The Effect of Autophagy Induction in Oncolytic Reovirus Replication in Mesenchymal Stem Cells

Background and Aims: Oncolytic reoviruses can infect and kill malignant cells while sparing their normal counterparts. Reoviral infection can induce or activate autophagy, even though metformin can induce autophagy. Identifying and regulating the cellular pathways important for reovirus replication and oncolysis can improve targeted-biological therapies for cancer. Here, the autophagic process was triggered via metformin, and we investigated the effect of autophagy activation on oncolytic reovirus replication in mesenchymal stem cells as primary cells and L929 cell lines. Materials and Methods: Adipose derived mesenchymal stem cells (AD-MSCs) and L929 cells were treated with metformin and reovirus type-3 strain Dearing (T3D). Twenty-four hours after infection, the viability of AD-MSCs and L929 cells were examined by MTT assay. Also, the effect of metformin-induced autophagy in the reovirus replication in these cells was determined by real-time polymerase-chain-reaction. Results: Our results show that treatment with metformin and reovirus reduced the viability of the cells compared to treatment with metformin or reovirus alone in both cells. Also, coadministration of metformin and reovirus significantly decreased the relative expression level of the Beclin-1 gene compared to treatment with metformin in both cells. However, the expression level of the reovirus L3 gene after treatment with metformin and reovirus in L929 cells increased significantly compared to AD-MSCs. Conclusion: Our data suggest that metformin-induced autophagy enhances reoviral replication in AD-MSCs and L929 cells. These findings represent the role of autophagy induction in facilitating reovirus replication and contribute to a better understanding of reovirus-host interactions.

Effect of Xuezhikang Therapy on Expression of Pulmonary Hypertension Related miR-638 in Patients With Low HDL-C Levels

Objective: Low plasma level of high-density lipoprotein cholesterol (HDL-C) associated with poor outcomes in several cardiovascular diseases, including pulmonary arterial hypertension (PAH). Regulation of miR-638 have been proved to be associated with PAH. The aim of this study was to evaluate the expression of miR-638 after Xuezhikang (XZK) therapy in patients with low HDL-C.Methods: Plasma levels of miR-638 were quantified by real-time polymerase chain reactions in 20 patients with PAH and 30 healthy controls. A total of 40 subjects with low HDL-C were assigned to receive an XZK therapy for 6 months. The miR-638 expression profiles were detected in PAH patients, XZK-treated subjects and lovastatin treated pulmonary arterial smooth muscle cells (PA-SMCs).Results: The relative expression level of miR-638 in the plasma was lower in the PAH patients than that in the controls (p < 0.001). An increase of 11.2% from baseline in the HDL-C level was found after XZK therapy (p < 0.001). The relative expression of miR-638 was increased after XZK treatment (p < 0.01). The changes of miR-638 were inversely associated with baseline HDL-C levels. A significantly reduction in miR-638 expression were found in PDGF-BB-treated hPA-SMCs compared to the control cells, and the pre-treatment of the cells with lovastatin significantly re-gain the expression levels in miR-638.Conclusion: In patients with low HDL-C levels, XZK therapy raised the expression of miR-638, suggesting that the potential therapeutic effect of XZK in PAH patients with low serum HDL-C levels deserves further exploration.

Effects of nitrogen topdressing and paclobutrazol at different stages on spike differentiation and yield of winter wheat

