Transcriptomic study of the role of MeFtsZ2-1 in pigment accumulation in cassava leaves.

MeFtsZ2-1 is a key gene for plant plastid division, but the mechanism by which MeFtsZ2-1 affects pigment accumulation in cassava (Manihot esculenta Crantz) through plastids remains unclear. We found that MeFtsZ2-1 overexpression in cassava (OE) exhibited darker colors of leaves, with increased levels of anthocyanins and carotenoids. Further observation via Transmission Electron Microscopy (TEM) revealed no apparent defects in chloroplast structure but an increase in the number of plastoglobule in OE leaves. RNA-seq results showed 1582 differentially expressed genes (DEGs) in leaves of OE. KEGG pathway analysis indicated that these DEGs were enriched in pathways related to flavonoid, anthocyanin, and carotenoid biosynthesis. This study reveals the role of MeFtsZ2-1 in cassava pigment accumulation from a physiological and transcriptomic perspective, providing a theoretical basis for improving cassava quality.

Downregulation of Rab23 inhibits hepatocellular carcinoma by repressing SHH signaling pathway.

Hepatocellular carcinoma (HCC) is the sixth most common malignant tumors and the third leading cause of cancer-related death worldwide. As an oncogene, Rab23 has been shown to be significantly related to the growth and migration of hepatocellular carcinoma in both in vitro and in vivo studies, but its underlying mechanism remains obscure. In the present study, we examined the effect of inhibiting Rab23 expression on the pathological progression of HCC. The correlation between liver Rab23 gene expression and survival probability in human HCC patients was analyzed using the TCGA database and CPTAC database. Rab23 knockdown hepatocellular carcinoma cell line was generated through lentiviral transduction, then we established a nude HCC xenograft model by subcutaneously implanting the transfected cells. The analysis of gene and protein expression was carried out using Western blot or RT-qPCR, respectively. Flow cytometry analysis was used to detect the level of apoptosis. The expression levels of key proteins involved in the Sonic Hedgehog (SHH) signaling pathway were assessed. The results showed that HCC patients with low levels of hepatic Rab23 mRNA and protein had a better survival tendency than those with higher levels of Rab23. Cell proliferations were reduced and apoptosis levels were increased after Knocking down Rab23 in HCC cell lines. Furthermore, in vivo studies have demonstrated that suppression of the Rab23 gene results in decreased tumor size, proliferation rate, and reduced levels of SHH-related proteins Smoothened and GLI-1. The above results suggest that Rab23 is involved in the pathological progression of HCC as an important regulator of the SHH signaling pathway, which also provides an important research basis for new therapeutic strategies for HCC.

Genome-wide identification and expression analysis of heat shock protein gene family in cassava.

Heat shock proteins are important molecular chaperones that are involved in plant growth and stress responses. However, members of the Hsp family have been poorly studied in cassava. In this study, 225 MeHsp genes were identified in the cassava genome, and their genetic structures exhibited relatively conserved features within each subfamily. The 225 MeHsp genes showed random chromosomal distribution, and at least 74 pairs of segmentally duplicated MeHsp genes. Eleven tandemly duplicated MeHsp genes were identified. Cis-element analysis revealed the importance of MeHsps in plant adaptations to the environment. The prediction of protein interactions suggested that MeHsp70-20 may play a critical regulatory role in the interactive network. Furthermore, the expression profiles of MeHsps in different tissues and cell subsets were analyzed using bulk transcriptomics and single-cell transcriptomic data. Several subfamily genes exhibited unique expression patterns in the transcriptome and were selected for detailed analysis of the single-cell transcriptome. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed the expression patterns of these genes under temperature stress, further supporting the prediction of cis-acting elements. This study provides valuable information for understanding the functional characteristics of MeHsp genes and the evolutionary relationships between MeHsps.

Single-cell RNA-sequencing profiles reveal the developmental landscape of the Manihot esculenta Crantz leaves.

Cassava (Manihot esculenta Crantz) is an important crop with a high photosynthetic rate and high yield. It is classified as a C3-C4 plant based on its photosynthetic and structural characteristics. To investigate the structural and photosynthetic characteristics of cassava leaves at the cellular level, we created a single-cell transcriptome atlas of cassava leaves. A total of 11,177 high-quality leaf cells were divided into 15 cell clusters. Based on leaf cell marker genes, we identified 3 major tissues of cassava leaves, which were mesophyll, epidermis, and vascular tissue, and analyzed their distinctive properties and metabolic activity. To supplement the genes for identifying the types of leaf cells, we screened 120 candidate marker genes. We constructed a leaf cell development trajectory map and discovered 6 genes related to cell differentiation fate. The structural and photosynthetic properties of cassava leaves analyzed at the single cellular level provide a theoretical foundation for further enhancing cassava yield and nutrition.

An fgf21-like gene from swamp eel (Monopterus albus): Recombinant expression and its potential roles in glucose and lipid homeostasis.

