Identifying the key genes of autism is of great significance for understanding its pathogenesis and improving the clinical level of medicine. In this paper, we use the structural parameters (average degree) of gene correlation networks to identify genes related to autism and study its pathogenesis. Based on the gene expression profiles of 82 autistic patients (the experimental group, E) and 64 healthy persons (the control group, C) in NCBI database, spearman correlation networks are established, and their average degrees under different thresholds are analyzed. It is found that average degrees of C and E are basically separable at the full thresholds. This indicates that there is a clear difference between the network structures of C and E, and it also suggests that this difference is related to the mechanism of disease. By annotating and enrichment analysis of the first 20 genes (MD-Gs) with significant difference in the average degree, we find that they are significantly related to gland development, cardiovascular development, and embryogenesis of nervous system, which support the results in Alter et al.'s original research. In addition, FIGF and CSF3 may play an important role in the mechanism of autism.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, disrupts conservation programs of threatened species such as the white rhinoceros (Ceratotherium simum). Interferon gamma release assays have been developed for the diagnosis of M. bovis infection in rhinoceros, however, the discovery of additional diagnostic biomarkers might improve the accuracy of case detection. The aim of this pilot study was therefore to evaluate a novel unbiased approach to candidate biomarker discovery and preliminary validation. Whole blood samples from twelve white rhinoceros were incubated in Nil and TB antigen tubes of the QuantiFERON® TB Gold (In-Tube) system after which RNA was extracted and reverse transcribed. Using the equine RT² profiler PCR array, relative gene expression analysis of samples from two immune sensitized rhinoceros identified CCL4, CCL8, IL23A, LTA, NODAL, TNF, CSF3, CXCL10 and GPI as upregulated in response to antigen stimulation. Novel gene expression assays (GEAs) were designed for selected candidates, i.e. CCL4, CXCL10 and IFNG, and analysis of QFT-processed samples showed the CXCL10 GEA could distinguish between five M. bovis-infected and five uninfected rhinoceros. These findings confirm the value of the equine RT² profiler PCR array as a useful tool for screening biomarkers for the diagnosis of M. bovis infection in rhinoceros.

Keywords: CCL4; CXCL10; Cytokines; Gene expression; Mycobacterium bovis; White rhinoceros (Ceratotherium simum).

Background: Chronic rhinosinusitis (CRS) without nasal polyps (CRSsNP) is a common disease and is characterized by multiple inflammatory endotypes. However, molecular mechanisms in CRSsNP are poorly understood compared to polypoid CRS.

Objective: To identify mechanisms and biomarkers associated with inflammatory endotypes underpinning CRSsNP.

Methods: Ethmoid tissues and nasal lavage fluids (NLF) were obtained from control patients and patients with CRS. The gene expression profiles were determined by microarray as well as quantitative RT-PCR and expression of proteins was measured by ELISA and Luminex.

Results: Microarray found that 126, 241, and 545 genes were >3-fold and significantly elevated in CRSsNP with type (T) 1 endotype (T1 CRSsNP), T2 CRSsNP, and T3 CRSsNP, respectively, compared to control tissue. Selected identified genes were confirmed by RT-PCR. Gene set enrichment analysis suggested that T1 CRSsNP was associated with IFN-γ-signaling and anti-viral immunity controlled by T cells (Th1 and CD8+), NK cells and antigen presenting cells (APC); T2 CRSsNP was associated with STAT6-signaling and IgE-mediated activation controlled by eosinophils, mast cells, Th2 cells, ILC2 and APC; and T3 CRSsNP was associated with IL-17-signaling, acute-inflammatory response, complement-mediated inflammation and infection controlled by neutrophils, Th17 cells, B cells and APC. Results suggest that T1 (CXCL9 and CXCL10), T2 (eosinophilic proteins and CCL26) and T3 (CSF3) endotypic biomarkers in NLF may be able to distinguish tissue endotypes in CRSsNP.

Conclusions: Inflammatory endotypes in CRSsNP were controlled by different molecular mechanisms. NLF biomarker assays may allow for more precise and personalized medical treatments in CRS.

Keywords: Biomarker; Chronic rhinosinusitis without nasal polyps; Endotype; Transcriptome.

