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The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin⁻green fluorescent protein (Puro-GFP, pf ) or puromycin⁻red fluorescent protein (Puro-RFP, pr ) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.

Simple Summary: In the present study, we aimed to explore the potential local regulatory role of the cocaine- and amphetamine-regulated transcript (CART) signaling pathway in granulosa cell (GC) apoptosis, which is a key mechanism promoting follicular atresia in several animal species, including buffalo. Our results showed how CART activity adversely influences buffalo GC viability by affecting estradiol production and enhancing apoptosis. The regulatory mechanism by which CART can affect GC apoptosis entails the modulation of the AKT/GSK3β/β-catenin pathway, a key intracellular signaling pathway essential for cell viability. In conclusion, this study provides valuable insights into the intricate mechanisms governing ovarian follicle development and granulosa cell function. These findings have implications for reproductive biology not only for buffalo but also for different species. Atresia is a process in ovarian follicles that is regulated by hormone-induced apoptosis. During atresia, granulosa cell (GC) apoptosis is a key mechanism orchestrated through diverse signaling pathways. Cocaine- and amphetamine-regulated transcript (CART) signaling within ovarian GCs has been demonstrated to play a key role in the regulation of follicular atresia in cattle, pigs, and sheep. The present work aimed to investigate the potential local regulatory role of CART in GC apoptosis-induced follicular atresia in buffalo, focusing on the modulation of the AKT/GSK3β/β-catenin signaling pathways, which are the intracellular signaling pathways involved in cell viability. Our findings revealed increased expression of CARTPT and BAX and decreased levels of AKT, β-catenin, and CYP19A1 genes in atretic follicles compared to healthy follicles. Subsequently, CART treatment in the presence of FSH inhibited the FSH-induced increase in GC viability by reducing estradiol production and increasing apoptosis. This change was accompanied by an increase in the gene expression levels of both CARTPT and BAX. At the protein level, treatment with CART in the presence of FSH negatively affected the activity of AKT, β-catenin, and LEF1, while the activity of GSK3β was enhanced. In conclusion, our study shows how CART negatively influences buffalo GC viability, underlying the modulation of the AKT/GSK3β/β-catenin pathway and promoting apoptosis—a key factor in follicular atresia. [ABSTRACT FROM AUTHOR]

Objective: To investigate the relation of clinicopathological characteristics and RCN1 and GRP78 expression levels in colorectal cancer, and explore their prognostic value. Methods: The expression of RCN1 and HSPA5 mRNA in colorectal cancer( CRC) was examined using the TCGA and UALCAN databases, and clinical samples were obtained for immunohistochemistry to confirm the expression of both. Then, the correlation with clinicopathological features and its prognostic value were analyzed. Results: According to a bioinformatics analysis, RCN1 and HSPA5 mRNA were both significantly overexpressed in CRC and had a positive correlation.The expression of RCN1 was correlated with N stage and TP53 mutations, the expression of HSPA5 was higher in mucinous adenocarcinomas. In CRC tissues, RCN1 and GRP78 protein were both strongly expressed and localized in the cytoplasm. GRP78 expression was significantly correlated with T stage. The Kaplan-Meier survival analysis curve indicates that patients with higher RCN1 expression had significantly shorter DFS than those with lower expression and a tendency toward shorter OS.According to COX regression analysis,DFS in CRC was found to be independently associated with tumor nerve invasion, TNM stage, and high RCN1 expression, Correlation analysis: RCN1 and GRP78 expression were positively associated in CRC. Conclusion: RCN1 is an independent risk factor for a poor prognosis in colorectal cancer (CRC) and has a positive correlation with GRP78 expression. [ABSTRACT FROM AUTHOR]

