Chemical-tag-based semi-annotated metabolomics facilitates gene identification and specialized metabolic pathway elucidation in wheat.

The importance of metabolite modification and species-specific metabolic pathways has long been recognized. However, linking the chemical structure of metabolites to gene function in order to explore the genetic and biochemical basis of metabolism has not yet been reported in wheat (Triticum aestivum). Here, we profiled metabolic fragment enrichment in wheat leaves and consequently applied chemical-tag-based semi-annotated metabolomics in a genome-wide association study in accessions of wheat. The studies revealed that all 1,483 quantified metabolites have at least one known functional group whose modification is tailored in an enzyme-catalyzed manner and eventually allows efficient candidate gene mining. A Triticeae crop-specific flavonoid pathway and its underlying metabolic gene cluster were elucidated in further functional studies. Additionally, upon overexpressing the major effect gene of the cluster TraesCS2B01G460000 (TaOMT24), the pathway was reconstructed in rice (Oryza sativa), which lacks this pathway. The reported workflow represents an efficient and unbiased approach for gene mining using forward genetics in hexaploid wheat. The resultant candidate gene list contains vast molecular resources for decoding the genetic architecture of complex traits and identifying valuable breeding targets and will ultimately aid in achieving wheat crop improvement.

Wnt/Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression.

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid ß-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila. Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid ß-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid ß-oxidation.

PMhub 1.0: a comprehensive plant metabolome database.

The Plant Metabolome Hub (PMhub), available at https://pmhub.org.cn, is a valuable resource designed to provide scientists with comprehensive information on plant metabolites. It offers extensive details about their reference spectra, genetic foundations, chemical reactions, metabolic pathways and biological functions. The PMhub contains chemical data for 188 837 plant metabolites gathered from various sources, with 1 467 041 standard/in-silico high-resolution tandem mass-spectrometry (HRMS/MS) spectra corresponding to these metabolites. Beyond its extensive literature-derived data, PMhub also boasts a sizable collection of experimental metabolites; it contains 144 366 detected features in 10 typical plant species, with 16 423 successfully annotated by using standard/in-silico HRMS/MS data, this collection is further supplemented with thousands of features gathered from reference metabolites. For each metabolite, the PMhub enables the reconstructed of a simulated network based on structural similarities and existing metabolic pathways. Unlike previous plant-specific metabolome databases, PMhub not only contains a vast amount of metabolic data but also assembles the corresponding genomic and/or transcriptomic information, incorporating multiple methods for the comprehensive genetic analysis of metabolites. To validate the practicality, we verified a synthetic pathway for N-p-coumaroyltyramine by in vitro enzymatic activity experiments. In summary, the robust functionality provided by the PMhub will make it an indispensable tool for studying plant metabolomics.

Distinct Gag interaction properties of HIV-1 RNA 5' leader conformers reveal a mechanism for dimeric genome selection.

During HIV-1 assembly, two copies of viral genomic RNAs (gRNAs) are selectively packaged into new viral particles. This process is mediated by specific interactions between HIV-1 Gag and the packaging signals at the 5' leader (5'L) of viral gRNA. 5'L is able to adopt different conformations, which promotes either gRNA dimerization and packaging or Gag translation. Dimerization and packaging are coupled. Whether the selective packaging of the gRNA dimer is due to favorable interactions between Gag and 5'L in the packaging conformation is not known. Here, using RNAs mimicking the two 5'L conformers, we show that the 5'L conformation dramatically affects Gag-RNA interactions. Compared to the RNA in the translation conformation (5'LT), the RNA in the packaging conformation (5'LP) can bind more Gag molecules. Gag associates with 5'LP faster than it binds to 5'LT, whereas Gag dissociates from 5'LP more slowly. The Gag-5'LP complex is more stable at high salt concentrations. The NC-SP2-p6 region of Gag likely accounts for the faster association and slower dissociation kinetics for the Gag-5'LP interaction and for the higher stability. In summary, our data suggest that conformational changes play an important role in the selection of dimeric genomes, probably by affecting the binding kinetics and stability of the Gag-5'L complex.

A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1.

Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1-4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyses the synthesis of cyclic GMP-AMP (cGAMP)9-12, which stimulates the induction of type I interferons through the STING-TBK1-IRF-3 signalling axis13-15. STING oligomerizes after binding of cGAMP, leading to the recruitment and activation of the TBK1 kinase8,16. The IRF-3 transcription factor is then recruited to the signalling complex and activated by TBK18,17-20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of type I interferons21. However, the precise mechanisms that govern activation of STING by cGAMP and subsequent activation of TBK1 by STING remain unclear. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for induction of IFNß after cGAMP stimulation. Moreover, we show that full-length STING oligomerizes after it binds cGAMP, and highlight this as an essential step in the activation of STING-mediated signalling. These findings provide a structural basis for the development of STING agonists and antagonists for the treatment of cancer and autoimmune disorders.

Development and evaluation of a human CD47/HER2 bispecific antibody for Trastuzumab-resistant breast cancer immunotherapy.

The treatment for trastuzumab-resistant breast cancer (BC) remains a challenge in clinical settings. It was known that CD47 is preferentially upregulated in HER2+ BC cells, which is correlated with drug resistance to trastuzumab. Here, we developed a novel anti-CD47/HER2 bispecific antibody (BsAb) against trastuzumab-resistant BC, named IMM2902. IMM2902 demonstrated high binding affinity, blocking activity, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and internalization degradation effects against both trastuzumab-sensitive and trastuzumab-resistant BC cells in vitro. The in vivo experimental data indicated that IMM2902 was more effective than their respective controls in inhibiting tumor growth in a trastuzumab-sensitive BT474 mouse model, a trastuzumab-resistant HCC1954 mouse model, two trastuzumab-resistant patient-derived xenograft (PDX) mouse models and a cord blood (CB)-humanized HCC1954 mouse model. Through spatial transcriptome assays, multiplex immunofluorescence (mIFC) and in vitro assays, our findings provided evidence that IMM2902 effectively stimulates macrophages to generate C-X-C motif chemokine ligand (CXCL) 9 and CXCL10, thereby facilitating the recruitment of T cells and NK cells to the tumor site. Moreover, IMM2902 demonstrated a high safety profile regarding anemia and non-specific cytokines release. Collectively, our results highlighted a novel therapeutic approach for the treatment of HER2+ BCs and this approach exhibits significant anti-tumor efficacy without causing off-target toxicity in trastuzumab-resistant BC cells.

Integrated analysis of transcriptome and miRNAome reveals the heat stress response of Pinellia ternata seedlings.

Pinellia ternata (Thunb.) Briet., a valuable herb native to China, is susceptible to the "sprout tumble" phenomenon because of high temperatures, resulting in a significant yield reduction. However, the molecular regulatory mechanisms underlying the response of P. ternata to heat stress are not well understood. In this study, we integrated transcriptome and miRNAome sequencing to identify heat-response genes, microRNAs (miRNAs), and key miRNA-target pairs in P. ternata that differed between heat-stress and room-temperature conditions. Transcriptome analysis revealed extensive reprogramming of 4,960 genes across various categories, predominantly associated with cellular and metabolic processes, responses to stimuli, biological regulation, cell parts, organelles, membranes, and catalytic and binding activities. miRNAome sequencing identified 1,597 known/conserved miRNAs that were differentially expressed between the two test conditions. According to the analysis, genes and miRNAs associated with the regulation of transcription, DNA template, transcription factor activity, and sequence-specific DNA binding pathways may play a major role in the resistance to heat stress in P. ternata. Integrated analysis of the transcriptome and miRNAome expression data revealed 41 high-confidence miRNA-mRNA pairs, forming 25 modules. MYB-like proteins and calcium-responsive transcription coactivators may play an integral role in heat-stress resistance in P. ternata. Additionally, the candidate genes and miRNAs were subjected to quantitative real-time polymerase chain reaction to validate their expression patterns. These results offer a foundation for future studies exploring the mechanisms and critical genes involved in heat-stress resistance in P. ternata.

The potential roles of salivary biomarkers in neurodegenerative diseases.