Various pathogens use differing strategies to evade host immune response including modulating the host's epigenome. Here, we investigate if EVs secreted from P. aeruginosa alter DNA methylation in human lung macrophages, thereby potentially contributing to a dysfunctional innate immune response. Using a genome-wide DNA methylation approach, we demonstrate that P. aeruginosa EVs alter certain host cell DNA methylation patterns. We identified 1,185 differentially methylated CpGs (FDR < 0.05), which were significantly enriched for distal DNA regulatory elements including enhancer regions and DNase hypersensitive sites. Notably, all but one of the 1,185 differentially methylated CpGs were hypomethylated in association with EV exposure. Significantly hypomethylated CpGs tracked to genes including AXL, CFB and CCL23. Gene expression analysis identified 310 genes exhibiting significantly altered expression 48 hours post P. aeruginosa EV treatment, with 75 different genes upregulated and 235 genes downregulated. Some CpGs associated with cytokines such as CSF3 displayed strong negative correlations between DNA methylation and gene expression. Our infection model illustrates how secreted products (EVs) from bacteria can alter DNA methylation of the host epigenome. Changes in DNA methylation in distal DNA regulatory regions in turn can modulate cellular gene expression and potential downstream cellular processes.

Keywords: Pseudomonas aeruginosa; DNA methylation; Lung macrophage; extracellular vesicles; innate immunity.

The granulocyte colony-stimulating factor (G-CSF), now referred to as CSF3, is a very important cell growth factor that supports the proliferation, survival, and differentiation of neutrophilic progenitor cells, and also is a strong immune regulator of T cells and a promising therapeutic tool in acute graft versus host disease (GVHD). G-CSF acts by binding to its receptor G-CSFR (also called CSF3R), a member of the cytokine receptor type I superfamily, which after binding with G-CSF activates the canonical Janus kinase (Jak)/signal transducer, activator of transcription (STAT)and Ras/Raf/MAP kinase pathways. G-CSF has been applied to the clinic to treat congenital and acquired neutropenia before or during courses of intensive chemotherapy. It has also been applied to mobilize hematopoietic stem cells into the peripheral blood for Auto-or allogeneic transplantation, and the priming strategies designed to enhance the sensitivity of leukemia stem cells to cytotoxic agents in protocols aimed to induce their differentiation, accompanying growth arrest, and cell death. With the rapid development of molecular genetics and clinical research, CSF3R mutations have been implicated in the progression of severe congenital neutropenia (SCN) to leukemia. Recently, CSF3R mutations have been discovered frequently in chronic neutrophilic leukemia (CNL). Such findings might provide the theoretical basis for the targeted therapy. In this review, the clinical application of G-CSF receptor in hematonosis is briefhy summarized.

Purpose: To explore the consequences of innate interference on intraocular inflammatory responses during Bacillus endophthalmitis.

Methods: Bacillus endophthalmitis was induced in mice. Innate immune pathway activation was interfered by injecting S layer protein-deficient (∆slpA) B. thuringiensis or by treating wild-type (WT)-infected mice with a TLR2/4 inhibitor (WT+OxPAPC). At 10 hours postinfection, eyes were harvested and RNA was purified. A NanoString murine inflammation panel was used to compare gene expression in WT-infected, WT+OxPAPC, ∆slpA-infected, and uninfected eyes.

Results: In WT-infected eyes, 56% of genes were significantly upregulated compared to uninfected controls. Compared to WT-infected eyes, the expression of 27% and 50% of genes were significantly reduced in WT+OxPAPC and ∆slpA-infected eyes, respectively. Expression of 61 genes that were upregulated in WT-infected eyes was decreased in WT+OxPAPC and ∆slpA-infected eyes. Innate interference resulted in blunted expression of complement factors (C3, Cfb, and C6) and several innate pathway genes (TLRs 2, 4, 6, and 8, MyD88, Nod2, Nlrp3, NF-κB, STAT3, RelA, RelB, and Ptgs2). Innate interference also reduced the expression of several inflammatory cytokines (CSF2, CSF3, IL-6, IL-1β, IL-1α, TNFα, IL-23α, TGFβ1, and IL-12β) and chemokines (CCL2, CCL3, and CXCLs 1, 2, 3, 5, 9, and 10). All of the aforementioned genes were significantly upregulated in WT-infected eyes.

Conclusions: These results suggest that interfering with innate activation significantly reduced the intraocular inflammatory response in Bacillus endophthalmitis. This positive clinical outcome could be a strategy for anti-inflammatory therapy of an infection typically refractory to corticosteroid treatment.

Neutropenia and infection are major dose-limiting side effects of chemotherapy. The risk of initial infection and subsequent complications are directly related to the depth and duration of neutropenia. Recent genome-wide association studies identified variants in DARC and CXCL2 genes, and in ORMDL3-GSDMA-CSF3 locus on chromosome 17q21 that influence white blood cell and neutrophil counts in healthy individuals. To investigate whether polymorphisms in these loci in conjunction with chemotherapy may modulate risk of treatment complications, we analyzed 21 SNPs across these genes for an association with chemotherapy-related neutropenia and infection in 286 Caucasian children with acute lymphoblastic leukemia. After correction for multiple testing, DARC polymorphism rs3027012 in 5'-UTR was associated with higher risk of low absolute phagocyte count (APC<500 and <1000 cells per microliter, P=0.001 and P<0.0005, respectively) and hospitalization due to febrile neutropenia (P=0.002). Protective effect was instead seen for DARC rs12075 A to G substitution (P=0.004). The SNP rs3859192 in the GSDMA were associated with hospitalization due to infection (P=0.004); infection was also modulated in the additive manner by the CXCL2 rs16850408 (P=0.002). This study shows for the first time that the variations in DARC, GSDMA and CXCL2 genes may play a role in the onset of chemotherapy complications.

