2D Gel Research Articles

Immunoglobulin E-specific allergens against leaf in serum of dogs with clinical features of grass leaf allergy.

Grass leaf has been suspected of causing immunoglobulin (Ig)E-mediated immediate hypersensitivity reactions in humans and dogs. However, most studies in this area are case-control studies without in vitro data showing the involvement of IgE in the reaction. Laboratory studies have demonstrated the reactivity to a 50-55 kDa protein with clinical signs immediately after contact with grass leaf material. The clinical findings of dogs with atopic-like dermatitis immediately after contact with grass leaf material suggest the involvement of grass leaves as the allergen source. This study was designed to test the IgE-reactivity of grass leaf proteins in dogs with clinical signs and positive scratch test results against grass leaf material. The serum of 41 patients with a history of allergy and suspected to grass leaf material was immunoblotted against grass leaf extracts from five suspected grass species. The IgE-positive blots were separated with 2D gel electrophoresis and analysed with mass spectrometry (MS). Commercially supplied proteins were used to validate immunoblot activity. The serum of 25 dogs diagnosed with grass dermatitis had positive IgE-specific immunoblot against one or more grass leaf extracts. The MS data indicated a reactive band at 55 kDa to be beta-amylase or RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit (RbLS). All tested dog sera showed IgE-reactivity with beta-amylase and some with RbLS. Canines with clinical signs of grass-related dermatitis had IgE-reactivity against grass leaf proteins. Serum IgE-reactivity to beta-amylase and RuBisCO large subunit may indicate that these proteins act as allergens, possibly causing pruritus and skin lesions.

Production and Characterization of ACE Inhibitory and Anti-Diabetic Peptides from Buffalo and Camel Milk Fermented with Lactobacillus and Yeast: A Comparative Analysis with In Vitro, In Silico, and Molecular Interaction Study.

The investigation aimed at assessing a comparative study on the production and characterization of ACE inhibitory, anti-diabetic, and anti-inflammatory activities, along with the production of ACE inhibitory and anti-diabetic peptides through the fermentation of buffalo and camel milk by Limosilactobacillus fermentum (KGL4) and Saccharomyces cerevisiae (WBS2A). The angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic properties were evaluated at particular time intervals (12, 24, 36, and 48 h) at 37 °C, and we discovered maximum activity at 37 °C after 48 h of incubation. The maximum ACE inhibitory, lipase inhibitory activities, alpha-glucosidase inhibitory, and alpha-amylase inhibitory activities were found in the fermented camel milk (77.96 ± 2.61, 73.85 ± 1.19, 85.37 ± 2.15, and 70.86 ± 1.02), as compared to the fermented buffalo milk (FBM) (75.25 ± 1.72, 61.79 ± 2.14, 80.09 ± 0.51, and 67.29 ± 1.75). Proteolytic activity was measured with different inoculation rates (1.5%, 2.0%, and 2.5%) and incubation times (12, 24, 36, and 48 h) to optimize the growth conditions. Maximum proteolysis was found at a 2.5% inoculation rate and at a 48 h incubation period in both fermented buffalo (9.14 ± 0.06) and camel milk (9.10 ± 0.17). SDS-PAGE and 2D gel electrophoresis were conducted for protein purification. The camel and buffalo milk that had not been fermented revealed protein bands ranging from 10 to 100 kDa and 10 to 75 kDa, respectively, whereas all the fermented samples showed bands ranging from 10 to 75 kDa. There were no visible protein bands in the permeates on SDS-PAGE. When fermented buffalo and camel milk were electrophoresed in 2D gel, 15 and 20 protein spots were detected, respectively. The protein spots in the 2D gel electrophoresis ranged in size from 20 to 75 kDa. To distinguish between different peptide fractions, water-soluble extract (WSE) fractions of ultrafiltration (3 and 10 kDa retentate and permeate) of fermented camel and buffalo milk were employed in RP-HPLC (reversed-phase high-performance liquid chromatography). The impact of fermented buffalo and camel milk on inflammation induced by LPS (lipopolysaccharide) was also investigated in the RAW 264.7 cell line. Novel peptide sequences with ACE inhibitory and anti-diabetic properties were also analyzed on the anti-hypertensive database (AHTDB) and bioactive peptide (BIOPEP) database. We found the sequences SCQAQPTTMTR, EMPFPK, TTMPLW, HPHPHLSFMAIPPK, FFNDKIAK, ALPMHIR, IPAVFK, LDQWLCEK, and AVPYPQR from the fermented buffalo milk and the sequences TDVMPQWW, EKTFLLYSCPHR, SSHPYLEQLY, IDSGLYLGSNYITAIR, and FDEFLSQSCAPGSDPR from the fermented camel milk.

