Changes In Protein Expression Profiles Research Articles

Quantitative proteomic landscape of the pathophysiology of adhesive arachnoiditis and its clinical significance: Structure and mechanism of TNC and RANBP1 proteins.

The pathophysiological mechanism of adhesive arachnoiditis (AA) is complex, involving the interaction of multiple proteins. In recent years, the development of quantitative proteomics technology has provided a new perspective to reveal its pathological mechanism. The main objective of this study was to reveal the changes of protein expression profiles in arachnoid tissue of patients with AA. Proteomic analysis of arachnoid tissue samples was performed by high-throughput mass spectrometry. Bioinformatics tools were used to process and analyze the data and screen out 712 differentially expressed proteins (DEPs). Specially, 2 hub DEPs (TNC and RANBP1) were found as potential diagnostic markers. The expression levels of TNC and RANBP1 proteins were significantly up-regulated in patients with AA. TNC protein played a key role in inflammation and extracellular matrix remodeling, while RANBP1 was involved in cytoplasmic transport and cell cycle regulation. The abnormal expression and modification of these two proteins were closely related to the pathophysiological process of the disease. Immunoinfiltration analysis found the pathological process of AA were related to the infiltration of memory B cells, activated NK cells and CD8(+) T cells. Additonally, weighted gene co-expression network analysis (WGCNA) showed the organization of the arachnoid proteome into a network of 28 biologically meaningful modules of co-expressed proteins. Of these, 2 modules were positively correlated to AA phenotypes. This study shows a distinct proteomic landscape of AA and non-adhesive arachnoiditis (nAA). These findings also provide valuable insights into the molecular changes associated with potential mechanisms underlying AA.

Proangiogenic effect of thyrotropin receptor stimulating antibody in human umbilical vein endothelial cells.

This study aims to investigate the role of TRAb in the angiogenesis associated with Graves' disease (GD) and to elucidate its underlying mechanisms. Human thyroid follicular epithelial cells (Nthy-ori 3-1) and human umbilical vein endothelial cells (HUVECs) were treated with the monoclonal thyroid-stimulating antibody M22 and thyroid-stimulating hormone (TSH) at various concentrations. Cell viability, migration, and tube formation were evaluated using CCK-8, wound healing, and tube formation assays, respectively. Protein expressions of TSHR receptor (TSHR) and phosphorylated AKT (p-AKT) in M22-induced HUVECs were quantified via Western blotting. Proteomic analysis was employed to identify changes in protein expression profiles and relevant signaling pathways in GD specimens. Immunofluorescence assays were conducted to detect and localize the expressions of CD34 and PROX1 in GD specimens and normal thyroid tissues. M22 stimulated the proliferation of Nthy-ori 3-1 cells and HUVECs in a dose-dependent manner, while TSH exhibited an inverted U-shaped dose-response effect. M22 also dose-dependently promoted angiogenesis, and more effectively induced tube formation in HUVECs compared to TSH, although the difference was not statistically significant. A total of 16 proteins were significantly upregulated and 24 were downregulated in M22-induced HUVECs. Notably, PROX1, the most significantly upregulated protein, is closely associated with angiogenesis. Immunofluorescence confirmed that PROX1 was significantly more expressed in thyroid tissues from GD patients compared to normal tissues adjacent to papillary thyroid cancer (PTC), and it co-localized with CD34. TRAb enhances angiogenesis and upregulates PROX1 expression in HUVECs, suggesting a novel possible mechanism for goiter formation in GD.

Impact of Resolvin-E1 and Maresin-1 on Bone Marrow Stem Cell Osteogenesis under Inflammatory Stress.