In middle-aged women, the decline of ovarian follicle reserve below a critical threshold marks menopause, leading to hormonal, inflammatory, and metabolic changes linked to disease. The highest incidence and mortality of sporadic ovarian cancer (OC) occur at post-menopause, while OC risk is reduced by full-term pregnancies during former fertile life. Herein, we investigate how parity history modulates the ovarian transcriptome related to such declining follicle pool and systemic inflammation in reproductively-aged mice. Female C57BL/6 mice were housed under multiparous and virgin (nulliparous) breeding regimens from adulthood until estropause. The ovaries were then subjected to follicle count and transcriptional profiling, while a cytokine panel was determined in the sera. As expected, the follicle number was markedly decreased just by aging. Importantly, a significantly higher count of primordial and total follicles was observed in aged multiparous relative to aged virgin ovaries. Consistently, among the 65 genes of higher expression in aged multiparous ovaries, 27 showed a follicle count-like pattern, 21 had traceable evidence of roles in follicular/oocyte homeostasis, and 7 were transforming-growth factor beta (TGF-β)/bone morphogenetic protein (BMP) superfamily members. The remaining genes were enriched in cell chemotaxis and innate-immunity, and resembled the profiles of circulating CXCL1, CXCL2, CXCL5, CSF3, and CCL3, chemokines detected at higher levels in aged multiparous mice. We conclude that multiparity during reproductive life promotes the retention of follicle remnants while improving local (ovarian) and systemic immune-innate surveillance in aged female mice. These findings could underlie the mechanisms by which pregnancy promotes the long-term reduced OC risk observed at post-menopause.

Background: RAS gene family mutations are the most prevalent in thyroid nodules with indeterminate cytology and are present in a wide spectrum of histological diagnoses. We evaluated differentially expressed genes and signaling pathways across the histological/clinical spectrum of RAS-mutant nodules to determine key molecular determinants associated with a high risk of malignancy.

Methods: Sixty-one thyroid nodules with RAS mutations were identified. Based on the histological diagnosis and biological behavior, the nodules were grouped into five categories indicating their degree of malignancy: non-neoplastic appearance, benign neoplasm, indeterminate malignant potential, low-risk cancer, or high-risk cancer. Gene expression profiles of these nodules were determined using NanoString PanCancer Pathways and IO360 panels, and Angiopoietin-2 level was determined by immunohistochemical staining.

Results: Unsupervised cluster analysis exhibited a relatively homogeneous expression pattern with no discrete groups. The analysis of genes that were differentially expressed in the 5 categories as supervising parameters unearthed a significant correlation between the degree of malignancy and genes involved in cell cycle and apoptosis (BAX, CCNE2, CDKN2A, CDKN2B, CHEK1, E2F1, GSK3B, NFKB1, and PRKAR2A), PI3K pathway (CCNE2, CSF3, GSKB3, NFKB1, PPP2R2C, and SGK2), and angiogenesis (ANGPT2 and DLL4). The expression of Angiopoietin-2 by immunohistochemistry also showed the same trend of increasing expression from non-neoplastic appearance to high-risk cancer (p-value <0.001).

Conclusions: The gene expression analysis of RAS-mutant thyroid nodules suggests increasing upregulation of key oncogenic pathways depending on their degree of malignancy and supports the concept of a stepwise progression. The utility of ANGPT2 expression as a potential diagnostic biomarker warrants further evaluation.

Research question: Do endometrial stromal cells from primary infertile patients with severe ovarian endometriosis display differential secretory profiles of inflammation-associated cytokines during the implantation window that may cause infertility?

Design: Forty-eight cytokines were measured in conditioned medium of isolated endometrial stromal cells obtained from primary infertile patients without endometriosis (control group, n = 12) or with stage IV ovarian endometriosis (ovarian endometriosis group, n = 14) using multiplex assays. Key cytokines showing differential secretory profiles were validated using Western immunoblotting. Cellular phenotypic validation was carried out in vitro by comparing proliferation and migration capacity between control (n = 6) and ovarian endometriosis (n = 7) groups.

