Tripterygium wilfordii Hook. F. has been effectively used in the treatment of rheumatoid arthritis, nephrotic syndrome, leprosy, Behcet's syndrome, leprosy reaction and autoimmune hepatitis. However, due to its narrow and limited treatment window, TGT-induced organ toxicity  has gained increasing attention. The present study aimed to clarify the cellular and molecular events underlying TGT-induced acute liver injury using single-cell RNA sequencing (scRNA-seq) technology. The TGT-induced acute liver injury mouse model was constructed through short-term TGT exposure and further verified by hematoxylin-eosin staining and liver function-related serum indicators, including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin. Using the mouse model, we identified 15 specific subtypes of cells in the liver tissue, including endothelial cells, hepatocytes, cholangiocytes, and hepatic stellate cells. Further analysis indicated that TGT caused a significant inflammatory response in liver endothelial cells at different spatial locations; led to marked inflammatory response, apoptosis and fatty acid metabolism dysfunction in hepatocytes; activated hepatic stellate cells; brought about the activation, inflammation, and phagocytosis of liver capsular macrophages cells; resulted in immune dysfunction of liver lymphocytes; disturbed the intercellular crosstalk in liver microenvironment by regulating various signaling pathways. Thus, these findings elaborate the mechanism underlying TGT-induced acute liver injury, provide new insights into the safe and rational applications in the clinic, and complement the identification of new biomarkers and therapeutic targets for liver protection.Tripterygium glycosides tablet (TGT), the classical commercial drug of   •TGT induced acute liver injury via the regulation of inflammatory response.•TGT treatment resulted in hepatocyte apoptosis and HSC activation.•TGT treatment caused activated LCM cells and the immune dysfunction of lymphocyte. F. (TWHF) has been regarded as a “China-pioneered novel herbal drug” in the treatment of autoimmune and inflammatory diseases, such as rheumatoid arthritis (RA). Among the representative commercial preparations, tripterygium glycosides tablet (TGT) has similar clinical efficacy as methotrexate [otrexate . TGT canotrexate . The pototrexate ,4.Based on current studies, the development and application of TWHF are limited by its marked association with liver injury characterized by abnormal lipid metabolism . In a prSingle-cell RNA sequencing (scRNA-seq) is a robust tool for decomposing the transcriptomes of complex tissues at the single-cell level. In addition, it has provided a foundation for the Human Cell Atlas Project that aims to define the molecular states of all cell types in the human body . As the In the present study, we employed scRNA-seq technology to dissect the alterations in liver gene expression and functional signaling pathways after TGT treatment. The changes in gene expression and modulations of different cell types in the liver tissue were elucidated to delve into the liver microenvironment that is substantially remodeled by TGT. Thus, we speculated that our findings provide novel insights into the cellular and molecular mechanisms of TGT-induced acute liver injury and complement the further identification of potential therapeutic targets to alleviate the burden of TGT toxicity.22.1The experiments on C57BL/6 mice were in line with the regulations of the Institutional Animal Care and Use Committee and Animal Ethics Committee of the Institute of Chinese Materia Medica . Male C57BL/6 mice (8-weeks-old) were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd . Mice were kept under specific pathogen-free (SPF) conditions and acclimatized for a week before randomly dividing into two groups (6 mice/group). The animals were administered normal saline or TGT Provided by Zhejiang DND Pharmaceutical Co., Ltd  at a concentration of 270 mg/kg  intragastrically, 18 h before they were sacrificed.2.2The serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), and total bilirubin (TBIL) were detected using the corresponding kits, according to manufacturer's instructions . The serum inflammatory factors interleukin (IL)-1β, IL-2, CXCL2, CCL2, CCL3, CCL4, and interferon (IFN)-α were detected by enzyme-linked immunosorbent assay (ELISA) kit .2.3Mouse liver tissues were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and cut into 5-μm-thick sections before HE staining for pathological observation. The images were captured through a microscope  at × 40 magnification.S100a8) and glucose regulated protein 78 (Grp78) antibodies at 4 °C for 12 h, respectively, washed three times with Tris-Buffered Saline and Tween 20 (TBST), and incubated with secondary antibodies at room temperature on a shaker for 1 h. Subsequently, the sections were washed with TBST, stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, and covered with an anti-fading fixative, and fluorescence images were captured.The liver sections were incubated with S100 calcium binding protein A8 . The red blood cells were removed using the Red Blood Cell Lysis Solution . The above samples were washed twice with PBS for single-cell suspension.The mouse liver tissues from the control and TGT groups were collected for scRNA-seq analysis. Briefly, two liver samples from each group were dissected, sliced into small pieces, and enzymatically digested using the Liver Dissociation Kit for mouse  for about 30 min, according to the manufacturer's protocol. Then, the dissociated cells were filtered through a 70-mm strainer and rinsed with 15 mL Dulbecco's modified Eagle medium (DMEM) until a single-cell suspension was obtained. Subsequently, the cells were collected