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Invasive micropapillary carcinoma (IMPC) is a special histological subtype of breast cancer, featured with extremely high rates of lymphovascular invasion and lymph node metastasis. Based on a previous series of studies, our team proposed the hypothesis of "clustered metastasis of IMPC tumor cells". However, the transcriptomics characteristics underlying its metastasis are unknown, especially in spatial transcriptomics (ST). In this paper, we perform ST sequencing on four freshly frozen IMPC samples. We draw the transcriptomic maps of IMPC for the first time and reveal its extensive heterogeneity, associated with metabolic reprogramming. We also find that IMPC subpopulations with abnormal metabolism are arranged in different spatial areas, and higher levels of lipid metabolism are observed in all IMPC hierarchical clusters. Moreover, we find that the stromal regions show varieties of gene expression programs, and this difference depends on their distance from IMPC regions. Furthermore, a total of seven IMPC hierarchical clusters of four samples share a common higher expression level of the SREBF1 gene. Immunohistochemistry results further show that high SREBF1 protein expression is associated with lymph node metastasis and poor survival in IMPC patients. Together, these findings provide a valuable resource for exploring the inter- and intra-tumoral heterogeneity of IMPC and identify a new marker, SREBF1, which may facilitate accurate diagnosis and treatment of this disease., (© 2021. The Author(s).)

Background: The quinone oxidoreductase 1 (NQO1) gene was involved in the pathophysiological process of illicit drugs abuse, and its polymorphisms might be associated with methamphetamine (METH) dependence susceptibility. The purpose of this study was to examine the NQO1 mRNA and protein levels and to analyze the 609C/T polymorphism (rs1800566) between METH-dependent patients and controls., Methods: A total of 392 METH-dependent patients (cases) and 669 healthy controls (controls) were enrolled in the study. The quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the relative expressions of NQO1 mRNA in PBMCs and protein levels in plasma, respectively. PCR-restriction fragment length polymorphism (RFLP-PCR) and direct-sequencing genotyping were used to detect the alleles and genotypes of NQO1 609C/T polymorphism., Results: The levels of NQO1 mRNA in cases (3.2650 ± 2.2943) was significantly higher than in controls (1.0125 ± 0.7959) (p < 0.001), the plasma protein in cases (0.2368 ± 0.1486) was significantly lower than in controls (0.5844 ± 0.1742) (p < 0.001). The T allele of the 609C/T polymorphism significantly increased the risk of METH dependence (p = 0.032, OR = 1.214, 95%CI = 1.017-1.450). The TC and TC/TT genotypes of 609C/T were observed significantly more frequently in cases than in controls, respectively (TC vs CC: p = 0.012, OR = 1.457, 95% CI = 1.087-1.952; TC/TT vs CC: p = 0.008, OR = 1.460, 95% CI = 1.102-1.935). Similar results were obtained after adjusting for age and sex. We failed to find that any genotype of 609C/T polymorphism affected the mRNA or plasma protein levels in controls, respectively (p > 0.05)., Conclusion: The findings suggested that NQO1 might play an important role in the pathophysiological process of METH dependence, and the 609C/T polymorphism might contribute to the susceptibility to METH dependence in a Chinese Han population., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

The choice of reference gene is crucial for quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay. To screen and determine the suitable reference genes in fetal rat pancreas, we selected eight candidate reference genes (Gapdh, Actb, Rn18 s, B2m, Rpl13a, Tbp, Ywhaz and Ubc), and evaluated the constancy of gene expression from fetal rat pancreases in non-pathological situation and prenatal dexamethasone exposure (PDE) model, using four algorithms: GeNorm, NormFinder, Bestkeeper and Comparative ΔCt method. In addition, the alteration of mRNA levels of pancreatic insulin was compared between control and PDE groups to validate the reliability of selected reference genes for data normalization of RT-qPCR. The comprehensive ranking of reference genes under physiological condition was as follow: Gapdh > Actb > Ywhaz > Ubc > Rn18s > Rpl13a > B2m > Tbp (female); Actb > Ywhaz > Gapdh > Ubc > B2m > Rpl13a > Rn18 s | Tbp (male). The top ranking reference genes were also stably expressed in PDE fetal pancreas. The best reference gene combinations are: Ywhaz+Actb for female and Ywhaz+Gapdh for male fetal rat pancreas, respectively. Compared with low ranking or single reference gene, the change trend of insulin mRNA normalized by the best reference gene combination between control and PDE groups was more significant and consistent with that of serum insulin level. In conclusion, our results provided the optimal combination of stable reference genes for RT-qPCR assay in pancreatic developmental toxicity study., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Integrating systems biology is an approach for investigating metabolic diseases in humans. However, few studies use this approach to investigate the mechanism by which Rhizoma coptidis (RC) reduces the effect of lipids and glucose on high-fat induced obesity in rats., Methods: Twenty-four specific pathogen-free (SPF) male Sprague-Dawley rats (80 ± 10 g) were used in this study. Serum metabolomics were detected by ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry. Liver tissue and cecum feces were used for RNA-Seq technology and 16S rRNA gene sequencing, respectively., Results: We identified nine potential biomarkers, which are differential metabolites in the Control, Model and RC groups, including linoleic acid, eicosapentaenoic acid, arachidonic acid, stearic acid, and L-Alloisoleucine (p < 0.01). The liver tissue gene expression profile indicated the circadian rhythm pathway was significantly affected by RC (Q ≤ 0.05). A total of 149 and 39 operational taxonomic units (OTUs), which were highly associated with biochemical indicators and potential biomarkers in the cecum samples (FDR ≤ 0.05), respectively, were identified., Conclusion: This work provides information to better understand the mechanism of the effect of RC intervention on hyperlipidemia and hypoglycemic effects in obese rats. The present study demonstrates that integrating systems biology may be a powerful tool to reveal the complexity of metabolic diseases in rats intervened by traditional Chinese medicine., (© 2021. The Author(s).)

Allergic rhinitis (AR) is a familiar respiratory allergic inflammatory disease with higher incidence. The pathogenesis of AR is particularly complex. Therefore, a lot of work is acquired to excavate deep mechanisms, thereby providing effective strategies for AR diagnose and treatment. AR mice model was induced by recombinant murine IL-33 (0.05 µg/µl) on days 1, 3, and 5. The lentiviral vectors carrying si-circ&#95;0067835, miR-155 mimic, si-NC or miR-NC were injected into AR mice. Thus, mice were divided into control, AR, AR + si-NC, AR + si-circ&#95;0067835, AR + si-circ&#95;0067835 + miR-NC, and AR + si-circ&#95;0067835 + miR-155 mimic groups. qRT-PCR experiment was used to measure the expression of circ&#95;0067835 and miR-155. Behavioral test result was quantified to assess AR mice model. Hematoxylin and eosin (HE) staining was performed to analyze histopathological changes. Helper T cell 2 (Th2) cytokines (IL-4, IL-5, IL-9 and IL-13) and percentage of type-2 innate lymphoid cells (ILC2s) in nasal mucosa tissues in AR mice model were evaluated needing western blot, ELISA, and flow cytometry. Besides, the targeting relationship between circ&#95;0067835 and miR-155, or between miR-155 and GATA3, was investigated via luciferase report assay. Circ&#95;0067835 expression levels were raised in the nasal mucosa tissues of AR mice. Inhibiting circ&#95;0067835 could reduce Type2 cytokines and ILC2s levels in AR mice model. Furthermore, circ&#95;0067835 targeted and positively regulated miR-155 expression, and GATA3 was a downstream target of miR-155 and adjusted by circ&#95;0067835/miR-155 axis. In addition, silencing circ&#95;0067835 inhibited cytokines and ILC2s levels by down-regulating miR-155. Circ&#95;0067835 effectively inhibited AR response in ILC2s through participation of miR-155/GATA3 axis.