Motivation From a systematic perspective, it is crucial to infer and analyze gene regulatory network (GRN) from high-throughput single-cell RNA sequencing data. However, most existing GRN inference methods mainly focus on the network topology, only few of them consider how to explicitly describe the updated logic rules of regulation in GRNs to obtain their dynamics. Moreover, some inference methods also fail to deal with the over-fitting problem caused by the noise in time series data. Results In this article, we propose a novel embedded Boolean threshold network method called LogBTF, which effectively infers GRN by integrating regularized logistic regression and Boolean threshold function. First, the continuous gene expression values are converted into Boolean values and the elastic net regression model is adopted to fit the binarized time series data. Then, the estimated regression coefficients are applied to represent the unknown Boolean threshold function of the candidate Boolean threshold network as the dynamical equations. To overcome the multi-collinearity and over-fitting problems, a new and effective approach is designed to optimize the network topology by adding a perturbation design matrix to the input data and thereafter setting sufficiently small elements of the output coefficient vector to zeros. In addition, the cross-validation procedure is implemented into the Boolean threshold network model framework to strengthen the inference capability. Finally, extensive experiments on one simulated Boolean value dataset, dozens of simulation datasets, and three real single-cell RNA sequencing datasets demonstrate that the LogBTF method can infer GRNs from time series data more accurately than some other alternative methods for GRN inference. Availability and implementation The source data and code are available at https://github.com/zpliulab/LogBTF. [ABSTRACT FROM AUTHOR]

Soil salinization is an important factor limiting food security and ecological stability. As a commonly used greening tree species, Robinia pseudoacacia often suffers from salt stress that can manifest as leaf yellowing, decreased photosynthesis, disintegrated chloroplasts, growth stagnation, and even death. To elucidate how salt stress decreases photosynthesis and damages photosynthetic structures, we treated R. pseudoacacia seedlings with different concentrations of NaCl (0, 50, 100, 150, and 200 mM) for 2 weeks and then measured their biomass, ion content, organic soluble substance content, reactive oxygen species (ROS) content, antioxidant enzyme activity, photosynthetic parameters, chloroplast ultrastructure, and chloroplast development-related gene expression. NaCl treatment significantly decreased biomass and photosynthetic parameters, but increased ion content, organic soluble substances, and ROS content. High NaCl concentrations (100–200 mM) also led to distorted chloroplasts, scattered and deformed grana lamellae, disintegrated thylakoid structures, irregularly swollen starch granules, and larger, more numerous lipid spheres. Compared to control (0 mM NaCl), the 50 mM NaCl treatment significantly increased antioxidant enzyme activity while upregulating the expression of the ion transport-related genes Na+/H+ exchanger 1(NHX 1) and salt overly sensitive 1 (SOS 1) and the chloroplast development-related genes psaA, psbA, psaB, psbD, psaC, psbC, ndhH, ndhE, rps7, and ropA. Additionally, high concentrations of NaCl (100–200 mM) decreased antioxidant enzyme activity and downregulated the expression of ion transport- and chloroplast development-related genes. These results showed that although R. pseudoacacia can tolerate low concentrations of NaCl, high concentrations (100–200 mM) can damage chloroplast structure and disturb metabolic processes by downregulating gene expression. [ABSTRACT FROM AUTHOR]

The present study aimed to explore the improvement of the bioactivity of ginger peel polysaccharides (GPs) by the modification of zinc after structural characterization. The obtained GP–Zn (II) complexes consisted dominantly of glucose and galactose in a mass proportion of 95.10:2.10, with a molecular weight of 4.90 × 105 Da and a Zn content of 21.17 mg/g. The chelation of GPs and Zn (II) was mainly involved in the O–H of hydroxyl groups, and this interaction reduced the crystallinity and decreased the asymmetry of GPs, with a slight effect on the thermal stability. The administration of GPs and their Zn (II) complexes effectively alleviated CuSO4-induced inflammatory response in zebrafish (Tg: zlyz-EGFP) via down-regulating the mRNA expression levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-12 and TNF-α) and upregulating the expression of anti-inflammatory cytokine (IL-10). Furthermore, the modification of Zn (II) enhanced the inflammation-inhibiting effect of polysaccharides. Therefore, GP–Zn (II) complexes could be applied as a candidate anti-inflammatory agent for the treatment of chronic inflammation-related diseases. [ABSTRACT FROM AUTHOR]

