Hematological System Development Research Articles

AMD3100-Mediated CXCR4 Inhibition Impairs Development of Primary Lymphoma of the Central Nervous System

A hallmark of primary lymphoma of the central nervous system (CNS; PCNSL) is the strong CXCR4 expression of the tumor cells, the function of which is still unknown. Invitro treatment of BAL17CNS lymphoma cells by AMD3100, which inhibits CXCR4-CXCL12 interactions, resulted in the significantly differential expression of 273 genes encoding proteins involved in cell motility, cell-cell signaling and interaction, hematological system development and function, and immunologic disease. Among the genes down-regulated was the one encoding CD200, a regulator of CNS immunologic activity. These data directly translated into the invivo situation; BAL17CNS CD200 expression was down-regulated by 89% (3% versus 28% CD200+ lymphoma cells) in AMD3100-treated versus untreated mice with BAL17CNS-induced PCNSL. Reduced lymphoma cell CD200 expression may contribute to the markedly increased microglial activation in AMD3100-treated mice. AMD3100 also maintained the structural integrity of blood-brain barrier tight junctions and the outer basal lamina of cerebral blood vessels. Subsequently, lymphoma cell invasion of the brain parenchyma was impaired, and maximal parenchymal tumor size was significantly reduced by 82% in the induction phase. Thus, AMD3100 qualified as a potentially attractive candidate to be included into the therapeutic concept of PCNSL. Beyond therapy, CXCR4-induced suppression of microglial activity is of general neuroimmunologic interest and identifies CD200 expressed by the lymphoma cells as a novel mechanism of immune escape in PCNSL.

Comparative Transcriptome Analyses of Leg Muscle during Early Growth between Geese (Anser cygnoides) Breeds Differing in Body Size Characteristics.

Goose is an important poultry commonly raised for meat. The early growth performance of geese significantly influences their market weight and slaughter weight, affecting the poultry industry's economic benefits. To identify the growth surge between the Shitou goose and the Wuzong goose, we collected the early growth body traits from 0 to 12 weeks. In addition, we investigated the transcriptomic changes in leg muscles at the high growth speed period to reveal the difference between the two geese breeds. We also estimated the growth curve parameters under three models, including the logistic, von Bertalanffy, and Gompertz models. The results showed that except for body length and keel length, the best-fitting model between the body weight and body size of the Shitou and Wuzong was the logistic model. The growth turning points of Shitou and Wuzong were 5.954 and 4.944 weeks, respectively, and the turning point of their body weight was 1459.01 g and 478.54 g, respectively. Growth surge occurred at 2-9 weeks in Shitou goose and at 1-7 weeks in Wuzong goose. The body size traits of the Shitou goose and Wuzong goose showed a trend of rapid growth in the early stage and slow growth in the later stage, and the Shitou goose growth was higher than the Wuzong goose. For transcriptome sequencing, a total of 87 differentially expressed genes (DEGs) were identified with a fold change ≥ 2 and a false discovery rate < 0.05. Many DEGs have a potential function for growth, such as CXCL12, SSTR4, FABP5, SLC2A1, MYLK4, and EIF4E3. KEGG pathway analysis identified that some DEGs were significantly enriched in the calcium signaling pathway, which may promote muscle growth. The gene-gene interaction network of DEGs was mainly related to the transmission of cell signals and substances, hematological system development, and functions. This study can provide theoretical guidance for the production and breeding management of the Shitou goose and Wuzong goose and help reveal the genetic mechanisms underlying diverse body sizes between two goose breeds.

Hematotoxic Effect of Respiratory Exposure to PHMG-p and Its Integrated Genetic Analysis.

Polyhexamethylene guanidine phosphate (PHMG-p), the main ingredient of humidifier disinfectants, circulates systemically through the lungs; however, its toxicological assessment has been primarily limited to pulmonary disease. Herein, we investigated the possible abnormalities in hematopoietic function 20 weeks after intratracheal instillation of PHMG-p in a rat model. Notable abnormalities were found out in the peripheral blood cell count and bone marrow (BM) biopsy, while RNA sequencing of BM tissue revealed markedly altered gene expression. Furthermore, signaling involved in hematopoietic dysfunction was predicted by analyzing candidate genes through Ingenuity Pathway Analysis (IPA) program. Respiratory PHMG-p exposure significantly decreased monocyte and platelet (PLT) counts and total protein, while significantly increasing hemoglobin and hematocrit levels in peripheral blood. Histopathological analysis of the BM revealed a reduced number of megakaryocytes, with no significant differences in spleen and liver weight to body weight. Moreover, PHMG-p exposure significantly activated estrogen receptor signaling and RHOA signaling, and inhibited RHOGDI signaling. In IPA analysis, candidate genes were found to be strongly related to 'hematological system development and function' and 'hematological disease.' Accordingly, our results suggest that PHMG-p could affect hematopoiesis, which participates in monocyte differentiation and PLT production, and may induce hematologic diseases via the respiratory tract.

