A chromosome-level genome reveals genome evolution and molecular basis of anthraquinone biosynthesis in Rheum palmatum.

BACKGROUND: Rhubarb is one of common traditional Chinese medicine with a diverse array of therapeutic efficacies. Despite its widespread use, molecular research into rhubarb remains limited, constraining our comprehension of the geoherbalism. RESULTS: We assembled the genome of Rheum palmatum L., one of the source plants of rhubarb, to elucidate its genome evolution and unpack the biosynthetic pathways of its bioactive compounds using a combination of PacBio HiFi, Oxford Nanopore, Illumina, and Hi-C scaffolding approaches. Around 2.8 Gb genome was obtained after assembly with more than 99.9% sequences anchored to 11 pseudochromosomes (scaffold N50 = 259.19 Mb). Transposable elements (TE) with a continuous expansion of long terminal repeat retrotransposons (LTRs) is predominant in genome size, contributing to the genome expansion of R. palmatum. Totally 30,480 genes were predicted to be protein-coding genes with 473 significantly expanded gene families enriched in diverse pathways associated with high-altitude adaptation for this species. Two successive rounds of whole genome duplication event (WGD) shared by Fagopyrum tataricum and R. palmatum were confirmed. We also identified 54 genes involved in anthraquinone biosynthesis and other 97 genes entangled in flavonoid biosynthesis. Notably, RpALS emerged as a compelling candidate gene for the octaketide biosynthesis after the key residual screening. CONCLUSION: Overall, our findings offer not only an enhanced understanding of this remarkable medicinal plant but also pave the way for future innovations in its genetic breeding, molecular design, and functional genomic studies.

A novel deep-learning based weighted feature fusion architecture for precise classification of pressure injury.

Introduction: Precise classification has an important role in treatment of pressure injury (PI), while current machine-learning or deeplearning based methods of PI classification remain low accuracy. Methods: In this study, we developed a deeplearning based weighted feature fusion architecture for fine-grained classification, which combines a top-down and bottom-up pathway to fuse high-level semantic information and low-level detail representation. We validated it in our established database that consist of 1,519 images from multi-center clinical cohorts. ResNeXt was set as the backbone network. Results: We increased the accuracy of stage 3 PI from 60.3% to 76.2% by adding weighted feature pyramid network (wFPN). The accuracy for stage 1, 2, 4 PI were 0.870, 0.788, and 0.845 respectively. We found the overall accuracy, precision, recall, and F1-score of our network were 0.815, 0.808, 0.816, and 0.811 respectively. The area under the receiver operating characteristic curve was 0.940. Conclusions: Compared with current reported study, our network significantly increased the overall accuracy from 75% to 81.5% and showed great performance in predicting each stage. Upon further validation, our study will pave the path to the clinical application of our network in PI management.

Transcriptomic divergence of the Rheum palmatum complex derived from top-geoherb and non-geoherb areas provides the insights into geoherbalism properties of rhubarb.

Geoherb usually represents high-quality medicinal herbs with better clinical therapeutic effects, and elucidating the geoherbalism is essential for the quality improvement of traditional Chinese Medicine. However, few researches were conducted to clarify the geoherbalism based on a large scale of transcriptomics. In the present study, we compared the transcriptomes of Rheum palmatum complex derived from top-geoherb and non-geoherb areas to show the geoherbalism properties of rhubarb. A total of 412.32 Gb clean reads were obtained with unigene numbers of 100,615 after assembly. Based on the obtained transcriptome datasets, key enzyme-encoding genes involved in the anthraquinones biosynthesis were also obtained. We also found that 21 anthraquinone-related unigenes were differentially expressed between two different groups, and some of these DEGs were correlated to the content accumulation of five free anthraquinones, indicating that the gene expression profiles may promote the geoherbalism formation of rhubarb. In addition, the selective pressure analyses indicated that most paired orthologous genes between these two groups were subject to negative selection, and only a low proportion of orthologs under positive selection were detected. Functional annotation analyses indicated that these positive-selected genes related to the functions such as gene expression, substance transport, stress response and metabolism, indicating that discrepant environment also enhanced the formation of geoherbalism. Our study not only provided insights for the genetic mechanism of geoherbalism of rhubarb, but also laid more genetic cues for the future rhubarb germplasms improvement and utilization.