Periodontal disease is characterized by inflammation and bone loss. Central to its pathogenesis is the dysregulated inflammatory response, complicating regenerative therapies. Mesenchymal stem cells (MSCs) hold significant promise in tissue repair and regeneration. This study investigated the effects of specialized pro-resolving mediators (SPMs), Resolvin E1 (RvE1) and Maresin 1 (MaR1), on the osteogenic differentiation of human bone marrow-derived MSCs under inflammatory conditions. The stem cells were treated with SPMs in the presence of lipopolysaccharide (LPS) to simulate an inflammatory environment. Osteogenic differentiation was assessed through alkaline phosphatase activity and alizarin red staining. Proteomic analysis was conducted to characterize the protein expression profile changes, focusing on proteins related to osteogenesis and osteoclastogenesis. Treatment with RvE1 and MaR1, both individually and in combination, significantly enhanced calcified deposit formation. Proteomic analysis revealed the differential expression of proteins associated with osteogenesis and osteoclastogenesis, highlighting the modulatory impact of SPMs on bone metabolism. RvE1 and MaR1 promote osteogenic differentiation of hBMMSCs in an inflammatory environment, with their combined application yielding synergistic effects. This study provides insights into the therapeutic potential of SPMs in enhancing bone regeneration, suggesting a promising avenue for developing regenerative therapies for periodontal disease and other conditions characterized by inflammation-induced bone loss.

Osmotic response in Leptospirillum ferriphilum isolated from an industrial copper bioleaching environment to sulfate

Iron and sulfur-oxidizing microorganisms play important roles in several natural and industrial processes. Leptospirillum (L.) ferriphilum, is an iron-oxidizing microorganism with a remarkable adaptability to thrive in extreme acidic environments, including heap bioleaching processes, acid mine drainage (AMD) and natural acidic water. A strain of L. ferriphilum (IESL25) was isolated from an industrial bioleaching process in northern Chile. This strain was challenged to grow at increasing concentrations of sulfate in order to assess changes in protein expression profiles, cells shape and to determine potential compatible solute molecules. The results unveiled changes in three proteins: succinyl CoA (SCoA) synthetase, isocitrate dehydrogenase (IDH) and aspartate semialdehyde dehydrogenase (ASD); which were notably overexpressed when the strain grew at elevated concentrations of sulfate. ASD plays a pivotal role in the synthesis of the compatible solute ectoine, which was identified along with hydroxyectoine by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The relationship between IDH, SCoA, and ectoine production could be due to the TCA cycle, in which both enzymes produce metabolites that can be utilized as precursors or intermediates in the biosynthesis of ectoine. In addition, distinct filamentous cellular morphology in L. ferriphilum IESL25 was observed when growing under sulfate stress conditions. This study highlights a new insight into the possible cellular responses of L. ferriphilum under the presence of high sulfate levels, commonly found in bioleaching of sulfide minerals or AMD environments.

Actin cytoskeletal proteins in mice cortical microvessels decreased with aging

Background: The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and differentiation. The interactions of actin microfilaments with plaque proteins zonula occludens (ZO)-1, -2, -3 are required ensuring the formation of a polarized brain endothelium which ultimately drives development of blood-brain barrier (BBB). In this study, we carried out an extensive evaluation of proteins involved in the structure and function of mouse brain cortical microvessels (MVs) using a discovery-based quantitative proteomic approach. Methods: Cortical MVs were isolated from equal number of age-matched, male and female, young (4–6 months), middle-aged (12–14 months), and old (20–21 months) mice obtained from Jackson Laboratory [Tg(Thy1-EGFP)MJrs/J] and bred in a C57B16J background (n = 6/group). The presence of end-arterioles, capillaries, and venules in MVs was confirmed by light microscopy and by alkaline phosphate staining. Proteomics analysis was performed using liquid chromatography/mass spectrometry. Results: We quantified 4746, 4216, and 4579 differentially expressed proteins by proteomics in brain cortical MVs of young, middle-aged, and old mice, respectively. We found that several actin cytoskeletal-related alpha actinin-1, -2, -3 (ACTN1/2/4), actin-related protein -2, -3, -3B (ACTR2/3/3B), actin-related protein 2/3 complex subunit -1A, -2, -4, -5 (ARPC1A/2/4/5), and [F-actin]-monooxygenase MICAL3 proteins were significantly downregulated in mice cortical MVs with aging. Under physiological conditions, cerebral endothelial cells are connected by tight junctions (TJs) and adherens junctions (AJs). The TJ and AJ proteins are anchored to the actin cytoskeleton by multiple accessory proteins, ZO-1, ZO-2 and ZO-3. Interestingly, we observed that several TJ, AJ, and their signaling (PKC-α/γ/ε, RHOA, RAB3 and YES1) proteins were significantly downregulated in mice cortical MVs with aging. Moreover, we previously documented that oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability and ATP synthesis capacity in mice. Conclusions: We suggest that increased oxidative stress during aging leads to protein expression profile changes of brain cortical MVs that affect ATP synthesis capacity, which leads to actin cytoskeletal dysregulation, and accelerating to endothelial and BBB dysfunction in the brain microvasculature with aging. Funding: AG075988, HL148836, AG063345, AG074489, NS114286, AG047296, HL141143, HL168568, and the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