Objective: To explore the effects of hnRNP E1 on the expression of early genes E2, E6 of HPV16 and the biological function in cervical cancer SiHa cell lines. Methods: The cell experiments in vitro were carried out in cervical cancer cell lines SiHa. The expression levels of E2, E6 mRNA and protein of HPV16 were detected by Real-time PCR and Western blot, respectively, before and after up-regulating hnRNP E1. Meanwhile, the cell proliferation, cycle and apoptosis were evaluated by CCK-8 and flow cytometry. Data analyses were performed using SPSS 22.0 and Graphpad Prism 7.0 software. Results: Compared with the blank and the blank plasmid group, the cells activity and proliferation decreased at 24, 48 and 72 h after up-regulating hnRNP E1 ( P <0.05), while the percentage of cells in G0/G1 phase increased and the percentage in S and G2/M phase and proliferation index decreased ( P <0.05). Moreover, the late apoptotic rate and the total apoptotic rate increased ( P <0.05). The expression levels of E6 mRNA and protein of HPV16 in hnRNP E1 up-regulated group were significantly lower than that in both blank group and blank plasmid group, the differences were significant ( P <0.05), showing the tendency of cells proliferation index decrease and total apoptotic rate increase with decreased HPV16 E6 expression. There were no significant differences in the expression of E2 mRNA of HPV16 among the three groups ( P =0.427), and no E2 protein of HPV16 was detected. Conclusions: hnRNP E1 could inhibit the transcription and translation of E6 oncogene of HPV16 and further inhibit the proliferation and promote apoptosis of cervical cancer cells, suggesting that hnRNP E1 might be a potential target marker to prevent cervical lesions. But no association between hnRNP E1 and HPV16 E2 was found in SiHa cells.

Introduction: Although three randomized control trials have proven mortality benefit of CT lung cancer screening (CTLS), <5% of eligible US smokers are screened. Some attribute this to fear of harm conveyed at shared decision visits, including the harm of overdiagnosis/overtreatment of indolent BAC-like adenocarcinoma., Methods: Since the frequency of indolent cancers has not been compared between CTLS and routinely detected cohorts, we compare pathology and RNA expression of 86 NCCN high-risk CTLS subjects to 83 high-risk (HR-R) and 51 low-risk (LR-R) routinely detected patients. Indolent adenocarcinoma was defined as previously described for low malignant potential (LMP) adenocarcinoma along with AIS/MIA. Exome RNA sequencing was performed on a subset of high-risk (CTLS and HR-R) FFPE tumor samples., Results: Indolent adenocarcinoma (AIS, MIA, and LMP) showed 100% disease-specific survival (DSS) with similar frequency in CTLS (18%) and HR-R (20%) which were comparatively lower than LR-R (33%). Despite this observation, CTLS exhibited intermediate DSS between HR-R and LR-R (5-year DSS: 88% CTLS, 82% HR-R, & 95% LR-R, p = 0.047), possibly reflecting a 0.4 cm smaller median tumor size and lower frequency of tumor necrosis compared to HR-R. WGCNA gene modules derived from TCGA lung adenocarcinoma correlated with aggressive histologic patterns, mitotic activity, and tumor invasive features, but no significant differential expression between CTLS and HR-R was observed., Conclusion: CTLS subjects are at no greater risk of overdiagnosis from indolent adenocarcinoma (AIS, MIA, and LMP) than risk-matched patients whose cancers are discovered in routine clinical practice. Improved outcomes likely reflect detection and treatment at smaller size., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Rearing system is a critical nongenetic factor influencing meat quality of ducks. In this study, a total of 360 birds were randomly allocated into floor rearing system (FRS) and net rearing system (NRS) to compare their effects on intramuscular fat (IMF) deposition, fatty acid composition, and related gene expression in muscles of Nonghua ducks. Sawdust bedding and stainless mesh bed were equipped in FRS and NRS, respectively. At the eighth week (8w) and 13th week (13w), the breast and thigh muscles of ducks were collected to determine the profiles of lipids composition and the expressions of lipid metabolism-related genes. The IMF content was higher in 13w-FRS than 8w-FRS and 8w-NRS in breast muscle, whereas it was higher in 13w-NRS than other groups in thigh muscle (P < 0.05). C16:1, C20:5(n-3) of muscles were higher in 8w-NRS than 8w-FRS, whereas C18:1(n-9)c, C18:2(n-6)c, Ʃ monounsaturated fatty acid (MUFA), and ƩMUFA/Ʃsaturated fatty acid (SFA) ratio of muscles were higher in 13w-NRS than 8w-FRS and 8w-NRS (P < 0.05). C22:6(n-3), C20:4(n-6) of breast muscle and C20:3(n-6) of thigh muscle were higher in 13w-NRS than 13w-FRS (P < 0.05). Fatty acids variation was studied by principal component analysis, exhibiting extensive positive loadings on principal components. SREBP1, ACADL, and FABP3 were downregulated in breast muscle, whereas PPARα and ELOVL5 were upregulated in thigh muscle of NRS ducks at 13w. Principal components were extensively correlated with lipids composition parameters, and principal components of breast muscle 1 and principal components of thigh muscle 1 were correlated with SREBP1 and PPARα, respectively (P < 0.05). In conclusion, with increasing age, FRS enhanced IMF deposition in breast muscle, and the same promotion in thigh muscle was because of NRS. The variation of fatty acids in muscles was uniform, and the change of single fatty acid was unable to distinguish NRS and FRS. However, as NRS downregulated SREBP1, ACADL and FABP3 in breast muscle and upregulated PPARα and ELOVL5 in thigh muscle, NRS could improve nutrient value and meat quality by increasing ƩMUFA, ƩMUFA/ƩSFA ratio, and important PUFA levels. Therefore, NRS was more recommended than FRS for Nonghua ducks during week 8 to 13 posthatching., (Copyright © 2020. Published by Elsevier Inc.)

This study aimed to investigate the molecular mechanism by which microRNA (miR)-96 regulates the progression of intervertebral disc degeneration (IDD). The expression of miR-96 in normal intervertebral discs and in IDD was detected by performing reverse transcription-quantitative PCR. CCK8 assay was applied to examine the proliferation of nucleus pulpous (NP) cells and flow cytometry was used to evaluate the cell apoptosis and cell cycle profile. In addition, the immunofluorescence analysis was employed to detect cell proliferation. The expressions of proteins were assessed by western blot analysis. TargetScan and miRDB were used to predict the target genes of miR-96. The results indicated that miR-96 expression was upregulated in IDD compared with normal intervertebral discs. Overexpression of miR-96 could significantly inhibit the proliferation of NP cells via inducing apoptosis and G1 arrest. In addition, fibroblast growth factor receptor substrate 2 (FRS2) was identified as the target of miR-96 and overexpression of FRS2 could revere the effect of miR-96 mimics in NP cells. Therefore, these findings demonstrated that miR-96 plays a critical role during the progression of IDD and miR-96 may serve as a target for the treatment of IDD.

Background: Alzheimer's disease (AD) is the most common type of dementia, and patients with advanced AD frequently lose the ability to identify family members. The fusiform gyrus (FUS) of the brain is critical in facial recognition. However, AD etiology in the FUS of AD patients is poorly understood. New analytical strategies are needed to reveal the genetic and epigenetic basis of AD in FUS., Results: A complex of new analytical paradigms that integrates an array of transcriptomes and methylomes of normal controls, AD patients, and "AD-in-dish" models were used to identify genetic and epigenetic signatures of AD in FUS. Here we identified changes in gene expression that are specific to the FUS in brains of AD patients. These changes are closely linked to key genes in the AD network. Profiling of the methylome (5mC/5hmC/5fC/5caC) at base resolution identified 5 signature genes (COL2A1, CAPN3, COL14A1, STAT5A, SPOCK3) that exhibit perturbed expression, specifically in the FUS and display altered DNA methylome profiles that are common across AD-associated brain regions. Moreover, we demonstrate proof-of-principle that AD-associated methylome changes in these genes effectively predict the disease prognosis with enhanced sensitivity compared to presently used clinical criteria., Conclusions: This study identified a set of previously unexplored FUS-specific AD genes and their epigenetic characteristics, which may provide new insights into the molecular pathology of AD, attributing the genetic and epigenetic basis of FUS to AD development.

The rs6647 variant G allele in SERPINA1 gene was reported to be associated with the risk of large artery atherosclerotic stroke (LAS), however, the mechanism remains unclear. Here, we performed a functional annotation of the rs6647 variant by using the software HaploReg version 4.1 (HaploReg v4.1). Next, the expression quantitative trait loci (eQTLs) analysis of multiple datasets was conducted for determining the association between the rs6647 and SERPINA1 expression in various tissues. Then, a case-control gene expression analysis was done using two independent ischemic stroke (IS) gene expression datasets. Finally, SERPINA1 expression in whole blood samples from 8 LAS patients and 14 healthy persons were compared. The functional annotation suggested that the rs6647 regulates gene expression in multiple human tissues especially in brain and blood. The eQTLs analysis revealed a significant association of the rs6647 G allele with increased expression of SERPINA1 gene only in whole blood. Compared with the controls, there was an increased expression of SERPINA1 gene in whole blood in both IS patients and LAS patients. SERPINA1 gene expression in whole blood bridges the rs6647 variant G allele with increased LAS risk, providing new insights into the mechanisms underlying role of the rs6647 in determining LAS risk., (© 2020 Federation of American Societies for Experimental Biology.)