Antimicrobial Peptides and Biomarkers Induced by Ultraviolet Irradiation Have the Potential to Reduce Endodontic Inflammation and Facilitate Tissue Healing.

Background: Ultraviolet (UV) irradiation can modulate host immune responses and this approach is a novel application for treating endodontic infections and inflammation in root canals. Methods: A dataset of UV-induced molecules was compiled from a literature search. A subset of this dataset was used to calculate expression log2 ratios of endodontic tissue molecules from HEPM cells and gingival fibroblasts after 255, 405, and 255/405 nm UV irradiation. Both datasets were analyzed using ingenuity pathway analysis (IPA, Qiagen, Germantown, MD, USA). Statistical significance was calculated using Fisher’s exact test and z-scores were calculated for IPA comparison analysis. Results: The dataset of 32 UV-induced molecules contained 9 antimicrobial peptides, 10 cytokines, 6 growth factors, 3 enzymes, 2 transmembrane receptors, and 2 transcription regulators. These molecules were in the IPA canonical pathway annotations for the wound healing signaling pathway (9/32, p = 3.22 × 10−11) and communication between immune cells (6/32, p = 8.74 × 10−11). In the IPA disease and function annotations, the 32 molecules were associated with an antimicrobial response, cell-to-cell signaling and interaction, cellular movement, hematological system development and function, immune cell trafficking, and inflammatory response. In IPA comparison analysis of the 13 molecules, the predicted activation or inhibition of pathways depended upon the cell type exposed, the wavelength of the UV irradiation used, and the time after exposure. Conclusions: UV irradiation activates and inhibits cellular pathways and immune functions. These results suggested that UV irradiation can activate innate and adaptive immune responses, which may supplement endodontic procedures to reduce infection, inflammation, and pain and assist tissues to heal.

Mitochondrial supplementation of Sus scrofa metaphase II oocytes alters DNA methylation and gene expression profiles of blastocysts

BackgroundMitochondrial DNA (mtDNA) copy number in oocytes correlates with oocyte quality and fertilisation outcome. The introduction of additional copies of mtDNA through mitochondrial supplementation of mtDNA-deficient Sus scrofa oocytes resulted in: (1) improved rates of fertilisation; (2) increased mtDNA copy number in the 2-cell stage embryo; and (3) improved development of the embryo to the blastocyst stage. Furthermore, a subset of genes showed changes in gene expression. However, it is still unknown if mitochondrial supplementation alters global and local DNA methylation patterns during early development.ResultsWe generated a series of embryos in a model animal, Sus scrofa, by intracytoplasmic sperm injection (ICSI) and mitochondrial supplementation in combination with ICSI (mICSI). The DNA methylation status of ICSI- and mICSI-derived blastocysts was analysed by whole genome bisulfite sequencing. At a global level, the additional copies of mtDNA did not affect nuclear DNA methylation profiles of blastocysts, though over 2000 local genomic regions exhibited differential levels of DNA methylation. In terms of the imprinted genes, DNA methylation patterns were conserved in putative imprint control regions; and the gene expression profile of these genes and genes involved in embryonic genome activation were not affected by mitochondrial supplementation. However, 52 genes showed significant differences in expression as demonstrated by RNAseq analysis. The affected gene networks involved haematological system development and function, tissue morphology and cell cycle. Furthermore, seven mtDNA-encoded t-RNAs were downregulated in mICSI-derived blastocysts suggesting that extra copies of mtDNA affected tRNA processing and/or turnover, hence protein synthesis in blastocysts. We also showed a potential association between differentially methylated regions and changes in expression for 55 genes due to mitochondrial supplementation.ConclusionsThe addition of just an extra ~ 800 copies of mtDNA into oocytes can have a significant impact on both gene expression and DNA methylation profiles in Sus scrofa blastocysts by altering the epigenetic programming established during oogenesis. Some of these changes may affect specific tissue-types later in life. Consequently, it is important to determine the longitudinal effect of these molecular changes on growth and development before considering human clinical practice.

