Hemoglobin Subunit Alpha Research Articles

Longitudinal transcriptomic analysis reveals persistent enrichment of iron homeostasis and erythrocyte function pathways in severe COVID-19 ARDS.

The acute respiratory distress syndrome (ARDS) is a common complication of severe COVID-19 and contributes to patient morbidity and mortality. ARDS is a heterogeneous syndrome caused by various insults, and results in acute hypoxemic respiratory failure. Patients with ARDS from COVID-19 may represent a subgroup of ARDS patients with distinct molecular profiles that drive disease outcomes. Here, we hypothesized that longitudinal transcriptomic analysis may identify distinct dynamic pathobiological pathways during COVID-19 ARDS. We identified a patient cohort from an existing ICU biorepository and established three groups for comparison: 1) patients with COVID-19 ARDS that survived hospitalization (COVID survivors, n = 4), 2) patients with COVID-19 ARDS that did not survive hospitalization (COVID non-survivors, n = 5), and 3) patients with ARDS from other causes as a control group (ARDS controls, n = 4). RNA was isolated from peripheral blood mononuclear cells (PBMCs) at 4 time points (Days 1, 3, 7, and 10 following ICU admission) and analyzed by bulk RNA sequencing. We first compared transcriptomes between groups at individual timepoints and observed significant heterogeneity in differentially expressed genes (DEGs). Next, we utilized the likelihood ratio test to identify genes that exhibit different patterns of change over time between the 3 groups and identified 341 DEGs across time, including hemoglobin subunit alpha 2 (HBA1, HBA2), hemoglobin subunit beta (HBB), von Willebrand factor C and EGF domains (VWCE), and carbonic anhydrase 1 (CA1), which all demonstrated persistent upregulation in the COVID non-survivors compared to COVID survivors. Of the 341 DEGs, 314 demonstrated a similar pattern of persistent increased gene expression in COVID non-survivors compared to survivors, associated with canonical pathways of iron homeostasis signaling, erythrocyte interaction with oxygen and carbon dioxide, erythropoietin signaling, heme biosynthesis, metabolism of porphyrins, and iron uptake and transport. These findings describe significant differences in gene regulation during patient ICU course between survivors and non-survivors of COVID-19 ARDS. We identified multiple pathways that suggest heme and red blood cell metabolism contribute to disease outcomes. This approach is generalizable to larger cohorts and supports an approach of longitudinal sampling in ARDS molecular profiling studies, which may identify novel targetable pathways of injury and resolution.

Use of tryptic peptide MALDI mass spectrometry imaging to identify the spatial proteomic landscape of colorectal cancer liver metastases.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. CRC liver metastases (CRLM) are often resistant to conventional treatments, with high rates of recurrence. Therefore, it is crucial to identify biomarkers for CRLM patients that predict cancer progression. This study utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially map the CRLM tumour proteome. CRLM tissue microarrays (TMAs) of 84 patients were analysed using tryptic peptide MALDI-MSI to spatially monitor peptide abundances across CRLM tissues. Abundance of peptides was compared between tumour vs stroma, male vs female and across three groups of patients based on overall survival (0-3 years, 4-6 years, and 7+ years). Peptides were then characterised and matched using LC-MS/MS. A total of 471 potential peptides were identified by MALDI-MSI. Our results show that two unidentified m/z values (1589.876 and 1092.727) had significantly higher intensities in tumours compared to stroma. Ten m/z values were identified to have correlation with biological sex. Survival analysis identified three peptides (Histone H4, Haemoglobin subunit alpha, and Inosine-5’-monophosphate dehydrogenase 2) and two unidentified m/z values (1305.840 and 1661.060) that were significantly higher in patients with shorter survival (0-3 years relative to 4-6 years and 7+ years). This is the first study using MALDI-MSI, combined with LC-MS/MS, on a large cohort of CRLM patients to identify the spatial proteome in this malignancy. Further, we identify several protein candidates that may be suitable for drug targeting or for future prognostic biomarker development.

Identification of key overlapping DEGs and molecular pathways under multiple stressors in the liver of Nile tilapia (Oreochromis niloticus)