Reproduction trait is one of the most important economic traits in poultry industry. This study was aimed to investigate the mRNA expression levels, single nucleotide polymorphisms (SNP) of POMC gene, and the association with reproduction traits in chickens. Five SNP (g.958 G > A, g.1374 G > C, g.1393 G > A, g.1817 C > T, and g.1918G > A) were detected in introns of POMC gene in 317 Zhenning yellow chickens. Association analysis revealed that g.958 G > A and g.1817 C > T showed significantly associations with fertilization rate, hatching rate of hatching eggs, and hatching rate of fertilized eggs in chickens. Simultaneously, g.1374 G > C and g.1918G > A were both associated with egg weight at 300 D of age (P < 0.05). The SNP of g.958 G > A, g.1393 G > A, and g.1817 C > T were all associated with E2 hormone levels (P < 0.05). The result of mRNA expression levels in different tissues showed that POMC mRNA expression level in the pituitary was higher than those in the other tissues and varied in different genotypes. In conclusion, the results in this study provided new evidences that polymorphisms of the POMC gene have potential effects on reproduction traits in chickens. The 5 SNP detected in this study could be potential markers for improving reproduction traits in chickens., (Copyright © 2020. Published by Elsevier Inc.)

Bombyx mori vitellogenin (BmVg) is highly upregulated during pupation, and the 20-hydroxyecdysone and amino acids may regulate stage-specific BmVg expression. However, previous studies showed that other factors may also affect stage-specific BmVg expression. Here, we characterized effective BmVg transcription factors by identifying the corresponding cis-regulatory elements (CREs). We prepared transgenic B. mori, in which DsRed was driven by various lengths of BmVg promoter. qRT-PCR analysis showed that DsRed expression driven by a 1.0-kb BmVg promoter (VgP1.0K) was consistent with endogenous BmVg. VgP1.0K specificity was closer to the endogenous BmVg promoter than that of VgP0.8K. These results suggest that CREs affecting stage-specific BmVg expression were localized to the 1.0-kb BmVg promoter. We investigated the effects of certain CREs that could influence the stage specificity of BmVg promoter on BmVg expression in transgenic B. mori. The relative DsRed expression was significantly reduced in transgenic female B. mori and the peak in DsRed expression was delayed after E-box CRE mutation. These results demonstrate that the E-box element enhanced BmVg expression and also affected stage-specific BmVg expression. Moreover, the relative DsRed expression was significantly increased in transgenic female of B. mori after 3×BD CRE mutation in BmVg promoter. However, the stage specificity of the mutated promoter was consistent with that of the endogenous BmVg promoter. The 3×BD element downregulated BmVg but had no effect on stage-specific BmVg expression. The present study promoted the process of elucidating the regulatory network for stage-specific BmVg expression and furnished a theoretical basis for the application of BmVg promoter., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Disinfection by-products (DBPs) are generated during chlorination of drinking water. Previous studies demonstrate that DBPs are cytotoxic, genotoxic and associated with an increased risk of human cancer. However, the molecular basis of DBPs-induced toxic effects remains unclear. Here, we chlorinated samples of algal-derived organic matter (AOM) and sediment organic matter (SOM) from a local drinking water reservoir. Chemical properties, toxicities and transcriptomic profiles of human Caco-2 cell exposed to AOM and SOM were compared before and after chlorination. We analyzed chlorination-caused distinct gene expression patterns between AOM and SOM, and identified a set of 22 differentially expressed genes under chlorination of AOM that are different from chlorinated SOM. Consequent network analysis indicates that differential CYP1A1, CYP1B1, ID1 and ID2 are common targets of the upstream regulators predicted in the AOM group, but not the SOM group. Through experimental validation and data integration from previous reports related to DBPs or environmental stressors, we found that CYP1A1 and CYP1B1 are specifically up-regulated after chlorinating AOM. Our study demonstrates that the two CYP1 genes likely act as novel biomarkers of AOM derived DBPs, and this would be helpful for testing drinking water DBPs toxicity and further monitoring drinking water safety., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

An 8-week feeding trial was conducted to investigate the effects of partial replacement of fish meal by soy protein concentrate (SPC) on the growth performance, immune responses, intestine morphology and relation gene expression of intestinal inflammation for juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) (initial weight 12.5 ± 0.00 g). Eight isonitrogenous and isolipidic diets (48.61% protein and 11.17% lipid) were formulated by replacing 0% (the control), 11%, 22%, 33%, 44%, 55%, 66%, and 77% of fish meal (FM) with SPC, respectively (the eight dietary be named FM, S11, S22, S33, S44, S55, S66, and S77, respectively). With the replacement level increased, the final body weight, weight gain ratio (WGR), specific growth rate (SGR), and survival rate of fish were significantly decreased (P < 0.05) compared with the group FM. By contrast, the feed conversion ratio (FCR) of fish was significantly increased (P < 0.05) when the replacement level up to 44%. Partial FM replacement by SPC (ranging from 11% to 77%) substantially reduced (P < 0.05) the serum total protein, albumin, and total cholesterol contents compared with the group FM. Liver total superoxide dismutase, glutathione peroxidase, catalase activities, and total antioxidant capacity showed the same trend of gradual increase first and then decrease. Their highest values were found in the replacement levels of 55%, 33%, 22%, and 55% and were significantly higher (P < 0.05) than the control group. The lowest malondialdehyde content was observed in group S77 and was significantly lower (P < 0.05) than that of the control group. The complements C3 and C4 contents of fish fed with experimental diets (replacement level ranged from 11% to 66%) were significantly higher (P < 0.05) than the group FM. The liver lysozyme activity of the control group was the lowest and was significantly lower than that of other dietary treatments (P < 0.05). Villus length and muscle thickness in the intestine of fish were significantly lower (P < 0.05) than other groups when the replacement level exceeded 44%. With dietary replacement levels increased, the TLR22, MyD88, p65, pro-inflammatory cytokines (IL-1β, TNF-α, IL-12P40 and INF-γ) and anti-inflammatory cytokines (TGF-β, IL-10, epinecidin, MHCIIβ and hepcidin) mRNA levels in the proximal intestine were significantly up-regulated (P < 0.05). The TLR22, MyD88, p65, pro-inflammatory cytokines (IL-1β, TNF-α, IL-12P40 and INF-γ) and anti-inflammatory cytokines (TGF-β, IL-10, MHCIIβ and hepcidin) mRNA levels in the mid intestine were significantly up-regulated (P < 0.05). The mRNA levels of TLR22, anti-inflammatory cytokines (IL-1β, TNF-α, IL-12P40, INF-γ) and anti-inflammatory cytokines (TGF-β, IL-10, epinecidin, MHCIIβ and hepcidin) in the distal intestine were significantly down-regulated (P < 0.05). The mRNA expression of MyD88 and p65 mRNA were showed a tend increased first and then decreased, and the highest values were observed in group S33 and S55 (P < 0.05), respectively. Based on the present work, the correlation between WGR and FM replacement level with SPC was described using the broken-line model, which estimated the optimum FM replacement to 37.23% for juvenile hybrid grouper dietary., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

High grade ovarian serous cancer (HGSC) is a malignant disease with high mortality. Glycosylation plays important roles in tumor invasion and immune evasion, but its effect on the immune microenvironment of HGSC remains unclear. This study examined the association of glycosyltransferase expression with HGSC prognosis and explored the underlying mechanism using clinical specimens and integrated bioinformatic analyses. We identified a cluster of 15 glycogenes associated with reduced overall survival, and GALNT10 was found to be an independent predictor of HGSC prognosis. The high GALNT10 expression was associated with increased regulatory CD4+ T cells infiltration and decreased granzyme B expression in CD8+ T cells. The expression of GALNT10 and its product, Tn antigen, in HGSC specimens was associated with the increased infiltration of M2 macrophages and neutrophils, and the decreased infiltration of CD3+ T cells, NK cells, and B cells. Taken collectively, high GALNT10 expression confers with immunosuppressive microenvironment to promote tumor progression and predicts poor clinical outcomes in HGSC patients.