Screening Serum Differential Proteins in Children with Acute Promyelocytic Leukemia Based on iTRAQ Technique

To screen the serum differentially expressed proteins of APL in children. Serum protein expression profiles from 20 cases of normal healthy controls, and 20 cases of APL patients were detected by iTRAQ (isobaric tag for relative and absolute quantification)labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2DLC-MS/MS), and analyzed by bioinformatics software. S100A8, LRG1 and SPARC were validated by ELISA. ROC was built by SPSS 20.0 software. Analysis identified 83 differentially expressed proteins in APL serum compared with control according to our defined criteria, of which 33 proteins were up-regulated and 50 proteins were down-regulated (P<0.05)．IPA analysis revealed that these differentially expressed proteins were related to the function of Cellular Movement, Immune Cell Trafficking, Hematological System Development and Function, Cell-To-Cell Signaling and Interaction, Tissue Development, and involved in a variety of signalling Pathways, the most representative pathways including LXR/RXR Activation and Acute Phase Response Signaling. S100A8 and LRG1 were found to be elevated and SPARC was markedly down-regulated in serum of childhood APL when compared to the normal controls as examined by ELISA (P<0.05), which was consistent with the iTRAQ result. The overall predictive accuracy of each protein was reflected by the area under the ROC curve(AUC), S100A8,LRG1 and SPARC with ROC areas of 0.841,1.000 and 0.944 respectively. S100A8,LRG1 and SPARC may be serve as serum candidate biomarkers for pediatric APL.

Endothelial Cell-Selective Adhesion Molecule (ESAM) Plays Important Roles in the Adult-Type Hemoglobin Synthesis during Fetal Erythropoiesis

Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker for HSCs and is highly expressed on the first definitive HSCs. In addition, ESAM expression is functionally essential for adult HSCs because ESAM deficiency causes life-threatening myelo-suppression, especially severe anemia, after administration of 5-FU. In the last ASH annual meeting, we reported that ESAM deletion perturbed the development of definitive hematopoiesis, especially definitive erythropoiesis in the fetal liver (FL). In this study, we precisely analyzed the mechanism via which ESAM deletion affects erythroid lineage development.Although the number and the size of ESAM homozygous (homo) knockout (KO) fetuses were comparable to that of wild type (WT) or heterozygous KO littermates until embryonic day (E) 14.5, some ESAM homo KO fetuses exhibited severe anemia after E15.5 and approximately half of the ESAM homo KO fetuses died before E17.5. Quantitative real-time PCR analyses for assessing the expression of erythropoiesis-related genes in E16.5 ESAM-null FLs revealed significant reduction in mRNA levels for adult globins (Hba and Hbb-b1) and the erythroid-specific isoenzyme of 5-aminolevulinic acid synthase 2 (Alas2). In contrast, transcript levels of erythropoietin (Epo), Epo-receptor, and several transcription factors essential for erythropoiesis, such as Gata1, Klf1, and c-myb, were not reduced by ESAM deletion.Although no apparent malformation was evident in the E14.5 ESAM-null fetuses, the developmental abnormality in erythropoiesis already existed, because the proportion of enucleated erythrocytes was smaller and the number of mature Ter119+ erythroid cells was significantly decreased.Next, we examined the quality of ESAM deficient HSCs in culture and in vivo transplantation. In methylcellulose cultures, although E14.5 LSK CD48- FL cells of ESAM-null mice produced sufficient numbers of BFU-E colonies, the mRNA expression levels of adult globins and Alas2 in the BFU-E colonies produced by ESAM-null HSCs were significantly reduced. In competitive repopulation assays, 4 × 102 LSK CD48- HSCs sorted from CD45.2+ E14.5 ESAM-null or WT FLs were transplanted into lethally irradiated CD45.1+ congenic WT mice with 2 × 105 CD45.1+ bone marrow cells and analyzed after 15 weeks. Although donor chimerism and the number of CD45.2+ HSCs did not differ between the two groups, peripheral blood hemoglobin level in ESAM-null HSC-transplanted recipient mice was significantly reduced.Next, to understand the molecular mechanisms involved in the developmental failure of definitive erythropoiesis in the ESAM-null FL, we conducted RNA-sequencing (RNA-seq) and compared the gene expression patterns of WT and ESAM-null HSCs. We found that the expression of many erythropoiesis-related genes was reduced in ESAM-null HSCs. Among them, the reduction of ALAS2 expression was most remarkable. In addition, transcripts for both adult globins (Hba-a1, Hba-a2 and Hbb-b1) and embryonic globins (Hbb-y and Hbb-bh1) were dramatically decreased. These results corroborated our hypothesis that hemoglobin synthesis is impaired by ESAM deficiency, and further suggested that the cause of developmental failure in the ESAM-null fetuses can be attributed to aberrant gene expression in ESAM-null “definitive” HSCs.We exploited an antibody cross-linking method to address whether ESAM directly affected gene expression profiles in HSCs. WT E14.5 LSK CD48- cells were either untreated or incubated with a rat mAb against mouse ESAM. Then, both cells were incubated with goat anti-rat IgG Abs-conjugated microbeads for 4 h, and were subsequently applied to RNA-seq. The IPA network analysis indicated that “Hematological System Development and Function, Increased Levels of Hematocrit, Molecular Transport” network, which were associated with hemoglobin synthesis including Hbb-bh1, were the most affected. We also found up-regulation of GDF15, which promotes hemoglobin synthesis by suppressing hepcidin secretion.In conclusion, we showed that approximately half of ESAM-null fetuses died between E15.5 and E17.5, mainly due to the disturbance of adult-type hemoglobin synthesis. Although indirect mechanisms could be involved in the anemic phenotype of ESAM-null fetuses, we propose that ESAM-mediated interactions directly modulate the expression pattern of erythropoiesis-related genes in the developing HSCs. DisclosuresYokota:MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Celgene: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; MOCHIDA PHARMACEUTICAL: Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Astellas: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Bristol Myers: Research Funding; Nippon Shinyaku: Research Funding; Kyowa Hakko Kirin: Research Funding; Eisai: Research Funding; Chugai Pharmaceutical: Research Funding; Fujimotoseiyaku: Research Funding; Pfizer: Research Funding; Shionogi: Research Funding; Toyama Chemical: Research Funding.