Current research efforts on neurodegenerative diseases are focused on identifying novel and reliable biomarkers for early diagnosis and insight into disease progression. Salivary analysis is gaining increasing interest as a promising source of biomarkers and matrices for measuring neurodegenerative diseases. Saliva collection offers multiple advantages over the currently detected biofluids as it is easily accessible, non-invasive, and repeatable, allowing early diagnosis and timely treatment of the diseases. Here, we review the existing findings on salivary biomarkers and address the potential value in diagnosing neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and Amyotrophic lateral sclerosis. Based on the available research, ß-amyloid, tau protein, α-synuclein, DJ-1, Huntington protein in saliva profiles display reliability and validity as the biomarkers of neurodegenerative diseases.

Determinants of the interaction between the 5'-leader of HIV-1 genome and human lysyl-tRNA synthetase in reverse transcription primer release process.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3' end of human tRNALys3. The primer tRNALys3 is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5' leader (5'L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNALys3 for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5'L responsible for LysRS binding, how the conformations and various hairpin structures of 5'L regulate 5'L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5'L significantly influences its binding affinity with LysRS. The 5'L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5'L conformation that is not selected for packaging. Additionally, dimerization of 5'L impairs LysRS-5'L interaction. Furthermore, among various regions of 5'L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5'L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.

High Thermoelectric Performance in Bismuth Telluride via Constructing MoSe2-2D Heterojunction.

Currently, the only thermoelectric (TE) materials commercially available at room temperature are those based on bismuth telluride. However, their widespread application is limited due to their inferior thermoelectric and mechanical properties. In this study, a strategy of growing a rigid second phase of MoSe2 is employed, in situ within the matrix phase to achieve n-type bismuth telluride-based materials with exceptional mechanical and thermoelectric properties. The in situ grown second phase contributes to both the electronic and lattice thermal conductivities. This is primarily attributed to the strong energy filtering effect, as the second phase forms a semi-common lattice interfacial structure with the matrix phase during growth. Furthermore, for composites containing 2 wt% MoSe2, a maximum zT value of 1.24 at 373 K can be achieved. On this basis, 8-pair TE module is fabricated and 1-pair TE module is optimized using a homemade p-type material. The optimized 1-pair TE module generates a maximum output power of 13.6 µW, which is twice that of the 8-pair TE module and four times more than the 8-pair TE module fabricated by commercial material. This work facilitates the development of the TE module by presenting a novel approach to obtaining bismuth telluride-based thermoelectric materials with superior thermoelectric and mechanical properties.

Deciphering the SOX4/MAPK1 regulatory axis: a phosphoproteomic insight into IQGAP1 phosphorylation and pancreatic Cancer progression.

OBJECTIVE: This study aims to elucidate the functional role of IQGAP1 phosphorylation modification mediated by the SOX4/MAPK1 regulatory axis in developing pancreatic cancer through phosphoproteomics analysis. METHODS: Proteomics and phosphoproteomics data of pancreatic cancer were obtained from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Differential analysis, kinase-substrate enrichment analysis (KSEA), and independent prognosis analysis were performed on these datasets. Subtype analysis of pancreatic cancer patients was conducted based on the expression of prognostic-related proteins, and the prognosis of different subtypes was evaluated through prognosis analysis. Differential analysis of proteins in different subtypes was performed to identify differential proteins in the high-risk subtype. Clinical correlation analysis was conducted based on the expression of prognostic-related proteins, pancreatic cancer typing results, and clinical characteristics in the pancreatic cancer proteomics dataset. Functional pathway enrichment analysis was performed using GSEA/GO/KEGG, and most module proteins correlated with pancreatic cancer were selected using WGCNA analysis. In cell experiments, pancreatic cancer cells were grouped, and the expression levels of SOX4, MAPK1, and the phosphorylation level of IQGAP1 were detected by RT-qPCR and Western blot experiments. The effect of SOX4 on MAPK1 promoter transcriptional activity was assessed using a dual-luciferase assay, and the enrichment of SOX4 on the MAPK1 promoter was examined using a ChIP assay. The proliferation, migration, and invasion functions of grouped pancreatic cancer cells were assessed using CCK-8, colony formation, and Transwell assays. In animal experiments, the impact of SOX4 on tumor growth and metastasis through the regulation of MAPK1-IQGAP1 phosphorylation modification was studied by constructing subcutaneous and orthotopic pancreatic cancer xenograft models, as well as a liver metastasis model in nude mice. RESULTS: Phosphoproteomics and proteomics data analysis revealed that the kinase MAPK1 may play an important role in pancreatic cancer progression by promoting IQGAP1 phosphorylation modification. Proteomics analysis classified pancreatic cancer patients into two subtypes, C1 and C2, where the high-risk C2 subtype was associated with poor prognosis, malignant tumor typing, and enriched tumor-related pathways. SOX4 may promote the occurrence of the high-risk C2 subtype of pancreatic cancer by regulating MAPK1-IQGAP1 phosphorylation modification. In vitro cell experiments confirmed that SOX4 promoted IQGAP1 phosphorylation modification by activating MAPK1 transcription while silencing SOX4 inhibited the proliferation, migration, and invasion of pancreatic cancer cells by reducing the phosphorylation level of MAPK1-IQGAP1. In vivo, animal experiments further confirmed that silencing SOX4 suppressed the growth and metastasis of pancreatic cancer by reducing the phosphorylation level of MAPK1-IQGAP1. CONCLUSION: The findings of this study suggest that SOX4 promotes the phosphorylation modification of IQGAP1 by activating MAPK1 transcription, thereby facilitating the growth and metastasis of pancreatic cancer.

