Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

A Milieu Molecule for TGF-ß Required for Microglia Function in the Nervous System.

Extracellular proTGF-ß is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-ß is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVß8-dependent TGF-ß activation. Lrrc33-/- mice lack CNS vascular abnormalities associated with deficiency in TGF-ß-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33-/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33-/- microglia in the same brain suggest that there is little spreading of TGF-ß activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-ß provide localized and selective activation of TGF-ß.

A male germ-cell-specific ribosome controls male fertility.

Ribosomes are highly sophisticated translation machines that have been demonstrated to be heterogeneous in the regulation of protein synthesis1,2. Male germ cell development involves complex translational regulation during sperm formation3. However, it remains unclear whether translation during sperm formation is performed by a specific ribosome. Here we report a ribosome with a specialized nascent polypeptide exit tunnel, RibosomeST, that is assembled with the male germ-cell-specific protein RPL39L, the paralogue of core ribosome (RibosomeCore) protein RPL39. Deletion of RibosomeST in mice causes defective sperm formation, resulting in substantially reduced fertility. Our comparison of single-particle cryo-electron microscopy structures of ribosomes from mouse kidneys and testes indicates that RibosomeST features a ribosomal polypeptide exit tunnel of distinct size and charge states compared with RibosomeCore. RibosomeST predominantly cotranslationally regulates the folding of a subset of male germ-cell-specific proteins that are essential for the formation of sperm. Moreover, we found that specialized functions of RibosomeST were not replaceable by RibosomeCore. Taken together, identification of this sperm-specific ribosome should greatly expand our understanding of ribosome function and tissue-specific regulation of protein expression pattern in mammals.

Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases.

Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family, including NSD1, NSD2 and NSD3, have crucial roles in chromatin regulation and are implicated in oncogenesis1,2. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling dimethylation of histone H3 at Lys36 (H3K36)3-7. However, the molecular basis that underlies this mechanism is largely unknown. Here we solve the cryo-electron microscopy structures of NSD2 and NSD3 bound to mononucleosomes. We find that binding of NSD2 and NSD3 to mononucleosomes causes DNA near the linker region to unwrap, which facilitates insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between NSD2 or NSD3 and the nucleosome precisely defines the position of the enzyme on the nucleosome, explaining the specificity of methylation to H3K36. Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3. NSDs that contain these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes the proliferation of cancer cells and the growth of xenograft tumours. Together, our research provides molecular insights into the nucleosome-based recognition and histone-modification mechanisms of NSD2 and NSD3, which could lead to strategies for therapeutic targeting of proteins of the NSD family.

CK2-HTATSF1-TOPBP1 signaling axis modulates tumor chemotherapy response.

Homologous recombination (HR) plays a key role in maintaining genomic stability, and the efficiency of the HR system is closely associated with tumor response to chemotherapy. Our previous work reported that CK2 kinase phosphorylates HIV Tat-specific factor 1 (HTATSF1) Ser748 to facilitate HTATSF1 interaction with TOPBP1, which in turn, promotes RAD51 recruitment and HR repair. However, the clinical implication of the CK2-HTATSF1-TOPBP1 pathway in tumorigenesis and chemotherapeutic response remains to be elucidated. Here, we report that the CK2-HTATSF1-TOPBP1 axis is generally hyperactivated in multiple malignancies and renders breast tumors less responsive to chemotherapy. In contrast, deletion mutations of each gene in this axis, which also occur in breast and lung tumor samples, predict higher HR deficiency scores, and tumor cells bearing a loss-of-function mutation of HTATSF1 are vulnerable to poly(ADP-ribose) polymerase inhibitors or platinum drugs. Taken together, our study suggests that the integrity of the CK2-HTATSF1-TOPBP1 axis is closely linked to tumorigenesis and serves as an indicator of tumor HR status and modulates chemotherapy response.

Hbxip is essential for murine embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1.

HBXIP, also named LAMTOR5, has been well characterized as a transcriptional co-activator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5, and that depletion of Hbxip compromises the self-renewal of embryonic stem cells (ESCs), with reduced expression of pluripotency genes, reduced cell proliferation and decreased colony-forming capacity. In addition, both Hbxip-/- ESCs and E7.5 embryos displayed defects in ectodermal and mesodermal differentiation. Mechanistically, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, displayed differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/- and p18-/- mice, lacking subunits of the Ragulator complex, also shared similar phenotypes, embryonic lethality and retarded growth around E7-E8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3.