Fibroblast growth factor 21 (FGF21) plays important roles in the regulation of glucose and lipid metabolism and energy balance in mammals. In this study, the full-length cDNA of swamp eel fgf21 was cloned. Sequence analysis showed that swamp eel FGF21 displayed high similarity with FGF21 of other vertebrates. Subsequently, a prokaryotic expression vector for swamp eel fgf21 was constructed, and recombinant FGF21 (rFGF21) was successfully induced and purified. To investigate the potential roles of swamp eel FGF21 in glucose and lipid metabolism, we examined the effects of rFGF21 on regulation of glucose and lipid homeostasis in type 1 diabetes mellitus (T1DM) mice as well as swamp eels under glucose stress. In T1DM mice, the levels of blood glucose, serum triglyceride (TG), liver TG, serum total cholesterol (TC), and liver TC were significantly downregulated after repeated daily injection of rFGF21 for 15 days. In addition, liver pathological section analysis indicated that rFGF21 alleviated the degree of damage to liver cells in T1DM mice. Furthermore, rFGF21 significantly upregulated the mRNA expression levels of peroxisome proliferators-activated receptor alpha (Pparα), ß-Klotho, fibroblast growth factor receptor 1 (Fgfr1), phosphoenolpyruvate carboxykinase (Pepck), glucose transporter 1 (Glut1), and glucose transporter 4 (Glut4) in T1DM mouse livers. Moreover, in swamp eels, rFGF21 significantly decreased blood glucose and liver TC levels under glucose stress and upregulated the mRNA expression levels of fgf21, pparα, ß-klotho, and fgfr1 in liver tissue. These results suggested that FGF21 plays important roles in the regulation of glucose and lipid homeostasis in swamp eel.

Single-cell transcriptome sequencing atlas of cassava tuberous root.

Introduction: Single-cell transcriptome sequencing (ScRNA-seq) has emerged as an effective method for examining cell differentiation and development. In non-model plants, it hasn't been employed very much, especially in sink organs that are abundant in secondary metabolites. Results: In this study, we sequenced the single-cell transcriptomes at two developmental phases of cassava tuberous roots using the technology known as 10x Genomics (S1, S2). In total, 14,566 cells were grouped into 15 different cell types, primarily based on the marker genes of model plants known to exist. In the pseudotime study, the cell differentiation trajectory was defined, and the difference in gene expression between the two stages on the pseudotime axis was compared. The differentiation process of the vascular tissue and cerebral tissue was identified by the trajectory. We discovered the rare cell type known as the casparian strip via the use of up-regulated genes and pseudotime analysis, and we explained how it differentiates from endodermis. The successful creation of a protoplast isolation technique for organs rich in starch was also described in our study. Discussion: Together, we created the first high-resolution single-cell transcriptome atlas of cassava tuberous roots, which made significant advancements in our understanding of how these roots differentiate and develop.

Genome-Wide Analysis of Tubulin Gene Family in Cassava and Expression of Family Member FtsZ2-1 during Various Stress.

Filamentous temperature-sensitive protein Z (Tubulin/FtsZ) family is a group of conserved GTP-binding (guanine nucleotide-binding) proteins, which are closely related to plant tissue development and organ formation as the major component of the cytoskeleton. According to the published genome sequence information of cassava (Manihot esculenta Crantz), 23 tubulin genes (MeTubulins) were identified, which were divided into four main groups based on their type and phylogenetic characteristics. The same grouping generally has the same or similar motif composition and exon-intron structure. Collinear analysis showed that fragment repetition event is the main factor in amplification of cassava tubulin superfamily gene. The expression profiles of MeTubulin genes in various tissue were analyzed, and it was found that MeTubulins were mainly expressed in leaf, petiole, and stem, while FtsZ2-1 was highly expressed in storage root. The qRT-PCR results of the FtsZ2-1 gene under hormone and abiotic stresses showed that indole-3-acetic acid (IAA) and gibberellin A3 (GA3) stresses could significantly increase the expression of the FtsZ2-1 gene, thereby revealing the potential role of FtsZ2-1 in IAA and GA3 stress-induced responses.

Heme oxygenase 1 plays a crucial role in swamp eel response to oxidative stress induced by cadmium exposure or Aeromonas hydrophila infection.