Background Optimal nitrogen (N) application and plant growth regulators can improve wheat productivity. This can help to improve yield level and ensure food security with limited resources in the Huang-Huai-Hai Plain of China (HPC). Methods A 2-year field experiment was conducted using a randomized block design with four treatments (TS-N topdressing at pseudostem erection stage ; TPS-N topdressing combined with paclobutrazol application at pseudostem erection stage; TJ-N topdressing at jointing stage; TPJ-N topdressing at combined with paclobutrazol application at jointing stage) in 2011–2013. Results The grain number per ear, thousand kernel weight and yield for the TJ and TPJ treatments were higher than those of the TS and TPS treatments. Grain number per ear, yield, and thousands kernel weigh for the TPJ treatment were significantly higher than for the TS and TPS in 2011–2012 (9.82% and 7.27%, 10.23% and 8.99%, 6.12% and 5.58%) and in 2012–2013 (10.21% and 11.55%, 8.00% and 6.58%, 0.00 and 0.00), respectively. Thousands kernel weight under TJ were significantly higher than those under TS and TPS by 13.21% and 14.03%, respectively in 2012–2013. The floret number, significantly correlated with cytokinin content, was also significantly increased under TJ and TPJ at connectivum differentiation stage. For TPJ treatment, the floret number was significantly higher than for the TS, TPS, and TJ by 19.92%, 10.21%, 6.10% in 2011–2012; it was higher than for the TS and TPS by 28.06% and 29.61% in 2012–2013, respectively. The relative expression level of cytokinin oxidase/dehydrogenase gene (TaCKX2.2) was improved during flowering, when cytokinin content was at high level and was also inhibited by paclobutrazol with different degrees. Conclusions Therefore, nitrogen topdressing at jointing stage had increased grain number per ear, thousand kernel weight, and grain yield of wheat. Paclobutrazol could delay spike differentiation and promote cytokinin accumulation that induced expression of TaCKX2.2, maintaining hormonal balance and affecting wheat spike morphogenesis.

Integrated Bioinformatics Analyses of PIN1, CKX, and Yield-Related Genes Reveals the Molecular Mechanisms for the Difference of Seed Number Per Pod Between Soybean and Cowpea

There is limited advancement on seed number per pod (SNPP) in soybean breeding, resulting in low yield in China. To address this issue, we identified PIN1 and CKX gene families that regulate SNPP in Arabidopsis, analyzed the differences of auxin and cytokinin pathways, and constructed interaction networks on PIN1, CKX, and yield-related genes in soybean and cowpea. First, the relative expression level (REL) of PIN1 and the plasma membrane localization and phosphorylation levels of PIN1 protein were less in soybean than in cowpea, which make auxin transport efficiency lower in soybean, and its two interacted proteins might be involved in serine hydrolysis, so soybean has lower SNPP than cowpea. Then, the CKX gene family, along with its positive regulatory factor ROCK1, had higher REL and less miRNA regulation in soybean flowers than in cowpea ones. These lead to higher cytokinin degradation level, which further reduces the REL of PIN1 and decreases soybean SNPP. We found that VuACX4 had much higher REL than GmACX4, although the two genes essential in embryo development interact with the CKX gene family. Next, a tandem duplication experienced by legumes led to the differentiation of CKX3 into CKX3a and CKX3b, in which CKX3a is a key gene affecting ovule number. Finally, in the yield-related gene networks, three cowpea CBP genes had higher RELs than two soybean CBP genes, low RELs of three soybean-specific IPT genes might lead to a decrease in cytokinin synthesis, and some negative and positive SNPP regulation were found, respectively, in soybean and cowpea. These networks may explain the SNPP difference in the two crops. We deduced that ckx3a or ckx3a ckx6 ckx7 mutants, interfering CYP88A, and over-expressed DELLA increase SNPP in soybean. This study reveals the molecular mechanism for the SNPP difference in the two crops, and provides an important idea for increasing soybean yield.

Evaluation of level of expression of microRNA-21 and let-7g in serum and stool of patients with colorectal cancer

Background MicroRNAs (miRs) are involved in the pathogenesis of various malignancies such as colorectal cancer through regulating multiple cellular processes, including cell proliferation, cell cycle, apoptosis and migrationMiR-21 and let-7 are two important genes that have confirmed in this pathway. The role of the let-7 gene as a gene tumor process in various cancers and the role of miR-21 in the development and progression of cancer has been conclusively identified also this gene has an oncogenic role in various cancers. In this study, the expression patterns of miR-21 and let-7 in serum and stool samples of colorectal cancer patients were evaluated. Materials and Methods During the present study, 120 samples including 40 serum samples of CRC and 40 stool samples from the same patients and 40 healthy samples were collected. After total RNA extraction, real-time PCR was used to measure changes in genes expression. Statistical analysis of data was performed with GraphPad Prism statistical software (Version 6.0) with a significance level of 5%. Results The relative expression level of miR-21 in the serum samples of CRC increased compared to the healthy group, which was statistically significant. On the other hand, the relative expression level of let-7g in the serum samples of CRC showed a significant decrease compared to the healthy sample. In stool samples, the expression changes of either of the two genes were not significant. Conclusion Our findings indicate that the relative expression of miR-21 and let-7g genes can be used as a diagnostic or predictive biomarker in colorectal cancer serum samples. While, this is not the case in stool samples. Moreover, further investigations at the protein level should be performed.