Porcine deltacoronavirus (PDCoV) has caused enormous economic losses to the global pig industry. However, the immune escape mechanism of PDCoV remains to be fully clarified. Transcriptomic analysis revealed a high abundance of interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) transcripts after PDCoV infection, which initially implied a correlation between IFIT3 and PDCoV. Further studies showed that PDCoV nsp5 could antagonize the host type I interferon signaling pathway by cleaving IFIT3. We demonstrated that PDCoV nsp5 cleaved porcine IFIT3 (pIFIT3) at Gln-406. Similar cleavage of endogenous IFIT3 has also been observed in PDCoV-infected cells. The pIFIT3-Q406A mutant was resistant to nsp5-mediated cleavage and exhibited a greater ability to inhibit PDCoV infection than wild-type pIFIT3. Furthermore, we found that cleavage of IFIT3 is a common characteristic of nsp5 proteins of human coronaviruses, albeit not alphacoronavirus. This finding suggests that the cleavage of IFIT3 is an important mechanism by which PDCoV nsp5 antagonizes IFN signaling. Our study provides new insights into the mechanisms by which PDCoV antagonizes the host innate immune response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a potential emerging zoonotic pathogen, and studies on the prevalence and pathogenesis of PDCoV are ongoing. The main protease (nsp5) of PDCoV provides an excellent target for antivirals due to its essential and conserved function in the viral replication cycle. Previous studies have revealed that nsp5 of PDCoV antagonizes type I interferon (IFN) production by targeting the interferon-stimulated genes. Here, we provide the first demonstration that nsp5 of PDCoV antagonizes IFN signaling by cleaving IFIT3, which affects the IFN response after PDCoV infection. Our findings reveal that PDCoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by deltacoronaviruses.

MYD88L265P augments proximal B-cell receptor signaling in large B-cell lymphomas via an interaction with DOCK8.

Diffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease with at least 5 recognized molecular subtypes. Cluster 5 (C5)/MCD tumors frequently exhibit concurrent alterations in the toll-like receptor (TLR) and B-cell receptor (BCR) pathway members, MYD88L265P and CD79B, and have a less favorable prognosis. In healthy B cells, the synergy between TLR and BCR signaling pathways integrates innate and adaptive immune responses and augments downstream NF-κB activation. In addition, physiologic TLR9 pathway engagement via MYD88, protein tyrosine kinase 2 (PYK2), and dedicator of cytokinesis 8 (DOCK8) increases proximal BCR signaling in healthy murine B cells. Although C5/MCD DLBCLs are selectively sensitive to Bruton tyrosine kinase (BTK) inhibition in in vitro studies and certain clinical trials, the role of mutated MYD88 in proximal BCR signaling remains undefined. Using engineered DLBCL cell line models, we found that concurrent MYD88L265P and CD79B alterations significantly increased the magnitude and duration of proximal BCR signaling, at the level of spleen tyrosine kinase and BTK, and augmented PYK2-dependent DOCK8 phosphorylation. MYD88L265P DLBCLs have significantly increased colocalization of DOCK8 with both MYD88 and the proximal BCR-associated Src kinase, LYN, in comparison with MYD88WT DLBCLs, implicating DOCK8 in MYD88L265P/proximal BCR cross talk. Additionally, DOCK8 depletion selectively decreased proximal BCR signaling, cellular proliferation, and viability of DLBCLs with endogenous MYD88L265P/CD79BY196F alterations and increased the efficacy of BTK blockade in these lymphomas. Therefore, MYD88L265P/DOCK8-enhanced proximal BCR signaling is a likely mechanism for the increased sensitivity of C5/MCD DLBCLs to BTK blockade.

A glimpse into novel acylations and their emerging role in regulating cancer metastasis.

Metastatic cancer is a major cause of cancer-related mortality; however, the complex regulation process remains to be further elucidated. A large amount of preliminary investigations focus on the role of epigenetic mechanisms in cancer metastasis. Notably, the posttranslational modifications were found to be critically involved in malignancy, thus attracting considerable attention. Beyond acetylation, novel forms of acylation have been recently identified following advances in mass spectrometry, proteomics technologies, and bioinformatics, such as propionylation, butyrylation, malonylation, succinylation, crotonylation, 2-hydroxyisobutyrylation, lactylation, among others. These novel acylations play pivotal roles in regulating different aspects of energy mechanism and mediating signal transduction by covalently modifying histone or nonhistone proteins. Furthermore, these acylations and their modifying enzymes show promise regarding the diagnosis and treatment of tumors, especially tumor metastasis. Here, we comprehensively review the identification and characterization of 11 novel acylations, and the corresponding modifying enzymes, highlighting their significance for tumor metastasis. We also focus on their potential application as clinical therapeutic targets and diagnostic predictors, discussing the current obstacles and future research prospects.