The epithelial-mesenchymal transition (EMT) and fibroblast activation are major events in idiopathic pulmonary fibrosis pathogenesis. Here, we investigated whether growth arrest-specific protein 6 (Gas6) plays a protective role in lung fibrosis via suppression of the EMT and fibroblast activation. rGas6 administration inhibited the EMT in isolated mouse ATII cells 14 days post-BLM treatment based on morphologic cellular alterations, changes in mRNA and protein expression profiles of EMT markers, and induction of EMT-activating transcription factors. BLM-induced increases in gene expression of fibroblast activation-related markers and the invasive capacity of primary lung fibroblasts in primary lung fibroblasts were reversed by rGas6 administration. Furthermore, the hydroxyproline content and collagen accumulation in interstitial areas with damaged alveolar structures in lung tissue were reduced by rGas6 administration. Targeting Gas6/Axl signaling events with specific inhibitors of Axl (BGB324), COX-2 (NS-398), EP1/EP2 receptor (AH-6809), or PGD2 DP2 receptor (BAY-u3405) reversed the inhibitory effects of rGas6 on EMT and fibroblast activation. Finally, we confirmed the antifibrotic effects of Gas6 using Gas6−/− mice. Therefore, Gas6/Axl signaling events play a potential role in inhibition of EMT process and fibroblast activation via COX-2-derived PGE2 and PGD2 production, ultimately preventing the development of pulmonary fibrosis.

Unveiling the Cell Wall-Targeting Mechanisms and Multifaceted Virulence Modulation by a Eugenol Glycoconjugate against Aspergillus fumigatus: Insights from in vitro and in ovo Studies.

The primary objective of this study was to elucidate the putative cell wall-associated targets of compound 6i, a glycoconjugate of eugenol, in Aspergillus fumigatus, while also evaluating its toxicity and assessing histopathologic alterations in the liver, heart, and kidney of compound 6i-treated embryos using an in ovo model. To achieve this aim, compound 6i was synthesized, and a series of biochemical assays were performed to determine its impact on the fungal cell wall. Additionally, qRT-PCR and LC-MS/MS analyses were conducted to investigate changes in gene and protein expression profiles associated with melanin biosynthesis, conidiation, siderophore production, transcriptional regulation of β-glucan biosynthesis, and calcineurin activity in A. fumigatus. The experimental findings revealed that compound 6i exhibited notable antifungal activity against A. fumigatus by perturbing cell wall integrity, hindering ergosterol, glucan, and chitin biosynthesis, and inhibiting catalase production. Moreover, relative gene expression and proteomic analyses demonstrated that compound 6i exerted both down-regulatory and up-regulatory effects on several crucial genes and proteins involved in the aforementioned fungal processes. Furthermore, increased expression of oxidative stress-related proteins was observed in the presence of compound 6i. Notably, the glycoconjugate of eugenol did not elicit cytotoxicity in the liver, heart, and kidney of chick embryos. The current investigation elucidated the multifaceted mechanisms by which compound 6i exerts its antifungal effects against A. fumigatus, primarily through targeting cell wall components and signaling pathways. These findings underscore the potential of the eugenol glycoconjugate as a promising antifungal candidate, warranting further exploration and development for combating A. fumigatus infections.

Long-term administration of morphine specifically alters the level of protein expression in different brain regions and affects the redox state

Long-term administration of morphine specifically alters the level of protein expression in different brain regions and affects the redox state

Microglial changes in the retina and optic nerve of Parkinson's disease patients

xsanchez@ua.esPatients that suffer from Parkinson disease (PD) also display non‐motor alterations. Among these non‐motor symptoms, there is clinical evidence of visual dysfunction in the pathology. Here, we will describe that there is retinal degeneration in these patients observed as a loss of retinal dopaminergic amacrine cells and intrinsically photosensitive retinal ganglion cells. Also, we describe in the ganglion cell layer the accumulation of phosphorylated alpha‐synuclein in axons and dendrites and the presence of Lewy bodies. Moreover, there is a degeneration of starburst amacrine cells, which are involved in motion direction selectivity. All these pathogenic processes are accompanied with an activation of microglia, which contribute to the retinal and optic nerve neurodegeneration providing a neuroinflammatory environment. By immunohistochemistry and bioinformatic analysis we have found that retinal and optic nerve microglia show morphological and protein expression profile changes in these patients. Taken together, these results demonstrate that in PD, microglia have a key role in the response to the retinal neurodegeneration.

