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Messenger RNA (mRNA)-based therapy shows immense potential for broad biomedical applications. However, the development of safe and efficacious mRNA delivery vectors remains challenging due to delivery barriers and inefficient intracellular payload release. Herein, we presented a simple strategy to boost the mRNA intracellular release by incorporation of anionic poly(γ-glutamic acid) (PGA) into an ionizable lipid-based LNP/mRNA. We systematically investigated the impact of PGA incorporation on mRNA transfection both in vitro and in vivo. The molecular weights and formulation ratios of PGA greatly affected the transfection efficacy of LNP/mRNA. From in vitro study, the optimized LNP/mRNA/PGA was formulated by incorporation of PGA with the molecular weight of 80 kDa or 200 kDa and the charge ratio (N/P/C) of 25/1/1. The optimized formulation achieved around 3-fold mRNA expression in HeLa cells compared to the bare LNP/mRNA. The intracellular releasing study using specific DNA probe revealed that this enhancement of transfection efficacy was attributed to the elevated mRNA release into cytoplasm. Moreover, the optimized LNP/mRNA/PGA achieved up to 5-fold or 3-fold increase of luciferase mRNA expression in vivo after being injected into mice systematically or intramuscularly, respectively. In addition, the incorporation of PGA did not significantly alter the biodistribution profile of the complexes on both organ and cellular levels. Therefore, our work provides a simple strategy to boost mRNA delivery, which holds great promise to improve the efficacy of mRNA therapeutics for various biomedical applications. The process of designing and screening potent mRNA carriers is complicated and time-consuming, while the efficacy is not always satisfying due to the delivery barriers and inefficient mRNA release. This work presented an alternative strategy to boost the mRNA delivery efficacy by incorporating an anionic natural polymer poly(γ-glutamic acid) (PGA) into LNP/mRNA complexes. The optimized LNP/mRNA/PGA achieved up to 3-fold and 5-fold increase in transfection efficacy in vitro and in vivo , respectively. Intracellular releasing analysis revealed that the enhancement of transfection efficacy was mainly attributed to the elevated intracellular release of mRNA. In addition, the incorporation of PGA did not alter the biodistribution or the biosafety profile of the complexes. These findings indicate that PGA incorporation is a promising strategy to improve the efficacy of mRNA therapeutics. [Display omitted] [ABSTRACT FROM AUTHOR]

Introduction: Low serum selenium and altered tumour RNA expression of certain selenoproteins are associated with a poor breast cancer prognosis. Selenoprotein expression stringently depends on selenium availability, hence circulating selenium may interact with tumour selenoprotein expression. However, there is no matched analysis to date. Methods: This study included 1453 patients with newly diagnosed breast cancer from the multicentric prospective Sweden Cancerome Analysis Network – Breast study. Total serum selenium, selenoprotein P and glutathione peroxidase 3 were analysed at time of diagnosis. Bulk RNA-sequencing was conducted in matched tumour tissues. Fully adjusted Cox regression models with an interaction term were employed to detect dose-dependent interactions of circulating selenium with the associations of tumour selenoprotein mRNA expression and mortality. Results: 237 deaths were recorded within ~ 9 years follow-up. All three serum selenium biomarkers correlated positively (p < 0.001). All selenoproteins except for GPX6 were expressed in tumour tissues. Single cell RNA-sequencing revealed a heterogeneous expression pattern in the tumour microenvironment. Circulating selenium correlated positively with tumour SELENOW and SELENON expression (p < 0.001). In fully adjusted models, the associations of DIO1, DIO3 and SELENOM with mortality were dose-dependently modified by serum selenium (p < 0.001, p = 0.020, p = 0.038, respectively). With increasing selenium, DIO1 and SELENOM associated with lower, whereas DIO3 expression associated with higher mortality. Association of DIO1 with lower mortality was only apparent in patients with high selenium [above median (70.36 µg/L)], and the HR (95%CI) for one-unit increase in log(FPKM + 1) was 0.70 (0.50–0.98). Conclusions: This first unbiased analysis of serum selenium with the breast cancer selenotranscriptome identified an effect-modification of selenium on the associations of DIO1, SELENOM, and DIO3 with prognosis. Selenium substitution in patients with DIO1-expressing tumours merits consideration to improve survival. [ABSTRACT FROM AUTHOR]