MicroRNAomes of Cattle Intestinal Tissues Revealed Possible miRNA Regulated Mechanisms Involved in Escherichia coli O157 Fecal Shedding.

Cattle have been suggested as the primary reservoirs of E. coli O157 mainly as a result of colonization of the recto-anal junction (RAJ) and subsequent shedding into the environment. Although a recent study reported different gene expression at RAJ between super-shedders (SS) and non-shedders (NS), the regulatory mechanisms of altered gene expression is unknown. This study aimed to investigate whether bovine non-coding RNAs play a role in regulating the differentially expressed (DE) genes between SS and NS, thus further influencing E. coli O157 shedding behavior in the animals through studying miRNAomes of the whole gastrointestinal tract including duodenum, proximal jejunum, distal jejunum, cecum, spiral colon, descending colon and rectum. The number of miRNAs detected in each intestinal region ranged from 390 ± 13 (duodenum) to 413 ± 49 (descending colon). Comparison between SS and NS revealed the number of differentially expressed (DE) miRNAs ranged from one (in descending colon) to eight (in distal jejunum), and through the whole gut, seven miRNAs were up-regulated and seven were down-regulated in SS. The distal jejunum and rectum were the regions where the most DE miRNAs were identified (eight and seven, respectively). The miRNAs, bta-miR-378b, bta-miR-2284j, and bta-miR-2284d were down-regulated in both distal jejunum and rectum of SS (log2fold-change: −2.7 to −3.8), bta-miR-2887 was down-regulated in the rectum of SS (log2fold-change: −3.2), and bta-miR-211 and bta-miR-29d-3p were up-regulated in the rectum of SS (log2fold-change: 4.5 and 2.2). Functional analysis of these miRNAs indicated their potential regulatory role in host immune functions, including hematological system development and immune cell trafficking. Our findings suggest that altered expression of miRNA in the gut of SS may lead to differential regulation of immune functions involved in E. coli O157 super-shedding in cattle.

Abstract 12683: Genome-wide Mrna Expression Analysis of Acute Psychological Stress Responses

Introduction: Previous studies have examined effects of acute psychological stress in humans on selected panels of genes. This exploratory study aimed to investigate genome-wide transcriptional activity changes in responses to acute psychological stress. Methods: The sample included 40 healthy women who participated in a previous study (22 who had a stress induced experience (experimental group) and 18 who did not (control group)). The psychological stress was induced by the Trier Social Stress Test (TSST). Psychological stress levels (measured by the Subjective Units of Distress Scale (SUDS) and the state anxiety subscale of the Spielberger State-Trait Anxiety Inventory) and hemodynamic changes (measured by blood pressure (BP) and heart rate (HR)) were assessed before and after the TSST. The peripheral blood samples obtained before and after the TSST were processed for mRNA-sequencing. Results: The mean age of the participants was 31 years (SD 11.6). The psychological and hemodynamic stress parameters indicated that the TSST successfully induced moderate but statistically significant levels of acute psychological stress in the participants of the experimental group. Compared with the participants in the control group, six genes ( BCL2L14, FAM3B, HCG26, HLA-F-AS1, LOC101928710 , and SLC22A16 ) were up-regulated and 9 genes ( ATP2C2, CA1, CRYBG3, FBXO9, HBD, SLC39A9, SNCA, STRADB , and TRMT12 ) were down-regulated among those who experienced stress induction. The IPA analysis identified one network of cell-to-cell signaling and interaction, cellular function and maintenance, and hematological system development and function. Thirteen genes out of 15 were found in this network. Conclusions: The identified genes including FAM3B, BCL2L14, SLC22A16 , and SNCA , have been proposed as the therapeutic targets of diabetes-related CVDs, certain cancers, and Parkinson’s disease, but have not previously been reported to be associated with psychological stress. Future studies are suggested to examine the pathological mechanisms by which the identified genes may mediate the association between psychological stress and adverse health outcome.