SIRT2 inhibitor SirReal2 enhances anti-tumor effects of PI3K/mTOR inhibitor VS-5584 on acute myeloid leukemiacells.

Acute myeloid leukemia (AML) is a highly aggressive form of cancer that is frequently diagnosed in adults and small molecule inhibitors have gained significant attention as a potential treatment option for AML. The up-regulated genes in AML were identified through bioinformatics analysis. Potential candidate agents were selected through pharmacogenomics analysis. Proteomic experiments were conducted to determine the molecular mechanism after inhibitor treatment. To evaluate drug synergy, both cellular functional experiments and an AML mouse model were used. Through bioinformatics analysis, we conducted a screening for genes that are highly expressed in AML, which led to the identification of nine small-molecule inhibitors. Among these inhibitors, the PI3K/mTOR inhibitor VS-5584 demonstrated significant effectiveness in inhibiting AML cell proliferation at low concentrations. Further testing revealed that VS-5584 induced apoptosis and cycle arrest of AML cells in a dose- and time-dependent manner. Proteomics analysis showed significant changes in protein expression profiles of AML cells after VS-5584 treatment, with 287 proteins being down-regulated and 71 proteins being up-regulated. The proteins that exhibited differential expression were primarily involved in regulating the cell cycle and apoptosis, as determined by GO analysis. Additionally, KEGG analysis indicated that the administration of VS-5584 predominantly affected the P53 and SIRT2 signaling pathways. The use of SIRT2 inhibitor SirReal2 alongside VS-5584 caused a significant reduction in the half-maximal inhibitory concentration (IC50 ) of VS-5584 on AML cells. In vivo, experiments suggested that VS-5584 combined with SirReal2 suppressed tumor growth in the subcutaneous model and extended the survival rate of mice injected with tumor cells via tail vein. Taken together, the PI3K/mTOR inhibitor VS-5584 was effective in suppressing AML cell proliferation. PI3K/mTOR inhibitor combined with SIRT2 inhibitor exhibited a synergistic inhibitory effect on AML cells. Our findings offer promising therapeutic strategies and drug candidates for the treatment of AML.

Neuroprotective mechanisms of defatted walnut powder against scopolamine-induced Alzheimer's disease in mice revealed through metabolomics and proteomics analyses

Ethnopharmacological relevanceDefatted walnut powder (DWP), the byproduct remaining after extracting oil from the walnut kernel, has the actions of nourishing liver and the kidney, replenishing blood, and calming the nerves, which is believed to be a brain-nourishing in Chinese medicine. DWP is rich in phenolic substances with demonstrated anti-inflammatory, antioxidant, lipid-lowering and neuroprotective effects. Despite these promising properties of DWP, its effectiveness in treating Alzheimer's disease (AD) remains unclear, and further research is needed to understand the mechanism of action. Aim of the studyThis study aimed to investigate the potential mechanism of DWP on AD by constructing the overall metabolic profile of mice with an anti-scopolamine AD model and verification of the highly correlated pathway. Materials and methodsThe neuroprotective efficacy of DWP in a mouse model of AD established by scopolamine injection was examined. Spatial memory performance in the Morris water maze (MWM), markers of cholinergic function in hippocampus and cortex, and neuropathological changes were compared among control, model, and DWP-consuming model group mice. In addition, combined metabolomic and proteomic analyses were conducted to investigate changes in metabolite and protein expression profiles in AD model mice induced by DWP consumption. Differentially expressed proteins and metabolites were then analyzed for KEGG pathway enrichment and results confirmed through targeted amino acid metabolomics. ResultsThe results showed that consumption of DWP improved spatial learning and memory in the MWM, enhanced cholinergic function, and reduced histopathological damage in the cortex and hippocampus of AD model mice. Based on differentially abundant metabolites and proteins, 43 metabolic pathways modulated by DWP were identified, mainly involving in amino acid metabolic pathways strongly associated with cellular energetics and antioxidant capacity, and targeted amino acid metabolomics confirmed that DWPE significantly elevated the levels of Arginine (Arg), Histidine (His), Proline (Pro), Serine (Ser), and Tyrosine (Tyr), while reducing the levels of Glutamate (Glu). This ultimately resulted in an improvement in the progression of AD. ConclusionThis study identified numerous metabolic networks modulated by DWP that can mitigate scopolamine-induced AD neuropathology and cognitive dysfunction. DWP is a promising resource to identify AD-related pathogenic pathways and therapeutic strategies.