The identification of key genes and molecular pathways that are involved in the response to stressors is crucial for controlling stress in fish and sustainable aquaculture. Environmental stressors can induce stress responses in aquatic animals, resulting in compromised immune function, inhibited growth, and increased mortality rates. mRNA-seq analysis provides a powerful tool to identify key genes and pathways associated with stress response. In the present study, mRNA-seq analysis was employed to identify key overlapping differentially expressed genes (DEGs) and molecular pathways under salinity, nitrite, copper, and pH stress in the liver of Nile tilapia (Oreochromis niloticus). The pathways associated with the immune response, oxygen transport, homeostasis, and oxidative stress were enriched across all stressors. The top KEGG pathways were complement and coagulation cascades, PPAR signaling pathway, and cardiac muscle contraction. The top GO enrichment terms were oxidoreductase activity, aerobic respiration, endopeptidase inhibitor activity, endopeptidase regulator activity, heme binding, and iron ion binding. The complement genes (C3, C4, C5, factor B, and factor H), alpha-2-macroglobulin (A2M), hemoglobin subunit epsilon (HBE), hemoglobin subunit alpha (HBA), coagulation factor genes (XI and X) and the cytochrome c oxidase (COX) gene family (cox1, cox2, cox3, cytochrome P450) were identified as key shared genes across multiple stressors. The discovery of these genes and molecular pathways provided a better understanding of the molecular mechanism underlying the stress response in Nile tilapia. The results of the present study can facilitate the development of stress management strategies in Nile tilapia.

Proteomic research on new urinary biomarkers of renal disease in canine leishmaniosis: Survival and monitoring response to treatment

The objective of our study was to search for survival biomarkers (SB) and treatment response monitoring biomarkers (TRMB) in the urinary proteome of dogs with renal disease secondary to canine leishmaniosis (CanL), using UHPLC–MS/MS. The proteomic data are available via ProteomeXchange with identifier PXD042578. Initially, a group of 12 dogs was evaluated and divided into survivors (SG; n = 6) and nonsurvivors (NSG; n = 6). A total of 972 proteins were obtained from the evaluated samples. Then, bioinformatic analysis reduced them to 6 proteins like potential SB increased in the NSG, specifically, Haemoglobin subunit Alpha 1, Complement Factor I, Complement C5, Fibrinogen beta chain (fragment), Peptidase S1 domain-containing protein, and Fibrinogen gamma chain. Afterwards, SG was used to search for TRMB, studying their urine at 0, 30, and 90 days, and 9 proteins that decreased after treatment were obtained: Apolipoprotein E, Cathepsin B, Cystatin B, Cystatin-C-like, Lysozyme, Monocyte differentiation CD14, Pancreatitis-associated precursor protein, Profilin, and Protein FAM3C. Finally, enrichment analysis provided information about the biological mechanisms in which these proteins are involved. In conclusion, this study provides 15 new candidate urinary biomarkers and an improved understanding of the pathogenesis of kidney disease in CanL.

RNA sequencing suggests that non-coding RNAs play a role in the development of acquired haemophilia.

Acquired haemophilia (AH) is a rare disorder characterized by bleeding in patients with no personal or family history of coagulation/clotting-related diseases. This disease occurs when the immune system, by mistake, generates autoantibodies that target FVIII, causing bleeding. Small RNAs from plasma collected from AH patients (n = 2), mild classical haemophilia (n = 3), severe classical haemophilia (n = 3) and healthy donors (n = 2), for sequencing by Illumina, NextSeq500. Based on bioinformatic analysis, AH patients were compared to all experimental groups and a significant number of altered transcripts were identified with one transcript being modified compared to all groups at fold change level. The Venn diagram shows that haemoglobin subunit alpha 1 was highlighted to be the common upregulated transcript in AH compared to classical haemophilia and healthy patients. Non-coding RNAs might play a role in AH pathogenesis; however, due to the rarity of HA, the current study needs to be translated on a larger number of AH samples and classical haemophilia samples to generate more solid data that can confirm our findings.

URINARY PROTEOMIC SIGNATURE OF THE RETINAL MICROVASCULATURE

Objective: The retinal vasculature provides a window into the body's microcirculation. Variations in retinal vessel diameters have been associated with adverse cardiovascular outcomes, such as stroke and coronary heart disease. As the pathogenesis of retinal microvascular diameter changes remain poorly understood, this study aimed to provide novel insights by characterizing the urinary proteomic signature of the retinal microvasculature. Design and method: The central retinal arteriolar equivalent (CRAE), central retinal venular equivalent (CRVE), and arteriole-to venule-ratio (AVR) were measured from digitized retinal photographs of 562 randomly recruited Flemish participants (mean age, 48.9 years; 50.9% women). The urinary proteome was analyzed using capillary electrophoresis coupled with mass spectrometry. Multivariable linear regression analysis identified significant urinary peptides with adjustment for age, sex, body mass index, smoking, hypertension, plasma glucose, total and HDL cholesterol, eGFR, and history of cardiovascular disease. Reactome pathway enrichment analysis was performed to interpret the proteomic signature. Results: Table 1 shows participant characteristics. Of the 359 peptides analyzed, 19 were significantly associated with CRAE after adjusting for potential confounders and multiple testing (Table 2). In the proteomic profile, 14 peptides (74%) were fragments of collagen I, III, and XI proteins, while the remaining peptides were fragments of the fibrinogen alpha chain (11%), xylosyltransferase 1 (5%), S100 calcium-binding protein A9 (5%), and hemoglobin subunit alpha (5%) proteins. Pathway analysis showed that these proteins were predominantly involved in endothelial cell proliferation and migration, collagen formation and degradation, and cell-extracellular matrix interactions (Figure 1). No peptides were significantly associated with CRVE and AVR following adjustment. Conclusions: This study revealed a novel urinary proteomic signature as a potential biomarker of retinal arteriolar narrowing. The signature suggests fibrosis and neovascularization may be involved in retinal microvascular diameter changes. Further investigation may help elucidate the pathophysiologic mechanisms underlying the relationship between microvascular and macrovascular disease.

