The silk gland proteome of Stenopsyche angustata provides insights into the underwater silk secretion.

Caddisworms (Trichoptera) spin adhesive silks to construct a variety of underwater composite structures. Many studies have focused on the fibroin heavy chain of caddisworm silk and found that it contains heavy phosphorylation to maintain a stable secondary structure. Besides fibroins, recent studies have also identified some new silk proteins within caddisworm silk. To better understand the silk composition and its secretion process, this study reports the silk gland proteome of a retreat-building caddisworm, Stenopsyche angustata Martynov (Trichoptera, Stenopsychidae). Using liquid chromatography tandem mass spectrometry (LC-MS/MS), 2389 proteins were identified in the silk gland of S. angustata, among which 192 were predicted as secreted silk proteins. Twenty-nine proteins were found to be enriched in the front silk gland, whereas 109 proteins were enriched in the caudal silk gland. The fibroin heavy chain and nine uncharacterized silk proteins were identified as phosphorylated proteins. By analysing the sequence of the fibroin heavy chain, we found that it contains 13 Gly/Thr/Pro-rich regions, 12 Val/Ser/Arg-rich regions and a Gly/Arg/Thr-rich region. Three uncharacterized proteins were identified as sericin-like proteins due to their larger molecular weights, signal peptides and repetitive motifs rich in serine. This study provides valuable information for further clarifying the secretion and adhesion of underwater caddisworm silk.

Identification of key genes and miRNA-mRNA regulatory networks associated with bone marrow immune microenvironment regulations in multiple myeloma by integrative bioinformatics analysis.

The deregulation of microRNAs (miRNAs) and genes in the bone marrow microenvironment have been involved with the pathogenesis of multiple myeloma (MM). However, the exploration of miRNA-mRNA regulatory networks in MM remains lacking. We used GSE125363, GSE125361, GSE47552, GSE2658, GSE136324, GSE16558, and GSE13591 datasets for this bioinformatics study. We identified 156 downregulated and 13 upregulated differentially expressed miRNAs (DEmiRs) in MM. The DEmiRs are associated with the enrichment of pathways mainly involved with cancers, cellular signaling, and immune regulations. We identified 112 hub genes associated with five significant clusters in MM. Moreover, we identified 9 upregulated hub genes (such as IGF1, RPS28, UBA52, CDKN1A, and CDKN2A) and 52 downregulated hub genes (such as TP53, PCNA, BRCA1, CCNB1, and MSH2) in MM that is targeted by DEmiRs. The expression of DEmiRs targeted two hub genes (CDKN2A and TP53) are correlated with the survival prognosis of MM patients. Furthermore, the expression level of CDKN2A is correlated with immune signatures, including CD4+ Regulatory T cells, T cell exhaustion, MHC Class I, immune checkpoint genes, macrophages, neutrophils, and TH2 cells in the TME of MM. Finally, we revealed the consistently deregulated expression level of key gene CDKN2A and its co-regulatory DEmiRs, including hsa-mir-192, hsa-mir-10b, hsa-mir-492, and hsa-mir-24 in the independent cohorts of MM. Identifying key genes and miRNA-mRNA regulatory networks may provide new molecular insights into the tumor immune microenvironment in MM.

Bioinformatics analysis deciphering the transcriptomic signatures associated with signalling pathways and prognosis in the myelodysplastic syndromes.

BACKGROUND: Several studies scatteredly identified the myelodysplastic syndromes' transcriptomic profiles (MDS). However, the exploration of transcriptional signatures, key signalling pathways, and their association with prognosis and diagnosis in the integrated multiple datasets remains lacking. METHODS: We integrated the GSE4619, GSE19429, GSE30195, and GSE58831 microarray datasets of CD34 + cells for identifying the differentially expressed genes (DEGs) in the MDS. The series of bioinformatics methods are applied to identify the key hub genes, gene clusters, prognostic hub genes, and genes associated with diagnostic efficacy. Finally, we validated the expression differences of hub genes in the GSE114922 dataset. RESULTS: We explored the DEGs related to gene ontology enrichment and KEGG pathways. We identified significant hub genes, including 168 upregulated hub genes (such as STAT1, IFIH1, EPRS, GRB2, RAC2, MAPK14, CASP1, and SPI1) and 52 downregulated hub genes (such as CREBBP, HIF1A, PIK3CA, EZH2, PIK3R1, MDM2, IRF4, CXCR4, PCNA, and CD19) in the MDS. In addition, we identified six significant molecular complex detection (MCODE)-derived upregulated gene clusters and one downregulated gene cluster, respectively. Moreover, we found that the higher expression level of MX2, GBP2, PXN, IFI44, FDXR, PLCB2, ASS1, ERCC4, PML, and RRAGD and the lower expression level of CD19, PAX5, TCF3, LEF1, NUSAP1, and TIMELESS hub genes are significantly correlated with shorter survival times of MDS patients. Furthermore, the area value under the ROC curve (AUC) of PXN, FDXR, PLCB2, PML, CD19, PAX5, and LEF1 prognostic genes are more than 0.80, indicating that these genes could be effectively used for the diagnostic efficacy of MDS patients. CONCLUSIONS: Identifying key hub genes and their association with the prognosis and diagnostic efficacy may provide substantial clues for the treatment and diagnosis of MDS patients.