Comparative analysis of high altitude hypoxia induced erythropoiesis and iron homeostasis in Indian and Kyrgyz lowlander males

Abstract Lowland residents acclimatize to reduced oxygen availability at high altitude by certain functional alterations in body which vary according to the different lifestyle, geographical location, ethnicity etc. In our study, two different ethnic lowlander groups acclimatization response was investigated during high altitude exposure. Overnight fasting blood of Indian (n = 20) and Kyrgyz (n = 20) at baseline and high altitude (4,111 m) day 3, 7, 14, 21 was collected and analysed. High altitude induced erythropoiesis was higher in Indians compared to Kyrgyz. Hemoglobin, hematocrit, red blood cells increased till high altitude day 21 and erythropoietin till day 7 in Indians. Hepcidin decrease was double in Indians compared with Kyrgyz and iron, ferritin and transferrin receptor were found synchronized to maintain circulatory iron for erythropoiesis. Further to validate, RNA sequencing analysis of differentially expressed gene by DAVID hit hematopoietic cell lineage among top up regulated pathway in Indian vs Kyrgyz. IPA analysis disclosed GATA1 as second most top upstream regulator in Indians as compared to Kyrgyz at high altitude day7 and activation of haematological system development and function at HAD14 in Indian vs Kyrgyz. Valedictory qRT-PCR for putative genes like HAMP, BMP6, EPOR, etc. indicated differences in erythroid and iron regulation in both. Our data strongly reflected different erythropoietic response towards altitude exposure in two different ethnic lowlanders.

Cytotoxicity of apigenin toward multiple myeloma cell lines and suppression of iNOS and COX-2 expression in STAT1-transfected HEK293 cells

BackgroundApigenin is one of the most abundant dietary flavonoids that possesses multiple bio-functions. PurposeThis study was designed to determine the influence of apigenin on gene expressions, cancer cells, as well as STAT1/COX-2/iNOS pathway mediated inflammation and tumorigenesis in HEK293-STAT1 cells. Furthermore, the cytotoxic activity toward multiple myeloma (MM) cell lines was investigated. MethodsBioinformatic analyses were used to predict the sensitivity and resistance of tumor cells toward apigenin and to determine cellular pathways influenced by this compound. The cytotoxic and ferroptotic activity of apigenin was examined by the resazurin reduction assay. Additionally, we evaluated apoptosis, and cell cycle distribution, induction of reactive oxygen species (ROS) and loss of integrity of mitochondrial membrane (MMP) by using the flow cytometry analysis. DAPI staining was used to detect characteristic apoptotic features. Furthermore, we verified its anti-inflammatory and additional mechanism of cell death by western blotting. ResultsCOMPARE and hierarchical cluster analyses exhibited that 29 of 55 tumor cell lines were sensitive against apigenin (p < 0.001). The Ingenuity Pathway Analysis data showed that important bio-functions affected by apigenin were: gene expression, cancer, hematological system development and function, inflammatory response, and cell cycle. The STAT1 transcription factor was chosen as target protein on the basis of gene promoter binding motif analyses. Apigenin blocked cell proliferation of wild-type HEK293 and STAT1 reporter cells (HEK293-STAT1), promoted STAT1 suppression and subsequent COX-2 and iNOS inhibition. Apigenin also exhibited synergistic activity in combination with doxorubicin toward HEK293-STAT1 cells. Apigenin exerted excellent growth-inhibitory activity against MM cells in a concentration-dependent manner with the greatest activity toward NCI-H929 (IC50 value: 10.73 ± 3.21 μM). Apigenin induced apoptosis, cell cycle arrest, ferroptosis and autophagy in NCI-H929 cells. ConclusionApigenin may be a suitable candidate for MM treatment. The inhibition of the STAT1/COX-2/iNOS signaling pathway by apigenin is an important mechanism not only in the suppression of inflammation but also in induction of apoptosis.

Alterations in innate immunity and epithelial cell differentiation are the molecular pillars of hidradenitis suppurativa.