Spatial analysis of the ancient proteome of archeological teeth using mass spectrometry imaging.

Proteins extracted from archaeological bone and teeth are utilised for investigating the phylogeny of extinct and extant species, the biological sex and age of past individuals, as well as ancient health and physiology. However, variable preservation of proteins in archaeological materials represents a major challenge. To better understand the spatial distribution of ancient proteins preserved within teeth, we applied matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) for the first time to bioarchaeological samples to visualise the intensity of proteins in archaeological teeth thin sections. We specifically explored the spatial distribution of four proteins (collagen type I, of whichthe chains alpha-1 and alpha-2, alpha-2-HS-glycoprotein, haemoglobin subunit alpha and myosin light polypeptide 6). We successfully identified ancient proteins in archaeological teeth thin sections using mass spectrometry imaging. The data are available via ProteomeXchange with identifier PXD038114. However, we observed that peptides did not always follow our hypotheses for their spatial distribution, with distinct differences observed in the spatial distribution of several proteins, and occasionally between peptides of the same protein. While it remains unclear what causes these differences in protein intensity distribution within teeth, as revealed by MALDI-MSI in this study, we have demonstrated that MALDI-MSI can be successfully applied to mineralised bioarchaeological tissues to detect ancient peptides. In future applications, this technique could be particularly fruitful not just for understanding the preservation of proteins in a range of archaeological materials, but making informed decisions on sampling strategies and the targeting of key proteins of archaeological and biological interest.

Saliva proteomic profile of early childhood caries and caries-free children

Objective Saliva plays an important antimicrobial role and it is related to the pathogenesis of early childhood caries (ECC). The aim of this study was to compare the proteomic profile of unstimulated saliva of children aged 3–5 years who had ECC and caries-free (CF) children. Materials and methods After the saliva collection from 20 children (ECC: n = 10; CF: n = 10), the samples were processed for proteomic analysis on a mass spectrometer. Results 1638 proteins were identified, of which 355 were present in both groups. A total of 579 proteins were exclusively identified in the CF group and included Leucine-rich alpha-2-glycoprotein, Protein S100-A5, Protein S100-A8 and Mucin-2. Moreover, 704 proteins were exclusively identified in the ECC group, including Enamelin. The differential expression analysis revealed that 112 proteins were up-regulated in the CF group. Among these proteins, we highlighted Hemoglobin subunit gamma-1 (343-fold increase), gamma-2 (336-fold increase) and alpha (40-fold increase). Conclusions The proteomic profile of the saliva varied substantially between the groups. Hemoglobin subunit gamma-1, gamma-2 and alpha may play a protective role in children with ECC. These proteins should be evaluated in future studies, because they may be possible good candidates to be included in anti-caries dental products.

Experimental Bothropsatrox Envenomation: Blood Plasma Proteome Effects after Local Tissue Damage and Perspectives on Thromboinflammation.

The clinical manifestations of Bothrops atrox envenoming involve local and systemic changes, among which edema requires substantial attention due to its ability to progress to compartmental syndromes and sometimes cause tissue loss and amputations. However, the impact of edema on the poisoned body’s system has not been explored. Thus, the present study aimed to explore the systemic pathological and inflammatory events that are altered by intraplantar injection of B. atrox venom in a mouse model through hematologic, lipidic, and shotgun proteomics analysis. Plasma samples collected showed a greater abundance of proteins related to complement, coagulation, lipid system, platelet and neutrophil degranulation, and pathways related to cell death and ischemic tolerance. Interestingly, some proteins, in particular, Prdx2 (peroxiredoxin 2), Hba (hemoglobin subunit alpha), and F9 (Factor IX), increased according to the amount of venom injected. Our findings support that B. atrox venom activates multiple blood systems that are involved in thromboinflammation, an observation that may have implications for the pathophysiological progression of envenomations. Furthermore, we report for the first time a potential role of Prdx2, Hba, and F9 as potential markers of the severity of edema/inflammation in mice caused by B. atrox.