Lipidomic studies revealing serological markers associated with the occurrence of retinopathy in type 2 diabetes.

PURPOSE: The duration of type 2 diabetes mellitus (T2DM) and blood glucose levels have a significant impact on the development of T2DM complications. However, currently known risk factors are not good predictors of the onset or progression of diabetic retinopathy (DR). Therefore, we aimed to investigate the differences in the serum lipid composition in patients with T2DM, without and with DR, and search for potential serological indicators associated with the development of DR. METHODS: A total of 622 patients with T2DM hospitalized in the Department of Endocrinology of the First Affiliated Hospital of Xi'an JiaoTong University were selected as the discovery set. One-to-one case-control matching was performed according to the traditional risk factors for DR (i.e., age, duration of diabetes, HbA1c level, and hypertension). All cases with comorbid chronic kidney disease were excluded to eliminate confounding factors. A total of 42 pairs were successfully matched. T2DM patients with DR (DR group) were the case group, and T2DM patients without DR (NDR group) served as control subjects. Ultra-performance liquid chromatography-mass spectrometry (LC-MS/MS) was used for untargeted lipidomics analysis on serum, and a partial least squares discriminant analysis (PLS-DA) model was established to screen differential lipid molecules based on variable importance in the projection (VIP) > 1. An additional 531 T2DM patients were selected as the validation set. Next, 1:1 propensity score matching (PSM) was performed for the traditional risk factors for DR, and a combined 95 pairings in the NDR and DR groups were successfully matched. The screened differential lipid molecules were validated by multiple reaction monitoring (MRM) quantification based on mass spectrometry. RESULTS: The discovery set showed no differences in traditional risk factors associated with the development of DR (i.e., age, disease duration, HbA1c, blood pressure, and glomerular filtration rate). In the DR group compared with the NDR group, the levels of three ceramides (Cer) and seven sphingomyelins (SM) were significantly lower, and one phosphatidylcholine (PC), two lysophosphatidylcholines (LPC), and two SMs were significantly higher. Furthermore, evaluation of these 15 differential lipid molecules in the validation sample set showed that three Cer and SM(d18:1/24:1) molecules were substantially lower in the DR group. After excluding other confounding factors (e.g., sex, BMI, lipid-lowering drug therapy, and lipid levels), multifactorial logistic regression analysis revealed that a lower abundance of two ceramides, i.e., Cer(d18:0/22:0) and Cer(d18:0/24:0), was an independent risk factor for the occurrence of DR in T2DM patients. CONCLUSION: Disturbances in lipid metabolism are closely associated with the occurrence of DR in patients with T2DM, especially in ceramides. Our study revealed for the first time that Cer(d18:0/22:0) and Cer(d18:0/24:0) might be potential serological markers for the diagnosis of DR occurrence in T2DM patients, providing new ideas for the early diagnosis of DR.

Characteristics and Correlations of Wall Shear Stress and Flow Turbulence in the Carotid Bifurcation Evaluated Using an Ultrasound Vector Flow Imaging.