Several microRNAs (miRNAs) have been identified to play roles in adipocyte differentiation. However, little is known about their involvement in the differentiation of ovine intramuscular adipocytes. Here, the role of one such miRNA, miR-340-5p, in ovine adipocyte differentiation was investigated. Stromal vascular (SV) cells were isolated from skeletal muscle tissues of 1-month-old lambs and induced to differentiate into mature adipocytes. miRNA mimics and inhibitors were used for miR-340-5p overexpression and knockdown assays. For overexpression and knockdown of activating transcription factor 7 (ATF7), lentivirus infection was performed. Luciferase reporter assay was performed to determine the relationship between miR-340-5p and ATF7. The expression of adipogenesis marker genes, PPARγ, C/EBPα, FABP4, ADIPOQ, and ACC, and formation of lipid droplets were detected after the overexpression and inhibition of miR-340-5p, or upon overexpression or knockdown of ATF7. miR-340-5p inhibited the expression of the marker genes and the formation of lipid droplets. ATF7 positively regulated the expression of the marker genes and the formation of lipids. Thus, ATF7 is the target of miR-340-5p in sheep. Overall, these findings indicate that miR-340-5p acts as an inhibitor of the differentiation of intramuscular adipocytes by targeting ATF7. Our study provides a new theoretical basis for improving sheep meat quality., (© 2020 Japanese Society of Animal Science.)

The noble scallop Chlamys nobilis is an economically important marine bivalve cultivated in the southern sea of China since the 1980s. Unfortunately, mass mortality of this scallop species often occurs in summer. The present study was conducted to investigate whether the expression of heat shock protein 90 (HSP90) and level of carotenoids could enhance high-temperature stress resistance in scallop. First, the HSP90 homolog of C. nobilis (designated CnHSP90) was identified and cloned. The complete cDNA sequence of CnHSP90 was 2631 bp, including a 2181-bp open reading frame (ORF) encoding a 726 amino acid polypeptide with five HSP90 family signatures, and sharing high homology with members of the HSP90 family. CnHSP90 was ubiquitously expressed in all examined tissues including the intestine, kidney, adductor, mantle, gill, and gonad, with the highest in the gonad. Golden and brown scallops, which contain significantly different total carotenoid content (TCC), were subjected to acute thermal challenge, and the LTE 50 (semi-lethal temperature at 36 h heat shock) and LTI 50 (semi-lethal time after heat shock) as well as the correlation between CnHSP90 gene expression and TCC were determined. The LTE 50 of golden scallop (32.14 °C) was higher than that of brown scallops (31.19 °C), with longer LTI 50 at all tested temperatures, indicating that golden scallops were more resistant to thermal stress than brown scallops. Similarly, the mRNA expression levels of CnHSP90 in gill of golden scallops were significantly higher (P < 0.05) than that of brown scallops at 6, 12, 24, and 36 h, with a strong positive correlation between CnHSP90 expression level and TCC. This suggests that both carotenoids and HSP90 levels could improve thermal resistance in the noble scallops.

This study was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acids (n-3 HUFA) on growth performance, non-specific immunity, expression of some immune-related genes and resistance to Vibrio harveyi in juvenile hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatu). Six isoproteic and isolipidic experimental diets were formulated with graded levels of n-3 HUFA (0.65, 1.00, 1.35, 1.70, 2.05 and 2.40% of dry matter, respectively), and the 0.65% group was used as control group. Each diet was randomly allocated to triplicate groups of fish in 1000 L fiberglass tank, and each tank was stocked with 40 fish (initial weight 12.06 ± 0.01 g) for 8 weeks. Results showed that feed conversion ratio (FCR), survival rate (SR), hepatosomatic index (HSI) and condition factor (CF) were all not significantly affected by dietary n-3 HUFA levels (P > 0.05). Weight gain (WG) and specific growth rate (SGR) in 1.35% group were significantly higher than those in 2.40% group (P < 0.05). Crude lipid of body in 1.00% group was significantly lower than that in 1.70% and 2.40% groups (P < 0.05). Liver and muscle fatty acid profiles reflected that of diets. Before challenge with Vibrio harveyi, the activity of serum superoxide dismutase (SOD), catalase (CAT) and content of complement 3 (C3) in 1.35% and 1.70% groups significantly higher than those of control group (P < 0.05). After challenge with Vibrio harveyi, serum CAT, glutathione peroxidase (GSH-PX), lysozyme (LZM) and C3 all increased sharply, while SOD showed the opposite trend. Before challenge with Vibrio harveyi, the expression levels of intestine toll-like receptor 22 (TLR22) and myeloid differentiation factor 88 (MyD88) mRNA in 2.40% group were significantly increased, and the expression levels of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) mRNA in 2.05% group were significantly higher than those in 1.00% and 1.35% groups (P < 0.05). In addition, the TLR22 and IL-1β mRNA levels in kidney of 1.70% group were significantly lower than those in control group (P < 0.05). After challenge with Vibrio harveyi, the expression level of MyD88 mRNA in intestine of 1.35% group was significantly higher than that in 1.00% group and from 1.70% to 2.40% groups (P < 0.05), while TNF-α and IL-1β obtained minimum values in 1.70% group. In the kidney, the interleukin 10 (IL10) mRNA expression was significantly higher in 1.70% group than that in other groups, while the IL-1β expression in 1.70% group was on the contrary and significantly lower than that in 2.40% group (P < 0.05). Results of this study suggested that moderate dietary n-3 HUFA (1.47%-1.70% HUFA) could improve the growth performance, non-specific immunity and inhibit the inflammatory response of hybrid grouper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Paraquat (PQ) as a non-selective heterocyclic herbicide, has been applied worldwide for over a few decades. But PQ is very harmful to humans and rodents. The lung is the main target organ of PQ poisoning. It is an important event that lung epithelial cells are injured during PQ-induced acute lung injury and pulmonary fibrosis. As a regulator of mRNA expression, microRNA (miRNA) may play an important role in the progress. Our study was to investigate the mechanisms of PQ-induced injury of pulmonary epithelial cells through analyzing the profiling of miRNAs and their target genes. As a result, 11 differentially expressed miRNAs were screened, including 1 upregulated miRNA and 10 downregulated miRNAs in PQ-treated murine lung alveolar epithelial cells (MLE-12 cells). The bioinformatic analyses suggested that the target genes of these miRNAs were involved in mitochondrial apoptosis pathway and DNA methylation, and participated in the regulation of PI3K-Akt, mTOR, RAS, TNF, MAPK and other signal pathways which related to oxidative stress and apoptosis. This indicated that miRNAs were an important regulator of oxidative stress and apoptosis during PQ-induced injury of murine lung alveolar epithelial cells. The findings would deepen our understanding of the mechanisms of PQ-induced pulmonary injury and might provide new treatment targets for this disease.