Results: CCL3, CCL4, CCL5, CXCL10, FGF2, IFNG, IL1RN, IL5, TNFA, and VEGF could be detected only in the conditioned media of stromal cells obtained from the ovarian endometriosis group. Among other cytokines detected in the conditioned media of both groups, CCL2 (P = 0.0018), CSF3 (P = 0.0017), IL1B (P = 0.0066), IL4 (P = 0.036), IL6 (P = 0.0039) and IL13 (P = 0.036) were found to be higher, whereas the concentration of IL18 was lower (P = 0.023) in the ovarian endometriosis group. Concentrations of CCL2, IL1B, IL4 and IL13 in conditioned medium reflected significant diagnostic performance for predicting ovarian endometriosis. Cellular phenotypic validation in vitro revealed an enhanced proliferative phenotype (P = 0.046) with no change in cell migratory capacity of endometrial stromal cells from the ovarian endometriosis group.

Conclusions: Endometrial stromal cells derived from severe ovarian endometriosis samples displayed a hyperinflammatory and hyperproliferative bias in the endometrial stroma during the 'window of implantation' putatively causing loss of fecundability.

Keywords: Cytokines; Endometriosis; Endometrium; Infertility; Inflammation; Stromal cell.

Mixed lineage leukemia (MLL) arises from several KMT2A-gene chromosomal translocations. Shb gene deficiency has been found to exhibit pleiotropic effects in different models of leukemia, and consequently, this study aimed to investigate MLL-AF9-induced leukemia in Shb deficiency. Bone marrow cells from wild type and Shb knockout (KO) mice were transduced with the MLL-AF9 gene. Shb KO MLL-AF9 cells proliferated at an increased rate, exhibited altered expression of certain cytokine genes (Kitl, Csf3, IL6, IL1b) and higher expression of cell cycle genes (Ccnd2, Ccne1). Mice receiving Shb KO MLL-AF9 cells showed longer latency without displaying any difference in rates of leukemic cell proliferation, indicating a dichotomy between the in vitro and in vivo phenotypes. The mice with Shb deficient MLL-AF9 cells had a lower content of leukemic bone marrow cells allowing elevated normal hematopoiesis, explaining the longer latency. Finally, Shb knockout GFP-positive bone marrow cells showed a higher percentage of cells expressing myeloid markers. The result suggests a role of Shb in the progression of leukemia and that the relevance of the Shb gene is context-dependent as inferred from the differences between the in vivo and in vitro responses. These findings help to obtain an increased understanding of human MLL-AF9 leukemia.

Keywords: AML; Cell cycle; Cytokines; FAK; Hematopoiesis; IL-6; Latency; MLL-AF9; SHB.

The hydrogenation energies and singlet-triplet (S-T) splittings at the G3MP2 level of theory have been calculated for a wide range of carbenes. The carbene, :CXY with different substituents (X, Y=H, CN, NC, F, Cl, OH, OCH3 , CH3 , CF3 , SiH3 , SiMe3 , phenyl, CH=O, PH2 , and NH2 ) at the carbenic carbon center, immidazole-based carbenes, Bertrand's carbenes, and Seppelt's CF3 CSF3 were studied. The stable carbenes are singlets with large S-T splittings and with the least exothermic hydrogenation energies. The singlet ground state immidazole-based carbenes are calculated to have the least exothermic hydrogenation energies (-15 to -30 kcal mol-1 ) and the largest S-T gaps. The singlet ground states of the Bertrand carbenes have more exothermic hydrogenation (ca. -67 kcal mol-1 ) energies and much smaller S-T gaps. The more exothermic reaction energies arise due to the need to make the phosphorus planar in the carbene so that it can donate into the empty p-orbital at the carbene carbon center. The bending potential at the carbene carbon center in the Bertrand compounds is very flat with a large XC:Y angle. Seppelt's CF3 CSF3 appears to be energetically similar to the Bertrand system, probably due to the required adjustments for geometric distortions at the sulfur center.

Stress-responsive dihydroxy flavonoids exhibit capability to inhibit the accretion of reactive oxygen species (ROS). The formation of these dihydroxy flavonols is catalyzed by flavonoid hydroxylases which are among the rate limiting enzymes of flavonoid biosynthesis pathway. Although flavonoid hydroxylases have been identified in several plant species but their role in abiotic stress is not explicitly documented. In the present study we report identification of all the flavonoid biosynthesis pathway genes of Crocus sativus and their expression profiling. We also report functional characterization of flavonoid 3' hydroxylase (CsF3'H) and attempt to explore its physiological role in vitro and in planta. The results indicated that CsF3'H is 1608 bp long encoding 535 amino acids. Docking and enzyme kinetic studies revealed that CsF3'H catalyzes hydroxylation of naringenin and dihydrokaempferol to eriodictoyl and dihydroquercetin respectively, but exhibits higher affinity for naringenin. Further, CsF3'H showed comparatively higher expression in floral tissues particularly stigma and its expression was significantly enhanced in response to UV-B, dehydration and salinity stress indicative of its role in stress. The expression of CsF3'H was associated with concomitant accumulation of eriodictoyl and dihydroquercetin. Transient overexpression of CsF3'H in Nicotiana benthamiana leads to the accumulation of substantial amounts of eriodictoyl and dihydroquercetin. Further, it was observed that transient expression of CsF3'H conferred tolerance to UV-B and dehydration stress as was evident from higher chlorophyll and soluble sugar and lower MDA contents. Taken together, these results suggest that CsF3'H confers tolerance to UV-B and dehydration in planta through synthesis of dihydroflavonols.