INTRODUCTION: The aim of the study was to evaluate characteristics and provide the normal values of wall shear stress (WSS) and flow turbulence (Tur), and the relationship between them in the carotid bifurcation based on an ultrasound vector flow imaging (V Flow) in healthy adults. METHODS: Max and mean WSS and Tur values at three segments (initial segments of internal and external carotid arteries [IICA and IECA]; distal segment of common carotid artery [DCCA]), both in anterior and posterior walls, were successfully obtained in 56 healthy adults, using ultrasound V Flow function. Relationship between mean WSS and Tur was further explored. RESULTS: The mean WSS value was 0.71 Pa, 0.86 Pa, and 0.96 Pa at IICA, IECA, and DCCA, respectively (IICA < IECA < DCCA, p < 0.05). The mean Tur value was 13.85%, 5.46%, and 4.17% at IICA, IECA, and DCCA, respectively (IICA > IECA > DCCA, p < 0.05). A cutoff value (WSS = 0.4 Pa) was selected and Tur values were significantly higher in group with WSS cutoff value <0.4 Pa than group with WSS cutoff value ≥0.4 Pa (p < 0.01). CONCLUSION: WSS and Tur are moderately negatively correlated, which can be used in the quantitative evaluation of carotid bifurcation and could be a potential dual-parameter tool in the clinical research for early detection of carotid atherosclerosis.

Single-cell and spatial transcriptomic investigation reveals the spatiotemporal specificity of the beta-defensin gene family during mouse sperm maturation.

Low sperm motility is a significant contributor to male infertility. beta-defensins have been implicated in host defence and the acquisition of sperm motility; however, the regulatory mechanisms governing their gene expression patterns and functions remain poorly understood. In this study, we performed single-cell RNA and spatial transcriptome sequencing to investigate the cellular composition of testicular and epididymal tissues and examined their gene expression characteristics. In the epididymis, we found that epididymal epithelial cells display a region specificity of gene expression in different epididymal segments, including the beta-defensin family genes. In particular, Defb15, Defb18, Defb20, Defb25 and Defb48 are specific to the caput; Defb22, Defb23 and Defb26 to the corpus; Defb2 and Defb9 to the cauda of the epididymis. To confirm this, we performed mRNA fluorescence in situ hybridisation (FISH) targeting certain exon region of beta-defensin genes, and found some of their expression matched the sequencing results and displayed a close connection with epididimosome marker gene Cd63. In addition, we paid attention to the Sertoli cells and Leydig cells in the testis, along with fibroblasts and smooth muscle cells in the epididymis, by demonstrating their gene expression profile and spatial information. Our study provides a single-cell and spatial landscape for analysing the gene expression characteristics of testicular and epididymal environments and has important implications for the study of spermatogenesis and sperm maturation.

Capsaicin attenuates Porphyromonas gingivalis-suppressed osteogenesis of periodontal ligament stem cells via regulating mitochondrial function and activating PI3K/AKT/mTOR pathway.

BACKGROUND AND OBJECTIVE: Prevention of periodontal bone resorption triggered by Porphyromonas gingivalis (P. gingivalis) is crucial for dental stability. Capsaicin, known as the pungent ingredient of chili peppers, can activate key signaling molecules involved in osteogenic process. However, the effect of capsaicin on osteogenesis of periodontal ligament stem cells (PDLSCs) under inflammation remains elusive. METHODS: P. gingivalis culture suspension was added to mimic the inflammatory status after capsaicin pretreatment. The effects of capsaicin on the osteogenesis of PDLSCs, as well as mitochondrial morphology, Ca2+ level, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and osteogenesis-regulated protein expression levels were analyzed. Furthermore, a mouse experimental periodontitis model was established to evaluate the effect of capsaicin on alveolar bone resorption and the expression of osteogenesis-related proteins. RESULTS: Under P. gingivalis stimulation, capsaicin increased osteogenesis of PDLSCs. Not surprisingly, capsaicin rescued the damage to mitochondrial morphology, decreased the concentration of intracellular Ca2+ and ROS, enhanced MMP and activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. The in vivo results showed that capsaicin significantly attenuated alveolar bone loss and augmented the expression of bone associated proteins. CONCLUSION: Capsaicin increases osteogenesis of PDLSCs under inflammation and reduces alveolar bone resorption in mouse experimental periodontitis.