ADAR1 links R-loop homeostasis to ATR activation in replication stress response.

Unscheduled R-loops are a major source of replication stress and DNA damage. R-loop-induced replication defects are sensed and suppressed by ATR kinase, whereas it is not known whether R-loop itself is actively involved in ATR activation and, if so, how this is achieved. Here, we report that the nuclear form of RNA-editing enzyme ADAR1 promotes ATR activation and resolves genome-wide R-loops, a process that requires its double-stranded RNA-binding domains. Mechanistically, ADAR1 interacts with TOPBP1 and facilitates its loading on perturbed replication forks by enhancing the association of TOPBP1 with RAD9 of the 9-1-1 complex. When replication is inhibited, DNA-RNA hybrid competes with TOPBP1 for ADAR1 binding to promote the translocation of ADAR1 from damaged fork to accumulate at R-loop region. There, ADAR1 recruits RNA helicases DHX9 and DDX21 to unwind R-loops, simultaneously allowing TOPBP1 to stimulate ATR more efficiently. Collectively, we propose that the tempo-spatially regulated assembly of ADAR1-nucleated protein complexes link R-loop clearance and ATR activation, while R-loops crosstalk with blocked replication forks by transposing ADAR1 to finetune ATR activity and safeguard the genome.

Expanding DNA Origami Design Freedom with De Novo Synthesized Scaffolds.

The construction of DNA origami nanostructures is heavily dependent on the folding of the scaffold strand, which is typically a single-stranded DNA genome extracted from a bacteriophage (M13). Custom scaffolds can be prepared in a number of methods, but they are not widely accessible to a broad user base in the DNA nanotechnology community. Here, we explored new design and construction possibilities with custom scaffolds prepared in our cost- and time-efficient production pipeline. According to the pipeline, we de novo produced a variety of scaffolds of specified local and global sequence characteristics and consequent origami constructs of modular arrangement in morphologies and functionalities. Taking advantage of this strategy of template-free scaffold production, we also designed and produced three-letter-coded scaffolds that can fold into designated morphologies rapidly at room temperature. The expanded design and construction freedom immediately brings in many new research opportunities and invites many more on the horizon.

Development of a chemiluminescence assay for tissue plasminogen activator inhibitor complex and its applicability to gastric cancer.

BACKGROUND: Venous thromboembolism (VTE), is a noteworthy complication in individuals with gastric cancer, but the current diagnosis and treatment methods lack accuracy. In this study, we developed a t-PAIC chemiluminescence kit and employed chemiluminescence to detect the tissue plasminogen activator inhibitor complex (t-PAIC), thrombin-antithrombin III complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC) and thrombomodulin (TM), combined with D-dimer and fibrin degradation products (FDP), to investigate their diagnostic potential for venous thrombosis in gastric cancer patients. The study assessed variations in six indicators among gastric cancer patients at different stages. RESULTS: The t-PAIC reagent showed LOD is 1.2 ng/mL and a linear factor R greater than 0.99. The reagents demonstrated accurate results, with all accuracy deviations being within 5%. The intra-batch and inter-batch CVs for the t-PAIC reagent were both within 8%. The correlation coefficient R between this method and Sysmex was 0.979. Gastric cancer patients exhibited elevated levels of TAT, PIC, TM, D-D, FDP compared to the healthy population, while no significant difference was observed in t-PAIC. In the staging of gastric cancer, patients in III-IV stages exhibit higher levels of the six markers compared to those in I-II stages. The ROC curve indicates an enhancement in sensitivity and specificity of the combined diagnosis of four or six indicators. CONCLUSION: Our chemiluminescence assay performs comparably to Sysmex's method and at a reduced cost. The use of multiple markers, including t-PAIC, TM, TAT, PIC, D-D, and FDP, is superior to the use of single markers for diagnosing VTE in patients with malignant tumors. Gastric cancer patients should be screened for the six markers to facilitate proactive prophylaxis, determine the most appropriate treatment timing, ameliorate their prognosis, decrease the occurrence of venous thrombosis and mortality, and extend their survival.