A 56-day growth trial was conducted to investigate the effects of dietary yeast hydrolysate on the growth performance, antioxidation, immune response and resistance against Aeromonas hydrophila in largemouth bass. Four experimental diets were prepared with yeast hydrolysate levels of 0% (Y0), 1.5% (Y1.5), 3.0% (Y3.0) and 4.5% (Y4.5). Each diet was randomly assigned to triplicate 150-L tanks and each tank was stocked with 30 largemouth bass (initial body weight, IBW = 7.71 ± 0.02 g). A challenge test was carried out after the feeding trial by injecting A. hydrophila intraperitoneally for 4-day observation. The results showed that the FBW and WGR in Y1.5 group were significantly higher than those in Y0 group (P < 0.05) and the feed conversion ratio (FCR) got the lowest value in Y1.5 group. And the hydrolysate supplement significantly increased the 4-day cumulative survival rate after the bacterial challenge (P < 0.05). The plasma malondialdehyde was lower in the yeast hydrolysate supplement groups in both pre- and post-challenge test (P < 0.05), while the plasma C3 increased (P < 0.05). In post-challenge test, the plasma superoxide dismutase (SOD) and catalase (CAT) activities increased in the Y1.5 and Y3.0 groups respectively (P < 0.05), and plasma lysozyme in Y1.5 group and the plasma IgM in Y3.0 group were higher than those in others respectively (P < 0.05). For the q-PCR results, in post-challenge test, the hepatic hep2 expression level in Y1.5 and Y4.5 groups were both significantly higher than those in others (P < 0.05), as well as il-8 in Y3.0 group. The spleen hif-1alpha and tgf-beta1 expression levels in Y4.5 group were all significantly lower than those in others (P < 0.05), while the gilt was significantly higher (P < 0.05) in the post-challenge test. And the expression levels of spleen tnf-alpah1 in Y1.5 and Y3.0 groups and il-8 in Y3.0 group were all significantly higher than those in other groups (P < 0.05) in the post-challenge test. The head kidney gilt expression level was significantly higher in the yeast hydrolysate supplement groups compared with the Y0 group (P < 0.05), and the head kidney il-8 expression level in Y1.5 group was significant higher than those in other groups in post-challenge test (P < 0.05). The present results indicated dietary yeast hydrolysate improved the antioxidant ability and enhanced the immune response of largemouth bass without negative effect on growth. And 1.5% or 3.0% of dietary yeast hydrolysate was recommended for largemouth bass based on the present results., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Stocking density is a crucial factor in shellfish aquaculture that affects overall growth performance and health status. Present study analyzes the effects of stocking densities on growth, survival and hemolymph immune status of noble scallop Chlamys nobilis. The scallops with the same size were separately placed in the lantern cages (10 layers per cage) using high stocking density (500 scallops per cage) and low stocking density (100 scallops per cage) and cultivated in the same location for 60 days. The results indicated that the scallops cultivated at high stocking density had significantly higher mortality and slower growth than those cultivated at low stocking density. Moreover, the hemolymph of scallops cultivated at high density showed significantly higher bacterial load, higher reactive oxygen species (ROS), higher expression level of Nrf2 and lower expression level of Keap1, as well as lower antibacterial ability of Vibrio parahemolyticus than that of scallops cultivated at low density. The present results demonstrated that long-term overcrowding is detrimental for the scallops, which can not only lead to high mortality and slow growth, but also cause more vulnerable to pathogenic bacteria. Therefore, we speculated that high stocking density culture practice of scallops in China might be the root of infectious bacteria outbreaks., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Complement factor H‑related 3 (CFHR3) belongs to the human factor H protein family and is associated with various human diseases, including nephropathy, age‑related macular degeneration and atypical hemolytic uremic syndrome. However, to the best of our knowledge, the role of CFHR3 in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis were performed to determine mRNA and protein expression levels of CFHR3 in HCC and normal adjacent tissue. In addition, CFHR3 was overexpressed in Huh‑7 cells and cell counting kit‑8 assay was used to determine cell viability. Cell proliferation and apoptosis were assessed using flow cytometry, RT‑qPCR and western blotting. The results demonstrated that mRNA (2‑ΔΔCq) and protein expression levels of CFHR3 were significantly lower in tumor tissue compared with in adjacent tissue. Additionally, CFHR3 overexpression decreased cell viability, inhibited cell proliferation and significantly increased apoptosis. It was also identified that CFHR3 could downregulate the expression of Ki67. The results suggested that CFHR3 induced apoptosis by downregulating the expression of survivin and B cell lymphoma 2, upregulating the expression of Bcl‑2‑associated X and promoting caspase‑3 activity. Western blotting revealed that CFHR3 significantly inhibited the protein expression levels of phosphorylated (p)‑phosphoinositide 3‑kinase (PI3K), p‑protein kinase B (Akt) and p‑mammalian target of rapamycin (mTOR). Overexpression of CFHR3 suppressed proliferation and promoted apoptosis of HCC cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

There has been an increasing incidence rate of rice false smut in global rice cultivation areas. However, there is a dearth of studies on the environmental concentrations and hazards of ustiloxin A (UA), which is the major mycotoxin produced by a pathogenic fungus of the rice false smut. Here, the concentrations of UA in the surface waters of two paddy fields located in Enshi city, Hubei province, China, were measured, and its toxicity in T. Thermophila was evaluated. This is the first study to detect UA in the surface waters of the two paddy fields, and the measured mean concentrations were 2.82 and 0.26 μg/L, respectively. Exposure to 2.19, 19.01 or 187.13 μg/L UA for 5 days significantly reduced the theoretical population and cell size of T. thermophila. Furthermore, treatment with 187.13 μg/L UA changed the percentages of T. thermophila cells in different cell-cycle stages, and with an increased malformation rate compared with the control, suggesting the disruption of the cell cycle. The expressions of 30 genes involved in the enriched proteasome pathway, 7 cyclin genes (cyc9, cyc10, cyc16, cyc22, cyc23, cyc26, cyc33) and 2 histone genes (mlh1 and hho1) were significantly down-regulated, which might be the modes of action responsible for the disruption of cell cycling due to UA exposure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms. Neoseiulus barkeri Hughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACT and Hsp40 were the top two stable reference genes in developmental stages. The Hsp60 and Hsp90 were the most stable reference genes in various acaricides stress. For alterations in temperature, Hsp40 and α-TUB were the most suitable reference genes. About UV stress, EF1α and α-TUB were the best choice, and for the different prey stress, β-ACT and α-TUB were best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression in N. barkeri.

Peptide transporter 1 (SLC15A1, PepT1), excitatory amino acid transporter 3 (SLC1A1, EAAT3) and cationic amino acid transporter 1 (SLC7A1, CAT1) were identified as genes responsible for the transport of small peptides and amino acids. The tissue expression pattern of rabbit (SLC15A1, SLC7A1 and SLC1A1) across the digestive tract remains unclear. The present study investigated SLC15A1, SLC7A1 and SLC1A1 gene expression patterns across the digestive tract at different stages of development and in response to dietary protein levels. Real time-PCR results indicated that SLC15A1, SLC7A1 and SLC1A1 genes throughout the rabbits' entire development and were expressed in all tested rabbit digestive sites, including the stomach, duodenum, jejunum, ileum, colon and cecum. Furthermore, SLC7A1 and SLC1A1 mRNA expression occurred in a tissue-specific and time-associated manner, suggesting the distinct transport ability of amino acids in different tissues and at different developmental stages. The most highly expressed levels of all three genes were in the duodenum, ileum and jejunum in all developmental stages. All increased after lactation. With increased dietary protein levels, SLC7A1 mRNA levels in small intestine and SLC1A1 mRNA levels in duodenum and ileum exhibited a significant decreasing trend. Moreover, rabbits fed a normal level of protein had the highest levels of SLC15A1 mRNA in the duodenum and jejunum (P&lt;0.05). In conclusion, gene mRNA differed across sites and with development suggesting time and sites related differences in peptide and amino acid absorption in rabbits. The effects of dietary protein on expression of the three genes were also site specific.

Background: Enzalutamide, a novel androgen receptor (AR) signaling inhibitor, has been widely used to increase survival in patients with castration-resistant prostate cancer. However, resistance to enzalutamide invariably develops., Methods: To understand the underlying mechanisms of resistance to enzalutamide, we performed integrative analysis on multiple transcriptome datasets to identify those genes constantly up- or down-regulated in response to enzalutamide treatment., Results: There were 703 and 581 differentially expressed genes derived from enzalutamide-sensitive and -resistant cell lines, respectively. Functional enrichment analysis on these genes demonstrated that biological processes of cell proliferation and ubiquitin mediated proteolysis pathway are specifically disturbed in sensitive cell lines but not resistant ones. Such divergence explained why enzalutamide ineffective for resistant prostate cancer., Conclusions: Taken together, the present study revealed a set of critical genes, which can provide etiologic clues as to enzalutamide-resistant prostate cancer and guide novel therapeutic approaches.

Background: Nuclear factor-κB (NF-κB) signaling plays essential roles in inflammatory responses. However, whether the expression levels of NF-κB family genes affect inflammatory responses is unclear. Moreover, little is known regarding the association between NF-κB family genes expression and the pathogenesis of chronic obstructive pulmonary disease (COPD). The present study was undertaken to assess the relationship between the expression levels of NF-κB family genes mRNA and of inflammatory markers relevant to COPD pathogenesis., Methods: A total of 186 unrelated patients with acute exacerbations of COPD and 180 healthy controls were recruited. Total RNA was extracted from the peripheral fasting blood of each subject using trizol reagent. The mRNA levels of NF-κB family genes ( NF-κB1 , NF-κB2 and c-REL ) were measured by real-time quantitative polymerase chain reaction. The serum levels of cyclooxygenase-2 (COX-2), C-reactive protein, interleukin (IL)-1β, IL-6, IL-8, IL-12 and tumor necrosis factor-α were measured with enzyme-linked immunosorbent assay kits., Results: The relative mRNA levels of the NF-κB family genes and the levels of inflammatory molecules were significantly higher in the COPD group than in the control group after adjustment for smoking. The IL-1β, IL-8 and COX-2 levels were significantly lower in the NF-κB2 under-expression subgroup as compared to the NF-κB2 over-expression subgroup. The COX-2 level was significantly lower ( P < 0.05) in the c-REL under-expression subgroup as compared to the c-REL over-expression subgroup., Conclusion: NF-κB2 over-expression was associated with IL-1β, IL-8 and COX-2 levels, whereas c-REL overexpression was associated with COX-2 level. Over-expression of both NF-κB2 and c-REL was found to be related to COPD., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background: The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest., Results: In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized., Conclusion: Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.