Whole-genome duplication often results in a reduction in the lignin content in autopolyploid plants compared with their diploid counterparts. However, the regulatory mechanism underlying variation in the lignin content in autopolyploid plants remains unclear. Here, we characterize the molecular regulatory mechanism underlying variation in the lignin content after the doubling of homologous chromosomes in Populus hopeiensis. The results showed that the lignin content of autotetraploid stems was significantly lower than that of its isogenic diploid progenitor throughout development. Thirty-six differentially expressed genes involved in lignin biosynthesis were identified and characterized by RNA sequencing analysis. The expression of lignin monomer synthase genes, such as PAL, COMT, HCT, and POD, was significantly down-regulated in tetraploids compared with diploids. Moreover, 32 transcription factors, including MYB61, NAC043, and SCL14, were found to be involved in the regulatory network of lignin biosynthesis through weighted gene co-expression network analysis. We inferred that SCL14, a key repressor encoding the DELLA protein GAI in the gibberellin (GA) signaling pathway, might inhibit the NAC043–MYB61 signaling functions cascade in lignin biosynthesis, which results in a reduction in the lignin content. Our findings reveal a conserved mechanism in which GA regulates lignin synthesis after whole-genome duplication; these results have implications for manipulating lignin production. [ABSTRACT FROM AUTHOR]

Through excellent absorption and transformation, the macrophyte Myriophyllum (M.) aquaticum can considerably remove phosphorus from wastewater. The results of changes in growth rate, chlorophyll content, and roots number and length showed that M. aquaticum could cope better with high phosphorus stress compared with low phosphorus stress. Transcriptome and differentially expressed genes (DEGs) analyses revealed that, when exposed to phosphorus stresses at various concentrations, the roots were more active than the leaves, with more DEGs regulated. M. aquaticum also showed different gene expression and pathway regulatory patterns when exposed to low phosphorus and high phosphorus stresses. M. aquaticum's capacity to cope with phosphorus stress was maybe due to its improved ability to regulate metabolic pathways such as photosynthesis, oxidative stress reduction, phosphorus metabolism, signal transduction, secondary metabolites biosynthesis, and energy metabolism. In general, M. aquaticum has a complex and interconnected regulatory network that deals efficiently with phosphorus stress to varying degrees. This is the first time that the mechanisms of M. aquaticum in sustaining phosphorus stress have been fully examined at the transcriptome level using high-throughput sequencing analysis, which may indicate the direction of follow-up research and have some guiding value for its future applications. [ABSTRACT FROM AUTHOR]

Leukemia caused by environmental chemical pollutants has attracted great attention, the malignant leukemic transformation model of TK6 cells induced by hydroquinone (HQ) has been previously found in our team. However, the type of leukemia corresponding to this malignant transformed cell line model needs further study and interpretation. Furthermore, the molecular mechanism of malignant proliferation of leukemic cells induced by HQ remains unclear. This study is the first to reveal the expression of aberrant genes in leukemic cells of HQ-induced malignant transformation, which may correspond to chronic lymphocytic leukemia (CLL). The expression of Linc01588, a long non-coding RNA (lncRNA), was significantly up-regulated in CLL patients and leukemic cell line model which previously described. After gain-of-function assays and loss-of-function assays, feeble cell viability, severe apoptotic phenotype and the increased secretion of TNF-α were easily observed in malignant leukemic TK6 cells with Linc01588 deletion after HQ intervention. The tumors derived from malignant TK6 cells with Linc01588 deletion inoculated subcutaneously in nude mice were smaller than controls. In CLL and its cell line model, the expression of Linc01588 and miR-9–5p, miR-9–5p and SIRT1 were negative correlation respectively in CLL and cell line model, while the expression of Linc01588 and SIRT1 were positive correlation. The dual-luciferase reporter assay showed that Linc01588 & miR-9–5p, miR-9–5p & SIRT1 could bind directly, respectively. Furthermore, knockdown of miR-9–5p successfully rescued the severe apoptotic phenotype and the increased secretion of TNF-α caused by the Linc01588 deletion, the deletion of Linc01588 in human CLL cell line MEC-2 could also inhibit malignant biological characteristics, and the phenotype caused by the deletion of Linc01588 could also be rescued after overexpression of SIRT1. Moreover, the regulation of SIRT1 expression in HQ19 cells by Linc01588 and miR-9–5 P may be related to the Akt/NF-κB pathway. In brief, Linc01588 deletion inhibits the malignant biological characteristics of HQ-induced leukemic cells via miR-9–5p/SIRT1, and it is a novel and hopeful clue for the clinical targeted therapy of CLL. • HQ-induced leukemic cells may also correspond to chronic lymphocytic leukemia (CLL). • Knockdown of miR-9–5p rescued the malignant phenotype caused by Linc01588 deletion. • Overexpression of SIRT1 triggers malignant phenotype caused by deletion of Linc01588. • Linc01588 deletion inhibits malignant features via miR-9–5p/SIRT1 axis. • Targeting Linc01588 or miR-9–5p may be a new clue for the treatment of CLL. [ABSTRACT FROM AUTHOR]