FeSA­Ir/Metallene Nanozymes Induce Sequential Ferroptosis­Pyroptosis for Multi­Immunogenic Responses Against Lung Metastasis.

For cancer metastasis inhibition, the combining of nanozymes with immune checkpoint blockade (ICB) therapy remains the major challenge in controllable reactive oxygen species (ROS) generation for creating effective immunogenicity. Herein, new nanozymes with light-controlled ROS production in terms of quantity and variety are developed by conjugating supramolecular-wrapped Fe single atom on iridium metallene with lattice-strained nanoislands (FeSA-Ir@PF NSs). The Fenton-like catalysis of FeSA-Ir@PF NSs effectively produced •OH radicals in dark, which induced ferroptosis and apoptosis of cancer cells. While under second near-infrared (NIR-II) light irradiation, FeSA-Ir@PF NSs showed ultrahigh photothermal conversion efficiency (𝜂, 75.29%), cooperative robust •OH generation, photocatalytic O2 and 1O2 generation, and caused significant pyroptosis of cancer cells. The controllable ROS generation, sequential cancer cells ferroptosis and pyroptosis, led 99.1% primary tumor inhibition and multi-immunogenic responses in vivo. Most importantly, the inhibition of cancer lung metastasis is completely achieved by FeSA-Ir@PF NSs with immune checkpoint inhibitors, as demonstrated in different mice lung metastasis models, including circulating tumor cells (CTCs) model. This work provided new inspiration for developing nanozymes for cancer treatments and metastasis inhibition.

Semi-automatic proximal humeral trabecular bone density assessment tool: technique application and clinical validation.

PURPOSE: This study aimed to apply a newly developed semi-automatic phantom-less QCT (PL-QCT) to measure proximal humerus trabecular bone density based on chest CT and verify its accuracy and precision. METHODS: Subcutaneous fat of the shoulder joint and trapezius muscle were used as calibration references for PL-QCT BMD measurement. A self-developed algorithm based on a convolution map was utilized in PL-QCT for semi-automatic BMD measurements. CT values of ROIs used in PL-QCT measurements were directly used for phantom-based quantitative computed tomography (PB-QCT) BMD assessment. The study included 376 proximal humerus for comparison between PB-QCT and PL-QCT. Two sports medicine doctors measured the proximal humerus with PB-QCT and PL-QCT without knowing each other's results. Among them, 100 proximal humerus were included in the inter-operative and intra-operative BMD measurements for evaluating the repeatability and reproducibility of PL-QCT and PB-QCT. RESULTS: A total of 188 patients with 376 shoulders were involved in this study. The consistency analysis indicated that the average bias between proximal humerus BMDs measured by PB-QCT and PL-QCT was 1.0 mg/cc (agreement range - 9.4 to 11.4; P > 0.05, no significant difference). Regression analysis between PB-QCT and PL-QCT indicated a good correlation (R-square is 0.9723). Short-term repeatability and reproducibility of proximal humerus BMDs measured by PB-QCT (CV: 5.10% and 3.41%) were slightly better than those of PL-QCT (CV: 6.17% and 5.64%). CONCLUSIONS: We evaluated the bone quality of the proximal humeral using chest CT through the semi-automatic PL-QCT system for the first time. Comparison between it and PB-QCT indicated that it could be a reliable shoulder BMD assessment tool with acceptable accuracy and precision. This study developed and verify a semi-automatic PL-QCT for assessment of proximal humeral bone density based on CT to assist in the assessment of proximal humeral osteoporosis and development of individualized treatment plans for shoulders.

Long-term outcomes with HLX01 (HanliKang®), a rituximab biosimilar, in previously untreated patients with diffuse large B-cell lymphoma: 5-year follow-up results of the phase 3 HLX01-NHL03 study.