Previous studies have identified the association between cartilage endplate (CEP) degeneration and abnormal mechanical loading. Several studies have reported that intermittent cyclic mechanical tension (ICMT) regulates CEP degeneration via various biological processes and signaling pathways. However, the functions of microRNAs in regulating the cellular responses of CEP chondrocytes to ICMT remain to be elucidated. The current study determined the differentially expressed microRNAs in human CEP chondrocytes exposed to ICMT using microarray analysis. A total 21 significantly upregulated and 62 downregulated miRNAs were identified compared with the control. The findings were subsequently partially validated by reverse transcription‑quantitative polymerase chain reaction. Potential target genes of the significantly differentially expressed miRNAs were predicted using bioinformatics analysis and were used for Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The present study revealed that the significantly differentially expressed microRNAs were involved in various signaling pathways and biological processes that are crucial to regulating the responses of CEP chondrocytes to ICMT. The current study provided a global view of microRNA expression in CEP chondrocytes under mechanical stimulation, suggesting that microRNAs are important for regulating the mechanical response of CEP chondrocytes. Additionally, it provided a novel insight into the association between mechanical stress and the establishment and progression of intervertebral disc degeneration.

Background and Aim: A considerable number of early-stage colorectal cancer (CRC) patients may develop cancer relapse or metastasis after curative surgery. Isolated tumor cells (ITC) and micrometastasis in lymph nodes (LNMM), which are undetectable by conventional pathological examination, may be one primary reason. Detection of ITC/LNMM is time-consuming and cost-ineffective; we aimed to find biomarkers in primary CRC tissues to help predicting ITC/LNMM status., Methods: We enrolled 137 node-negative patients with early-stage CRC in this study. Existence of ITC/LNMM was identified by immunohistological staining with cytokeratin 20 in resected lymph nodes. Expression of transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) in primary CRC tissues was also investigated. Chi-squared test was performed to reveal the correlations between ITC/LNMM and clinicopathological characteristics. Univariate and multivariate analyses were used to determine independent prognostic factors. Knockdown experiment together with proliferation and invasion assays were carried out to explore molecular mechanisms between TBL1XR1 and ITC/LNMM., Results: About 29.2% (40/137) patients were identified as ITC/LNMM positive, and most of them (32/40 cases, 80%) showed high TBL1XR1 expression in primary CRC tissues. Both ITC/LNMM and TBL1XR1 expression were independent prognostic factors for disease relapse or metastasis. In vitro experiments demonstrated that TBL1XR1 can regulate the expression of vascular endothelial growth factor C and epithelial-mesenchymal transition proteins, thus mediate the process of lymph node metastasis., Conclusions: Identification of ITC/LNMM is significant in evaluating clinical outcome and guiding adjuvant chemotherapy for early-stage CRC patients. TBL1XR1 overexpression in CRC tissues can serve as an efficient biomarker to predict the status of ITC/LNMM., (© 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Accumulating evidence demonstrates that the Wnt/β-catenin signaling pathway plays a dominant role in bone repair. However, the role of Wnt/β-catenin signaling in the remodeling phase during bone fracture healing is currently unknown. In the present study, β-catenin was activated at different levels or deleted in mice at the late stage of fracture healing, and the effects on healing quality were investigated. Deletion of β-catenin disturbed bone remodeling, as confirmed by increased bone resorption and decreased bone formation, and significantly decreased bone strength compared with wildtype mice. In addition, the constitutive activation of β-catenin significantly increased the bone mass and delayed the bone remodeling process, resulting in slightly impaired bone strength. In contrast, a slight activation of β-catenin significantly increased bone formation and slightly hindered bone resorption. These effects lead to improved bone fracture healing quality compared with wildtype mice. In summary, the present study provides the first demonstration showing that Wnt/β-catenin signaling should be maintained at a slightly activated level during the late stage of fracture healing to ensure better bone fracture repair.

Objective: To analyze the distribution of stress in the upper and lower plates of the prosthesis-bone interface, and the effect of interface pressure on osseointegration., Methods: CT scanning was performed on goats at 1 week after artificial cervical disc replacement to establish the finite element model of C 3, 4 . The stress distribution of the upper and lower plates of the interface was observed. At 6 and 12 months after replacement, Micro-CT scan and three dimensional reconstruction were performed to measure the bone volume fraction (BVF), trabecular number (Tb. N), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), bone mineral density (BMD), bone surface/bone volume (BS/BV), and trabecular pattern factor (Tb. Pf). The C 3 lower plate and C 4 upper plate of 4 normal goat were chosen to made the cylinder of the diameter of 2 mm. The gene expressions of receptor activator for nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), transforming growth factor β (TGF-β), and macrophage colony-stimulating factor (M-CSF) were detected by real time fluorescent quantitative PCR at immediate after cutting and at 24 and 48 hours after culture. The samples of appropriate culture time were selected to made mechanical loading, and the gene expressions of RANKL, OPG, M-CSF, and TGF-β were detected by real time fluorescent quantitative PCR; no mechanical loading samples were used as normal controls., Results: Under 25 N axial loading, the stress of the upper plate of C 3, 4 was concentrated to post median region, and the stress of the lower plate to middle-front region and two orbits. According to stress, the plate was divided into 5 regions. The Micro-CT scan showed that BMD, Tb.Th, BVF, and Tb.N significantly increased, and BS/BV, Tb.Sp, and Tb.Pf significantly decreased at 12 months after replacement when compared with ones at 6 months ( P <0.05). At 24 and 48 hours after culture, the gene expressions of RANKL, OPG, and TGF-β were signifi-cantly higher than those at immediate ( P <0.05), but no significant difference was found between at 24 and 48 hours after culture ( P >0.05). The mechanical loading test results at 24 hours after culture showed that the RANKL and OPG gene expressions and OPG/RANKL ratio in C 3 lower plate and C 4 upper plate were significantly up-regulated when compared with controls ( P <0.05), but no significant difference was shown in TGF-β and M-CSF gene expressions ( P >0.05)., Conclusion: Domestic artificial cervical disc endplate has different pressure distribution, the stress of lower plate is higher than that of upper plate. Pressure has important effect on local osseointegration; the higher pressure area is, the osseointegration is better. Under the maximum pressure in interface, the osteoblast proliferation will increase, which is advantageous to the local osseointegration.

The aim of this study was to investigate whether exogenous retinoic acid (RA) can upregulate the mRNA and protein expression of growth-associated protein 43 (GAP-43), thereby promoting brain functional recovery in a rat distal middle cerebral artery occlusion (MCAO) model of ischemia. A total of 216 male Sprague Dawley rats weighing 300-320 g were divided into 3 groups: sham-operated group, MCAO+vehicle group and MCAO+RA group. Focal cortical infarction was induced with a distal MCAO model. The expression of GAP-43 mRNA and protein in the ipsilateral perifocal region was assessed using qPCR and immunocytochemistry at 1, 3, 7, 14, 21, and 28 days after distal MCAO. In addition, an intraperitoneal injection of RA was given 12 h before MCAO and continued every day until the animal was sacrificed. Following ischemia, the expression of GAP-43 first increased considerably and then decreased. Administration of RA reduced infarction volume, promoted neurological functional recovery and upregulated expression of GAP-43. Administration of RA can ameliorate neuronal damage and promote nerve regeneration by upregulating the expression of GAP-43 in the perifocal region after distal MCAO.

Currently, there is limited information on the toxicity of low concentration of metal mixtures in the environment. Of particular interest is the effect of low levels of metal mixtures on neurodevelopment of aquatic organisms. This study reports the neurological gene expressions after exposing zebrafish embryos to low concentration toxic heavy metals, 120 h post fertilization (hpf). Embryos were exposed to low concentration individual and mixtures of lead (Pb), mercury (Hg), arsenic (As), and cadmium (Cd). Quantitative real-time PCR was used to assess gene expressions. The findings of this study confirmed that exposure to low concentration heavy metals upregulated N-methyl-D-aspartate (NMDA) receptor subunits NMDAR2A (NR2A), NMDAR2B (NR2B), and NMDAR2D (NR2D) and B cell lymphoma (Bcl-2) genes. NR2A genes were significantly upregulated by 90 and 74%, respectively, on exposure to Pb + As and Pb + Cd. NR2B genes were upregulated by 85.3, 68.6, 62.7, and 62.7% on exposure to As, Pb + Hg, Pb + As, and Pb + Cd, respectively. Exposure to As, Pb + Cd, and Pb + Hg + As significantly upregulated Bcl-2 genes by 2.01-, 1.84-, and 1.80-fold, respectively. NR1A and C-fos gene expressions were not significantly different from control. Upregulation of NMDAR subunits and Bcl-2 genes in this study was largely a counter measure against insults from exposure to low concentration heavy metals. Principal component analysis confirmed the influence of low concentration individual and mixtures of Pb, Hg, As, and Cd on gene expression of NMDAR subunits and Bcl-2. These data suggest that altered expression of NMDA receptor subunits and Bcl-2 genes may explain toxicity of low concentration individual and mixtures of Pb, Hg, As, and Cd.