Procine recombinant NK-lysin inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 and inhibiting oxidative phosphorylation and glycolysis: a proteomic analysis

To investigate the potential mechanisms that mediate the inhibitory effect of porcine recombinant NKlysin (prNK-lysin) against liver cancer cell metastasis. HPLC-tandem mass spectrometry was used to identify the differentially expressed proteins in prNK-lysin-treated hepatocellular carcinoma SMMOL/LC-7721 cells in comparison with the control and PBS-treated cells. GO functional annotation and KEGG pathway analysis of the differentially expressed proteins were performed using GO and KEGG databases. RT-qPCR was used to determine the mRNA expression levels of polypeptide-N-acetylgalactosaminotransferase 13 (GALNT13), transmembrane protein 51 (TMEM51) and FKBP prolyl isomerase 3 (FKBP3) in the cells, and the protein expression of FKBP3 was verified using Western blotting. Proteomic analysis identified 1989 differentially expressed proteins in prNK-lysin-treated cells compared with the control cells, and 2753 compared with PBS-treated cells. Fifteen proteins were differentially expressed between PBS-treated and the control cells, and 1909 were differentially expressed in prNK- lysin group compared with both PBS and control groups. These differentially expressed proteins were involved mainly in the viral process, translational initiation and RNA binding and were enriched mainly in ribosome, protein process in endoplasmic reticulum, and RNA transport pathways. RT-qPCR showed that compared with the control group, prNK-lysin treatment significantly increased the mRNA expressions of GALNT13 (P < 0.05) and TMEM51 (P < 0.01) and lowered FKBP3 mRNA expression (P < 0.05). Western blotting also showed a significantly decreased expression of FKBP3 protein in prNK-lysin-treated cells (P < 0.001). Treatment with prNK-lysin causes significant changes in protein expression profile of SMMOL/LC-7721 cells and inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 protein and affecting the cellular oxidative phosphorylation and glycolysis pathways.

Tear proteomics in dry eye disease.

Dry eye disease (DED) is a multi-factorial ocular surface condition driven by compromised ocular lubrication and inflammation which leads to itching, dryness, and vision impairment. The available treatment modalities primarily target the acquired symptoms of DED including tear film supplements, anti-inflammatory drugs, mucin secretagogues, etc., However, the underlying etiology is still an area of active research, especially in regard to the diverse etiology and symptoms. Proteomics is a robust approach that has been playing major role in understanding the causative mechanism and biochemical changes in DED by identifying the changes in protein expression profile in tears. Tears are a complex fluid composed of several biomolecules such as proteins, peptides, lipids, mucins, and metabolites secreted from lacrimal gland, meibomian gland, cornea, and vascular sources. Over the past two decades, tears have emerged as a bona-fide source for biomarker identification in many ocular conditions because of the minimally invasive and simple sample collection procedure. However, the tear proteome can be altered by several factors, which increases the complexity of the approach. The recent advancements in untargeted mass spectrometry-based proteomics could overcome such shortcomings. Also, these technological advancements help to distinguish the DED profiles based on its association with other complications such as Sjogren's syndrome, rheumatoid arthritis, diabetes, and meibomian gland dysfunction. This review summarizes the important molecular profiles found in proteomics studies to be altered in DED which have added to the understanding of its pathogenesis.