PD36-09 DIFFERENTIAL EXPRESSION OF SEMINAL PLASMA PROTEINS IN RESPONDERS AND NON-RESPONDERS TO VARICOCELE TREATMENT

You have accessJournal of UrologyCME1 May 2022PD36-09 DIFFERENTIAL EXPRESSION OF SEMINAL PLASMA PROTEINS IN RESPONDERS AND NON-RESPONDERS TO VARICOCELE TREATMENT Timur Bitsoev, Vitaliy Chagovets, Taras Shatylko, Alisa Tokareva, Safar Gamidov, Natalia Starodubtseva, Alina Popova, and Vladimir Frankevich Timur BitsoevTimur Bitsoev More articles by this author , Vitaliy ChagovetsVitaliy Chagovets More articles by this author , Taras ShatylkoTaras Shatylko More articles by this author , Alisa TokarevaAlisa Tokareva More articles by this author , Safar GamidovSafar Gamidov More articles by this author , Natalia StarodubtsevaNatalia Starodubtseva More articles by this author , Alina PopovaAlina Popova More articles by this author , and Vladimir FrankevichVladimir Frankevich More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002594.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite large amounts of evidence linking varicocele to male infertility, the role of varicocele repair in infertile men is controversial. Even after technically perfect operation not all couples achieve pregnancy or at least have a significant improvement in semen parameters. Probably, there are cases of infertility which share a causal relationship with varicocele, and there are cases when idiopathic infertility co-exists with harmless varicocele. Unfortunately, there is no way to predict response to varicocele treatment among infertile males. Since sperm are transcriptionally inert, the proteome of seminal plasma is relatively stable and may reflect the true extent of deleterious effects of varicocele, such as oxidative stress and hyperthermia. Our goal was to assess whether proteomic signature of seminal plasma may help to distinguish responders and non-responders to varicocele repair. METHODS: We used mass-spectrometry to perform a proteomic analysis of seminal plasma specimens obtained from 10 infertile patients (age: 25–44 years) with bilateral varicocele. All patients underwent routine semen analysis and had at least two altered core parameters out of three (total sperm count, progressive motility, percentage of sperm with normal morphology). Microsurgical bilateral subinguinal varicocelectomy was performed in all patients. Routine semen analysis and proteomic analysis were repeated at follow-up, which was scheduled at 6 months after the surgery. Responders were defined as patients whose spouse became pregnant during follow-up period or whose total motile sperm count increased by at least 50%. Proteomic profiles of responders and non-responders were compared. RESULTS: Five patients were responders to varicocele repair (partners of two of them became pregnant). Proteomic analysis identified 18 proteins with differential expression in responders and non-responders: melanophilin, kallikrein-2, alpha-actinin-4, Kunitz-type protease inhibitor 3, alpha-1-acid glycoprotein 1, CD177 antigen, alpha-1-acid glycoprotein 2, protein-glutamine gamma-glutamyltransferase 4, phosphatidylethanol amine-binding protein 4, acyl-CoA-binding protein, hemoglobin subunit alpha, antileukoproteinase, collagen alpha-1(XVIII) chain, vitronectin, serine protease HTRA1, V-type proton ATPase catalytic subunit A, endoplasmic reticulum resident protein 44, NADP-dependent malic enzyme. CONCLUSIONS: If validated, semen proteomics may become a useful tool to predict response to varicocele repair and avoid unnecessary surgery. Source of Funding: N/A © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e636 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Timur Bitsoev More articles by this author Vitaliy Chagovets More articles by this author Taras Shatylko More articles by this author Alisa Tokareva More articles by this author Safar Gamidov More articles by this author Natalia Starodubtseva More articles by this author Alina Popova More articles by this author Vladimir Frankevich More articles by this author Expand All Advertisement PDF DownloadLoading ...

The possible role of hypoxia in the affected tissue of relapsed clubfoot

Our aim was to study the expression of hypoxia-related proteins as a possible regulatory pathway in the contracted side tissue of relapsed clubfoot. We compared the expression of hypoxia-related proteins in the tissue of the contracted (medial) side of relapsed clubfoot, and in the tissue of the non-contracted (lateral) side of relapsed clubfoot. Tissue samples from ten patients were analyzed by immunohistochemistry and image analysis, Real-time PCR and Mass Spectrometry to evaluate the differences in protein composition and gene expression. We found a significant increase in the levels of smooth muscle actin, transforming growth factor-beta, hypoxia-inducible factor 1 alpha, lysyl oxidase, lysyl oxidase-like 2, tenascin C, matrix metalloproteinase-2, matrix metalloproteinase-9, fibronectin, collagen types III and VI, hemoglobin subunit alpha and hemoglobin subunit beta, and an overexpression of ACTA2, FN1, TGFB1, HIF1A and MMP2 genes in the contracted medial side tissue of clubfoot. In the affected tissue, we have identified an increase in the level of hypoxia-related proteins, together with an overexpression of corresponding genes. Our results suggest that the hypoxia-associated pathway is potentially a factor contributing to the etiology of clubfoot relapses, as it stimulates both angioproliferation and fibroproliferation, which are considered to be key factors in the progression and development of relapses.