Stem bark methanolic extract of Bauhinia thonningii (Schum) demonstrates gastroprotective properties against in vivo ethanol-induced injury

Background: Stomach ulcer is one of the diseases that many patients suffer from all over the world, and it causes damage to the intestinal wall. Some research has shown that Bauhinia thonningii extract, which has been traditionally used to treat a variety of ailments, including stomach ulcers, has powerful antioxidant and mucus-increasing properties. Objective:This research is aimed at studying the mechanism(s) through which methanolic (MBST) and chloroform (CBST) extracts of B. thonningii stem bark protects against ethanol-induced stomach damage in rats. Material and Methods: MBST and CBST were evaluated in vivo for their cytoprotective qualities employing stomach acidity, mucus content, macroscopic and microscopic methods, lipid peroxidation, nitric oxide, mass spectroscopy, 2D gel electrophoresis, and biochemical indicators. These extracts were tested in vitro on lipopolysaccharide/interferon-stimulated rat cells. Additionally, in-vivo, and in-vitro toxicity investigations were conducted. FRAP, DPPH, and ORAC tests were used to determine the antioxidant activity of MBST and CBST. Chemical and spectroscopic approaches were used to analyze the phytochemistry of MBST and CBST.Results: The gross and histological characteristics of B. thonningii proved its anti-ulcerogenic effects. MBST has gastroprotective effects by modulating lipid peroxidation and proteomics indicators [heat shock protein (Hsp60), disulfide isomerase (ERp60), protein disulfide-isomerase, creatine kinase B, cytosolic malate dehydrogenase, peroxiredoxin-6, and ATP synthase]. MBST and CBST have substantial impacts on nitric oxide both in-vitro and in-vivo. In comparison to CBST, methanolic extract (MBST) demonstrated greater gastroprotective properties, polyphenolic content, and antioxidant activity. The plant extracts did not show any toxicity in-vitro or in-vivo. MBST shows anti-ulcer action, which may be ascribed to its ability to reduce ethanol-triggered oxidative injury, to intervene in proteomic mechanisms, and to modulate nitric oxide.Conclusion:The present study found that stem bark extracts of Bauhinia thonningii Schum have cytoprotective activities against in vivo ethanol-induced damage due to its powerful antioxidant and mucus-increasing capabilities. Bauhinia thonningii warrants more attention since it may provide an exciting novel medication for the treatment of acute erosive gastropathy.Keywords: Bauhinia thonningii Schum;polar and non-polar extraction; peptic ulcer; nitric oxide; proteomics

Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene (Igh6), IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

Hyperextended telomeres promote formation of C-circle DNA in telomerase positive human cells

Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to ∼100 kb). cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect circular DNA with extrachromosomal telomere repeats. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. We observed similar cECTR results in two other LTT+ cell lines, HeLa1.3 (∼23 kb telomeres) and HeLaE1 (∼50 kb telomeres). In LTT+ cells, telomerase activity did not directly impact C-circle signal; instead, C-circle signal correlated with telomere length. LTT+ cell lines were less sensitive to hydroxyurea than ALT+ cell lines, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, the DNA repair-associated protein FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.

FungiProteomeDB: a database for the molecular weight and isoelectric points of the fungal proteomes.