Global and Comparative Proteome Analysis of Nitrogen-Stress Responsive Proteins in the Root, Stem and Leaf of Brassica napus

Nitrogen (N) is one of the basic nutrients and signals for plant development and deficiency of it would always limit the productions of crops in the field. Quantitative research on expression of N-stress responsive proteins on a proteome level remains elusive. In order to gain a deep insight into the proteins responding to nitrogen stress in rapeseed (Brassica napus L.), comparative proteomic analysis was performed to investigate changes of protein expression profiles from the root, stem and leaf under different N concentrations, respectively. More than 200 differential abundance proteins (DAPs) were detected and categorized into groups according to annotations, including “binding and catalytic activity”, “involved in primary metabolism and cellular processes”, “stress-response” and so on. Variation in chlorophyll (Chl) content and antioxidant activities further revealed that oxidative stress raised with the increase of N concentration. Bioinformatics analysis based on the expression level of total proteins suggested these DAPs might play important roles in adaptation to N-stress conditions. Generally, these results provides a new aspect into N-stress responding proteins in Brassica plants.

Quantitative Proteomics Reveals Metabolic Reprogramming in Host Cells Induced by Trophozoites and Intermediate Subunit of Gal/GalNAc Lectins from Entamoeba histolytica.

ABSTRACTEntamoeba histolytica is an intestinal protozoan parasite with remarkable ability to kill and phagocytose host cells, causing amoebic colitis and extraintestinal abscesses. The intermediate subunit (Igl) of galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-specific lectins is considered an important surface antigen involved in the pathogenesis of E. histolytica. Here, we applied mass spectrometry-based quantitative proteomics technology to analyze the protein expression profile changes occurring in host Caco2 cells incubated with E. histolytica trophozoites or stimulated by purified native Igl protein. The expression levels of 1,490 and 489 proteins were significantly altered in the E. histolytica-treated and Igl-treated groups, respectively, among 6,875 proteins totally identified. Intriguingly, central carbon metabolism of host cells was suppressed in both E. histolytica-treated and Igl-treated groups, with evidence of decreased expression levels of several key enzymes, including pyruvate kinase muscle type 2, presenting a Warburg-like effect in host cells. Besides, Igl had potential physical interactions with central carbon metabolism enzymes and the proteolytic degradation family members proteasome subunit alpha and beta, which may be responsible for the degradation of key enzymes in carbon metabolism. These results provided a novel perspective on the pathogenic mechanism of E. histolytica and compelling evidence supporting the important role of Igl in the virulence of E. histolytica.IMPORTANCE Metabolic reprogramming is considered a hallmark of some infectious diseases. However, in amoebiasis, a neglected tropical disease caused by protozoan parasite E. histolytica, metabolic changes in host cells have yet to be proven. In this study, advanced data-independent acquisition mass spectrometry-based quantitative proteomics was applied to investigate the overall host cellular metabolic changes as high-throughput proteomics could measure molecular changes in a cell or tissue with high efficiency. Enrichment analysis of differentially expressed proteins showed biological processes and cellular pathways related to amoeba infection and Igl cytotoxicity. Specifically, central carbon metabolism of host cells was dramatically suppressed in both E. histolytica-treated and Igl-treated groups, indicating the occurrence of a Warburg-like effect induced by trophozoites or Igl from E. histolytica. Distinct differences in ubiquitin-mediated proteolysis, rapamycin (mTOR) signaling pathway, autophagy, endocytosis, and tight junctions provided novel perspectives on the pathogenic mechanism of E. histolytica.

Exposure of Microglia to Interleukin-4 Represses NF-κB-Dependent Transcription of Toll-Like Receptor-Induced Cytokines.

Interleukin (IL)-4 is a cytokine that affects both adaptive and innate immune responses. In the central nervous system, microglia express IL-4 receptors and it has been described that IL-4-exposed microglia acquire anti-inflammatory properties. We here demonstrate that IL-4 exposure induces changes in the cell surface protein expression profile of primary rhesus macaque microglia and enhances their potential to induce proliferation of T cells with a regulatory signature. Moreover, we show that Toll like receptor (TLR)-induced cytokine production is broadly impaired in IL-4-exposed microglia at the transcriptional level. IL-4 type 2 receptor-mediated signaling is shown to be crucial for the inhibition of microglial innate immune responses. TLR-induced nuclear translocalization of NF-κB appeared intact, and we found no evidence for epigenetic modulation of target genes. By contrast, nuclear extracts from IL-4-exposed microglia contained significantly less NF-κB capable of binding to its DNA consensus site. Further identification of the molecular mechanisms that underlie the inhibition of TLR-induced responses in IL-4-exposed microglia may aid the design of strategies that aim to modulate innate immune responses in the brain, for example in gliomas.