A comprehensive and comparative proteomic analysis of horse serum proteins in colitis

Equine colitis is a diarrhoeal disease caused by inflammation of the large bowel and can potentially be life-threatening due to its rapid progression. Pathogenesis is multifactorial and pathophysiology is highly complicated, therefore, reliable diagnostic biomarkers are needed in the veterinary field. Serum is one of the most commonly used diagnostic tools in equine clinical investigation. To discover diagnostic or prognostic protein markers for colitis in horse serum, comprehensive and comparative proteomic analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Case-control study. Serum samples were collected from 36 healthy Thoroughbreds and 12 Thoroughbreds with colitis. Serum from each horse suffering from colitis was collected daily until death or recovery. Collected sera were digested with trypsin. Peptides obtained from serum proteins were measured by Q-Exactive HF Orbitrap mass spectrometer. The identification and quantification of peptides were performed using Proteome Discoverer version 2.2. On day 1 of treatment, eight proteins in the colitis group were upregulated (P<.05, more than a twofold change) compared with the healthy group. Among the eight proteins, biliverdin reductase B was significantly upregulated (P<.05) in the non-survivor group (n=5) compared with the survivor group (n=7). On the last day of the treatment, haemoglobin subunit alpha, clusterin, glyceraldehyde-3-phosphate dehydrogenase, lipopolysaccharide-binding protein, and biliverdin reductase B showed significant increases (P<.05) in the non-survivor group. The number of the identified proteins is limited due to the existence of abundant proteins. Measuring the changes of these proteins together may enable a potential prognosis or early diagnosis of horses suffering from colitis.

Comparison of Traditional and Industrial Sausages Baranjski Kulen and Kulenova Seka Using Comprehensive Proteome, Peptidome and Metabolome Techniques

SUMMARYResearch backgroundBaranjski kulen is one of the most popular fermented meat sausages originating from Croatia. It has protected geographical indication, and is traditionally produced in the Baranja region of Croatia. Kulenova seka is a fermented sausage very similar to baranjski kulen, but it has a different calibre and consequently, a shorter time of production. In recent decades, due to the high demand and popularity of these products, industrially produced baranjski kulen and kulenova seka have become available on the market. This work aims to identify specific characteristics of traditional and industrial sausages baranjski kulen and kulenova seka on proteome, peptidome and metabolome level, which could potentially lead to better optimization of the industrial production process in order to obtain an equivalent to the traditional product.Experimental approachProtein profiles of baranjski kulen and kulenova seka (traditional and industrial) were analysed using two-dimensional gel electrophoresis followed by differential display analysis and protein identification by mass spectrometry. Peptidomic profile was analysed via liquid chromatography-tandem mass spectrometry. Furthermore, aroma profiles were investigated via headspace solid phase microextraction and gas chromatography-mass spectrometry.Results and conclusionsThe major identified characteristics of each product were: industrial baranjski kulen: specific degradation of myosin-1 and titin, overabundance of stress-related proteins and increased phenylalanine degradation; traditional baranjski kulen: decreased concentration of phenylalanine and overabundance of fructose-bisphosphate aldolase A and carbonic anhydrase 3; industrial kulenova seka - specific myosin-4 and haemoglobin subunit alpha degradation process; traditional kulenova seka - overabundance of dihydropyrimidine dehydrogenase [NADP(+)] and myosin light chain 1/3, skeletal muscle isoform, degradation of albumin and myoglobin, decreased concentrations of almost all free amino acids and increased amounts of smoke-derived volatile compounds. Presented results showed that potential product type-specific quality markers for each sausage could be found.Novelty and scientific contributionIn this preliminary communication, the first insights into protein degradation processes and generation of peptides, free amino acids and aroma compounds of industrial and traditional baranjski kulen and kulenova seka are presented. Although further research is needed to draw general conclusions, the specific profile of proteins, peptides, amino acids and volatile compounds represents the first step in the industrial production of sausages that meet the characteristics of traditional flavour.

Molecular Docking of 6-shogaol and Curcumin on DNMT1 and LSD1 As Potential Agents for Thalassemia Treatment