BACKGROUND

Interleukin-23 (IL-23) is required for T helper 17 (Th17) cell responses and IL-17 production in hepatitis B virus infection. A previous study showed that the IL-23/IL-17 axis aggravates immune injury in patients with chronic hepatitis B virus infection. However, the role of IL-23 in acute liver injury remains unclear.

OBJECTIVE

The purpose of this study was to determine the role of the inflammatory cytokine IL-23 in lipopolysaccharide/d-galactosamine (LPS/GalN)-induced acute liver injury in mice.

METHODS

Serum IL-23 from patients with chronic hepatitis B virus (CHB), acute-on-chronic liver failure (ACLF) and healthy individuals who served as healthy controls (HCs) was measured by ELISA. An IL-23p19 neutralizing antibody or an IL-23p40 neutralizing antibody was administered intravenously at the time of challenge with LPS (10μg/kg) and GalN (400mg/kg) in C57BL/6 mice. Hepatic pathology and the expression of Th17-related cytokines, including IL-17 and TNF-α; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and the stabilization factor Csf3 were assessed in liver tissue.

RESULTS

Serum IL-23 was significantly upregulated in ACLF patients compared with CHB patients and HCs (P<0.05 for both). Serum IL-23 was significantly upregulated in the non-survival group compared with the survival group of ACLF patients, which was consistent with LPS/GalN-induced acute hepatic injury in mice (P<0.05 for both). Moreover, after treatment, serum IL-23 was downregulated in the survival group of ACLF patients (P<0.001). Compared with LPS/GalN mice, mice treated with either an IL-23p19 neutralizing antibody or an IL-23p40 neutralizing antibody showed less severe liver tissue histopathology and significant reductions in the expression of Th17-related inflammatory cytokine, including IL-17 and TNF-α; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and stabilization factors Csf3 within the liver tissue compared with LPS/GalN mice (P<0.05 for all).

CONCLUSIONS

High serum IL-23 was associated with mortality in ACLF patients and LPS/GalN-induced acute liver injury in mice. IL-23 neutralizing antibodies attenuated liver injury by reducing the expression of Th17-related inflammatory cytokines, neutrophil chemoattractants and stabilization factors within the liver tissue, which indicated that IL-23 likely functions upstream of Th17-related cytokine and chemokine expression to recruit inflammatory cells into the liver.

This study aimed at evaluate the effects of different aperture-sized type I collagen/silk fibroin (CSF) scaffolds on the proliferation and differentiation of human dental pulp cells (HDPCs). The CSF scaffolds were designed with 3D mapping software Solidworks. Three different aperture-sized scaffolds (CSF1-<em>CSF3</em>) were prepared by low-temperature deposition 3D printing technology. The morphology was observed by scanning electron microscope (SEM) and optical coherence tomography. The porosity, hydrophilicity and mechanical capacity of the scaffold were detected, respectively. HDPCs (third passage, 1 × 10⁵ cells) were seeded into each scaffold and investigated by SEM, CCK-8, alkaline phosphatase (ALP) activity and HE staining. The CSF scaffolds had porous structures with macropores and micropores. The macropore size of CSF1 to <em>CSF3</em> was 421 ± 27 μm, 579 ± 36 μm and 707 ± 43 μm, respectively. The porosity was 69.8 ± 2.2%, 80.1 ± 2.8% and 86.5 ± 3.3%, respectively. All these scaffolds enhanced the adhesion and proliferation of HDPCs. The ALP activity in the CSF1 group was higher than that in the <em>CSF3</em> groups (<i>P </i><<i> </i>0.01). HE staining showed HDPCs grew in multilayer within the scaffolds. CSF scaffolds significantly improved the adhesion and ALP activity of HDPCs. CSF scaffolds were promising candidates in dentine-pulp complex regeneration.

Keywords: collagen; dentine-pulp complex regeneration; odontogenic differentiation; silk fibroin; tissue engineering.

A flavonoid 3'-hydroxylase from tea plant was engineered to synthesize B-3',4'-dihydroxylated flavones such as eriodictyol, dihydroquercetin and quercetin.