Development of genetic markers in Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica represents a potential microbial cell factory for the recombinant production of various valuable products. Currently, the commonly used selection markers for transformation in Y. lipolytica are limited, and successive genetic manipulations are often restricted by the number of available selection markers. In our study, we developed a dominant marker, dsdA, which encodes a D-serine deaminase for genetic manipulation in Y. lipolytica. In Y. lipolytica, this marker confers the ability to use D-serine as a nitrogen source. In addition, the selection conditions of several infrequently used dominant markers including bleoR (zeocin resistance), kanMX (G418 resistance), and guaB (mycophenolic acid resistance) were also analyzed. Our results demonstrated that these selection markers can be used for the genetic manipulation of Y. lipolytica and their selection conditions were different for various strains. Ultimately, the selection markers tested here will be useful to expand the genetic toolbox of Y. lipolytica. KEY POINTS: • The dsdA from Escherichia coli was developed as a dominant marker. • The applicability of several resistance markers in Y. lipolytica was determined. • We introduced the Cre/mutant lox system for marker recycling.

Comprehensive analysis of EphA2 in pan-cancer: A prognostic biomarker associated with cancer immunity.

BACKGROUND: Several studies have reported a significant relationship between Ephrin receptor A2 (EphA2) and malignant progression in numerous cancers. However, there is a lack of comprehensive pan-cancer analysis on the prognostic value, mutation status, methylation landscape, and potential immunological function of EphA2. METHOD: Using The Cancer Genome Atlas, Genotype Tissue Expression Database and GEO data, we analysed the differences in EphA2 expression between normal and tumour tissues and the effects of EphA2 on the prognosis of different tumours. Furthermore, using GSCALite, cBioPortal, TISDB, ULCLAN and TIMER 2.0 databases or platforms, we comprehensively analysed the potential oncogenic mechanisms or manifestations of EphA2 in 33 different tumour types, including tumour mutation status, DNA methylation status and immune cell infiltration. The correlation of EphA2 with immune checkpoints, tumour mutational burden, DNA microsatellite instability and DNA repair genes was also calculated. Finally, the effects of EphA2 inhibitors on the proliferation of human glioma and lung cancer cells were verified in cellular experiments. RESULTS: EphA2 is differentially expressed in different tumours, and patients with overexpression have poorer overall survival. In addition, gene mutations, gene copy number variation and DNA/RNA methylation of EphA2 have been identified in various tumours. Moreover, EphA2 is positively associated with immune infiltration involving macrophages and CD8+ T cells. Further, EphA2 mRNA expression is significantly associated with immune checkpoint in various cancers, especially programmed death-ligand 1. Finally, the EphA2 inhibitor ALW-II-41-27 shows potent anti-tumour activity. CONCLUSION: Our first pan-cancer study of EphA2 provides insight into the prognostic and immunological roles of EphA2 in different tumours, suggesting that EphA2 might be a potential biomarker for poor prognosis and immune infiltration in cancer.

Quantitative evaluation of disc degeneration using dual-energy CT: advantages of R-VH, D-VH values and the IVNCa + CT model.

OBJECTIVE: To evaluate the correlation between dual-energy CT (DECT) virtual calcium free (VNCA), CT attenuation, the ratio and difference of VNCA to CT attenuation, and Pfirrmann grading of lumbar disc degeneration. METHODS: A retrospective analysis on 135 intervertebral discs from 30 patients who underwent DECT and MR. Discs was graded using the Pfirrmann system. ROIs on the sagittal plane assessed HU value, VNCA value, Rho value, Z value, R-VH value, and D-VH value. Correlation, grade differences, and multivariate regression models were assessed. Diagnostic performance and cut-off values were determined using AUC. RESULTS: VNCA (r = 0.589, P < 0.001), R-VH (r = 0.622, P < 0.001), and D-VH (r = 0.613, P < 0.001) moderately correlated with Pfirrmann grading. HU (r = 0.388, P < 0.001), Rho (r = 0.142, P = 0.102), and Z (r = -0.125, P = 0.153) showed a weak correlation. R-VH, D-VH, and VNCA had significantly higher correlation than HU. Statistically significant differences were observed in P values of VNCA, HU, R-VH, and D-VH in relative groups (P < 0.05), but not in Rho and Z values (P > 0.05). R-VH and D-VH had significant differences between Pfirrmann grades 1 and 2, and grades 2 and 3 (early stage) (P < 0.05). AUC readings of R-VH and D-VH (≥2, ≥3, ≥4) were higher. The multivariate model IVNCa + CT had the highest AUC. CONCLUSION: The new quantitative indices R-VH value and D-VH value of DECT have advantages over VNCA value and HU value in evaluating early-stage disc degeneration (≥2 grades, ≥3 grades). The multivariate model IVNCa + CT has the best AUC values for evaluating disc degeneration at all stages.