1. In a previous study, a laying-related single nucleotide polymorphism (SNP) Record-106975 (A-G) was found in the intron of Membrane-associated Guanylate Kinase with Inverted Domain Structure-1 (MAGI1) gene by restriction site-associated DNA sequencing. This study was performed to further identify the function of MAGI1 on laying performance in Yangzhou geese. 2. The complete MAGI1 cDNA sequence was cloned from the ovary of the Yangzhou goose. A total of 4 alternative splice variants were identified, and the characterisation of their nucleic acid and amino acid (AA) sequences were analysed. The AA sequence of goose MAGI1 was highly conserved with other avian species. 3. MAGI1 was ubiquitously expressed in 11 tested tissues, with greater amounts of mRNA in the granulosa cell layer and ovary. GG (n = 14) and GA (n = 6) genotypes of SNP Record-106975 showed significantly lower MAGI1 mRNA levels than those of the AA genotype. 4. Over-expression and knockdown experiments revealed that MAGI1 mRNA expression was associated with caspase-3 mRNA levels in granulosa cells, and may play a role in regulating apoptosis of granulosa cells.

Nonhuman primate (NHP) induced pluripotent stem cells (iPSCs) offer the opportunity to investigate the safety, feasibility, and efficacy of proposed iPSC-derived cellular delivery in clinically relevant in vivo models. However, there is need for stable, robust, and safe labeling methods for NHP iPSCs and their differentiated lineages to study survival, proliferation, tissue integration, and biodistribution following transplantation. Here we investigate the utility of the adeno-associated virus integration site 1 (AAVS1) as a safe harbor for the addition of transgenes in our rhesus macaque iPSC (RhiPSC) model. A clinically relevant marker gene, human truncated CD19 (hΔCD19), or GFP was inserted into the AAVS1 site in RhiPSCs using the CRISPR/Cas9 system. Genetically modified RhiPSCs maintained normal karyotype and pluripotency, and these clones were able to further differentiate into all three germ layers in vitro and in vivo. In contrast to transgene delivery using randomly integrating viral vectors, AAVS1 targeting allowed stable transgene expression following differentiation. Off-target mutations were observed in some edited clones, highlighting the importance of careful characterization of these cells prior to downstream applications. Genetically marked RhiPSCs will be useful to further advance clinically relevant models for iPSC-based cell therapies., (Published by Elsevier Inc.)

Anthocyanins, a class of flavonoids, are responsible for the orange to blue coloration of flowers and act as visual attractors to aid pollination and seed dispersal. Malonyl-CoA is the precursor for the formation of flavonoids and anthocyanins. Previous studies have suggested that malonyl-CoA is formed almost exclusively by acetyl-CoA carboxylase, which catalyzes the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate. In the present study, the full-length cDNA of Petunia hybrida acyl-activating enzyme 13 (PhAAE13), a member of clade VII of the AAE superfamily that encodes malonyl-CoA synthetase, was isolated. The expression of PhAAE13 was highest in corollas and was down-regulated by ethylene. Virus-induced gene silencing of petunia PhAAE13 significantly reduced anthocyanin accumulation, fatty acid content, and cuticular wax components content, and increased malonic acid content in flowers. The silencing of PhAAE3 and PhAAE14, the other two genes in clade VII of the AAE superfamily, did not change the anthocyanin content in petunia flowers. This study provides strong evidence indicating that PhAAE13, among clade VII of the AAE superfamily, is specifically involved in anthocyanin biosynthesis in petunia flowers.

Bone morphogenetic protein 2 (BMP-2) and transforming growth factor β (TGF-β) isoforms are important in advancing bone regeneration. The aim of the present study was to investigate the positive and reciprocal effect of TGF‑β3, one of the three TGF‑β isoforms, on BMP‑2 in promoting osteogenic differentiation. Exogenous BMP‑2 and TGF‑β3 genes were separately, and in combination, overexpressed in rabbit bone marrow‑derived mesenchymal stem cells (rBMSCs). Expression levels of BMP‑2 and TGF‑β3 were evaluated using reverse‑transcription‑polymerase chain reaction (RT‑PCR) and Western blotting assays. Furthermore, the osteogenic differentiation capacities of BMSCs were assessed by measuring Alizarin Red S staining, an alkaline phosphatase activity assay, and quantification of the osteogenic‑specific genes, Runt‑related transcription factor 2 (Runx2) and Osterix (Osx). Using lentiviral‑mediated transfection, robust co‑transfection efficiency of >90% was achieved. RT‑PCR and immunoblotting results indicated a marked elevated expression of BMP‑2 and TGF‑β3 in rBMSCs undergoing co‑transfection, compared with transfection with BMP‑2 or TGF‑β3 alone, indicating that BMP‑2 and TGF‑β3 are synergistically expressed in rBMSCs. Furthermore, enhanced osteogenic differentiation was observed in rBMSCs co‑transfected with BMP‑2/TGF‑β3. The present study successfully delivered BMP‑2 together with TGF‑β3 into rBMSCs with high efficiency for the first time. Furthermore, TGF‑β3 overexpression was demonstrated to enhance the osteogenic efficacy of BMP‑2 in rBMSCs, and vice versa. This provides a potential clinical therapeutic approach for regenerating the function of osseous tissue, and may present a promising strategy for bone defect healing.

Hypopharyngeal cancer is a distinct type of malignant head and neck tumor, which exhibits low sensitivity to anti-cancer drugs. The importance of developing methods for reducing chemotherapy resistance, and improving and enhancing prognosis has previously been emphasized and is considered a challenge for effective clinical treatment of hypopharyngeal cancer. The current study investigated the effects of co‑expression of inhibitor of growth protein 4 (ING4) and P53, a tumor suppressor gene, on chemosensitivity to cisplatin in human hypopharyngeal cancer xenografts in vivo, and the potential molecular mechanisms involved. A tumor model was established by injecting athymic nude mice with FADU human hypopharyngeal cancer cells. Five days after intratumoral and peritumoral injections of an empty adenoviral vector (Ad), Ad‑ING4‑P53, cisplatin, or a combination of Ad‑ING4‑P53 and cisplatin (Ad‑ING4‑P53 + cisplatin) every other day for 5 days, the mice were euthanized and their tumors, livers, and kidneys were removed. The tumor weights were used to calculate the inhibition rate, and the expression levels of ING4 and P53 were detected by reverse transcription‑polymerase chain reaction. Additionally, apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunohistochemistry determined the levels ING4, P53, B‑cell lymphoma‑2 (Bcl‑2) and Bcl‑2 associated X protein (Bax) protein expression. The results demonstrated increased expression of ING4 and P53 in the Ad‑ING4‑P53 groups compared with PBS and Ad groups, indicating successful introduction of the genes into the tumor cells. Notably, the Ad‑ING4‑P53 + cisplatin group exhibited a higher inhibition rate compared with the four other groups. The results of immunohistochemistry analysis demonstrated that Bax expression was increased and Bcl‑2 was decreased in the Ad‑ING4‑P53 + cisplatin group. This suggested that the enhanced cisplatin chemosensitivity with Ad-ING4-P53 gene therapy in hypopharyngeal cancer xenografts may be associated with apoptosis induction through upregulation of Bax expression and downregulation of Bcl‑2. The results of the present study indicated that gene therapy combined with cisplatin treatment may be a promising treatment for human hypopharyngeal cancer.

Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Previous studies have demonstrated that recombinant adenovirus serotype 5 (Ad5) may be a potential vaccine candidate because it can express JEV envelope epitopes and induce immune responses against JEV. Still, it will be necessary to develop an adjuvant that can enhance both humoral and cellular immune responses to the recombinant antigen delivered by non-replicating Ad5. In this study, we investigated the systemic immune responses of BALB/c mice immunized with recombinant adenovirus expressing JEV envelope epitopes in combination with porcine interleukin-6 (rAdE-IL-6).The rAdE-IL-6 immunized group had the highest titers of anti-JEV antibody as detected by an enzyme-linked immunosorbent assay (ELISA), as well as the highest levels of neutralizing antibody (1:75) as detected by a serum neutralization test. Similarly, higher concentrations of interferon-gamma (834.7pg/ml) and interleukin-6 (IL-6) (229.7pg/ml) were detected in the rAdE-IL-6 group using an ELISA assay. These data indicate that immunized BALB/c induce a strong cellular response against rAdE-IL-6. Furthermore, after challenge with the virulent JEV SCYA201201 strain, the rAdE-IL-6 group generated an immune protective response 70% greater than that of the control group, indicating that rAdE-IL-6 induced a protective immune response against JEV challenge in mice. The results from this study demonstrated that IL-6 is a strong adjuvant that can enhance both humoral and cellular immune responses in mice. Furthermore, a recombinant adenovirus coexpressing JEV envelope epitopes and porcine IL-6 protein may be an effective vaccine in animals., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Many studies have proved that DNA methylation can regulate gene expression and further affect skeletal muscle growth and development of pig, whereas the mechanisms of how DNA methylation or gene expression alteration ultimately lead to phenotypical differences between the cloned and natural mating pigs remain elusive. This study aimed to investigate genome-wide gene expression and DNA methylation differences between abnormally cloned and normally natural mating piglets and identify molecular markers related to skeletal muscle growth and development in pig. The DNA methylation and genome-wide gene expression in the two groups of piglets were analysed through methylated DNA immunoprecipitation binding high-throughput sequencing and RNA sequencing respectively. We detected 1493 differentially expressed genes between the two groups, of which 382 genes were also differentially methylated. The results of the integrative analysis between DNA methylation and gene expression revealed that the DNA methylation levels showed a significantly negative and monotonic correlation with gene expression levels around the transcription start site of genes. By contrast, no notable monotonic correlation was observed in other regions. Furthermore, we identified some interesting genes and signalling pathways (e.g. myosin, heavy chain 7 and mammalian target of rapamycin) which possibly play essential roles in skeletal muscle growth and development. The results of this study provide insights into the relationship of DNA methylation with gene expression in newborn piglets and into the mechanisms in abnormally cloned animals through somatic cell nuclear transfer., (© 2016 Stichting International Foundation for Animal Genetics.)

Reconstruction of networks from high-throughput expression data is an important tool to identify new regulatory relations. Given that nonlinear and complex relations exist between biological units, methods that can utilize nonlinear dependencies may yield insights that are not provided by methods using linear associations alone. We have previously developed a distance to measure predictive nonlinear relations, the Distance based on Conditional Ordered List (DCOL), which is sensitive and computationally efficient on large matrices. In this study, we explore the utility of DCOL in the reconstruction of networks, by combining it with local false discovery rate (lfdr)-based inference. We demonstrate in simulations that the new method named nlnet is effective in recovering hidden nonlinear modules. We also demonstrate its utility using a single cell RNA seq dataset. The method is available as an R package at https://cran.r-project.org/web/packages/nlnet.

Matrix metalloproteinase-3 (MMP-3) can mediate the occurrence and development of rheumatoid arthritis (RA). The MMP3 promoter gene exhibits polymorphism with 5A/6A alleles. We investigated the correlation between the expression of MMP3 gene polymorphism and RA to provide an objective basis for prognosis evaluation. We enrolled 80 RA patients and 80 healthy subjects. Enzyme-linked immunosorbent assay was used to detect MMP-3 serum levels, pyrosequencing was used to test MMP3 genotypes, and real-time polymerase chain reaction determined MMP-3 mRNA expression levels. Compared with the control group, the serum level of MMP-3 in the RA patients increased significantly (P < 0.05). The serum level of MMP-3 in RA patients in the active period was markedly elevated compared with that in patients in the relief period (P < 0.05). There was no statistically significant difference between MMP3 gene frequency distribution in the RA patients and the control group (P > 0.05). MMP-3 mRNA expression in the RA patients was markedly upregulated compared with the control group (P < 0.05), while RA patients in the active period exhibited higher MMP-3 mRNA expression (P < 0.05). There was no significant difference in MMP-3 mRNA expression between RA patients with or without the 6A/6A genotype (P > 0.05). RA patients exhibited higher serum MMP-3 levels and mRNA expression, which were more obvious in the active period. MMP-3 is associated with the occurrence and development of RA bone erosion, and its serum level and mRNA expression can be treated as important predictors of joint damage.

Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment.

Objective: We constructed highly efficient expression systems for agarase AgaD and optimized its culture conditions., Methods: First, the codon usage of AgaD was optimized to make it suitable for expression in E. coli. Then, the gene expression vector was transformed into different E. coli hosts. According to the "N-end rule" that is related to the in vivo half-life of a protein, a mutant was constructed. Finally, the effects of CaCl2 and glycine on enzyme production were evaluated., Results: A highly efficient expression system of agarase AgaD was constructed, named pET-22b (+)-optagaDx-AD494 ( E3). Replacing N-terminal second amino acid phenylalanine with alanine significantly improved agarase production and shortened the fermentation period. The extracellular enzyme activity was further up-regulated by CaCll and glycine. After optimization, the extracellular enzyme production raised from 20 U/L to 11300 U/L, more than 500 folds., Conclusion: The high expression system of AgaD provides good basis for further studying agarases.

Curcumin is a traditional Chinese medicine extracted from the rhizome of the herb Curcuma longa, which exhibits anti-human cytomegalovirus (HCMV) activity, however, the underlying mechanism remains to be elucidated. The present study reported that the pharmacogenomics of curcumin are similar to that of the antiviral drug, geldanamycin, which targets heat shock protein 90 (Hsp90). Comparative analysis of 3,000 clinical drugs demonstrated that curcumin had a positive association with the gene expression profiles of several drugs, among which the pharmacogenomics of the antiviral drug, geldanamycin, were most similar to that of curcumin. Molecular docking simulation analysis revealed that curcumin fit well in the binding pocket of Hsp90, with hydrogen bonds, hydrophobic interactions and conjugation to maintain adhesion. Consistently, HCMV infection of human embryonic lung fibroblast cells resulted in increased expression of Hsp90α, which was significantly inhibited by treatment with curcumin. These findings suggested that targeting Hsp90 contributed to the anti‑HCMV activity of curcumin.

Plumbagin is a natural compound that is isolated from the root of the medicinal plant Plumbago zeylanica L. Based on a previous in vitro study performed by our group, which demonstrated the effectiveness of plumbagin against glioma cells, we further ascertained whether plumbagin exhibits the same effectiveness against glioma cell xenografts in nude mice. Our results revealed that tumor volume was reduced by 54.48% in the plumbagin-treated group compared with the controls. Furthermore, there were no obvious signs of toxicity as assessed by the organ sizes and cell morphologies of the mice that were treated with plumbagin. Immunofluorescence assays further revealed that plumbagin significantly inhibited glioma cell proliferation and induced cell apoptosis. Importantly, we also determined that the expressions of FOXM1 and its downstream target effectors, including cyclin D1 and Cdc25B, were down-regulated in the treated group, while the expressions of p21 and p27 were increased; the latter findings corroborate the results of our previous in vitro study. Taken together, these findings indicate that plumbagin may be a natural downregulator of FOXM1 with potential therapeutic effectiveness for the treatment of gliomas., (Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Key Message: Conditions for the isolation and transfection of maize nucellus protoplasts were established. We demonstrated its utilization for protein expression, localization, protein-protein interaction, and the investigation of PCD-related processes. Plant protoplasts are an important and versatile cell system that is widely used in the analysis of gene characterization and diverse signaling pathways. Programmed cell death (PCD) occurs throughout the life of plants from embryogenesis to fertilization. The maize nucellus undergoes typical PCD during development of the embryo sac. The nucellus protoplast shows potential for use in research of PCD-related processes. No studies have reported previously the isolation and transfection of nucellus protoplasts. In this study, conditions for the isolation and transfection of maize nucellus protoplasts were established. The maize protoplast system can be used for protein expression, localization, and protein-protein interaction. We applied this system to investigate PCD-related processes. Quantitative real-time PCR analysis revealed that transient expression of MADS29 in the maize nucellus protoplast increases Cys-protease gene transcript level. In addition, β-glucuronidase and luciferase activity assays showed that MADS29 could enhance the promoter activities of the Cys-protease gene. Thus, we demonstrated the potential of a highly efficient maize nucellus protoplast system for transient gene expression and investigation of PCD-related processes.