Four articifical P450 constructs harboring both flavonoid 3'-hydroxylase gene from Camellia sinensis (CsF3'H) and P450 reductase gene from Arabidopsis thaliana (ATR1 or ATR2) were introduced into Escherichia coli strains TOP10, DH5α and BL21, resultantly engineering strains S1 to S12. The plasmid pYES-Dest52-CsF3'H harboring CsF3'H gene was introduced into yeast Saccharomyces cerevisiae WAT11 designated as strain S13. The plasmid pES-HIS-CsF3H::AtFLS 9 AA was constructed through fusing flavanone 3-hydroxylase gene from Camellia sinensis (CsF3H) and flavonol synthase gene from Arabidopsis thaliana (AtFLS). Plasmid pES-URA-CsF3'H and pES-HIS-CsF3H::AtFLS 9 AA were then co-introduced into yeast S. cerevisiae WAT11 designated as strain S14.

Strain S6 generated highest bioconversion efficiency at 25℃ among all E. coli strains during 24 h fernentation. Supplemented with 1000 μmol/L naringenin, dihydrokaempferol and kaempferol, the maximum amounts of eriodictyol, dihydroquercetin and quercetin produced by strain S13 were 734.32 μmol/L, 446.07 μmol/L and 594.64 μmol/L respectively. Supplemented with 5 mmol/L naringenin, the maximum amounts of eriodictyol, kaempferol, quercetin, dihydroquercetin and dihydrokaempferol produced by strain S14 were 1412.16 μmol/L, 490.25 μmol/L, 445.75 μmol/L, 66.75 μmol/L and 73.50 μmol/L during 36-48 h fermentaion respectively.

CsF3'H was engineered for biosynthesis of B-3',4'-dihydroxylated flavone.

Background & aims: Magnesium (Mg²⁺) is able to modulate the differentiation and proliferation of cells. Mg²⁺ restriction can trigger neutrophilia, but the processes that result in this change have yet to be investigated and are not fully understood. Hematopoiesis is a complex process that is regulated by many factors, including cytokines and growth factors, and is strongly influenced by nutrient availability. In this context, our objective was to investigate the impact of the short-term restriction of dietary Mg²⁺ on bone marrow hematopoietic and peripheral blood cells, especially in processes related to granulocyte differentiation and proliferation.

Methods: Male C57BL/6 mice were fed a Mg²⁺ restricted diet (50 mg Mg²⁺/kg diet) for 4 weeks. Cell blood count and bone marrow cell count were evaluated. Bone marrow cells were also characterized by flow cytometry. Gene expression and cytokine production were evaluated, and a colony-forming cell assay related to granulocyte differentiation and proliferation was performed.

Results: Short-term dietary restriction of Mg²⁺ resulted in peripheral neutrophilia associated with an increased number of granulocytic precursors in the bone marrow. Additionally, Mg²⁺ restriction resulted in an increased number of granulocytic colonies formed in vitro. Moreover, the Mg²⁺ restricted group showed increased expression of CSF3 and CEBPα genes as well as increased production of G-CSF in association with increased expression of STAT3 protein.

Conclusion: Short-term dietary restriction of Mg²⁺ induces granulopoiesis by increasing G-CSF production and activating the CEBPα and STAT-3 pathways, resulting in neutrophilia in peripheral blood.

Keywords: Granulocyte-colony stimulating factor; Granulopoiesis; Hematopoiesis; Magnesium.

Introduction: The impaired balance between cell proliferation and cell death, followed the inability to receive the death signals, cells push towards the neoplasia pathway. Fibulin 1 (FBLN1) plays a role as a co-factor in the mechanism of action of a protease such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1), which has important roles in angiogenesis, can also act as both tumor suppressor gene (TSG) and an oncogene in the main constituent of the extra-cellular matrix. This preliminary study has investigated the effects of silencing FBLN1 with siRNA on autophagy, proliferation, apoptosis pathways in the MSM cell line.

Material and methods: It was transfected siRNA specific to FBLN1 incubated MSM SPC212 cells, and compared with negative control siRNAs by a real-time polymerase chain reaction. It was determined apoptosis, proliferation, autophagy-related genes in mRNA levels.

Results: It was observed that increased anti-apoptosis genes, such as CASP2, CASP7, DDFA, and BCL2, anti-apoptotic gene, reduced APAF1, CASP8. Proliferation induced through while increased ADAMTS1, CDH1, CDH6, CLDN7, CSF3, MMP7, MMP13 genes. Autophagy increased via increasing MAP1LC3B, ATG-16L1 genes while decreased via suppressed ULK1, and ATG7 genes by silencing FBLN1 with siRNAs (p < 0.05).