[Characteristics and Clinical Significance of Gene Mutation in Patients with Myelodysplastic Syndrome].

OBJECTIVE: To investigate the characteristics of gene mutations in patients with myelodysplastic syndromes (MDS) and its prognostic significance. METHODS: High-throughput sequencing was used to detect 34 blood tumor-related genes in 210 patients with MDS, and the relationship with the revised International Prognostic Scoring System (IPSS-R) and the impact on prognosis of the patients were analyzed. RESULTS: Among the 210 MDS patients, 142 cases (67.6%) showed mutations, and the first six genes with the highest mutation detection rate were ASXL1(20.5%), TET2(17.1%), U2AF1(14.3%), DNMT3A (11.9%), TP53(10.5%) and RUNX1(10.0%). The gene mutation rate of the patients in IPSS-R relatively high-risk group was higher than those in relatively low-risk group (P=0.001). Both TP53 and BCOR genes showed higher mutation rates in the higher risk group than in the lower risk group (P<0.05). Survival time of the patients in TP53 mutant group was lower than those in non-mutant group (P<0.001), survival time of patients in SF3B1 mutant group was higher than those in non-mutant group (P=0.018). According to the number of gene mutations, the patients could be divided into groups with 0-1, 2 and ≥3 gene mutations, and the median OS of the three groups were not reached, 43 and 27 months, respectively (P=0.004). The Multivariate analysis showed that the increasing number of gene mutations and TP53 mutation was the independent risk factors affecting prognosis of the patients, while SF3B1 mutation was the independent protective factor for the prognosis of the patients. CONCLUSION: The gene mutation rate was higher in MDS patients. And the increasing numbers of gene mutation, TP53 and SF3B1 were the influence factors of prognosis in the patients.

Synergistic inhibitory effect of Smo inhibitor jervine and its combination with decitabine can target Hedgehog signaling pathway to inhibit myelodysplastic syndrome cell line.

OBJECTIVE: Hypomethylating agents (HMAs) have been reported to target the Sonic Hedgehog (Shh) signaling pathway in myelodysplastic syndrome (MDS). However, the synergistic inhibitory effect of Smo inhibitor jervine and its combination with decitabine in MUTZ-1 cell lines remains lacking. METHODS: We used a CCK-8 assay to detect the in-vitro proliferation rate of MUTZ-1 cell lines. Besides, the Annexin V-FITC/PI double staining flow cytometry was utilized to detect the apoptosis rate and cell cycle changes. The expression levels of mRNA were quantified by using qRT-PCR, and the western blot was employed to detect the expression of proteins. RESULTS: We found that the single-agent jervine or decitabine can significantly inhibit the proliferation rate of MUTZ-1 cell lines, and this inhibitory effect is time-dependent and concentration-dependent. The combined intervention of the jervine and decitabine can more significantly inhibit cell proliferation, induce cell apoptosis, and block the G1 phase of the cell cycle. The combined intervention of the two drugs significantly reduced Smo and G1i-1 mRNA expression in MUTZ-1 cells. Furthermore, after combining both of the drug treatments, the proteins levels of Smo, G1i-1, PI3K, p-AKT, Bcl2, and Cyclin Dl were significantly downregulated, and Caspase-3 is upregulated, indicating that jervine with its combination of decitabine might be effective for controlling the proliferation, apoptosis, and cell cycle. CONCLUSION: The Smo inhibitor jervine and its combination with decitabine have a synergistic effect on the proliferation, cell cycle, and apoptosis of MUTZ-1 cells, and its mechanism may be achieved by interfering with the Shh signaling pathway.

[Expression and Significance of Shh Signaling Pathway in Bone Marrow CD34+ Cells of Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia with Myelodysplasia-Related Changes].