The large unmet need of hidradenitis suppurativa/acne inversa (HS) therapy requires the elucidation of disease-driving mechanisms and tissue targeting. Robust characterization of the underlying HS mechanisms and detection of the involved skin compartments. Hidradenitis suppurativa/acne inversa molecular taxonomy and key signalling pathways were studied by whole transcriptome profiling. Dysregulated genes were detected by comparing lesional and non-lesional skin obtained from female HS patients and matched healthy controls using the Agilent array platform. The differential gene expression was confirmed by quantitative real-time PCR and targeted protein characterization via immunohistochemistry in another set of female patients. HS-involved skin compartments were also recognized by immunohistochemistry. Alterations to key regulatory pathways involving glucocorticoid receptor, atherosclerosis, HIF1α and IL17A signalling as well as inhibition of matrix metalloproteases were detected. From a functional standpoint, cellular assembly, maintenance and movement, haematological system development and function, immune cell trafficking and antimicrobial response were key processes probably being affected in HS. Sixteen genes were found to characterize HS from a molecular standpoint (DEFB4, MMP1, GJB2, PI3, KRT16, MMP9, SERPINB4, SERPINB3, SPRR3, S100A8, S100A9, S100A12, S100A7A (15), KRT6A, TCN1, TMPRSS11D). Among the proteins strongly expressed in HS, calgranulin-A, calgranulin-B and serpin-B4 were detected in the hair root sheath, koebnerisin and connexin-32 in stratum granulosum, transcobalamin-1 in stratum spinosum/hair root sheath, small prolin-rich protein-3 in apocrine sweat gland ducts/sebaceous glands-ducts and matrix metallopeptidase-9 in resident monocytes. Our findings highlight a panel of immune-related drivers in HS, which influence innate immunity and cell differentiation in follicular and epidermal keratinocytes as well as skin glands.

Prediction of triptolide targets in rheumatoid arthritis using network pharmacology and molecular docking

Network pharmacology is a novel approach that uses bioinformatics to predict and identify multiple drug targets and interactions in disease. Here, we used network pharmacology to investigate the mechanism by which triptolide acts in rheumatoid arthritis (RA). We first searched public databases for genes and proteins known to be associated with RA, as well as those predicted to be targets of triptolide, and then used Ingenuity Pathway Analysis (IPA) to identify enriched gene pathways and networks. Networks and pathways that overlapped between RA-associated proteins and triptolide target proteins were then used to predict candidate protein targets of triptolide in RA. The following proteins were found to occur in both RA-associated networks and triptolide target networks: CD274, RELA, MCL1, MAPK8, CXCL8, STAT1, STAT3, c-JUN, JNK, c-Fos, NF-κB, and TNF-α. Docking studies suggested that triptolide can fit in the binding pocket of the six top candidate triptolide target proteins (CD274, RELA, MCL1, MAPK8, CXCL8 and STAT1). The overlapping pathways were activation of Th1 and Th2 cells, macrophages, fibroblasts and endothelial cells in RA, while the overlapping networks were involved in cellular movement, hematological system development and function, immune cell trafficking, cell-to-cell signaling and interaction, inflammatory response, cellular function and maintenance, and cell death and survival. These results show that network pharmacology can be used to generate hypotheses about how triptolide exerts therapeutic effects in RA. Network pharmacology may be a useful method for characterizing multi-target drugs in complex diseases.

Exposure of Larval Zebrafish to the Insecticide Propoxur Induced Developmental Delays that Correlate with Behavioral Abnormalities and Altered Expression of hspb9 and hspb11.

Recent studies suggest that organophosphates and carbamates affect human fetal development, resulting in neurological and growth impairment. However, these studies are conflicting and the extent of adverse effects due to pesticide exposure warrants further investigation. In the present study, we examined the impact of the carbamate insecticide propoxur on zebrafish development. We found that propoxur exposure delays embryonic development, resulting in three distinct developmental stages: no delay, mild delay, or severe delay. Interestingly, the delayed embryos all physically recovered 5 days after exposure, but behavioral analysis revealed persistent cognitive deficits at later stages. Microarray analysis identified 59 genes significantly changed by propoxur treatment, and Ingenuity Pathway Analysis revealed that these genes are involved in cancer, organismal abnormalities, neurological disease, and hematological system development. We further examined hspb9 and hspb11 due to their potential roles in zebrafish development and found that propoxur increases expression of these small heat shock proteins in all of the exposed animals. However, we discovered that less significant increases were associated with the more severely delayed phenotype. This raises the possibility that a decreased ability to upregulate these small heat shock proteins in response to propoxur exposure may cause embryos to be more severely delayed.

2149-P: Mitochondrial Akt Modulated Human Embryonic Stem Cell Differentiation Lineages—Analysis with Single Cell RNA Sequencing