&lt;p&gt;Beta-thalassemia therapy is developed by increasing γ-globin production which binds to α-globin to form haemoglobin fetal (HbF). Meanwhile, DNA methyltransferase 1 (DNMT1) and lysine specific demethylase 1 (LSD1) play an important role in silencing the &lt;em&gt;HbF&lt;/em&gt; gene by inhibiting the production of HbF and inducing haemoglobin subunit alpha (HbA) expression. 6-Shogaol and curcumin induce HbF by inhibiting signal transducer and activator of transcription 3 (STAT3) expression. Therefore, this study predicts the interaction between 6-shogaol and curcumin on DNMT1 and LSD1. The protein structure of DNMT1 (3SWR) and LSD1 (6KGP) was prepared by removing the water molecules, while the validation step was performed by separating protein from native ligands (sinefungin for 3SWR and flavine-adenine dinucleotide (FAD) for 6KGP) in new protein data bank files. Furthermore, the protein was docked with a native ligand to obtain grid box coordinates, while the root means standard deviation (RMSD) was calculated from the conformation results of the validation process. 6-Shogaol and curcumin were docked with coordinates of the validation results, and the best conformation was visualized with Discovery Studio. The validation step results in the RMSD value of 0.861Å and 1.410Å for DNMT1 and LSD1, respectively. The binding affinity of 6-shogaol and curcumin on DNMT1 was -6.5 kcal/mol and -8.0 kcal/mol, respectively. Furthermore, the binding affinity of 6-shogaol and curcumin on LSD1 was -8.2 kcal/mol and -10.1 kcal/mol, respectively. Amino acid residues found in DNMT1 interaction include Gly1147, Phe1145, Glu1168, Asn1278, Pro1225, Leu1151, Val1580, Ala1579, Asn1578, Trp1170, and Ala1579; meanwhile, Val288, Ser289, Arg310, Gly285, Thr624, Leu659, Lys661, Arg316, Leu625, Tyr761, Trp751, Gly330, and Leu659 were found in LSD1. This study showed that curcumin has the potential to inhibit DNMT1 as well as LSD1 proven by lower bonding energy and stronger bond types compared to sinefungin and FAD native ligands and other DNMT1 and LSD1 inhibitors.&lt;/p&gt;

Proteomic analysis of lacrimal gland adenoid cystic carcinoma with high-grade transformation

Objective: To analyze the protein expression differences of lacrimal gland adenoid cystic carcinoma (LACC) with high-grade transformation (HGT). Methods: Experimental study. A total of 8 paraffin tissue samples were collected in Tianjin Medical University Eye Hospital from December 2012 to January 2019. According to pathological examination, the samples were divided into the LACC group and the LACC-HGT group, with 4 cases in each group. The LACC group included 2 male samples and 2 female samples, with an average age of 53 years. The LACC-HGT group included 2 male samples and 2 female samples, with an average age of 44 years. Primary cells were cultured from fresh tumor tissue. Isobaric tags for relative and absolute quantification techniques were used to screen the differentially expressed proteins between the two groups, and bioinformatics analysis was conducted for the differentially expressed proteins. Microarray was used to screen differentially expressed mRNAs between LACC and LACC-HGT primary cells. The mass spectrum data were intersected with mRNA microarray data, and quantitative real-time (qRT) PCR was performed to verify the results. Proteomics and microarray data were compared using the independent sample t test. The qRT-PCR data were compared pairwise by one-way analysis of variance. Results: A total of 105 HGT-related differential proteins were detected in this study, including 50 up-regulated proteins and 55 down-regulated proteins. The significantly up-regulated proteins included hemoglobin subunit beta, hemoglobin subunit alpha 1, and collagen type Ⅵ alpha 2 chain; the significantly down-regulated proteins included Cereblon, adenosylhomocysteinase like 2, and ribosomal protein L39 pseudogene 5. Gene ontology analysis results showed that the LACC-HGT differential proteins were mainly located in the cytoplasm, vesicle cavity, and extracellular matrix, had organic acid binding and molecular carrier activity, and participated in the regulation of extracellular matrix composition, immunity, inflammation, apoptosis, and other biological processes. Pathway analysis showed that the LACC-HGT differential proteins were mainly involved in signal pathways such as mitogen-activated protein kinase signal pathway and extracellular matrix proteoglycans and glycan metabolism signal pathway. Protein complex prediction analysis screened out 4 up-regulated protein complexes and 1 down-regulated protein complex. There were 15 LACC-HGT differential proteins that overlapped with mRNA chip differential genes, of which 6 were tumor-related proteins including collagen type XIV alpha 1 chain (COL14A1), EMAP like 4 (EML4), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), NDRG family member 2 (NDRG2), osteoglycin (OGN) an Ras homolog family member C (RhoC). The main function was the movement and migration of tumor cells. The qRT-PCR results showed that the relative expression levels of COL14A1, EML4, ITIH4, NDRG2, OGN, and RhoC in primary LACC-1, LACC-2, LACC-HGT-1, and LACC-HGT-2 cells were significantly different (F=1 675.98, 38.53, 27.37, 16.47, 13.38, 25.22, all P<0.01). For example, the relative expression of COL14A1 in primary LACC-HGT-1 (16.09±0.51) and LACC-HGT-2 (9.96±0.34) cells was significantly higher than that in primary LACC-1 (1.00±0.13) and LACC-2 (0.67±0.08) cells (all P<0.05). Conclusion: There are differentially expressed proteins between LACC-HGT and LACC, among which COL14A1, EML4, ITIH4, NDRG2, OGN, and RhoC may play an important role in LACC-HGT and can be used as potential targets of LACC-HGT in further study. (Chin J Ophthalmol, 2021, 57: 531-539).