OBJECTIVE: To study the expression of Shh singaling related gene, including Shh, Ptch1, Smo and Gli1 in bone marrow CD34+ cells of patients with myelodysplastic syndrome(MDS) and acute myeloid leukemia with myelodysplasia-related changes(AML-MRC), and to explore their clinical significance. METHODS: The count of CD34+ cells in bone marrow was detected by flow cytometry in 53 patients with MDS and 30 patients with AML-MRC. Magnetic beads were used to separate CD34+ cells. The expression of Shh, Ptch1,Smo and Gli1 on CD34+ cells was detected by qRT-PCR, the effect of the 4 gene expression on prognosis of patients in MDS and AML-MRC group was compared, 25 patients with iron-deficiency anemia were used as controls. RESULTS: The expression levels of Shh, Smo and Gli1 of patients in MDS and AML-MRC group were significantly higher than those in control group (P＜0.05), moreover increased with disease progression(P<0.05). The expression of Ptch1 was not statistically significantly different between 3 groups（P＞0.05）. In comparison of prognosis, the expression of Smo and Gli1 in the patients of relatively high risk groups and AML-MRC groups were significantly increased (P<0.05). The median overall survival time of patients in MDS and AML-MRC groups was 12（7.5，16.5） and 6（3.0，9.0） months (P=0.000) respectively. The median survival time of MDS and AML-MRC patients with high expression of Smo and Gli1 was significantly shorter than that of MDS and AML-MRC patients with low expression of Smo and Gli1(P<0.05). CONCLUSION: Shh signaling pathway in the patients with MDS is activated, which is involved in the progress and prognosis of MDS.

[Effect of Sonic Hedgehog Signal Pathway Inhibitor Jervine on Myelodysplastic Syndromes MUTZ-1 Cells].

OBJECTIVE: To study the effect of SMO inhibitor (Jervine) on proliferation, apoptosis and cell cycle of MDS cell line MUTZ-1, and its mechanism. METHODS: The effect of different concentrations Jervine on proliferation of MUTZ-1 cells was detected by CCK-8 method. Apoptosis and cell cycle of MUTZ-1 cells were detected by flow cytometry. Western blot was used to detect the changes of Shh signaling pathway effecting proteins BCL2 and CyclinD1. The expression levels of Smo and Gli1 gene were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). RESULTS: Jervine inhibited MUTZ-1 cell proliferation in a concentration dependent manner (24 h, r=-0.977), the apoptosis rate of MUTZ-1 cells increased with the enhancement of concentration of Jervine in MUTZ-1 cells (P＜0.001), the cell proportion of G1 phase increased and the cell number of S phase decreased with enhancement of concentration (P＜0.001). The result of RT-qPCR and Western blot showed that the expression of Smo, Gli1 mRNA and BCL2, CyclinD1 proteins decreased (P＜0.05). CONCLUSION: SMO inhibitor can effectively inhibit the growth of MDS cell line MUTZ-1 improve the cell apoptosis and induce cell cycle arrest. Its action mechanism may be related with dowm-regulating the expression of BCL2 and CyclinD1.

[Expression Level and Clinical Significance of Gli1 in Patients with Myelodysplastic Syndrome].

OBJECTIVE: To study the expression level and clinical significance of Gli1 gene in patients with myelodysplastic syndrome(MDS). METHODS: The positive rate of bone marrow CD34+ cells was detected by flow cytometry in 53 patients with MDS.Magnetic beads were used to separate CD34+ cells. The expression of Gli1 on CD34+ cells was detected by RT-qPCR, 25 patients with iron deficiency anemia were selected as controls. The relationship of Gli1 expression with clinical characteristics were analyzed. RESULTS: The expression of Gli1 in patients with MDS (0.73±1.26) was significantly higher than that in the control group (0.07±0.46) (P＜0.05). The expression of Gli1 significantly correlated with platelet count, chromosome grouping and IPSS risk stratification (P＜0.05). The median overall survival time of patients in high and low expression groups were 7 and 20 months respectively (P＜0.05). Multivariate analysis showed that Gli1 and chromosome grouping were 2 independent poor prognostic factors (P＜0.05). CONCLUSION: The expression of Gli1 is high in MDS. Abnormal expression of Gli1 positively correlates with clinical characteristics and prognosis of patients.Gli1 may be involved in the occurrence and development of MDS.

Endoplasmic reticulum stress-induced iRhom2 up-regulation promotes macrophage-regulated cardiac inflammation and lipid deposition in high fat diet (HFD)-challenged mice: Intervention of fisetin and metformin.