Single-cell RNA-sequencing (scRNA-seq) is a recent high-throughput technique that can measure gene expression, reveal cell heterogeneity, rare and complex cell populations, and discover cell types and their relationships. The analysis of scRNA-seq data is challenging because of transcripts sparsity, replication noise, and outlier cell populations. A gene coexpression network (GCN) analysis effectively deciphers phenotypic differences in specific states by describing gene–gene pairwise relationships. The underlying gene modules with different coexpression patterns partially bridge the gap between genotype and phenotype. This study presents a new framework called scGENA (single-cell gene coexpression network analysis) for GCN analysis based on scRNA-seq data. Although there are several methods for scRNA-seq data analysis, we aim to build an integrative pipeline for several purposes that cover primary data preprocessing, including data exploration, quality control, normalization, imputation, and dimensionality reduction of clustering as downstream of GCN analysis. To demonstrate this integrated workflow, an scRNA-seq dataset of the human diabetic pancreas with 1600 cells and 39,851 genes was implemented to perform all these processes in practice. As a result, scGENA is demonstrated to uncover interesting gene modules behind complex diseases, which reveal biological mechanisms. scGENA provides a state-of-the-art method for gene coexpression analysis for scRNA-seq data. [ABSTRACT FROM AUTHOR]

Background. Invasive breast carcinoma (BRCA) is a common type of breast cancer with a high clinical incidence. Thus, it is significant to find effective biomarkers for BRCA diagnosis and treatment. Although some members of armadillo (ARM) repeat family of proteins are confirmed to be biomarkers in cancers, the role of armadillo repeat-containing 1 (ARMC1) in BRCA remains unknown. Methods. We firstly analyzed the ARMC1 expression in normal breast tissues and BRCA samples and its association with overall survival by the public database. Next, the χ 2 test was used to evaluate the prognostic significance of ARMC1 expression in TCGA-BRCA patient samples. The ARMC1 mutations in BRCA were explored in the cBioportal database. Besides, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to explore the biological functions of ARMC1 in BRCA. Finally, immunohistochemistry and immunofluorescence staining were performed to validate the ARMC1 expression in BRCA. Results. ARMC1 expression in tumor samples was significantly higher than that in normal tissues, and higher expression of ARMC1 was related to lower survival. Moreover, the tumor stage and histology of BRCA patients were associated with ARMC1 expression. ARMC1 genetic mutations occurred in 32% of BRCA patients, and the amplification and high expression of ARMC1 accounted for most of them. Furthermore, functional enrichment analysis suggested that ARMC1 might be involved in the cell cycle in BRCA. Ultimately, increased ARMC1 expression was found in clinical breast carcinoma tissues by our confirmatory experiments. Conclusions. ARMC1 may play a significant role in BRCA and act as a biomarker, which provides valuable clues for the treatment and diagnosis of BRCA. [ABSTRACT FROM AUTHOR]

This study evaluated the effects of dietary myo-inositol (MI) on growth performance, antioxidant status and lipid metabolism of juvenile Chinese mitten crab (Eriocheir sinensis) fed different percentage of lipid. Crabs (4·58 (sem 0·05) g) were fed four diets including a normal lipid diet (N, containing 7 % lipid and 0 mg/kg MI), N with MI supplementation (N + MI, containing 7 % lipid and 1600 mg/kg MI), a high lipid diet (H, containing 13 % lipid and 0 mg/kg MI) and H with MI supplementation (H + MI, containing 13 % lipid and 1600 mg/kg MI) for 8 weeks. The H + MI group showed higher weight gain and specific growth rate than those in the H group. The dietary MI could improve the lipid accumulations in the whole body, hepatopancreas and muscle as a result of feeding on the high dietary lipid (13 %) in crabs. Besides, the crabs fed the H + MI diets increased the activities of antioxidant enzymes but reduced the malondialdehyde content in hepatopancreas compared with those fed the H diets. Moreover, dietary MI enhanced the expression of genes involved in lipid oxidation and exportation, yet reduced lipid absorption and synthesis genes expression in the hepatopancreas of crabs fed the H diet, which might be related to the activation of inositol 1,4,5-trisphosphate receptor (IP3R)/calmodulin-dependent protein kinase kinase-β (CaMKKβ)/adenosine 5'-monophosphate-activated protein kinase (AMPK) signalling pathway. This study demonstrates that MI could increase lipid utilisation and reduce lipid deposition in the hepatopancreas of E. sinensis fed a high lipid diet through IP3R/CaMKKβ/AMPK activation. This work provides new insights into the function of MI in the diet of crustaceans. [ABSTRACT FROM AUTHOR]