HLX01 (HanliKang®) is a rituximab biosimilar that showed bioequivalence to reference rituximab in untreated CD20-positive diffuse large B-cell lymphoma (DLBCL) in the phase 3 HLX01-NHL03 study. Here, we report the 5-year follow-up results from the open-label extension part. Patients were randomised to either rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) or HLX01 plus CHOP (H-CHOP) every 21 days for up to six cycles. The primary efficacy endpoint was overall survival (OS), and secondary efficacy endpoint was progression-free survival (PFS). Of the 407 patients enrolled in HLX01-NHL03, 316 patients (H-CHOP = 157; R-CHOP = 159) were included in the 5-year follow-up for a median duration of 65.1 (range, 2.2-76.5) months. 96.5% of the patients had an International Prognostic Index (IPI) of 1 or 2, and 17.7% had bone marrow involvement. The 5-year OS rates were 81.0% (95% CI: 74.9-87.5%) and 75.4% (95% CI: 68.9-82.6%)( HR: 0.75, 95% CI 0.47-1.20; p = 0.23) while 5-year PFS rates were 77.7% (95% CI: 71.4-84.6%) and 73.0% (95% CI: 66.3-80.3%) (HR: 0.84, 95% CI 0.54-1.30; p = 0.43) in the H-CHOP and R-CHOP groups, respectively. Treatment outcomes did not differ between groups regardless of IPI score and were consistent with the primary analysis. H-CHOP and R-CHOP provided no significant difference in 5-year OS or PFS in previously untreated patients with low or low-intermediate risk DLBCL.

Development and validation of a novel risk stratification model and a survival rate calculator for diffuse large B-cell lymphoma in the rituximab era: a multi-institutional cohort study.

BACKGROUND: This study aimed to develop and validate a novel risk stratification model and a web-based survival rate calculator to improve discriminative and predictive accuracy for diffuse large B-cell lymphoma (DLBCL) in the rituximab era. METHODS: We retrospectively collected pre-treatment data from 873 primary DLBCL patients who received R-CHOP-based immunochemotherapy regimens at the Cancer Hospital, Chinese Academy of Medical Sciences, from January 1, 2005, to December 31, 2018. An independent cohort of 175 DLBCL patients from Fujian Cancer Hospital was used for external validation. FINDINGS: Age, ECOG PS, number of extranodal sites, Ann Arbor stage, bulky disease, and LDH levels were screened to develop the nomogram and web-based survival rate calculator. The C-index of the nomogram in the training, internal validation, and external validation cohorts was 0.761, 0.758, and 0.768, respectively. The risk stratification model generated based on the nomogram effectively stratified patients into three distinct risk groups. K-M survival curves demonstrated that the novel risk stratification model exhibited a superior level of predictive accuracy compared to IPI, R-IPI, and NCCN-IPI both in training and two validation cohorts. Additionally, the area under the curve (AUC) value of the novel model (0.763) for predicting 5-year overall survival rates was higher than those of IPI (0.749), R-IPI (0.725), and NCCN-IPI (0.727) in the training cohort. Similar results were observed in both internal and external validation cohort. CONCLUSIONS: In conclusion, we have successfully developed and validated a novel risk stratification model and a web-based survival rate calculator that demonstrated superior discriminative and predictive accuracy compared to IPI, R-IPI, and NCCN-IPI in the rituximab era.

The mTORC1-eIF4F axis controls paused pluripotency.

Mouse embryonic stem cells (mESCs) can self-renew indefinitely and maintain pluripotency. Inhibition of mechanistic target of rapamycin (mTOR) by the kinase inhibitor INK128 is known to induce paused pluripotency in mESCs cultured with traditional serum/LIF medium (SL), but the underlying mechanisms remain unclear. In this study, we demonstrate that mTOR complex 1 (mTORC1) but not complex 2 (mTORC2) mediates mTOR inhibition-induced paused pluripotency in cells grown in both SL and 2iL medium (GSK3 and MEK inhibitors and LIF). We also show that mTORC1 regulates self-renewal in both conditions mainly through eIF4F-mediated translation initiation that targets mRNAs of both cytosolic and mitochondrial ribosome subunits. Moreover, inhibition of mitochondrial translation is sufficient to induce paused pluripotency. Interestingly, eIF4F also regulates maintenance of pluripotency in an mTORC1-independent but MEK/ERK-dependent manner in SL, indicating that translation of pluripotency genes is controlled differently in SL and 2iL. Our study reveals a detailed picture of how mTOR governs self-renewal in mESCs and uncovers a context-dependent function of eIF4F in pluripotency regulation.

Renal damage and old age: risk factors for thrombosis in patients with ANCA-associated vasculitis.