Proteins' molecular weight (MW) and isoelectric point (pI) are crucial for their subcellular localization and subsequent function. These are also useful in 2D gel electrophoresis, liquid chromatography-mass spectrometry and X-ray protein crystallography. Moreover, visualizations like a virtual 2D proteome map of pI vs. MW are worthwhile to discuss the proteome diversity among different species. Although the genome sequence data of the fungi kingdom improved enormously, the proteomic details have been poorly elaborated. Therefore, we have calculated the MW and pI of the fungi proteins and reported them in, FungiProteomeDB, an online database (DB) https://vision4research.com/fungidb/. We analyzed the proteome of 685 fungal species that contain 7 127 141 protein sequences. The DB provides an easy-to-use and efficient interface for various search options, summary statistics and virtual 2D proteome map visualizations. The MW and pI of a protein can be obtained by searching the name of a protein, a keyword or a list of accession numbers. It also allows querying protein sequences. The DB will be helpful in hypothesis formulation and in various biotechnological applications. Database URL https://vision4research.com/fungidb/.

Proteomic landscape presents cues for vegetative to reproductive transition in mango

Proteome-based vegetative and flower bud formation characterization was utilized to identify and differentiate protein species with significant variable abundance during floral transition in mango cv. Dashehari using 2DE and corroborated the identified protein spots using gene expression analysis. Total soluble proteins were phenol-extracted from mango cv's vegetative and floral flush. Dashehari and separated on 2D gels at pH 4-7. The protein spots with variable intensity were identified through SameSpots software. The protein sequences of differentially accumulated spots were identified based on PI and MW using Citrus sinensis proteome isoelectric focusing database. Furthermore, these protein sequences were used to conduct (tBLASTn) against Mangifera indica to predict the protein. Real time gene expression was done to corroborate identified proteins. Total 301 spots were detected, out of which 16 were identified as differentially expressed (P?0.05) and a 2-fold change. These 16 protein spots were identified on the basis of in silico comparative mapping protein against genome of mango and citrus, a close relative. They were classified into eight categories: transcriptional regulation, phenylpropanoid pathway and cell wall /cytoskeleton metabolism-related proteins, hormone signalling, flowering time, signal-transduction, transport and protein synthesis to flowering. Five genes coding for shortlisted proteins were used for validation of results using gene expression analysis. SAM (S adenosyl methonine synthase) was found up-regulated in floral flush, involved in the biosynthesis of polyamines has association with flowering, and stress responses. Furthermore, ARF (Auxin Response Factor), serine/threonine kinase gene members were also found to play critical role in determining floral development process in mango, consistent with results obtained through 2DE. Protein species that are putatively involved in phenylpropanoid pathway were also identified, showing the process of mango flowering from a new perspective beyond the conventional view. This flowering related proteomics study provides an overview of the biological pathways and regulatory mechanisms associated with flowering developmental physiology.

Discovery of a Missense Mutation (Q222K) of the APOE Gene from the Australian Imaging, Biomarker and Lifestyle Study.

After age, polymorphisms of the Apolipoprotein E (APOE) gene are the biggest risk factor for the development of Alzheimer's disease (AD). During our investigation to discovery biomarkers in plasma, using 2D gel electrophoresis, we found an individual with and unusual apoE isoelectric point compared to APOE ɛ2, ɛ3, and ɛ4 carriers. Whole exome sequencing of APOE from the donor confirmed a single nucleotide polymorphism (SNP) in exon 4, translating to a rare Q222K missense mutation. The apoE ɛ4 (Q222K) mutation did not form dimers or complexes observed for apoE ɛ2 & ɛ3 proteins.

Icos gene disruption in NOD mice elicits myositis associated with anti-troponin T3 autoantibodies.