Aberrant reprogramming of metabolism has been considered a hallmark in various malignant tumors. The metabolic changes of amino acid not only have dramatic effects in cancer cells but also influence their immune-microenvironment in gliomas. However, the features of the amino acid metabolism-related and immune-associated gene set have not been systematically described. The expression level of mRNA was obtained from The Cancer Genome Atlas database and the Chinese Glioma Genome Atlas database, which were used as training set and validation set, respectively. Different bioinformatics and statistical methods were combined to construct a robust amino metabolism-related and immune-associated risk signature for distinguishing prognosis and clinical pathology features. Constructing the nomogram enhanced risk stratification and quantified risk assessment based on our gene model. Besides this, the biological mechanism related to the risk score was investigated by gene set enrichment analysis. Hub genes of risk signature were identified by the protein–protein interaction network. The amino acid metabolism-related and immune-associated gene signature recognized high-risk patients, defined as an independent risk factor for overall survival. The nomogram exhibited a high accuracy in predicting the overall survival rate for glioma patients. Furthermore, the high risk score hinted an immunosuppressive microenvironment and a lower sensitivity of immune checkpoint blockade therapy and also identified PSMC5 and PSMD3 as novel biomarkers in glioma. In conclusion, a novel amino acid metabolism-related and immune-associated risk signature for predicting prognosis in glioma has been constructed and identified as two potential novel biomarkers. [ABSTRACT FROM AUTHOR]

Objective. To determine the role of sodium butyrate in intestinal inflammation via regulation of high-mobility group box-1 (HMGB1), we analyzed the potential mechanism in necrotizing enterocolitis (NEC) in a neonatal mouse model. Methods. A NEC model was created with hypoxia and cold exposure and artificial overfeeding. C57BL/6 neonatal mice were randomized into three groups: the control, untreated NEC, and sodium butyrate (150 mM)-pretreated NEC groups. Pathological variations in ileocecal intestinal tissue were observed by HE staining and scored in a double-blind manner. The mRNA expression levels of HMGB1, Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and inflammatory cytokines in intestinal tissues were determined by quantitative real-time PCR. The protein levels of HMGB1 and associated cytokines in intestinal tissues were evaluated using ELISA. The relative protein expression levels of TLR4 and NF-κB in intestinal tissues were quantified by western blot. Results. Sodium butyrate administration improved the body weight and survival rate of NEC mice; relieved intestinal pathological injury; reduced the intestinal expression of HMGB1, TLR4, NF-κB, interleukin- (IL-) 1β, IL-6, IL-8, and TNF-α; and increased the intestinal expression of IL-10 (P < 0.05). Treatment with butyrate decreased the proportion of opportunistic Clostridium_sensu_stricto_1 and Enterococcus and increased the proportion of beneficial Firmicutes and Lactobacillus in the NEC model. Conclusions. Sodium butyrate intervention relieves intestinal inflammation and partially corrects the disrupted intestinal flora in mice with NEC. [ABSTRACT FROM AUTHOR]