P052 Comprehensive proteomic profiling of stool from UC and CD patients reveals expected and novel mucosal pathobiology, enabling identification of candidate non-invasive biomarkers to monitor mucosal disease activity

Abstract Background Inflammatory bowel disease (IBD) is a multifactorial disorder characterized by chronic gastrointestinal (GI) inflammation. In clinical practice, physicians urgently need biomarkers to monitor changes in mucosal disease to manage treatment. Non-invasive tools such as fecal biomarkers could allow for frequent monitoring of mucosal disease activity and may reflect inflammation of the entire GI tract. Towards identification of novel non-invasive fecal biomarkers, we performed a comprehensive, unbiased, analysis of the human fecal proteome from ulcerative colitis (UC), Crohn’s disease (CD) patients, and healthy controls (HC), focusing on human secreted proteins using proteomic approaches. Methods Fecal extraction processes optimized for recovery of secreted proteins were applied to stool samples of mild to severe UC and CD patients and HCs, 20 samples per group. An unbiased survey of the human fecal proteome of the IBD and HC stool extracts was performed using both data-dependent acquisition and data-independent acquisition methods on QExactive HF and Fusion Lumos mass spectrometers. Data were analyzed in Spectronaut for peptide identification, followed by analysis in MS Stats for protein quantification. Fecal calprotectin (fCAL) levels were measured in these stool samples using the Buhlmann EK-CAL immunoassay kit to correlate fCAL levels to proteomic calprotectin (S100A8/S100A9 heterodimer). Results We identified and quantified 594 differentially expressed proteins in stool using proteomics, including known proteins perturbed in IBD pathobiology. Elevated levels of neutrophilic proteins, e.g. fCal, neutrophil elastase, lactoferrin and leucine-rich Α-2 glycoprotein 1 were detected as well as proteins linked to rectal bleeding and ulceration e.g. hemoglobin subunit alpha 1 and beta. Pancreatic enzymes, suggested to be associated with active disease, were also detected. Overall, UC and CD contained many proteins not detected in HC. Differential abundances were observed between UC and CD patients, possibly reflecting differences in mucosal pathophysiology. The abundance of proteomic calprotectin correlated highly with the fCAL levels as determined by immunoassay (Spearman rank order &amp;gt; 0.9 for S100A8 and S100A9 to fCAL), validating the fecal proteomic approach to uncover known and novel proteins of IBD biology. Conclusion We developed and qualified methods to perform fecal proteomics on IBD stool samples, enabling identification of known and novel proteins of mucosal UC and CD disease biology. In depth analysis of these proteomic datasets may reveal novel fecal biomarker candidates, targeted to reflect mucosal disease activity and potentially serve as non-invasive surrogates for endoscopy and help guide treatment in IBD patients.

Proteomic Analysis of the Vitreous Body in Proliferative and Non-Proliferative Diabetic Retinopathy

Background: Diabetic Retinopathy (DR), one of the major microvascular complications commonly occurring in diabetic patients, can be classified into Proliferative Diabetic Retinopathy (PDR) and Non-Proliferative Diabetic Retinopathy (NPDR). Currently available therapies are only targeted for later stages of the disease in which some pathologic changes may be irreversible. Thus, there is a need to develop new treatment options for earlier stages of DR through revealing pathological mechanisms of PDR and NPDR. Objective: The purpose of this study was to characterize proteomes of diabetic through quantitative analysis of PDR and NPDR. Methods: Vitreous body was collected from three groups: control (non-diabetes mellitus), NPDR, and PDR. Vitreous proteins were digested to peptide mixtures and analyzed using LC-MS/MS. MaxQuant was used to search against the database and statistical analyses were performed using Perseus. Gene ontology analysis, related-disease identification, and protein-protein interaction were performed using the differential expressed proteins. Results: Twenty proteins were identified as critical in PDR and NPDR. The NPDR group showed different expressions of kininogen-1, serotransferrin, ribonuclease pancreatic, osteopontin, keratin type II cytoskeletal 2 epidermal, and transthyretin. Also, prothrombin, signal transducer and activator of transcription 4, hemoglobin subunit alpha, beta, and delta were particularly up-regulated proteins for PDR group. The up-regulated proteins related to complement and coagulation cascades. Statherin was down-regulated in PDR and NPDR compared with the control group. Transthyretin was the unique protein that increased its abundance in NPDR compared with the PDR and control group. Conclusion: This study confirmed the different expressions of some proteins in PDR and NPDR. Additionally, we revealed uniquely expressed proteins of PDR and NPDR, which would be differential biomarkers: prothrombin, alpha-2-HS-glycoprotein, hemoglobin subunit alpha, beta, and transthyretin.

