunction and peripheral neuropathy found in human patients with metabolic syndrome. Similarly, endocrinopathic laminitis has been shown to partially result from vascular dysfunction. However, despite extensive research, the pathogenesis of this disease is not well elucidated and laminitis remains without an effective treatment. Here, we sought to identify novel proteins and pathways underlying the development of equine endocrinopathic laminitis. Healthy Standardbred horses (n = 4/group) were either given an electrolyte infusion, or a 48-h euglycemic-hyperinsulinemic clamp. Cardiac and lamellar tissues were analyzed by mass spectrometry (FDR = 0.05). All hyperinsulinemic horses developed laminitis despite being previously healthy. We identified 514 and 709 unique proteins in the cardiac and lamellar proteomes, respectively. In the lamellar tissue, we identified 14 proteins for which their abundance was significantly increased and 13 proteins which were significantly decreased in the hyperinsulinemic group as compared to controls. These results were confirmed via real-time reverse-transcriptase PCR. A STRING analysis of protein-protein interactions revealed that these increased proteins were primarily involved in coagulation and complement cascades, platelet activity, and ribosomal function, while decreased proteins were involved in focal adhesions, spliceosomes, and cell-cell matrices. Novel significant differentially expressed proteins associated with hyperinsulinemia-induced laminitis include talin−1, vinculin, cadherin-13, fibrinogen, alpha-2-macroglobulin, and heat shock protein 90. In contrast, no proteins were found to be significantly differentially expressed in the heart of hyperinsulinemic horses compared to controls. Together, these data indicate that while hyperinsulinemia induced, in part, microvascular damage, complement activation, and ribosomal dysfunction in the lamellae, a similar effect was not seen in the heart. In brief, this proteomic investigation of a unique equine model of hyperinsulinemia identified novel proteins and signaling pathways, which may lead to the discovery of molecular biomarkers and/or therapeutic targets for endocrinopathic laminitis.

A proteomic approach towards understanding crypoprotective action of Me2SO on the CHO cell proteome

Chinese hamster ovary (CHO) cell lines are the most widely used in vitro cells for research and production of recombinant proteins such as rhGH, tPA, and erythropoietin. We aimed to investigate changes in protein profiles after cryopreservation using 2D-DIGE MALDI-TOF MS and network pathway analysis. The proteome changes that occur in CHO cells between freshly prepared cells and cryopreserved cells with and without Me2SO were compared to determine the key proteins and pathways altered during recovery from cryopreservation. A total of 54 proteins were identified and successfully matched to 37 peptide mass fingerprints (PMF). 14 protein spots showed an increase while 23 showed decrease abundance in the Me2SO free group compared to the control. The proteins with increased abundance included vimentin, heat shock protein 60 kDa, mitochondrial, heat shock 70 kDa protein 9, protein disulfide-isomerase A3, voltage-dependent anion-selective channel protein 2. Those with a decrease in abundance were myotubularin, glutathione peroxidase, enolase, phospho glyceromutase, chloride intracellular channel protein 1. The main canonical functional pathway affected involved the unfolded protein response, aldosterone Signaling in Epithelial Cells, 14-3-3-mediated signaling. 2D-DIGE MALDI TOF mass spectrometry and network pathway analysis revealed the differential proteome expression of FreeStyle CHO cells after cryopreservation with and without 5% Me2SOto involve pathways related to post-translational modification, protein folding and cell death and survival (score = 56, 22 focus molecules). This study revealed, for the first time to our knowledge the proteins and their regulated pathways involved in the cryoprotective action of 5% Me2SO. The use of 5% Me2SO as a cryoprotectant maintained the CHO cell proteome in the cryopreserved cells, similar to that of fresh CHO cells.

Prognostic implication and functional annotations of APOBEC3G expression in patients with Melanoma.

Aim: Skin cutaneous melanoma (SKCM) is one of the most life-threatening malignancies damaging human health. APOBEC3G (A3G) has been found in several cancers; however, the role of A3G in SKCM is rarely studied. This study aimed to investigate the expression of A3G in tumor tissue and its prognostic value in SKCM patients.Method: A total of 512 SKCM patients from the First Affiliated Hospital of Soochow University (FAHSU) and the Cancer Genome Atlas (TCGA) database were consecutively recruited in analyses. Differential transcriptional and proteome expression profiles were obtained from multiple datasets. GEPIA was used to assess the survival analysis between distinguished groups. Both univariate and multivariate Cox regression analysis was performed to address the influence of independent factors on disease-free survival (RFS) and overall survival (OS). In addition, 31 SKCM and 31 nevus tissues were collected for immunohistochemical (IHC) staining and evaluation. STRING, DAVID and Gene Set Enrichment Analysis (GSEA) was utilized to conduct a network of related genes and significant pathways. Furthermore, we investigated the relationship of A3G with tumor-infiltrating immune cells (TIICs) by TIMER and TISIDB.Result: We found both transcriptional and proteomics expressions of A3G were elevated in SKCM. Survival analysis and ROC curves showed significant diagnostic and prognostic ability of A3G. IHC results showed increased expression of A3G in SKCM compared to nevus tissues. Importantly, A3G expression was closely associated with the immune-infiltrating levels of B cells, CD4+ T, CD8+ T, neutrophils, macrophages and dendritic cells.Conclusion: In summary, our study first reveals that elevated A3G expression is significantly correlated with better prognosis in SKCM patients. The role of A3G in SKCM demonstrated that it might be a prognostic and immunotherapeutic biomarker for SKCM.