Simple Summary: Melanoma is a growing health issue in the 21st century. Due to early metastasis and the development of resistance, it still goes along with a poor prognosis. ADCK protein kinases have been shown to play a role during cancer development and metastasis. Here, we investigated the role of ADCK2 in melanoma. In our study, we showed that higher levels of intratumoral ADCK2 benefit patient survival, while a low expression of ADCK2 was associated with a higher motility and a dedifferentiated state of melanoma cells, which facilitates metastasis. Our results could give new insights into melanoma metastasis, and ADCK2 could qualify as a prognostic marker or a target for melanoma therapy in the future. Background: ADCK2 is a member of the AarF domain-containing kinase family, which consists of five members, and has been shown to play a role in CoQ metabolism. However, ADCKs have also been connected to cancer cell survival, proliferation and motility. In this study, we investigated the role of ADCK2 in melanoma. Methods: The effect of ADCK2 on melanoma cell motility was evaluated by a scratch assay and a transwell invasion assay upon siRNA-mediated knockdown or stable overexpression of ADCK2. Results: We found that high levels of intratumoral ADCK2 and MYL6 are associated with a higher survival rate in melanoma patients. Knocking down ADCK2 resulted in enhanced cell migration of melanoma cells. Moreover, ADCK2-knockdown cells adopted a more dedifferentiated phenotype. A gene expression array revealed that the expression of ADCK2 correlated with the expressions of MYL6 and RAB2A. Knocking down MYL6 in ADCK2-overexpressing cells could abrogate the effect of ADCK2 overexpression and thus confirm the functional connection between ADCK2 and MYL6. Conclusion: ADCK2 affects melanoma cell motility, most probably via MYL6. Our results allow the conclusion that ADCK2 could act as a tumor suppressor in melanoma. [ABSTRACT FROM AUTHOR]

Meat quality is a critical aspect of pig breeding. In addition to genetics, meat quality is also influenced by nutritional and environmental factors. In this study, three pig breeds, Shengxianhua, Jiaxing, and Qinglian Black (SXH, JXB and QLB), were used as experimental animals. Transcriptional analysis was performed on the longissimus thoracis (LT) muscle to investigate variations in intramuscular fat (IMF), inosine monophosphate (IMP), amino acids, and muscle fiber morphology across different breeds. Ingenuity canonical pathway analysis (IPA) identified biological processes and key driver genes related to metabolism and muscle development. Additionally, weighted gene co-expression network analysis (WGCNA) revealed gene modules associated with IMP. KEGG and GO analyses identified specific biological processes and signaling pathways related to IMP, including the Oxidative Phosphorylation pathway and rRNA Metabolic Processes. These findings provide novel insights into the molecular regulatory mechanisms underlying meat quality variations among pig breeds. • Comparison of amino acid content in the longissimus thoracis (LT) muscle of three pig breeds. • Determination of inosine monophosphate and intramuscular fat content in the LT muscle. • Hematoxylin-eosin (H&E) staining revealed myofiber morphology in the LT muscle. • Identified canonical pathways and driver genes associated with meat quality traits. • WGCNA highlights gene modules related to meat quality traits. • GO and KEGG analyses elucidated the functions of differentially expressed genes in the gene modules. [ABSTRACT FROM AUTHOR]