Oxidative stress contributes a lot to initiation and progression of pathological conditions. Heme oxygenase 1 (HO1), a cytoprotective enzyme, is usually upregulated to alleviate oxidative stress in vivo. The function of teleost HO1 in the response to oxidative stress induced by heavy metal exposure and in pathogenic bacterial infection remains uncertain. In the present study, both complementary DNA and genomic sequence of a HO1-like gene cloned from the liver of swamp eel (Monopterus albus) are reported. Sequence analysis showed that the putative amino acid sequence contained a conserved heme oxygenase signature and displayed higher similarity to HO1 genes of other teleosts. Expression profile of swamp eel HO1 was investigated in healthy tissues and in tissues following stimulation with pathogenic bacteria (Aeromonas hydrophila) or cadmium chloride (CdCl2) exposure. Results demonstrated that HO1 messenger RNA (mRNA) was highly expressed in the liver and relatively less in other tissues. Bacterial infection with A. hydrophila significantly changed HO1 mRNA expression in the liver, spleen, and kidney, and the mRNA expression of HO1 and Nrf2 in the liver was elevated after the fish were exposed to CdCl2. Subsequently, the swamp eel HO1 was subcloned into a pET28a expression vector and transformed into Escherichia coli BL21 (DE3). Recombinant HO1 (rHO1) was successfully induced by 0.1 mmol/l IPTG and purified by Ni-NTA His Bind Resin purification system. To determine whether the rHO1 could confer stress tolerance in vitro, the viability of control and HO1-expressing E. coli under CdCl2 stress was compared by spot assay. The rHO1 protein significantly increased survival rates of the bacterial hosts. To evaluate whether intraperitoneal injection with rHO1 protected the liver of swamp eel against A. hydrophila-induced oxidative stress, mRNA expression of HO1, Nrf2, hepcidin, and IL-1ß as well as the oxidative stress-related parameters (ROS and total antioxidant capacity (T-AOC)) in the liver were examined. The results showed that exogenous rHO1 could significantly upgrade the mRNA expression of HO1 and hepcidin, coupled with increased ROS and T-AOC levels. However, Nrf2 and IL-1ß expression levels were significantly downregulated and upregulated, respectively. These results suggested that HO1 should not only play a protective role in oxidative stress response and its adverse effects deserved further investigation.

RRM2 elicits the metastatic potential of breast cancer cells by regulating cell invasion, migration and VEGF expression via the PI3K/AKT signaling.

Breast cancer is the second leading primary cause for cancer-related mortality among women and metastasis to the brain is a disastrous event for patients with increasing incidence. A previous study confirmed the critical function of RRM2 in breast cancer cell growth. Unfortunately, the role and fundamental molecular mechanism of RRM2 in breast cancer metastasis remains elusive. In the current study, higher RRM2 expression was validated in breast cancer tissues, especially in the brain metastasis group. Simultaneously, the expression of RRM2 was increased in breast cancer cells relative to the normal breast epithelial cell line MCF-10A, concomitant with higher levels of RRM2 in the highly metastatic MDA-MB-231 cell line relative to the weakly metastatic MCF-7 cell line. Knockdown of RRM2 by small interfering-RRM2 transfection notably suppressed the malignant metastatic behavior of breast cancer cells, including invasion and migration. Simultaneously, RRM2 downregulation also restrained the transcription and release of vascular endothelial growth factor (VEGF) in breast cancer cells. Moreover, inhibition of RRM2 dampened the activation of phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) signaling by decreasing phosphorylated-AKT and downstream matrix metalloproteinases-2 expression. Intriguingly, reactivation of the PI3K/AKT pathway with its agonist insulin-like growth factor-1 reversed the adverse effects of RRM2 suppression on cancer cell invasion, migration and VEGF expression. Together, these findings suggest that RRM2 may act as a pro-metastatic factor to facilitate breast cancer metastasis by evoking cell invasion, migration and VEGF expression through the PI3K/AKT signaling pathway. This study may provide an attractive target for metastatic intervention in breast cancer.

NLRP3 inflammasome inactivation driven by miR­223­3p reduces tumor growth and increases anticancer immunity in breast cancer.

MicroRNA­233­3p (miR­223­3p) is considered an important cancer­associated marker. The NACHT, LRR and PYD domains­containing protein 3 (NLRP3) inflammasome represents a novel potential target for the treatment of breast cancer. Therefore, it was hypothesized that miR­223­3p may affect tumor growth and immunosuppression in breast cancer by inhibiting the NLRP3 inflammasome. In the present study, an increased expression level of NLRP3 was detected in three breast cancer cell lines compared with normal mammary epithelial cells (HMEC). Suppressing the expression of NLRP3 in MCF­7 cell lines increased the apoptotic rate of breast cancer cells and reduced the proliferative capacity. NLRP3 was identified to be a direct target of miR­233­3p using a luciferase assay. In addition, miR­233­3p mimics inhibited the NLRP3­dependent processes in cancer cells by suppressing the NLRP3 expression level and the protein expression levels of its downstream factors, including PYD and CARD domain containing protein, interleukin­1ß and interleukin­18. In vivo experiments demonstrated the suppressive effect of miR­233­3p in tumor growth and immunosuppression. Collectively these findings suggested that the inactivation of the NLRP3 inflammasome driven by miR­223­3p reduced the growth and immunosuppression of breast cancer in vitro and in vivo, and may represent a novel therapeutic strategy in treating breast cancer.