Proteomic Protocols for Differential Protein Expression Analyses.

Protocols are described for preparation of various types of samples for differential protein expression proteomics using nano liquid chromatography-tandem mass spectrometry (nLCMS/MS). Fresh frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, cells, pull-downs, immunoprecipitations, and plasma or serum can be processed. For transplantation studies, it should be noted that samples must be comparable; thus, similar cells or organ systems from the same species can be compared when only a minority of the proteins change expression levels. For xenotransplantation studies, caution should be taken, as different species possess proteins with differences in primary structure. The sample preparation technique is based on spin filter devices using sodium deoxycholate (SDC) as detergent for extraction of proteins. The detergent is removed using phase extraction with ethyl acetate, and samples can be used for label-free quantification (LFQ) analysis or peptides can be labeled with tandem mass tags (TMT).

The Role of Serine Peptidase Inhibitor Kazal Type 13 (SPINK13) as a Clinicopathological and Prognostic Biomarker in Patients with Clear Cell Renal Cell Carcinoma.

BackgroundThe serine peptidase inhibitor Kazal type 13 (SPINK13) gene has tumor suppressor activity, but its role in renal cell carcinoma (RCC) remains unknown. This study aimed to investigate mRNA expression of SPINK13 in clear cell renal cell carcinoma (CCRCC) in human tissue and to use bioinformatics data to investigate the role of SPINK13 expression as a clinicopathological and prognostic biomarker for patients with CCRCC.Material/MethodsPatients with CCRCC (N=533) with available RNA sequence data from The Cancer Genome Atlas (TCGA)-CCRCC database were analyzed with patients who had a tissue diagnosis of CCRCC (N=305) at the Fudan University Shanghai Cancer Center (FUSCC). Differential transcriptional and proteome expression profiles were obtained from the ONCOMINE cancer microarray database, TCGA, and the Human Protein Atlas (HPA) database. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) measured SPINK13 mRNA expression in 305 samples of CCRCC tissue from the FUSCC. The effects of clinicopathological parameters on progression-free survival (PFS) and overall survival (OS) were analyzed using the Kaplan-Meier and log-rank test.ResultsTranscriptional and proteome expression of SPINK13 were significantly increased CCRCC tissue samples. Increased SPINK13 mRNA expression was significantly associated with reduced PFS and OS in 838 patients with CCRCC patients from the two independent cohorts, the FUSCC and the TCGA-CCRCC cohorts (p<0.01). Gene set enrichment analysis (GSEA) showed that SPINK13 expression was involved in complement, apical junction, epithelial-mesenchymal transition (EMT), glycolysis, hypoxia, and inflammation signaling pathways.ConclusionsIncreased expression of SPINK13 was associated with poor prognosis in patients with CCRCC.

Prognostic implications of Aquaporin 9 expression in clear cell renal cell carcinoma

BackgroundGrowing evidence has demonstrated immune reactivity as a confirmed important carcinogenesis and therapy efficacy for clear cell renal cell carcinoma (ccRCC). Aquaporin 9 (AQP9) is involved in many immune-related signals; however, its role in ccRCC remains to be elucidated. This study investigated AQP9 expression in tumor tissues and defined the prognostic value in ccRCC patients.MethodsA total of 913 ccRCC patients with available RNA-sequence data from the Cancer Genome Atlas (TCGA) database and Fudan University Shanghai Cancer Center (FUSCC) were consecutively recruited in analyses. Differential transcriptional and proteome expression profiles were obtained and validated using multiple datasets. A partial likelihood test from Cox regression analysis was developed to address the influence of independent factors on progression-free survival (PFS) and overall survival (OS). The Kaplan–Meier method and log-rank test were performed to assess survival. Receiver operating characteristic (ROC) curves were used to describe binary classifier value of AQP9 using area under the curve (AUC) score. Functional enrichment analyses and immune infiltration analysis were used to describe significantly involved hallmark pathways of hub genes.ResultsSignificantly elevated transcriptional and proteomic AQP9 expressions were found in ccRCC samples. Increased AQP9 mRNA expression was significantly associated with advanced clinicopathological parameters and correlated with shorter PFS and OS in TCGA and FUSCC cohorts (p < 0.001). ROC curves suggested the significant diagnostic and prognostic ability of AQP9 (PFS, AUC = 0.823; OS, AUC = 0.828). Functional annotations indicated that AQP9 is involved in the most significant hallmarks including complement, coagulation, IL6/JAK–STAT3, inflammatory response and TNF-alpha signaling pathways.ConclusionOur study revealed that elevated AQP9 expression was significantly correlated with aggressive progression, poor survival and immune infiltrations in ccRCC patients, and we validated its prognostic value in a real-world cohort. These data suggest that AQP9 may act as an oncogene and a promising prognostic marker in ccRCC.