Cite-Seq Reveals Distinct Patterns and Potential Mechanisms of Relapse in Pediatric AML

Introduction - Acute myeloid leukemia (AML) is an aggressive disease with a relapse rate of approximately 40% in children. Progress in improving cure rates has been slow, in part because AML is very heterogeneous. Molecular studies consistently show that most cases are comprised of distinct subclones that diminish or expand over the course of therapy. Single-cell profiling methods now allow parsing of the leukemic population into subsets based on gene and/or protein expression patterns. We hypothesized that comparing the features of the subsets that are dominant at relapse with those that are dominant at diagnosis would reveal mechanisms of treatment failure.Methods - We profiled diagnosis-relapse pairs from 6 pediatric AML patients by Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq). All patients were treated at Texas Children's Cancer Center and consented to banking of tissue for research. CITE-Seq was performed by Immunai (New York, NY) using a customized panel of 65 oligonucleotide-tagged antibodies, the 10x Genomics Chromium system for single-cell RNA library generation, and the Novaseq 6000 for sequencing. After data cleanup and normalization, clustering by scRNA-seq was done using the Seurat package. Cell-type identification of clusters was facilitated by published healthy bone marrow scRNA-seq datasets (van Galen et al, Cell 2019). Differentially expressed genes (DEGs) and proteins (DEPs) between diagnosis and relapse were determined using Wilcoxin ranked sum tests.Results - We generated single-cell transcriptomes for a total of 28,486 cells from 12 samples, with a mean of 2373 cells and 1416 genes per sample. Samples were integrated with batch effect correction, producing 30 distinct clusters (cell types) in total (Figure 1A). Cell types with expression profiles consistent with lymphocytes and erythroid precursors were identified in multiple patients, whereas AML cell types tended to be specific to individual patients (Figure 1B). For patients TCH1, TCH2 and TCH3, the most abundant cell types at diagnosis were rare at relapse, and cell types that were rare at diagnosis became dominant at relapse. For these 3 cases, we identified DEGs between the dominant diagnosis cell types and dominant relapse cell types. We found 18 genes that were upregulated at relapse in at least 2 of the cases. Several genes related to actin polymerization were enriched (ARPC1B, ACTB, PFN1), possibly reflecting an enhanced capacity for adhesion and migration. Also of note, macrophage migration inhibitory factor (MIF) and its receptor CD74 were upregulated at relapse, suggesting a role in chemoresistance. For patients TCH4, TCH5 and TCH6, the same cell types that were abundant at diagnosis were also abundant at relapse, and few genes were significantly altered between diagnosis and relapse in multiple cases. Only SRGN, which encodes the proteoglycan serglycin, and GAPDH were altered in 2 of these 3 cases, and both were downregulated at relapse. We performed similar comparisons to identify proteins that were differentially expressed between diagnosis and relapse pairs. The number of DEPs between the dominant diagnosis and relapse cell types ranged from 0 (TCH1 and TCH6) to 5 (TCH2). The only protein altered in more than one case was CD7, which was enriched at relapse in TCH2, TCH3 and TCH4.Conclusions - From CITE-Seq profiling of 6 pediatric AML cases we identified two distinct patterns of relapse. For 3 cases, relapse occurred by expansion of a subset that was small but present at diagnosis. Enrichment of genes associated with adhesion and survival signaling suggests that these cells survived because they were well-equipped to take advantage of interactions with the microenvironment. For 3 other cases, the population that was dominant at diagnosis persisted and expanded at relapse with few substantial changes in gene or protein expression profiles. This pattern suggests that these AML cells were a priori equipped to survive chemotherapy, even though bulk disease levels were transiently reduced below the limit of detection. Most profiled proteins did not change substantially between diagnosis and relapse. An exception is CD7, which was enriched at relapse in 50% of our cases and represents a potential therapeutic target. Analysis of more cases will refine these relapse patterns, reveal potential mechanisms of chemoresistance and inform the development of novel therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Changes in Metabolite and Protein Expression Profiles of Atypical Dark-cutting and Normal-pH Beef