Attenuation of the Apoptosis Stimulating Protein of TP53-1 (ASPP1) Is a Predictive Marker for Therapy Resistance and Overall Survival in Acute Myeloid Leukemia (AML)

ASPP1 (PPP1R13B) belongs to a family of p53-binding proteins and enhances apoptosis by stimulation of p53-transactivation of selected proapoptotic target genes. It is preferentially expressed in hematopoietic stem cells (HSC) and together with p53 preserves the genomic integrity of the HSC pool. We have previously demonstrated that ASPP1 is attenuated in AML, which is linked to methylation of the promoter region. ASPP1-interferference models suggest a functional role in leukemogenesis and therapy response. We now provide evidence that ASPP1 is highly altered in AML and attenuated expression levels associate with inferior outcomes: mRNA expression patterns of ASPP1 were assessed in an unselected patient cohort with newly diagnosed AML (n=39) - and were found to be significantly lower compared to bone marrow aspirates of 12 healthy donors (p < 0.0001, Mann-Whitney test) with a median relative expression level of 0.33 (ASPP1 AML) vs. 0.95 (ASPP1 donor). Subcohort analysis by genetic risk profile according to the European LeukemiaNet (ELN) 2017 genetic risk stratification revealed significantly lower expression levels of the intermediate/adverse risk group (n=27) compared to the favorable risk group (n=10, p=0,013) with a median relative expression level of 0.21 (ASPP1 int/adv) vs. 0.46 (ASPP1 good). In addition, analysis of patients undergoing induction chemotherapy demonstrated a significantly lower complete remission (CR) rate in patients with attenuated ASPP1 levels (p=0.0015) with a median relative expression level of 0.13 (ASPP1 nonCR) vs. 0.51 (ASPP1 CR). Of note, patients with a >10x fold decrease of ASPP2 expression were exclusively found in the patient cohort failing induction therapy. To validate these observations in an independent patient set, we next performed a database RNAseq screen on a defined, unselected AML cohort (n=142, FAB M3 were excluded). Together, ASPP1 was independently confirmed as an adverse prognostic factor: Overall survival (OS) was longer in the ASPP1 high cohort vs. the ASPP1 low group with a hazard ratio for death of 0.63. Even though this analysis closely failed significance (0.082) due to lack of power, probability to be alive after 70 months was 45% (ASPP1 high) vs. 10% (ASPP1 low) with a median survival of 19.23 months (ASPP1 low) vs. 26.4 months (ASPP1 high). Most interestingly, looking at the prognostic good risk population: attenuation of ASPP1 resulted in a dramatic decrease of survival with a hazard ratio of 0.29 and a probability to be alive after 70 months of 25% (ASPP1 low) vs. 85% (ASPP1 high). Consequently, screening for ASPP1 expression may define a patient cohort likely to fail (induction) therapy - which might be a target population for hypomethylating agents (HMA) to reconstitute ASPP1 and sensitize towards (chemo)therapy. To address this hypothesis, MOLM-14 acute leukemia cells and patient derived freshly-isolated AML samples were treated with decitabine - resulting in upregulation of ASPP1 mRNA expression levels in all assessed samples. As expected, cells were more chemo-susceptible when exposed to decitabine followed by daunorubicin and cytarabine in an ASPP1-dependent manner: MOLM-14.ASPP1i cell line strains did not reveal a significant change of proapoptotic efficacy when compared to mock strains which significantly benefitted from this approach with more than doubled proapoptotic rates (**** p < 0.0001, t-test), again confirming ASPP1 target specificity. Our results demonstrate that dysfunctional regulation of ASPP1 expression is frequently observed in AML, which associates with therapy responses and survival outcome. Prospective clinical studies are warranted to evaluate the role as a biomarker for risk stratification. Further, we provide a rationale to re-sensitize high-risk patients (defined as ASPP1 low-expressors) towards chemotherapy using HMA priming upfront - and this strategy should be followed in future trials. Disclosures Schittenhelm: BMS: Other: advisory board; Takeda: Other: advisory board; Astellas: Other: advisory board; University of Tuebingen: Patents & Royalties: patent for ASPP2k. Kampa-Schittenhelm: University of Tuebingen: Patents & Royalties: patent related to ASPP2k.