Application of proteomics to understand the molecular mechanisms determining meat quality of beef muscles during postmortem aging

Proteomics profiling disclosed the molecular mechanism underlying beef poor meat quality. This study aimed to identify protein markers indicating the quality of beef during postmortem storage at 4°C. Beef longissimus dorsi samples were stored at 4°C. The meat water holding capacity (WHC), pH value and moisture content were determined at different time points during the storage period. The iTRAQ MS/MS approach was used to determine the proteomics profiling at 0, 3.5 and 7 d during storage at 4°C. Bioinformatics analysis was performed to investigate the potential correlated proteins associated with meat quality. Storage at 4°C gradually decreased the pH value, WHC, and hence the moisture content. The iTRAQ proteomic analysis revealed that a cluster of glycolytic enzymes including malate dehydrogenase, cytoplasmic, L-lactate dehydrogenase, phosphoglycerate mutase and pyruvate kinase, and another cluster of proteins involved in oxygen transport and binding (myoglobin) and hemoglobin complex (including Globin A1 and hemoglobin subunit alpha) were decreased during the postmortem storage. These results suggest that the decreased glycolysis, oxygen, and heme-binding activities might be associated with the beef muscle low quality and the decline of tenderness during postmortem storage at 4°C.

The modulation of the burn wound environment by negative pressure wound therapy: Insights from the proteome

Negative pressure wound therapy has been used to promote wound healing in a variety of settings, including as an adjunct to silver-impregnated dressings in the acute management of paediatric burns. Fluid aspirated by the negative pressure wound therapy system represents a potentially insightful research matrix for understanding the burn wound microenvironment and the intervention's biochemical mechanisms of action. The aim of this study was to characterize the proteome of wound fluid collected using negative pressure wound therapy from children with small-area thermal burns. Samples were obtained as part of a randomized controlled trial investigating the clinical efficacy of adjunctive negative pressure wound therapy. They were compared with blister fluid specimens from paediatric burn patients matched according to demographic and injury characteristics. Protein identification and quantification were performed via liquid chromatography tandem mass spectrometry and sequential window acquisition of all theoretical mass spectra data-independent acquisition. Proteins and biological pathways that were unique to or enriched in negative pressure wound therapy fluid samples were evaluated using principal components, partial least squares-discriminant, and gene ontology enrichment analyses. Eight viable samples of negative pressure wound therapy fluid were collected and analyzed with eight matched blister fluid samples. A total of 502 proteins were quantitatively profiled in the negative pressure wound therapy fluid, of which 444 (88.4%) were shared with blister fluid. Several proteins exhibited significant abundance differences between fluid types, with negative pressure wound therapy fluid showing a higher abundance of matrix metalloproteinase-9, arginase-1, low affinity immunoglobulin gamma Fc region receptor III-A, filamin-A, alpha-2-macroglobulin, and hemoglobin subunit alpha. The results lend support to the hypothesis that negative pressure wound therapy augments wound healing through the modulation of factors involved in the inflammatory response, granulation tissue synthesis, and extracellular matrix maintenance. Data are available via ProteomeXchange with identifier PXD023168.

The identification and characterisation of novel bioactive peptides derived from porcine liver

Bioactive peptides (BAPs) can be derived from a variety of sources; these could be from dietary proteins which are then broken down in the gastrointestinal tract to release BAPs, or they can be isolated from various sources ex vivo. Sources include plant-based proteins such as soy, and chickpeas, and animal proteins from waste from the meat industry and from fish skin. Bioinformatics is also a useful approach to assess the peptides released from digests due to the great number of possible sequences that can be isolated from proteins. Therefore, an in silico analysis of peptides could potentially lead to a more rapid discovery of BAPs. This article investigates a “crude” liver peptide mixture derived from papain hydrolysis of porcine liver and purified peptides derived from the hydrolysates following HPLC fractionation and in silico digestion of the host proteins identified using LC-MS/MS. This allowed the identification of two proteins (cytosol aminopeptidase and haemoglobin subunit alpha) present in the “crude” mixture after LC-MS/MS. In silico hydrolysis of these proteins identified that several peptides were predicted to be both present in the crude mixture using the BIOPEP database and to have potential bioactivity using the Peptide Ranker tool. Peptides (FWG, MFLG and SDPPLVFVG) with the greatest potential bioactivity and which had not previously been reported in the literature were then synthesised. The results indicated that the predicted bioactivity of the synthetic peptides would likely include antioxidant activity. FWG and MFLG derived from the in silico papain hydrolysis of cytosol aminopeptidase showed activity better or comparable to Trolox in the Oxygen Radical Absorbance Capacity (ORAC) assay. The use of these in silico tools, alongside a robust range of biochemical assays which cover a wider range of bioactivities would be a way of improving the discovery of novel bioactive peptides.