Nucleocytoplasmic trafficking and turnover mechanisms of BRASSINAZOLE RESISTANT1 in Arabidopsis thaliana.

Regulation of the nucleocytoplasmic trafficking of signaling components, especially transcription factors, is a key step of signal transduction in response to extracellular stimuli. In the brassinosteroid (BR) signal transduction pathway, transcription factors from the BRASSINAZOLE RESISTANT1 (BZR1) family are essential in mediating BR-regulated gene expression. The subcellular localization and transcriptional activity of BZR1 are tightly regulated by reversible protein phosphorylation; however, the underlying mechanism is not well understood. Here, we provide evidence that both BZR1 phosphorylation and dephosphorylation occur in the nucleus and that BR-regulated nuclear localization of BZR1 is independent from its interaction with, or dephosphorylation by, protein phosphatase 2A. Using a photoconvertible fluorescent protein, Kaede, as a living tag to distinguish newly synthesized BZR1 from existing BZR1, we demonstrated that BR treatment recruits cytosolic BZR1 to the nucleus, which could explain the fast responses of plants to BR. Additionally, we obtained evidence for two types of protein turnover mechanisms that regulate BZR1 abundance in plant cells: a BR- and 26S proteosome-independent constitutive degradation mechanism and a BR-activated 26S proteosome-dependent proteolytic mechanism. Finally, treating plant cells with inhibitors of 26S proteosome induces the nuclear localization and dephosphorylation of BZR1, even in the absence of BR signaling. Based on these results, we propose a model to explain how BR signaling regulates the nucleocytoplasmic trafficking and reversible phosphorylation of BZR1.

Study on the Prevention of Fall Risk in Elderly Stroke Patients Based on an Intelligent Model of Rehabilitation Care.

Background: Fall is a public health problem that cannot be ignored by elderly stroke patients, and rehabilitation care plays an important role in the rehabilitation process of elderly stroke patients. Objective: To investigate the prevention effect of fall risk in elderly stroke patients under the intelligent model of rehabilitation care. Methods: The general data of elderly patients who were diagnosed as stroke and admitted to our hospital between June 2021 and June 2022 were retrospectively analyzed, with exclusion like unclear clinical data or combined with other severe organ insufficiency. A total of 150 of them were selected for the study, and the patients were divided into a fall group and a non-fall group according to whether they had a fall or not. The factors associated with falls in stroke patients were analyzed univariately, and the rehabilitation care intelligence model of the predictive model of falls in stroke patients was established using multiple covariance ridge regression analysis to observe the predictive value of patients' risk of falling in the rehabilitation care intelligence model. Results: Results of multiple covariance ridge regression analysis to build the model showed age (P < .001), low MNA-SF score (P < .001), hypertension (P = .035), anaemia(P = .048), gout (P < .001), assistive devices (P = .002), visual impairment (P = .033), elevated ALB (P < .001), and elevated HGB (P < .001) as risk factors for falls in stroke patients. The diagnostic threshold for screening elderly stroke patients for falls based on risk factors was 0.272, with a sensitivity of 90.7%, specificity of 98.1% and an area under the ROC curve of 0.976 (P < .05), which was superior to other single indicators in terms of diagnostic value. The calibration of the prediction model, based on the Hosmer and Lemeshow test of goodness of fit, showed P = 1.14, indicating a high calibration of the prediction model. Conclusion: There are many risk factors for falls in stroke elderly patients, such as low MNA-SF score, gout, elevated ALB, and elevated HGB. Building a rehabilitation nursing intelligent model based on the above inducement factors can reduce the risk of patients falling to a certain extent, and the prediction model has a high degree of calibration. Therefore, a simple and standardized intelligent rehabilitation nursing model for stroke patients in the early stage can effectively prevent the occurrence of falls.