The Role of TLR4 Inducing Macrophage Pyroptosis in the Pathogenesis of Severe Aplastic Anemia

Introduction：To investigate the expression level of macrophage pyroptosis in patients with severe aplastic anemia (SAA) and its effect on downstream effector T cells, the study explored the mechanism of Toll like receptor 4 (TLR4) inducing macrophage pyroptosis and maintaining immune homeostasis of SAA through pyruvate kinase M2 (PKM2) at cellular and molecular levels, which provided a theoretical basis for the targeted therapy of macrophages in SAA. Three groups of bone marrow macrophages of new diagnosed SAA patients, remission patients and healthy controls were induced and cultured in vitro. qRT-PCR and Western Blot were used to detect pyroptosis-related indicators IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD expression level; ELISA to detect the concentration of IL-1β and IL-18 in the culture supernatant. Results: 1. The mRNA and protein levels of macrophages IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD in the new diagnosed SAA group were significantly higher than those in healthy controls, and the IL-1β and IL-18 concentration in the macrophage culture supernatant increased significantly. The relative mRNA expression levels of macrophages IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD in the new diagnosed group were significantly negatively correlated with WBC, RBC, Hb and PLT. 2. The relative expression level of TLR4 mRNA in macrophages in the new diagnosed group was significantly higher than that in the remission group and healthy controls, and the protein level of TLR4 was significantly increased; at the same time, the relative expression level of TLR4 mRNA was positively correlated with IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD in macrophages. After knocking down TLR4 or adding TLR4 inhibitors, the mRNA and protein expression levels of IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD were significantly lower than those in healthy controls, and the concentration of IL-1β and IL-18 in the culture supernatant reduced significantly. 3. The expression rate of PKM2 in macrophages of new diagnosed group by flow cytometry was significantly higher than that in the remission group and heathy controls, and the levels of PKM2 mRNA and protein were significantly increased. At the same time, the relative expression level of PKM2 mRNA was positively correlated with the expression levels of IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD. After knocking down PKM2, the mRNA and protein expression levels of IL-1β, IL-18, NLRP3, Caspase-1, GSDMD were significantly lower than those healthy controls, and the concentration of IL-1β and IL-18 in the culture supernatant was also significantly reduced. After knocking down TLR4 or adding TLR4 inhibitor, PKM2 mRNA and protein expression levels in macrophages were significantly lower than those in healthy controls. 4. When CD8+ T cells were co-cultured with macrophages which were knocked down TLR4 or added with inhibitors, the expressions of perforin and granzyme B in CD8+ T cells were significantly reduced. CD8+ T cells were further co-cultured with K562 and the apoptosis level of K562 was reduced. Summary：The level of macrophage pyroptosis in SAA patients increased, and the expression levels of macrophages TLR4 and PKM2 increased and positively correlated with the level of pyroptosis. Inhibiting the expression of TLR4 and PKM2, respectively, reduced the level of macrophage pyroptosis, and at the same time inhibiting TLR4 expression reduced the expression of PKM2. Macrophages that inhibited TLR4 expression reduced the killing function of CD8+ T cells. Therefore, TLR4 may induce pyroptosis of macrophages in SAA patients through PKM2 and enhance the killing function of CD8+ T cells. Disclosures No relevant conflicts of interest to declare.

Expression analysis and single-nucleotide polymorphisms of MLPH and PMEL17 genes associated with melanin deposition in Xuefeng black-bone chickens