How signaling pathways control embryonic stem cell (ESC) differentiation is poorly understood. Recent studies suggested mitochondria metabolism may play a role in the regulation of stem cell differentiation. In this study, we found a novel role for mitochondrial Akt1 (mito-Akt) that modulated human ESC (hESC) differentiation lineage. We observed that Akt was activated and translocated into mitochondria in hESC upon growth factor stimulation. To determine the effect of mito-Akt on hESC differentiation, hESC were transduced with His-tagged mitochondrial-targeting dominant negative Akt1/GFP (Ad-mdnAkt) or GFP adenoviral vector (Ad-GFP). The transgene was expressed in &amp;gt;90% cells, and mitochondria-targeting was verified. Ad-mdnAkt altered hESC respiration, as measured with Seahorse extracellular flux analyzer, and mitochondria proton leak was increased. After 21 days of differentiation in vitro, H&amp;E stain showed Ad-mdnAkt transduced cells formed more organized cell clusters than control ones. Bulk RNA was extracted for qRT-PCR with primer array of representative marker genes, and the results suggested that marker genes for mesoderm and endoderm were up-regulated whereas marker genes for ectoderm were down-regulated when mito-Akt was inhibited by Ad-mdnAkt in hESCs. To further dissect how mito-Akt modulates cell lineage, single-cell RNA seq was analyzed with 10X Genomics platform. Among the 24 clusters identified by t-SNE plot, Ad-mdnAkt cells were over-represented in 5 clusters and under-represented in 4 clusters when compared to the controls. Interestingly, hESCs with Ad-mdnAkt favored lineages toward hematological system and lymphoid tissue development instead of pancreatic progenitor cells. In summary, mito-Akt is a novel modulator of stem cell differentiation, altering mito-Akt signaling during the early phase of hESC differentiation may impact later embryonic development. Disclosure H. Lee: None. Y. Chen: None. H.Y. Lin: None. A. Ta: None. P.H. Wang: None. Y. Chen: None. B. Andersen: None.

Label-free tissue proteomics can classify oral squamous cell carcinoma from healthy tissue in a stage-specific manner

ObjectivesNo prognostic or predictive biomarkers for oral squamous cell carcinoma (OSCC) exist. We aimed to discover novel proteins, altered in OSCC, to be further investigated as potential biomarkers, and to improve understanding about pathways involved in OSCC. Materials and MethodsProteomic signatures of seven paired healthy and OSCC tissue samples were identified using ultra-definition quantitative mass spectrometry, then analysed and compared using Anova, principal component analysis, hierarchical clustering and OPLS-DA modelling. A selection of significant proteins that were also altered in the serum from a previous study (PMID: 28632724) were validated immunohistochemically on an independent cohort (n = 66) to confirm immunopositivity and location within tumour tissue. Ingenuity Pathways Analysis was employed to identify altered pathways. ResultsOf 829 proteins quantified, 257 were significant and 72 were able to classify healthy vs OSCC using OPLS-DA modelling. We identified 19 proteins not previously known to be upregulated in OSCC, including prosaposin and alpha-taxilin. KIAA1217 and NDRG1 were upregulated in stage IVa compared with stage I tumours. Altered pathways included calcium signalling, cellular movement, haematological system development and function, and immune cell trafficking, and involved NF-kB and MAPK networks. ConclusionsWe found a set of proteins reliably separating OSCC tumour from healthy tissue, and multiple proteins differing between stage I and stage IVa OSCC. These potential biomarkers can be studied and validated in larger cohorts.

A Proteomics Based Approach Reveals Differential Regulation of Visceral Adipose Tissue Proteins between Metabolically Healthy and Unhealthy Obese Patients.

Obesity and the metabolic disorders that constitute metabolic syndrome are a primary cause of morbidity and mortality in the world. Nonetheless, the changes in the proteins and the underlying molecular pathways involved in the relevant pathogenesis are poorly understood.In this study a proteomic analysis of the visceral adipose tissue isolated from metabolically healthy and unhealthy obese patients was used to identify presence of altered pathway(s) leading to metabolic dysfunction. Samples were obtained from 18 obese patients undergoing bariatric surgery and were subdivided into two groups based on the presence or absence of comorbidities as defined by the International Diabetes Federation. Two dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was carried out. A total of 28 proteins were identified with a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p ≤ 0.05) between the groups. 11 proteins showed increased abundance while 17 proteins were decreased in the metabolically unhealthy obese compared to the healthy obese. The differentially expressed proteins belonged broadly to three functional categories: (i) protein and lipid metabolism (ii) cytoskeleton and (iii) regulation of other metabolic processes. Network analysis by Ingenuity pathway analysis identified the NFκB, IRK/MAPK and PKC as the nodes with the highest connections within the connectivity map. The top network pathway identified in our protein data set related to cellular movement, hematological system development and function, and immune cell trafficking. The VAT proteome between the two groups differed substantially between the groups which could potentially be the reason for metabolic dysfunction.

Platelets Proteomic Profiles of Acute Ischemic Stroke Patients.