Conclusions: Proliferation can be induction with silencing of FBLN1 with siRNA in processing mechanism MSM. It was concluded that FBLN1 could be act as pleiotropic on autophagy, and apoptosis pathways in proliferation processing for MSM. Therefore we think that FBLN1 acts like a TSG. FBLN1 can be considered as a targeted treatment option in advanced stage MSM.

Keywords: apoptosis; autophagy; fibulin 1; mesothelioma; proliferation.

Albizia julibrissin saponin active fraction (AJSAF) is a promising adjuvant candidate, but its innate immune response mechanisms remain unclear. Here, the quadriceps muscles from the mice injected intramuscularly with AJSAF alone or in combination with ovalbumin and avian influenza vaccine (rL-H5) were subjected to gene microarray. Antigen- and AJSAF-related modules with intramodular hub genes were identified and functionally analyzed using weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA). AJSAF induced early innate immune responses at the injection site, characterized by cytokine production and neutrophil recruitment. AJSAF mainly elicited the expression of "Th1 immune response" and "Neutrophils" genes such as CCL2, CXCL1, CXCL5, IL-1β, IL-6, IL-33, S100A8, and S100A9, whereas these two gene sets were negatively enriched for rL-H5. AJSAF-specific long noncoding RNAs MIRT1 and MIRT2 could function as inflammatory mediators, whereas function unknown TINCR was co-expressed with immune response genes including CCL2, CCL4, CCL7, CSF3, CXCL5, IL-33, S100A8, and S100A9. Finally, the innate immune molecular mechanisms of adjuvant action of AJSAF and the potential signatures were proposed. These findings expanded the current knowledge on the mechanisms of action of saponin-based adjuvants.

Keywords: Adjuvant; Albizia julibrissin saponin; Avian influenza vaccine; Innate immune response; Long noncoding RNA; Weighted gene co-expression network analysis.

Esophageal achalasia is characterized by abnormal peristaltic movements of the esophageal body and impaired relaxation of the lower esophageal sphincter (LES). However, its etiology remains unknown. In our previous study, it was shown that in the LES of patients with achalasia, hsv1-miR-H1 was overexpressed, ATG16L1 expression was downregulated and interleukin (IL)-1β levels were upregulated. However, systemic features were not evaluated. Herein, the plasma cytokine levels in patients with achalasia were determined. Plasma was collected from patients at Nagasaki University Hospital between February 2013 and March 2016, both before and after peroral endoscopic myotomy (POEM). Cytokine analysis was performed using plasma collected from 10 healthy individuals (control group) and 12 patients with achalasia using the Bio-Plex Pro™ Human Cytokine 27-plex assay kit. The levels of IL-17, IL-1β, C-C motif chemokine ligand 2, IL-4, IL-5, IL-1ra, IL-7, IL-12, interferon-γ, IL-2, fibroblast growth factor-2, colony-stimulating factor (CSF)2 and CSF3 were significantly higher in patients with achalasia compared with the control subjects. However, the levels did not differ between plasma samples collected before and after POEM. Thus, the occurrence of a cytokine storm was confirmed in the patients with achalasia.

Keywords: achalasia; cytokine storm; peroral endoscopic myotomy.

Laminin-332 pemphigoid is a rare and severe autoimmune blistering disease, caused by IgG autoantibodies targeting laminin-332 in the dermal-epidermal basement zone. Laminin-332 pemphigoid is characterized by variable inflammatory infiltrate and the predominance of non-complement-fixing antibodies. Given these findings, we hypothesized that IgG autoantibodies to laminin-332 directly resulted in keratinocyte expression of inflammatory factors. We performed RNA-seq on primary human keratinocytes treated with IgG from patients with laminin-332 pemphigoid. Genes for numerous cytokines and chemokines were upregulated, including CSF2, CSF3, CXCL1, CXCL5, CXCL3, CXCL8, CXCL10, CXCL1, IL6, IL7, IL15, IL23, IL32, IL37, TGFB2 as well as metalloproteases. Considering the pro-inflammatory and proteolytic effect of autoantibodies from patients with laminin-332 pemphigoid identified in our initial experiment, we next questioned whether the reactivity against specific laminin subunits dictates the inflammatory and proteolytic keratinocyte response. Then, we treated keratinocytes with IgG from a separate cohort of patients with reactivity against individual subunits of laminin-332. We identified upregulation of IL-1α, IL-6, IL-8, CXCL1, MMP9, TSLP, and GM-CSF at the protein level, most notably in keratinocytes treated with IgG from laminin β3-reactive patients. We for the first time demonstrated a pro-inflammatory response, similar to that described in keratinocytes treated with IgG autoantibodies from patients with bullous pemphigoid, providing novel insight into the pathogenesis of laminin-332 pemphigoid and laminin-332 biology.

Keywords: RNA-seq; autoimmune blistering diseases; immunobullous disease; keratinocyte; laminin-332 pemphigoid.