Comparative Transcriptome Analyses of Different Rheum officinale Tissues Reveal Differentially Expressed Genes Associated with Anthraquinone, Catechin, and Gallic Acid Biosynthesis.

Rheum officinale Baill. is an important traditional Chinese medicinal herb, its dried roots and rhizomes being widely utilized to cure diverse diseases. However, previous studies mainly focused on the active compounds and their pharmacological effects, and the molecular mechanism underlying the biosynthesis of these ingredients in R. officinale is still elusive. Here, we performed comparative transcriptome analyses to elucidate the differentially expressed genes (DEGs) in the root, stem, and leaf of R. officinale. A total of 236,031 unigenes with N50 of 769 bp was generated, 136,329 (57.76%) of which were annotated. A total of 5884 DEGs was identified after the comparative analyses of different tissues; 175 and 126 key enzyme genes with tissue-specific expression were found in the anthraquinone, catechin/gallic acid biosynthetic pathway, respectively, and some of these key enzyme genes were verified by qRT-PCR. The phylogeny of the PKS III family in Polygonaceae indicated that probably only PL_741 PKSIII1, PL_11549 PKSIII5, and PL_101745 PKSIII6 encoded PKSIII in the polyketide pathway. These results will shed light on the molecular basis of the tissue-specific accumulation and regulation of secondary metabolites in R. officinale, and lay a foundation for the future genetic diversity, molecular assisted breeding, and germplasm resource improvement of this essential medicinal plant.

Proinflammatory cytokines induce rapid, NO-independent apoptosis, expression of chemotactic mediators and interleukin-32 secretion in human pluripotent stem cell-derived beta cells.

AIMS/HYPOTHESIS: The aim of this study was to examine the effects of proinflammatory cytokines on cells of different developmental stages during the generation of stem cell-derived beta cells (SC-beta cells) from human pluripotent stem cells (hPSCs). We wanted to find out to what extent human SC-beta cells are suitable as an experimental cellular model and, with regard to a possible therapeutic use, whether SC-beta cells have a comparable vulnerability to cytokines as bona fide beta cells. METHODS: hPSCs were differentiated towards pancreatic organoids (SC-organoids) using a 3D production protocol. SC-beta cells and non-insulin-producing cells were separated by FACS and differential gene expression profiles of purified human SC-beta cells, progenitor stages and the human beta cell line EndoC-ßH1, as a reference, were determined after 24 h incubation with the proinflammatory cytokines IL-1ß, TNF-α and IFN-γ via a transcriptome microarray. Furthermore, we investigated apoptosis based on caspase cleavage, the generation of reactive oxygen species and activation of mitogen-activated protein-kinase (MAPK) stress-signalling pathways. RESULTS: A 24 h exposure of SC-beta cells to proinflammatory cytokines resulted in significant activation of caspase 3/7 and apoptosis via the extrinsic and intrinsic apoptosis signalling pathways. At this time point, SC-beta cells showed a markedly higher sensitivity towards proinflammatory cytokines than non-insulin-producing cells and EndoC-ßH1 cells. Furthermore, we were able to demonstrate the generation of reactive oxygen species and rule out the involvement of NO-mediated stress. A transient activation of stress-signalling pathways p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinase (JNK) was already observed after 10 min of cytokine exposure. The transcriptome analysis revealed that the cellular response to proinflammatory cytokines increased with the degree of differentiation of the cells. Cytokines induced the expression of multiple inflammatory mediators including IL-32, CXCL9 and CXCL10 in SC-beta cells and in non-insulin-producing cells. CONCLUSIONS/INTERPRETATION: Our results indicate that human SC-beta cells respond to proinflammatory cytokines very similarly to human islets. Due to the fast and fulminant cellular response of SC-beta cells, we conclude that SC-beta cells represent a suitable model for diabetes research. In light of the immaturity of SC-beta cells, they may be an attractive model for developmentally young beta cells as they are, for example, present in patients with early-onset type 1 diabetes. The secretion of chemotactic signals may promote communication between SC-beta cells and immune cells, and non-insulin-producing cells possibly participate in the overall immune response and are thus capable of amplifying the immune response and further stimulating inflammation. We demonstrated that cytokine-treated SC-organoids secrete IL-32, which is considered a promising candidate for type 1 diabetes onset. This underlines the need to ensure the survival of SC-beta cells in an autoimmune environment such as that found in type 1 diabetes.