Abstract The color of meat is an important deciding factor in consumers’ assessment of meat quality. To meat buyers, the bright cherry-red color of meat indicates freshness and wholesomeness. However, atypical dark-cutting beef represents dark-colored meat with a muscle pH between 5.6 and 5.8. Although previous studies have indicated that the ultimate pH of atypical dark-cutting beef is greater than normal, the mechanistic basis for the occurrence is not clear. Therefore, the objective of this study was to identify protein and metabolite profiles of normal and atypical dark-cutting beef. Longissimus thoracic (LT) muscles from 12 different animals (6 atypical dark-cutters and 6 normal-pH beef) were analyzed by comparing changes in metabolites and protein profiles using GC-MS non-targeted metabolomics and LC-MS/MS-based proteomics, respectively. Metabolomics analysis showed down-regulation of glycolytic metabolites such as glucose-6-phosphate, lactose, and glucose, indicating a reduced capacity to accumulate lactate postmortem in atypical dark-cutters compared with normal-pH beef. Mass spectrometry analysis identified 66 proteins with significant changes in protein expression profiles (P &amp;lt; 0.05) between atypical and normal-pH beef. Of these, 22 proteins were up-regulated while 14 were down-regulated in atypical dark-cutting beef. Functional annotation and protein-protein interaction network analyses revealed that up-regulated proteins such as NADH ubiquinone oxidoreductase subunit A7, ATPase Na+/K+ transporting subunit alpha 2, 3-oxoacid CoA-transferase 1, cytochrome b5 reductase 3, actinin alpha 4, tropomyosin 2, heat shock protein beta-7, and DnaJ heat shock protein family (Hsp40) member B4 are involved in energy synthesis, muscle contraction and stress responses. By contrast, down-regulated proteins, including phosphofructokinase, glycogen phosphorylase, amylo-alpha-1,6-glucosidase, phosphorylase kinase regulatory subunit alpha and gamma are involved in glycogen metabolism. In summary, lower glycolytic metabolites and glycogen degradation enzymes are associated with differences in muscle pH and color of atypical dark-cutting beef.

Cardiac adaptation and cardioprotection against arrhythmias and ischemia-reperfusion injury in mammalian hibernators

Hibernation allows animals to enter an energy conserving state to survive severe drops in external temperatures and a shortage of food. It has been observed that the hearts of mammalian hibernators exhibit intrinsic protection against ischemia-reperfusion (I/R) injury and cardiac arrhythmias in the winter whether they are hibernating or not. However, the molecular and ionic mechanisms for cardioprotection in mammalian hibernators remain elusive. Recent studies in woodchucks (Marmota monax) have suggested that cardiac adaptation occurs at different levels and mediates an intrinsic cardioprotection prior to/in the winter. The molecular/cellular remodeling in the winter (with or without hibernation) includes (1) an upregulation of transcriptional factor, anti-apoptotic factor, nitric oxide synthase, protein kinase C-ε, and phosphatidylinositol-4,5-bisphosphate 3-kinase; (2) an upregulation of antioxidant enzymes (e.g. superoxide dismutase and catalase); (3) a reduction in the oxidation level of Ca2+/calmodulin-dependent protein kinase II (CaMKII); and (4) alterations in the expression and activity of multiple ion channels/transporters. Therefore, the cardioprotection against I/R injury in the winter is most likely mediated by enhancement in signaling pathways that are shared by preconditioning, reduced cell apoptosis, and increased detoxification of reactive oxygen species (ROS). The resistance to cardiac arrhythmias and sudden cardiac death in the winter is closely associated with an upregulation of the antioxidant catalase and a downregulation of CaMKII activation. This remodeling of the heart is associated with a reduction in the incidence of afterdepolarizations and triggered activities. In this short review article, we will discuss the seasonal changes in gene and protein expression profiles as well as alterations in the function of key proteins that are associated with the occurrence of cardioprotection against myocardial damage from ischemic events and fatal arrhythmias in a mammalian hibernator. Understanding the intrinsic cardiac adaptive mechanisms that confer cardioprotection in hibernators may offer new strategies to protect non-hibernating animals, especially humans, from I/R injury and ischemia-induced fatal cardiac arrhythmias.

Characterization of Clozapine-Responsive Human T Cells.

Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vβ 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.