Melanin deposition related genes such as MLPH and PMEL17 play an important role in black-bone chicken. This study was aimed to identify and associate SNPs in the MLPH and PMEL17 genes with melanin content of pectoral muscle (MCPM) in Xuefeng black-bone chicken. A total of 120 Xuefeng black-bone chickens at 120-day-old were randomly selected to measure blackness of pectoral muscle (BPM), according to the degree of BPM selected 22 high blackness (HB) and 22 low blackness (LB) chickens to determine the MCPM, and extract DNA and mRNA. The results indicated that the MCPM in the HB group was higher than in the LB group (P < 0.01), and the L value in the HB group was lower than in the LB group (P < 0.01). And we measured the mRNA expression levels of MLPH and PMEL17 genes in pectoral muscle by quantitative real-time PCR. The results revealed that the mRNA expression levels of MLPH gene (P < 0.05) and PMEL17 gene (P < 0.01) in the HB group was higher than in the LB group, and the mRNA relative expression level of MLPH and PMEL17 genes with MCPM was positive correlation (P < 0.01). And the sequencing results found that a total of 17 SNPs were found in MLPH gene, the C-1411T was associated with MCPM (P< 0.05), there was no difference in MCPM among other locus (P> > 0.05). And there were 10 SNPs in PMEL17 gene, the G-1843C, C-2812T, and G-2794A were associated with MCPM (P < 0.05), there was no difference in the MCPM among other locus (P > 0.05). These SNPs could be molecular markers for breeding selection of blackness traits.

Applying HPLC to Screening QTLs for BLB Resistance in Rice

Bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae and is a major cause of rice yield reductions around the world. When diseased, plants produce a variety of metabolites to resist pathogens. In this study, the various defense metabolites were quantified using high-performance liquid chromatography (HPLC) after Xoo inoculation in a 120 Cheongcheong/Nagdong double haploid (CNDH) population. Quantitative trait locus (QTL) mapping was conducted using the concentration of the plant defense metabolites. HPLC analyzes the concentration of substances according to the severity of disease symptoms. Searching for BLB resistance candidate genes by applying this analysis method is very effective when mapping related genes. These resistance genes can be mapped directly to the causative pathogens. A total of 17 metabolites were detected by means of HPLC analysis after Xoo inoculation in the 120 CNDH population. QTL mapping of the metabolite concentrations resulted in the detection of the BLB resistance candidate gene, OsWRKYq6, in RM3343 of chromosome 6. OsWRKYq6 has a very high homology sequence with WRKY transcription factor 39, and when inoculated with Xoo, the relative expression level of the resistant population was higher than that of the susceptible population. Resistance genes have previously been detected using only phenotypic change data. In this study, resistance candidate genes were detected using the concentration of metabolites produced in plants after inoculation with pathogens. This newly developed analysis method can be used to effectively detect and identify genes directly involved in disease resistance for future studies.

CircRNA_0079586 and circRNA_RanGAP1 are involved in the pathogenesis of intracranial aneurysms rupture by regulating the expression of MPO

Several circRNAs have been reported to be dysregulated in human endothelial cells through sponging miRNAs. Previous reports demonstrated that MPO not only contributed to the formation and rupture of cerebral aneurysm but was also correlated with the degenerative remodeling predisposition to saccular intracranial aneurysm wall rupture, although its underlying mechanisms remain to be explored. Microarray screening was performed to compare the differential expression of circRNAs in the endothelial cells collected from UIAs and RIAs patients. Luciferase assays were used to explore the regulatory relationship between circRNAs and miRNAs, and between miRNAs and their target genes. Microarray screening analysis found a batch of up-regulated circRNAs in the endothelial cells harvested from RIAs patients, including circRNA-0079586 and circRNA-RanGAP1. Luciferase assays revealed the suppressive role of miR-183-5p/miR-877-3p in the expression of circRNA-0079586/circRNA-RanGAP1/MPO. And the expression of circRNA-0079586 and circRNA-RanGAP1 was respectively suppressed by the overexpression of miR-183-5p and miR-877-3p. And both the transfection of miR-183-5p and miR-877-3p mimics suppressed the relative expression level of MPO mRNA. The expression of circRNA-0079586, circRNA-RanGAP1 and MPO was significantly activated in the endothelial cells collected from RIAs patients when compared with UIAs patients, whereas the expression of miR-183-5p and miR-877-3p was remarkably suppressed in the endothelial cells collected from RIAs patients when compared with UIAs patients. We further altered the expression of circRNA-0079586 and circRNA-RanGAP1 using siRNA and overexpression in HUVECS, and the expression of circRNA-0079586 and circRNA-RanGAP1 was significantly and negatively correlated with the expression of miR-183-5p and miR-877-3p, but positively correlated with the expression of MPO under different conditions. In this study, we established two MPO-modulating signaling pathways of circRNA_0079586/miR-183-5p/MPO and circRNA_RanGAP1/miR-877-3p/MPO. These two signaling pathways are involved in the pathogenesis of intracranial aneurysms rupture.