Mineral nutrient supply can affect the hydraulic property of roots. The aim of the present work on sheepgrass (Leymus chinensis L.) plants was to test whether any changes in root hydraulic conductivity (Lp; exudation analyses) in response to a growth‐limiting supply of phosphate (P) are accompanied by changes in (1) cell Lp via measuring the cell pressure, (2) the aquaporin (AQP) gene expression by performing qPCR and (3) the formation of apoplastic barriers, by analyzing suberin lamella and Casparian bands via cross‐sectional analyses in roots. Plants were grown hydroponically on complete nutrient solution, containing 250 µM P, until they were 31–36 days old, and then kept for 2–3 weeks on either complete solution, or transferred on solution containing 2.5 µM (low‐P) or no added P (no‐P). Phosphate treatments caused significant decreases in root and cell‐Lp and AQP gene expression, while the formation of apoplastic barriers increased, particularly in lateral roots. Experiments using the AQP inhibitor mercury (Hg) suggested that a significant portion of radial root water uptake in sheepgrass occurs along a path involving AQPs, and that the Lp of this path is reduced under low‐ and no‐P. It is concluded that a growth‐limiting supply of phosphate causes parallel changes in (1) cell Lp and aquaporin gene expression (decrease) and (2) apoplastic barrier formation (increase), and that the two may combine to reduce root Lp. The reduction in root Lp, in turn, facilitates an increased root‐to‐shoot surface area ratio, which allocates resources to the root, sourcing the limiting nutrient. [ABSTRACT FROM AUTHOR]

Background Mineral nutrient limitation affects the water flow through plants. We wanted to test on barley whether any change in root-to-shoot ratio in response to low supply of nitrogen and phosphate is accompanied by changes in root and cell hydraulic properties and involves changes in aquaporin (AQP) gene expression and root apoplastic barriers (suberin lamellae, Casparian bands). Methods Plants were grown hydroponically on complete nutrient solution or on solution containing only 3.3 % or 2.5 % of the control level of nutrient. Plants were analysed when they were 14–18 d old. Results Nutrient-limited plants adjusted water flow to an increased root-to-shoot surface area ratio through a reduction in root hydraulic conductivity (Lp) as determined through exudation analyses. Cortex cell Lp (cell pressure probe analyses) decreased in the immature but not the mature region of the main axis of seminal roots and in primary lateral roots. The aquaporin inhibitor HgCl2 reduced root Lp most in nutrient-sufficient control plants. Exchange of low-nutrient for control media caused a rapid (20–80 min) and partial recovery in Lp, though cortex cell Lp did not increase in any of the root regions analysed. The gene expression level (qPCR analyses) of five plasma membrane-localized AQP isoforms did not change in bulk root extracts, while the formation of apoplastic barriers increased considerably along the main axis of root and lateral roots in low-nutrient treatments. Conclusions Decrease in root and cortex cell Lp enables the adjustment of root water uptake to increased root-to-shoot area ratio in nutrient-limited plants. Aquaporins are the prime candidate to play a key role in this response. Modelling of water flow suggests that some of the reduction in root Lp is due to increased formation of apoplastic barriers. [ABSTRACT FROM AUTHOR]