Idiopathic inflammatory myopathies (IIM) are autoimmune inflammatory disorders leading to skeletal muscle weakness and disability. The pathophysiology of IIM is poorly understood due to the scarcity of animal disease models. Genetic deletion of Icos or Icosl (Inducible T-cell co-stimulator deficient/ligand) in non-obese diabetic (NOD) mice leads to muscle disease. Our aim was to characterise Icos-/- NOD myopathy and to search for novel autoantibodies (aAbs) in this model. Diabetes, weight, myopathy incidence/clinical score and grip strength were assessed over time. Locomotor activity was analysed with the Catwalk XT gait analysis system. Muscle histology was evaluated in haematoxylin/eosin and Sirius red-stained sections, and immune infiltrates were characterised by immunofluorescence and flow cytometry. 2D gel electrophoresis of muscle protein extracts and mass spectrometry were used to identify novel aAbs. NOD mice were immunised with troponin T3 (TNNT3) in incomplete Freund's adjuvant (IFA) and R848. An addressable laser-bead immunoassay (ALBIA) was developed to measure aAb IgG serum levels. Icos-/- NOD mice did not exhibit diabetes but developed spontaneous progressive myositis with decreased muscle strength and altered locomotor activity. Muscle from these mice exhibited myofibre necrosis, myophagocytosis, central nuclei, fibrosis and perimysial and endomysial cell infiltrates with macrophages and T cells. We identified anti-TNNT3 aAbs in diseased mice. Immunisation of NOD mice with murine TNNT3 protein led to myositis development, supporting its pathophysiological role. These data show that Icos-/- NOD mice represent a spontaneous model of myositis and the discovery of anti-TNNT3 aAb suggests a new autoantigen in this model.

A comparative study of fermented buffalo and camel milk with anti-inflammatory, ACE-inhibitory and anti-diabetic properties and release of bio active peptides with molecular interactions: In vitro, in silico and molecular study

The aim of this study is to compare ACE inhibitory, anti-diabetic and Anti-inflammatory activity of buffalo and camel milk using Lacticaseibacillus paracasei (M11) in combination with yeast Saccharomyces cerevisiae (WBS2A) and to release peptides with antihypertensive and anti-diabetic properties. Fermented buffalo and camel milk were considered for the evaluation of Angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic activities at specific time intervals (12, 24, 36 and 48 h) at 37 °C. For the growth condition optimization, proteolytic activity was evaluated with specific inoculation rates (1.5%, 2.0%, and 2.5%) and at specific incubation periods (12, 24, 36, and 48 h). The highest peptides were produced at 2.5% inoculation rate and at 48 h of incubation in both buffalo and camel milk at 37 °C. Further, camel and buffalo milk fermented with M11+WBS2A significantly lower the overproduction of TNF-α, IL-6, IL-1β and Nitric oxide (NO), that was induced by lipopolysaccharide (LPS) stimulation in RAW 267.4 cells. For protein purification, 2D gel electrophoresis and SDS-PAGE were used. Camel and buffalo milk that had not been fermented displayed protein bands between 10 and 100 kDa and 10 and 75 kDa respectively, whereas all the fermented samples showed bands between 10 and 75 kDa. On SDS-PAGE, in permeates, there were no apparent protein bands. When fermented buffalo and camel milk were electrophoresed in 2D gel, a total of 26 and 25 protein spots were found. In 2D gel electrophoresis, protein spots had sizes between 10 and 37 kDa. In RP-HPLC, water-soluble extracts (WSEs) of ultra-filtered fractions were used to distinguish different fractions of a peptide. Peakview software was used to characterize the LC/MS results. Fourier transform-infrared spectroscopy (FTIR) was used to evaluate various functional groups of fermented milks. In molecular docking study, the peptide sequences identified from the fermented buffalo milk (FBM) and camel milk (FCM) were examined for their binding affinity against the binding sites of hPAM and hMGA.

The Phenotypic Polymorphism of Saliva From Dental Caries Patients

Introduction: The tooth decay or dental caries is a disease that results in imbalance in mineralization process of the tooth, cavitation and sensitivity as well as pulp infection that leading to inflammation and eventually tooth loose. Saliva which is in direct contact with the dentine, can serve as a medium for analysis of components or factors that show the difference in normal healthy and disease conditions. Methodology: Here, a biochemical analysis of individual saliva samples from healthy control and caries patients were carried out to see the differences in protein profiles. The unstimulated saliva samples were analyzed through sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), two-dimensional gel electrophoresis (2D gel), zymography and secretary IgA (sIgA) antibody ELISA procedure. Results: Differences were observed in individual samples not only in polymorphic band pattern on SDS-PAGE gels but also in activity of proteases in the saliva and the concentration of sIgA antibody. Conclusion: These were the initial results obtained from the study that was further optimized and evaluated with accuracy and reported elsewhere with statistical figures.