Endoplasmic reticulum stress (ERS) has been implicated in obesity-associated cardiac remodeling and dysfunction. Inactive rhomboid protein 2 (iRhom2), also known as Rhbdf2, is an inactive member of the rhomboid intramembrane proteinase family, playing an essential role in regulating inflammation. Nevertheless, the role of ERS-meditated iRhom2 pathway in metabolic stress-induced cardiomyopathy remains unknown. In the study, we showed that 4-PBA, as an essential ERS inhibitor, significantly alleviated high fat diet (HFD)-induced metabolic disorder and cardiac dysfunction in mice. Additionally, lipid deposition in heart tissues was prevented by 4-PBA in HFD-challenged mice. Moreover, 4-PBA blunted the expression of iRhom2, TACE, TNFR2 and phosphorylated NF-κB to prevent HFD-induced expression of inflammatory factors. Further, 4-PBA restrained HFD-triggered oxidative stress by promoting Nrf-2 signaling. Importantly, 4-PBA markedly suppressed cardiac ERS in HFD mice. The anti-inflammation, anti-ERS and anti-oxidant effects of 4-PBA were verified in palmitate (PAL)-incubated macrophages and cardiomyocytes. In addition, promoting ERS could obviously enhance iRhom2 signaling in vitro. Intriguingly, our data demonstrated that PAL-induced iRhom2 up-regulation apparently promoted macrophage to generate inflammatory factors that could promote cardiomyocyte inflammation and lipid accumulation. Finally, interventions by adding fisetin or metformin significantly abrogated metabolic stress-induced cardiomyopathy through the mechanisms mentioned above. In conclusion, this study provided a novel mechanism for metabolic stress-induced cardiomyopathy pathogenesis. Therapeutic strategy to restrain ROS/ERS/iRhom2 signaling pathway could be developed to prevent myocardial inflammation and lipid deposition, consequently alleviating obesity-induced cardiomyopathy.

Prolonged PM2.5 exposure elevates risk of oxidative stress-driven nonalcoholic fatty liver disease by triggering increase of dyslipidemia.

An increasing number of studies have shown that air pollution containing particulate matter (PM) ≤ 2.5 µm (PM2.5) plays a significant role in the development of metabolic disorder and other chronic diseases. Inflammation and oxidative stress caused by metabolic syndrome are widely determined to be critical factors in the development of nonalcoholic fatty liver disease (NAFLD) pathogenesis. However, there is no direct evidence of this, and the underlying molecular mechanism is still not fully understood. In this study, we investigated the role of inflammation and oxidative stress caused by prolonged PM2.5 exposure in dyslipidemia-associated chronic hepatic injury, and further determined whether an increase in hepatic inflammation and oxidative stress promoted lipid accumulation in the liver, ultimately increasing the risk of NAFLD. Therefore, we studied changes in indicators of metabolic disorder and in symbolic indices of NAFLD. We confirmed increases in insulin resistance, glucose tolerance, peripheral inflammation and dysarteriotony in PM2.5-induced mice. Oxidative stress and inflammatory response in the liver caused by PM2.5 inhalation contributed to abnormal hepatic function, further promoting lipid accumulation in the liver. Moreover, we observed inhibition of oxidative stress and inflammatory response by pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) in vitro, suggesting that oxidative stress and inflammatory in liver cells aggravated by PM2.5 contributed to hepatic injury by altering normal lipid metabolism. These results indicate a new goal for preventing and treating air pollution-induced diseases: suppression of oxidative stress and inflammatory response.

Activated iRhom2 drives prolonged PM2.5 exposure-triggered renal injury in Nrf2-defective mice.