Recently, identifying robust biomarkers or signatures from gene expression profiling data has attracted much attention in computational biomedicine. The successful discovery of biomarkers for complex diseases such as spontaneous preterm birth (SPTB) and high-grade serous ovarian cancer (HGSOC) will be beneficial to reduce the risk of preterm birth and ovarian cancer among women for early detection and intervention. In this paper, we propose a stable machine learning-recursive feature elimination (StabML-RFE for short) strategy for screening robust biomarkers from high-throughput gene expression data. We employ eight popular machine learning methods, namely AdaBoost (AB), Decision Tree (DT), Gradient Boosted Decision Trees (GBDT), Naive Bayes (NB), Neural Network (NNET), Random Forest (RF), Support Vector Machine (SVM) and XGBoost (XGB), to train on all feature genes of training data, apply recursive feature elimination (RFE) to remove the least important features sequentially, and obtain eight gene subsets with feature importance ranking. Then we select the top-ranking features in each ranked subset as the optimal feature subset. We establish a stability metric aggregated with classification performance on test data to assess the robustness of the eight different feature selection techniques. Finally, StabML-RFE chooses the high-frequent features in the subsets of the combination with maximum stability value as robust biomarkers. Particularly, we verify the screened biomarkers not only via internal validation, functional enrichment analysis and literature check, but also via external validation on two real-world SPTB and HGSOC datasets respectively. Obviously, the proposed StabML-RFE biomarker discovery pipeline easily serves as a model for identifying diagnostic biomarkers for other complex diseases from omics data. The source code and data can be found at https://github.com/zpliulab/StabML-RFE. [Display omitted] • A stable feature selection method (StabML-RFE) is proposed to screen robust biomarkers. • StabML-RFE employs some popular ML-RFE methods and integrates them into an aggregation-like framework. • StabML-RFE ensembles multiple optimal feature subsets by aggregating AUC values and stability indices. • The robustness of screened biomarker genes is measured by the Stability metric based on Hamming distance. [ABSTRACT FROM AUTHOR]

Leaf coloration changes evoke different photosynthetic responses among different poplar cultivars. The aim of this study is to investigate the photosynthetic difference between a red leaf cultivar (ZHP) and a green leaf (L2025) cultivar of Populus deltoides. In this study, 'ZHP' exhibited wide ranges and huge potential for absorption and utilization of light energy and CO2 concentration which were similar to those in 'L2025' and even showed a stronger absorption for weak light. However, with the increasing light intensity and CO2 concentration, the photosynthetic capacity in both 'L2025' and 'ZHP' was gradually restricted, and the net photosynthetic rate (Pn) in 'ZHP' was significantly lower than that in 'L2025'under high light or high CO2 conditions, which was mainly attributed to stomatal regulation and different photosynthetic efficiency (including the light energy utilization efficiency and photosynthetic CO2 assimilation efficiency) in these two poplars. Moreover, the higher anthocyanin content in 'ZHP' than that in 'L2025' was considered to be closely related to the decreased photosynthetic efficiency in 'ZHP'. According to the results from the JIP-test, the capture efficiency of the reaction center for light energy in 'L2025' was significantly higher than that in 'ZHP'. Interestingly, the higher levels of light quantum caused relatively higher accumulation of QA- in 'L2025', which blocked the electron transport and weakened the photosystem II (PSII) performance as compared with 'ZHP'; however, the decreased capture of light quantum also could not promote the utilization of light energy, which was the key to the low photosynthetic efficiency in 'ZHP'. The differential expressions of a series of photosynthesis-related genes further promoted these specific photosynthetic processes between 'L2025' and 'ZHP'. [ABSTRACT FROM AUTHOR]

Background: Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC O157 encounters varied microenvironments during infection, and can efficiently adapt to these using the two-component system (TCS). Recently, a functional TCS, RstAB, has been implicated in the regulation of virulence of several bacterial pathogens. However, the regulatory function of RstAB in EHEC O157 is poorly understood. This study aimed at providing insights into the global effects of RstA on gene expression in EHEC O157. Results: In the present study, we analyzed gene expression differences between the EHEC O157 wild-type strain and a ΔrstA mutant using RNA-seq technology. Genes with differential expression in the ΔrstA mutant compared to that in the wild-type strain were identified and grouped into clusters of orthologous categories. RstA promoted EHEC O157 LEE gene expression, adhesion in vitro, and colonization in vivo by indirect regulation. We also found that RstA could bind directly to the promoter region of hdeA and yeaI to enhance acid tolerance and decrease biofilm formation by modulating the concentration of c-di-GMP. Conclusions: In summary, the RstAB TCS in EHEC O157 plays a major role in the regulation of virulence, acid tolerance, and biofilm formation. We clarified the regulatory function of RstA, providing an insight into mechanisms that may be potential drug targets for treatment of EHEC O157-related infections. [ABSTRACT FROM AUTHOR]