Effects of Flupyradifurone and Two Reference Insecticides Commonly Used in Toxicological Studies on the Larval Proteome of the Honey bee Apis mellifera.

The western honey bee Apis mellifera is globally distributed due to its beekeeping advantages and plays an important role in the global ecology and economy. In recent decades, several studies have raised concerns about bee decline. Discussed are multiple reasons such as increased pathogen pressure, malnutrition or pesticide use. Insecticides are considered to be one of the major factors. In 2013, the use of three neonicotinoids in the field was prohibited in the EU. Flupyradifurone was introduced as a potential successor; it has a comparable mode of action as the banned neonicotinoids. However, there is a limited number of studies on the effects of sublethal concentrations of flupyradifurone on honey bees. Particularly, the larval physiological response by means of protein expression has not yet been studied. Hence, the larval protein expression was investigated via 2D gel electrophoresis after following a standardised protocol to apply sublethal concentrations of the active substance (flupyradifurone 10 mg/kg diet) to larval food. The treated larvae did not show increased mortality or an aberrant development. Proteome comparisons showed clear differences concerning the larval metabolism, immune response and energy supply. Further field studies are needed to validate the in vitro results at a colony level.

Lectin-Based Affinity Enrichment and Characterization of N-Glycoproteins from Human Tear Film by Mass Spectrometry.

The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals (n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.

Proteoform Identification in 2D Electrophoresis Maps by Using Isoelectric Point Prediction

The possibility of identifying specific protein proteoforms with post-translational modifications (PTM) by analyzing two-dimensional (2D) gel electrophoresis maps and using the prediction of the isoelectric point of proteins (pI) has been investigated. The pI values were predicted using the pIPredict 3 program, supporting a wide range of chemical and post-translational modifications. Eleven 11 proteins (albumin, alpha-1-microglobulin, annexin A2, apolipoprotein E, gastric triacylglycerol lipase, mitochondrial isocitrate dehydrogenase, clusterin, plasmin, prothrombin, endoplasmic reticulum chaperone, S-adenosylmethionine synthase type 1) identified on six 2D electrophoresis maps were used as examples. Various options for selecting hypotheses are considered. These take into consideration the following available information about a particular protein: possible modification sites, processing features, variability of the amino acid composition. The obtained results indicate that the use of predicting the pI value for proteins with hypothetical PTMs can form a set of hypotheses about specific proteoform occurrence on 2D electrophoresis maps.

Upregulation of Brain's Calcium Binding Proteins in Mitragynine Dependence: A Potential Cellular Mechanism to Addiction.

Background: Mitragyna speciosa Korth or Kratom is widely used traditionally for its medicinal values. The major alkaloid content of kratom leaves is mitragynine, which binds to opioid receptors to give opioid-like effects. This study aimed to analyse the brain proteome of animals that displayed addictive behaviors. Design and Methods: Six groups (n=6-8) of rats made up of negative control, positive control using morphine (10 mg/kg), and treatment groups at low (1mg/kg) and high doses of mitragynine (30 mg/kg) for 1 and 4 days. The rats' behaviors were evaluated and subsequently the rats' brains were harvested for proteomic analysis that was performed by using 2D gel electrophoresis and LC/MS/MS. Results: The rats developed physical dependence only on day 4 following morphine and mitragynine (1 and 30mg/kg) treatments. Among the proteins that were up-regulated in treatment groups were four calcium-binding proteins, namely calretinin, F-actin, annexin A3 and beta-centractin. Conclusions: Upregulation of calretinin acted as low Ca2+ buffering upon the blockage of Ca2+ ion channel by mitragynine in the brain, which subsequently caused a reduction of GABA released and inversely increased the dopamine secretions that contributed to dependence indicators.

Comparative proteomic analysis of patients with acute myeloid leukemia before and after induction therapy.

Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells, characterized by accumulation of immature blasts in the bone marrow and peripheral blood of affected patients. Response to chemotherapy treatment in patients with AML is wide-ranging, and to date there are no adequate molecular biomarkers used to predict clinical outcome. The aim of this study was to identify potential protein biomarkers which could help predict response to induction treatment in AML patients. Peripheral blood samples were obtained from 15 AML patients both before and after treatment. A comparative proteomic analysis was performed using 2D gel electrophoresis followed by Mass Spectrometry. This comparative proteomic study, combined with a protein network analysis, revealed several proteins that could be considered potential biomarkers of poor prognosis in AML: GAPDH which favors increased glucose metabolism; eEF1A1 and Annexin A1 that promote proliferation and migration, cofilin 1 which plays a role in the activation of apoptosis; and GSTP1 which is involved in the processes of detoxification and chemoresistance. This study gives an insight into a panel of protein biomarkers with prognostic potential that should be further investigated.

Proteomic‐Based Discovery of Predictive Biomarkers for Drug Therapy Response and Personalized Medicine in Chronic Immune Thrombocytopenia

Purpose. ITP is the most prevalent autoimmune blood disorder. The lack of predictive biomarkers for therapeutic response is a major challenge for physicians caring of chronic ITP patients. This study is aimed at identifying predictive biomarkers for drug therapy responses. Methods. 2D gel electrophoresis (2‐DE) was performed to find differentially expressed proteins. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometer (MALDI‐TOF MS) analysis was performed to identify protein spots. The Cytoscape software was employed to visualize and analyze the protein‐protein interaction (PPI) network. Then, enzyme‐linked immunosorbent assays (ELISA) were used to confirm the results of the proteins detected in the blood. The DAVID online software was used to explore the Gene Ontology and pathways involved in the disease. Results. Three proteins, including APOA1, GC, and TF, were identified as hub‐bottlenecks and confirmed by ELISA. Enrichment analysis results showed the importance of several biological processes and pathway, such as the PPAR signaling pathway, complement and coagulation cascades, platelet activation, vitamin digestion and absorption, fat digestion and absorption, cell adhesion molecule binding, and receptor binding. Conclusion and Clinical Relevance. Our results indicate that plasma proteins (APOA1, GC, and TF) can be suitable biomarkers for the prognosis of the response to drug therapy in ITP patients.

Alterations in the pH of pancreatic juice are associated with chymotrypsin C inactivation and lithostathine precipitation in chronic pancreatitis patients: a proteomic approach

BackgroundThe progression of chronic pancreatitis (CP), an inflammatory disease of the pancreas, causes pancreatic stones to form within the pancreatic ductal lumen/parenchyma, which occurs via protein plug formation. Pain is the most common symptom that necessitates clinical attention, and pain relief is the therapeutic goal for these patients. Endoscopic therapy and surgery are complimentary forms of therapy for pain relief. This study was envisaged to clarify the mechanism by which protein plug/soft stones form in pancreatic ducts prior to undergoing calcification.MethodsProtein plugs were obtained from twenty CP patients undergoing therapeutic ERCP for stone removal. Pancreatic juice was obtained from five CP patients without stones. Proteins were isolated by TCA/acetone precipitation, SDS PAGE and 2-D gel electrophoresis to determine the protein profile. Protein spots from the 2-D gel were excised and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) for identification. The effect of altered pH and elevated concentrations of trypsin on pancreatic juice protein was assessed by SDS‒PAGE to determine the protein profile. Differentially expressed protein bands were excised and subjected to MALDI-TOF. In silico analysis was performed by docking lithostathine with the calcite molecule using AutoDock Vina and PyMOL to clarify their interaction during stone formation.ResultsTwenty-three and twenty-nine spots from 2D gels of protein plugs and pancreatic juice, respectively, revealed that lithostathine (Reg1A) was the only protein in the protein plugs, whereas digestive enzymes and lithostathine were identified in pancreatic juice. Altered pH levels and increased trypsin concentrations in the pancreatic juice caused a protein to degrade via an unknown mechanism, and this protein was identified as chymotrypsin C (CTRC) by MALDI-TOF. Docking studies showed that the binding affinity of calcite was higher with the cleaved lithostathine, explaining the deposition of calcium that was observed around the protein plugs after calcified stones were formed through precipitation.ConclusionOur results suggest that chymotrypsin C (CTRC) is degraded in an acidic environment, leading to the precipitation of lithostathine in the ductal lumen.Graphical