INTRODUCTION: Thrombosis in ANCA-associated vasculitis (AAV) was prevalent and has been neglected in Chinese patients. This study tried to describe the clinical characteristics, identify the risk factors, and investigate the causal relationship between AAV and venous thromboembolism (VTE) by two-sample Mendelian randomization (MR) analysis. METHODS: In this retrospective, observational study, we included all hospitalized AAV patients from Jan 2013 to Apr 2022 in Peking Union Medical College Hospital. We collected their clinical data for multivariate regression analysis to determine the risk factors for thrombosis. The nomogram was constructed by applying these risk factors to predict thrombosis in AAV patients. As for MR analysis, we selected single nucleotide polymorphisms (SNPs) related to AAV from published genome-wide association studies and extracted the outcome data containing deep vein thrombosis (DVT) and pulmonary embolism (PE) from the UK biobank. RESULTS: 1203 primary AAV patients were enrolled, and thrombosis occurred in 11.3%. Multivariate regression suggested that older than 65 years, EGPA, neurological involvement, lung involvement, significantly elevated serum creatinine (> 500µmol/L), and elevated D-dimer were associated with thrombosis in AAV patients. The model demonstrated satisfied discrimination with an AUC of 0.769 (95% CI, 0.726-0.812). MR analysis showed that EGPA could increase the risk of developing DVT and PE (OR = 1.0038, 95%CI = 1.0035-1.0041, P = 0.009). CONCLUSION: Thrombosis was not rare in Chinese patients with AAV. Renal damage and old age emerged as critical risk factors for thrombosis. EGPA might have a potential causal relationship with DVT and PE.

Minor Hydroxylated Triterpenoids Produced in Engineered Yeast by the Enzymes OSC and CYP716s from the Plant Enkianthus chinensis and Their Anti-Inflammatory and Hepatoprotective Activities.

Triterpenoids are a type of specialized metabolites that exhibit a wide range of biological activities. However, the availability of some minor triterpenoids in nature is limited, which has hindered our understanding of their pharmacological potential. To overcome this limitation, heterologous biosynthesis of triterpenoids in yeast has emerged as a promising and time-efficient production platform for obtaining these minor compounds. In this study, we analyzed the transcriptomic data of Enkianthus chinensis to identify one oxidosqualene cyclase (EcOSC) gene and four CYP716s. Through heterologous expression of these genes in yeast, nine natural pentacyclic triterpenoids, including three skeleton products (1-3) produced by one multifunctional OSC and six minor oxidation products (4-9) catalyzed by CYP716s, were obtained. Of note, we discovered that CYP716E60 could oxidize ursane-type and oleanane-type triterpenoids to produce 6ß-OH derivatives, marking the first confirmed C-6ß hydroxylation in an ursuane-type triterpenoid. Compound 9 showed moderate inhibitory activity against NO production and dose-dependently reduced IL-1ß and IL-6 production at the transcriptional and protein levels. Compounds 1, 2, 8, and 9 exhibited moderate hepatoprotective activity with the survival rates of HepG2 cells from 61% to 68% at 10 µM.

Isoproterenol induces MD2 activation by ß-AR-cAMP-PKA-ROS signalling axis in cardiomyocytes and macrophages drives inflammatory heart failure.

Cardiac inflammation contributes to heart failure (HF) induced by isoproterenol (ISO) through activating ß-adrenergic receptors (ß-AR). Recent evidence shows that myeloid differentiation factor 2 (MD2), a key protein in endotoxin-induced inflammation, mediates inflammatory heart diseases. In this study, we investigated the role of MD2 in ISO-ß-AR-induced heart injuries and HF. Mice were infused with ISO (30 mg·kg-1·d-1) via osmotic mini-pumps for 2 weeks. We showed that MD2 in cardiomyocytes and cardiac macrophages was significantly increased and activated in the heart tissues of ISO-challenged mice. Either MD2 knockout or administration of MD2 inhibitor L6H21 (10 mg/kg every 2 days, i.g.) could prevent mouse hearts from ISO-induced inflammation, remodelling and dysfunction. Bone marrow transplantation study revealed that both cardiomyocyte MD2 and bone marrow-derived macrophage MD2 contributed to ISO-induced cardiac inflammation and injuries. In ISO-treated H9c2 cardiomyocyte-like cells, neonatal rat primary cardiomyocytes and primary mouse peritoneal macrophages, MD2 knockout or pre-treatment with L6H21 (10 µM) alleviated ISO-induced inflammatory responses, and the conditioned medium from ISO-challenged macrophages promoted the hypertrophy and fibrosis in cardiomyocytes and fibroblasts. We demonstrated that ISO induced MD2 activation in cardiomyocytes via ß1-AR-cAMP-PKA-ROS signalling axis, and induced inflammatory responses in macrophages via ß2-AR-cAMP-PKA-ROS axis. This study identifies MD2 as a key inflammatory mediator and a promising therapeutic target for ISO-induced heart failure.