Research suggests that particulate matter (PM2.5) is a predisposing factor for metabolic syndrome-related systemic inflammation and oxidative stress injury. TNF-α as a major pro-inflammatory cytokine was confirmed to participate in various diseases. Inactive rhomboid protein 2 (iRhom2) was recently determined as a necessary regulator for shedding of TNF-α in immune cells. Importantly, kidney-resident macrophages are critical to inflammation-associated chronic renal injury. Podocyte injury can be induced by stimulants and give rise to nephritis, but how iRhom2 contributes to PM2.5-induced renal injury is unclear. Thus, we studied whether PM2.5 causes renal injury and characterized iRhom2 with respect to TNF-α release in mice macrophages and renal tissues in long-term PM2.5-exposed mouse models. After long-term PM2.5 exposures, renal injury was confirmed via inflammatory cytokine, chemokine expression, and reduced antioxidant activity. Patients with kidney-related diseases had increased TNF-α, which may contribute to renal injury. We observed up-regulation of serum creatinine, serum urea nitrogen, kidney injury molecule 1, uric acid, TNF-α, MDA, H2O2, and O2- in PM2.5-treated mice, which was greater than that found in Nrf2-/- mice. Meanwhile, increases in metabolic disorder-associated indicators were involved in PM2.5-induced nephritis. In vitro, kidney-resident macrophages were observed to be critical to renal inflammatory infiltration and function loss via regulation of iRhom2/TACE/TNF-α signaling, and suppression of Nrf2-associated anti-oxidant response. PM2.5 exposure led to renal injury partly by inflammation-mediated podocyte injury. Reduced SOD1, SOD2, Nrf2 activation, and increased XO, NF-κB activity, TACE, iNOS, IL-1ß, TNF-α, IL-6, MIP-1α, Emr-1, MCP-1, and Cxcr4, were also noted. Long-term PM2.5 exposure causes chronic renal injury by up-regulation of iRhom2/TACE/TNF-α axis in kidney-resident macrophages. Overexpression of TNF-α derived from macrophages causes podocyte injury and kidney function loss. Thus, PM2.5 toxicities are related to exposure duration and iRhom2 may be a potential therapeutic renal target.

Multicombination Approach Suppresses Listeria monocytogenes-Induced Septicemia-Associated Acute Hepatic Failure: The Role of iRhom2 Signaling.

The mortality rate of acute liver failure significantly increases due to fatal septicemia. Inactive rhomboid protein 2 (iRhom2) is an essential regulator of shedding TNF-α by trafficking with TNF-α converting enzyme (TACE). Fisetin, a flavonoid present in various fruits and plants, possesses anti-oxidative stress and anti-inflammatory activities. Here, multi-combination nanoparticles Fe@Au conjugated with fisetin, iRhom2 small interfering RNA (siRNA), and TNF-α inhibitor (FN) are prepared to examine their effects on fatal septicemia-associated hepatic failure induced by Listeria monocytogenes (LM) in mice and to reveal the underlying mechanisms. After LM infection, upregulation of glutamic-oxalacetic transaminease, glutamic-pyruvic transaminase, alkaline phosphatase, TNF-α, malondialdehyde, H2 O2 , and O2- is observedcompared to FN-treated mice. The iRhom2/TACE/TNF-α signals are enhanced in vivo and in vitro, resulting in oxidative stress, which is especially associated with the activation of kupffer cells and other macrophages. Decrease in Nrf2 activation and increase of inflammation-associated regulators are also noted in vivo and in vitro. Furthermore, overexpression of TNF-α derived from macrophages aggravates hepatic failure. Inversely, the processes above are restored by FN nanoparticles through the regulation of the iRhom2/TACE/TNF-α axis and Nrf2 activation. These findings suggest that FN may be a potential approach to protect against bacterial septicemia-related diseases by targeting iRhom2.

iRhom2 deficiency relieves TNF-α associated hepatic dyslipidemia in long-term PM2.5-exposed mice.

Accumulating researches reported that particulate matter (PM2.5) is a risk factor for developing various diseases, including metabolic syndrome. Recently, inactive rhomboid protein 2 (iRhom2) was considered as a necessary modulator for shedding of tumor necrosis factor-α (TNF-α) in immune cells. TNF-α, a major pro-inflammatory cytokine, was linked to various pathogenesis of diseases, including dyslipidemia. Here, wild type (WT) and iRhom2-knockout (iRhom2-/-) mice were used to investigate the effects of iRhom2 on PM2.5-induced hepatic dyslipidemia. The hepatic histology, inflammatory response, glucose tolerance, serum parameters and gene expressions were analyzed. We found that long-term inhalation of PM2.5 resulted in hepatic steatosis. And a significant up-regulation of iRhom2 in liver tissues was observed, accompanied with elevated TNF-α, TNF-α converting enzyme (TACE), TNFα receptor (TNFR)2 and various inflammatory cytokines expressions. Additionally, PM2.5 treatment caused TG and TC accumulation in serum and liver, probably attributed to changes of genes modulating lipid metabolism. Intriguingly, hepatic injury and dyslipidemia were attenuated by iRhom2-/- in mice with PM2.5 challenge. In vitro, iRhom2-knockdwon reduced TNF-α expressions and its associated inflammatory cytokines in Kupffer cells, implying that liver-resident macrophages played an important role in regulating hepatic inflammation and lipid metabolism in cells treated with PM2.5. The findings indicated that long-term PM2.5 exposure caused hepatic steatosis and dyslipidemia through triggering inflammation, which was, at least partly, dependent on iRhom2/TNF-α pathway in liver-resident macrophages.