Sphingosine-1 Phosphate Lyase Regulates Sensitivity of Pancreatic Beta-Cells to Lipotoxicity.

Elevated levels of free fatty acids (FFAs) have been related to pancreatic beta-cell failure in type 2 diabetes (T2DM), though the underlying mechanisms are not yet fully understood. FFAs have been shown to dysregulate formation of bioactive sphingolipids, such as ceramides and sphingosine-1 phosphate (S1P) in beta-cells. The aim of this study was to analyze the role of sphingosine-1 phosphate lyase (SPL), a key enzyme of the sphingolipid pathway that catalyzes an irreversible degradation of S1P, in the sensitivity of beta-cells to lipotoxicity. To validate the role of SPL in lipotoxicity, we modulated SPL expression in rat INS1E cells and in human EndoC-ßH1 beta-cells. SPL overexpression in INS1E cells (INS1E-SPL), which are characterized by a moderate basal expression level of SPL, resulted in an acceleration of palmitate-mediated cell viability loss, proliferation inhibition and induction of oxidative stress. SPL overexpression affected the mRNA expression of ER stress markers and mitochondrial chaperones. In contrast to control cells, in INS1E-SPL cells no protective effect of oleate was detected. Moreover, Plin2 expression and lipid droplet formation were strongly reduced in OA-treated INS1E-SPL cells. Silencing of SPL in human EndoC-ßH1 beta-cells, which are characterized by a significantly higher SPL expression as compared to rodent beta-cells, resulted in prevention of FFA-mediated caspase-3/7 activation. Our findings indicate that an adequate control of S1P degradation by SPL might be crucially involved in the susceptibility of pancreatic beta-cells to lipotoxicity.

MCPIP1 is a novel link between diabetogenic conditions and impaired insulin secretory capacity.

During diabetes development insulin production and glucose-stimulated insulin secretion (GSIS) are defective due to inflammation-related, yet not fully understood mechanisms. MCPIP1 (monocyte chemotactic protein-induced protein-1) is a strong regulator of inflammation, and acts predominantly as a specific RNase. The impact of MCPIP1 on insulin secretory capacity is unknown. We show that the expression of the ZC3H12A gene, which encodes MCPIP1, was induced by T1DM- and by T2DM-simulating conditions, with a stronger effect of cytokines. The number of MCPIP1-positive pancreatic islet-cells, including beta-cells, was significantly higher in diabetic compared to nondiabetic individuals. In the 3'UTR regions of mRNAs coding for Pdx1 (pancreatic and duodenal homeobox 1), FoxO1 (forkhead box protein O1), and of a novel regulator of insulin handling, Grp94 (glucose-regulated protein 94), MCPIP1-target structures were detected. Overexpression of the wild type MCPIP1wt, but not of the mutant MCPIP1D141N (lacking the RNase activity), decreased the expression of genes involved in insulin production and GSIS. Additionally INS1-E-MCPIP1wt cells exhibited a higher Ire1 (inositol-requiring enzyme 1) expression. MCPIP1wt overexpression blunted GSIS and glucose-mediated calcium influx with no deleterious effects on glucose uptake or glucokinase activity. We identify MCPIP1 as a new common link between diabetogenic conditions and beta-cell failure. MCPIP1 may serve as an interesting target for novel beta-cell protective approaches.