Heterocyclic amines (HCAs) are a set of food contaminants that may exert a cytotoxic effect on human peripheral blood mononuclear cells (PBMC). However, the genetic mechanism underlying the cytotoxicity of HCAs on PBMC has not been investigated. In the study, bioinformatic analysis on gene dataset GSE19078 was performed. The results of weighted correlation network analysis and linear models for microarray and RNA-seq data analysis showed that four gene modules were relevant to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) exposure while one gene module was correlated with 2-amino-3-methyl-3H-imidazo[4,5f]quinoline (IQ) exposure. Gene functional analysis showed that the five modules were annotated mainly with mRNA transcriptional regulation, mitochondrial function, RNA catabolic process, protein targeting, and immune function. Five genes, MIER1, NDUFA4, MLL3, CD53 and CSF3 were recognized as the feature genes for each hub gene network of the corresponding gene module, and the expression of feature genes was observed with a significant difference between the PhIP/IQ samples and the other samples. Our results provide novel genes and promising mechanisms for exploration on the genetic mechanism of HCAs on PBMC.

Keywords: IQ; PBMC; PhIP; WGCNA; hub gene.

Objectives: To analyze the gene expression profile of peripheral blood monocytes in different stages of coronary artery disease (CAD) by transcriptome sequencing, and to explore potential genes and pathway involved in CAD pathogenesis.

Methods: According to the screening of coronary angiography and quality control of blood samples, eight intermediate coronary lesion patients were selected, then eight patients with acute myocardial infarction, and eight patients with normal coronary angiography were matched by age and gender. Transcriptomics sequencing was conducted for the peripheral blood monocytes of these 24 samples by using the Illumina HiSeq high-throughput platform. Then, differentially expressed genes (DEGs) were analyzed. Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, and protein-protein interaction (PPI) network were applied to annotate the potential functions of DEGs.

Results: Compared with the normal coronary angiography group, we identified a total of 169 DEGs in the intermediate coronary lesion group, which were significantly enriched in 59 GO terms and 17 KEGG pathways. Compared with the normal coronary angiography group, we found a total of 2,028 DEGs, which were significantly enriched in 311 GO terms and 20 KEGG pathways in the acute myocardial infarction group. The cross-comparison between normal versus intermediate coronary lesion group, and normal versus acute myocardial infarction group included 98 differential genes with 65 up regulated and 33 down regulated genes, which were significantly enriched in 46 GO terms and 10 KEGG pathways. During the progression of CAD, there was a significant up-regulated expression of CSF3, IL-1A, CCR7, and IL-18, and down-regulated expression of MAPK14. Besides GO items such as inflammatory response was significantly enriched, KEGG analysis showed the most remarkable enrichments in IL-17 signaling pathway and cytokine-cytokine receptor interactions.

Conclusions: Transcriptomics profiles vary in patients with different severity of CAD. CSF3, IL-1A, CCR7, IL-18, and MAPK14, as well as IL-17 signaling pathway and cytokine and cytokine receptor interaction signaling pathway related with inflammatory response might be the potential biomarker and targets for the treatment of coronary artery disease.

Keywords: RNA sequencing; acute myocardial infarction; coronary artery disease; differentially expressed genes; intermediate coronary lesions.

The bacteria-mediated inflammatory conditions adversely affect the osseointegration process of endosseous implants, which can even lead to implant malfunction or failure. Local drug delivery has been designed to exert anti-inflammatory and antibacterial activities, but whether this strategy has an effect on the compromised osseointegration under inflammation has rarely been studied. The present study focused on the osteoinductive efficacy of two known phytoestrogens (bergapten (BP) and quercetin (QE)) on implant sites under multiple bacteria-infected conditions in situ. Furthermore, the gene expression profiles of rat bone mesenchymal stem cells (rBMSCs) treated with BP and QE in the presence of Porphyromonas gingivalis-derived lipopolysaccharide were identified. The results showed that both drugs, especially QE, had significant potentiating effects on promoting osteogenic differentiation of rBMSCs, resisting multiple pathogens, and reducing inflammatory activity. Meanwhile, RNA sequencing analysis highlighted the enriched gene ontology terms and the differentially expressed genes (Vps25, Il1r2, Csf3, Efemp1, and Ccl20) that might play essential roles in regulating the above tri-effects, which provided the basis for the drug delivery system to be used as a novel therapeutic strategy for integrating peri-implant health. Overall, our study confirmed that QE appeared to outperform BP in osteogenesis and bacterial killing but not in anti-inflammation. Moreover, both drugs possess favorable tri-effects and can serve as the pivotal agents for the drug delivery system to boost osseointegration at inflammatory implant sites.

Keywords: anti-inflammation; bergapten; multispecies biofilm; osteogenesis; quercetin.