Platelets play a crucial role in the pathogenesis of stroke and antiplatelet agents exist for its treatment and prevention. Through the use of LC-MS based protein expression profiling, platelets from stroke patients were analyzed and then correlated with the proteomic analyses results in the context of this disease. This study was based on patients who post ischemic stroke were admitted to hospital and had venous blood drawn within 24 hrs of the incidence. Label-free protein expression analyses of the platelets’ tryptic digest was performed in triplicate on a UPLC-ESI-qTOF-MS/MS system and ProteinLynx Global Server (v2.5, Waters) was used for tandem mass data extraction. The peptide sequences were searched against the reviewed homo sapiens database (www.uniprot.org) and the quantitation of protein variation was achieved through Progenesis LC-MS software (V4.0, Nonlinear Dynamics). These Label-free differential proteomics analysis of platelets ensured that 500 proteins were identified and 83 of these proteins were found to be statistically significant. The differentially expressed proteins are involved in various processes such as inflammatory response, cellular movement, immune cell trafficking, cell-to-cell signaling and interaction, hematological system development and function and nucleic acid metabolism. The expressions of myeloperoxidase, arachidonate 12-Lipoxygenase and histidine-rich glycoprotein are involved in cellular metabolic processes, crk-like protein and ras homolog gene family member A involved in cell signaling with vitronectin, thrombospondin 1, Integrin alpha 2b, and integrin beta 3 involved in cell adhesion. Apolipoprotein H, immunoglobulin heavy constant gamma 1 and immunoglobulin heavy constant gamma 3 are involved in structural, apolipoprotein A-I, and alpha-1-microglobulin/bikunin precursor is involved in transport, complement component 3 and clusterin is involved in immunity proteins as has been discussed. Our data provides an insight into the proteins that are involved in the platelets’ activation response during ischemic stroke. It could be argued that this study lays the foundation for future mechanistic studies.

Lung IL-33 overexpression does not cause inflammation, but increases allergen sensitivity and disrupts lung morphogenesis

Abstract IL-33 is an IL-1 cytokine family member that promotes inflammatory responses via activation of many immune cell types. IL-33 is expressed in epithelial barrier tissues such as lung epithelium under both resting and inflammatory conditions. Secreted IL-33 plays an important role in type 2 innate immunity and adaptive immune responses. To characterize the roles of IL-33 in tissue and immune homeostasis in lung epithelium, we generated a novel transgenic mouse in which IL-33 was overexpressed only in lung epithelial cells driven by the tetracycline-sensitive Club cell secretory protein (CCSP) promoter. When IL-33 expression was induced in adult mice, overexpression of IL-33 protein approximately 3-fold above normal levels produced no pathological or immunological changes in the lungs. However, these animals developed significant increases in innate type 2 immune responses when exposed to natural or model allergens. When IL-33 expression was induced in pre- and post-natal mice, these mice had increased mortality, which was accompanied by lung hemorrhage and lung lobe malformation without detectable signs of inflammation and immune responses. Furthermore, IL-33 transgenic mice that survived to adulthood exhibited simplified and enlarged alveoli resembling bronchopulmonary dysplasia. Correspondingly, the epithelial cells from IL-33 transgenic mice displayed altered expression of genes involved in tissue and hematological system development. These data indicate dual effects of IL-33 overexpression depending on the developmental stage of the lungs, namely impairment of lung morphogenesis in the neonatal period and heightened sensitivity of innate type 2 immunity in adult mice.

Dietary intake alters gene expression in colon tissue: possible underlying mechanism for the influence of diet on disease.

Although the association between diet and disease is well documented, the biologic mechanisms involved have not been entirely elucidated. In this study, we evaluate how dietary intake influences gene expression to better understand the underlying mechanisms through which diet operates. We used data from 144 individuals who had comprehensive dietary intake and gene expression data from RNAseq using normal colonic mucosa. Using the DESeq2 statistical package, we identified genes that showed statistically significant differences in expression between individuals in high-intake and low-intake categories for several dietary variables of interest adjusting for age and sex. We examined total calories, total fats, vegetable protein, animal protein, carbohydrates, trans-fatty acids, mutagen index, red meat, processed meat, whole grains, vegetables, fruits, fiber, folate, dairy products, calcium, and prudent and western dietary patterns. Using a false discovery rate of less than 0.1, meat-related foods were statistically associated with 68 dysregulated genes, calcium with three dysregulated genes, folate with four dysregulated genes, and nonmeat-related foods with 65 dysregulated genes. With a more stringent false discovery rate of less than 0.05, there were nine meat-related dysregulated genes and 23 nonmeat-related genes. Ingenuity pathway analysis identified three major networks among genes identified as dysregulated with respect to meat-related dietary variables and three networks among genes identified as dysregulated with respect to nonmeat-related variables. The top networks (Ingenuity Pathway Analysis network score >30) associated with meat-related genes were (i) cancer, organismal injury, and abnormalities, tumor morphology, and (ii) cellular function and maintenance, cellular movement, cell death, and survival. Among genes related to nonmeat consumption variables, the top networks were (i) hematological system development and function, nervous system development and function, tissue morphology and (ii) connective tissue disorders, organismal injury, and abnormalities. Several dietary factors were associated with gene expression in our data. These findings provide insight into the possible mechanisms by which diet may influence disease processes.