Hydroquinone (HQ), a major metabolite of benzene and known hematotoxic carcinogen. MicroRNA 1246 (miR-1246), an oncogene, regulates target genes in carcinogenesis including leukemia. This study investigates the impact of exosomal derived miR-1246 from HQ-transformed (HQ19) cells on cell-to-cell communication in recipient TK6 cells. RNA sequencing was used to identify differentially expressed exosomal miRNAs in HQ19 cells and its phosphate buffered solution control cells (PBS19), which were then confirmed using qRT-PCR. The impact of exosomal miR-1246 derived from HQ-transformed cells on cell cycle distribution was investigated in recipient TK6 cells. RNA sequencing analysis revealed that 34 exosomal miRNAs were upregulated and 158 miRNAs were downregulated in HQ19 cells compared with PBS19 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses predicted that their targets are enriched in cancer development-related pathways, such as MAPK signaling, microRNAs in cancer, apoptosis, PI3K-Akt signaling, cell cycle, Ras signaling, and Chronic myeloid leukemia. Eleven miRNAs were confirmed to have differential expression through qRT-PCR, with 6 upregulated (miR-140–3p, miR-551b-3p, miR-7-5p, miR-1290, miR-92a-3p, and miR-1246) and 5 downregulated (miR-183–5p, miR-26a-5p, miR-30c-5p, miR-205–5p, and miR-99b-3p). Among these, miR-1246 exhibited the highest expression level. HQ exposure resulted in a concentration-dependent increase in miR-1246 levels and decrease Cyclin G2 (CCNG2) levels in TK6 cells. Similarly, exosomes from HQ19 exhibited similar effects as HQ exposure. Dual luciferase reporter gene assays indicated that miR-1246 could band to CCNG2. After HQ exposure, exosomal miR-1246 induced cell cycle arrest at the S phase, elevating the expression of genes like pRb, E2F1, and Cyclin D1 associated with S phase checkpoint. However, silencing miR-1246 caused G2/M-phase arrest. HQ-transformed cells' exosomal miR-1246 targets CCNG2, regulating TK6 cell cycle arrest, highlighting its potential as a biomarker for HQ-induced malignant transformation. The high expression of miR-1246 in exosomes derived from HQ-induced malignant transformed cells effectively suppresses the expression of CCNG2 in recipient TK6 cells following HQ treatment, ultimately leading to cell cycle arrest. [Display omitted] • MiR-1246 was one of highest expressed miRNAs in exosomes from HQ-transformed cells. • Exosomal miR-1246 regulated cell proliferation and cell cycle arrest in TK6 cells via Cyclin G2. • MiR-1246 may be a potential biomarker of HQ-induced malignant transformation. [ABSTRACT FROM AUTHOR]

Oxidative stress is the major culprits responsible for ovarian dysfunction by damaging granulosa cells (GCs). Ferritin heavy chain (FHC) may participate in the regulation of ovarian function by mediating GCs apoptosis. However, the specific regulatory function of FHC in follicular GCs remains unclear. Here, 3-nitropropionic acid (3-NPA) was utilized to establish an oxidative stress model of follicular GCs of Sichuan white geese. To explore the regulatory effects of FHC on oxidative stress and apoptosis of primary GCs in geese by interfering or overexpressing FHC gene. After transfection of siRNA- FHC to GCs for 60 h, the expressions of FHC gene and protein decreased significantly (P < 0.05). After FHC overexpression for 72 h, the expressions of FHC mRNA and protein upregulated considerably (P < 0.05). The activity of GCs was impaired after interfering with FHC and 3-NPA coincubated (P < 0.05). When overexpression of FHC combined with 3-NPA treatment, the activity of GCs was remarkably enhanced (P < 0.05). After interference FHC and 3-NPA treatment, NF-κB and NRF2 gene expression decreased (P < 0.05), the intracellular reactive oxygen species (ROS) level increased greatly (P < 0.05), BCL-2 expression reduced, BAX/BCL-2 ratio intensified (P < 0.05), the mitochondrial membrane potential decreased notably (P < 0.05), and the apoptosis rate of GCs aggravated (P < 0.05). While overexpression of FHC combined with 3-NPA treatment could promote BCL-2 protein expression and reduce BAX/BCL-2 ratio, indicating that FHC regulated the mitochondrial membrane potential and apoptosis of GCs by mediating the expression of BCL-2. Taken together, our research manifested that FHC alleviated the inhibitory effect of 3-NPA on the activity of GCs. FHC knockdown could suppress the expression of NRF2 and NF-κB genes, reduce BCL-2 expression and augment BAX/BCL-2 ratio, contributing to the accumulation of ROS and jeopardizing mitochondrial membrane potential, as well as exacerbating GCs apoptosis. [ABSTRACT FROM AUTHOR]