Abstract 12624: Smooth Muscle Cell Alpha-Actin Pathogenic Variant That Predisposes to Moyamoya-Like Cerebrovascular Disease Alters Smooth Muscle Cell Phenotype and Metabolism

Pathogenic variants in ACTA2 , which encodes smooth muscle α-actin ( α-SMA ), predispose to thoracic aortic disease. ACTA2 variants disrupting arginine 179 ( R179 ) also predispose to moyamoya-like ( MMD ) cerebrovascular disease, characterized by occlusion of the distal internal carotid arteries by neointimal cells that stain positive for smooth muscle cell ( SMC ) markers. Mice with conditional knockin of the Acta2 R179C mutation specifically in SMCs were generated ( Acta2 SMC-R179C/+ ). RNA sequencing and 2D gel electrophoresis confirmed presence of the mutation. Acta2 SMC-R179C/+ SMCs are not fully differentiated, maintain stem cell marker expression, and proliferate and migrate faster than wildtype ( WT ) SMCs. Seahorse analyses show increased glycolysis and decreased oxidative respiration ( OXPHOS ) in Acta2 SMC-R179C/+ SMCs, which have reduced mitochondrial mass and complex I activity, suggestive of deficient electron transport chain activity. Nicotinamide riboside ( NR ) augments complex I activity to boost OXPHOS without altering mitochondrial mass in Acta2 SMC-R179C/+ SMCs. NR also increases differentiation and reduces migration in Acta2 SMC-R179C/+ SMCs. With left carotid artery ligation ( LCAL ), 33% of Acta2 SMC-R179C/+ mice die within 4 days due to strokes, and 100% of surviving Acta2 SMC-R179C/+ mice at postop day 21 have occlusive lesions in the injured artery (8% in WT mice) containing α-SMA+ cells and fibrin deposits. Acta2 SMC-R179C/+ mice also exhibit markedly increased left carotid artery diameter and increased number, diameter, and tortuosity of left-sided collateral vessels compared to WT mice at postop day 21. NR treatment reduces deaths of LCAL-injured Acta2 SMC-R179C/+ mice to 8%, completely resolves occlusive lesion formation in 50% of mutant mice, and attenuates collateral formation and remodeling. These data suggest that immature SMCs with the ACTA2 R179C variant proliferate and migrate into the neointima causing MMD-like occlusive disease. Shifting metabolism to OXPHOS with NR drives differentiation and reduces migration, which diminishes arterial lesions in Acta2 SMC-R179C/+ mice. These data support the use of NR as a therapeutic to prevent MMD-like lesions and ischemic strokes in children with ACTA2 p.R179 variants.

Inhibition mechanisms of secretome proteins from Paenibacillus polymyxa Kp10 and Lactococcus lactis Gh1 against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus

Objective: To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10 (Kp10) and Lactococcus lactis Gh1 (Gh1) against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). Methods: The sensitivity and viability of MRSA and VRE treated with secretome proteins of Kp10 and Gh1 were determined using minimal inhibitory concentration, minimum bactericidal concentration, and time-to-kill assays. The morphological changes were observed using scanning electron microscopy and transmission electron microscopy. To elucidate the antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and VRE, 2D gel proteomic analysis using liquid chromatography-mass spectrometry was run by comparing upregulated and downregulated proteins, and the proton motive force study including the efflux of ATP, pH gradient, and the membrane potential study were conducted. Results: MRSA and VRE were sensitive to Kp10 and Gh1 secretome protein extracts and displayed apparent morphological and internal composition changes. Several proteins associated with cellular component functions were either downregulated or upregulated in treated MRSA and VRE by changing the membrane potential gradient. Conclusions: Kp10 and Gh1 secretome proteins reduce the growth of VRE and MRSA by damaging the cell membrane. Cell division, cell wall biosynthesis, and protein synthesis are involved in the inhibition mechanism.