by centrifugation at 300 2.5The single-cell suspensions collected from the above step were used for scRNA-seq library construction following the instruction of the Single Cell 3′ Reagent Kit v3.1 . The scRNA-seq libraries were constructed using the Chromium Next GEM Single Cell 3′ Kit v3.1 from 10× Genomics, according to the manufacturer's instructions. Briefly, single cells were diluted to a final concentration of 1,000 cells/μL. Then, approximately 10,000 cells were captured in droplets to generate nanoliter-scale Gel beads in EMulsion (GEMs). Then, GEMs were reverse transcribed before cell barcoding, the emulsions were broken, and the cDNA was separated and further purified before PCR amplification. Next, the obtained amplified cDNA was used for library construction. To build-up the RNA-seq library, the amplified cDNA was fragmented and end-repaired, followed by double-sided size selection and PCR amplification with sample index primers. The libraries were further purified and profiled for quality evaluation and sequenced on the Illumina NovaSeq 6000  platform.2.6The raw gene expression matrix was produced by the Cell ranger (version 6.0.1) pipeline along with the mouse reference genome (mm10) and analyzed with the Seurat R package (version 4.0.3). The low-quality cells were filtered by the sample-specific criteria due to different data qualities, and the remaining high-quality cells were selected for further analysis. Seurat's SCTransform function was used for the normalization and scaling of the samples. We integrated high-quality cells into a matrix and performed cell clustering based on common features. The last step was scRNA-seq visualization and analysis of all the clustered cells by uniform manifold approximation and projection (UMAP) algorithm.2.7Different types of cells were identified by the expression levels of known canonical markers in each cluster using Seurat's FindAllMarkers function. The cell subtype identification was confirmed by principal components analysis (PCA), clustering, and annotation.2.8P-value < 0.05. The violin plots or heatmap using Seurat VlnPlot function and R packages pheatmap (v1.0.12) were generated for further visualization of markers. Gene Ontology (GO) analysis was conducted with the clusterProfiler R package (version 3.18.1) based on the up- and down-regulated genes among DEGs between different groups. The pathway enrichment analysis with adjusted P-value < 0.05 was further visualized by the R package ggplot2 (v3.3.3).The DEG analysis for each cell type between the control and TGT groups was conducted by the Seurat FindMarkers function with the selection standard of |avg_log2FC| > 0.25, min.pct > 0.1, and adjusted 2.9To investigate the cell-state transitions of hepatocytes and HSCs, we performed pseudo-temporal analysis by the Monocle2 R package (version 2.18.0). Briefly, we converted Seurat object to CDS object, followed by the confirmation of differential cell states based on the identification of significantly changed genes by the differential GeneTest function. Subsequently, the plot lineage trajectories were performed using the plot_cell trajectory function.2.10P-value < 0.05 were considered significant ligand-receptor pairs. The results were displayed by bubble plots and chord diagrams.The ligand-receptor interaction analysis was performed using CellChat R package (version 1.1.3) with the recommended parameters. The tool first utilized the normalized gene expression data and cell type meta information from the TGT and control groups to generate different CellChat objects. The two objects were merged using the mergeCellChat function for subsequent analysis. The communication probability value and permutation test were calculated in the ligand-receptor pairs of all cell types between the TGT and control groups. Results with a 2.11P-value < 0.05  was considered significant in the Seurat's AddModuleScore function.In this study, results are presented as mean ± standard error of the mean. One-way analysis of variance (ANOVA) was used to analyze the statistical differences between the control and TGT groups. The evaluation of gene set scores with 33.1P < 0.05, A TGT-induced acute liver injury mouse model was constructed and evaluated with serum and organ indices before performing single-cell sequencing and cell type identification A. Accord3.2n = 2) with 10× Genomics technology. After quality control , hepatocytes , cholangiocytes , HSCs , Kupffer cells , liver capsular macrophages , conventional dendritic cells , plasmacytoid dendritic cells , T cells , B cells , natural killer cells , neutrophils , monocytes , basophils , and Prolif.cells . Further function enrichment indicated that endothelial cells were involved in endothelial cell migration, hepatocytes in the organic acid catabolic process, HSCs in the extracellular matrix organization, T/B cells in cell differentiation, and LCMs in myeloid leukocyte activation  has increased pro-inflammatory and pro-fibrotic activities [Liver endothelial cells consist of major cell types, including liver vascular endothelial cells (LVECs) and liver sinusoidal endothelial cells (LSECs), and play a major role in liver functions, such as liver regeneration and fibrosis under both physiological and pathological conditions . As showand Selp B. Among and Selp C. Notabland Selp  and S2C.and Selp E, while and Selp F. To furmulation . These cand CCL4 . Moreovetivities  in all e3.4Ass1, Gsta3, Cdo1, Acaa2, and Asl, Hep2 by Lyz2, Fcer1g, Tyrobp, Srgn, and Actg1, and Hep3 by Pzp, Kng1, Vtn, Apob, and Cyp2f2 (P < 0.001). Moreover, one of the mechanisms of TGT-induced hepatotoxicity is to inhibit the expression of cytochrome P450 enzymes (CYPs), thereby reducing the metabolic capacity of the liver. In this study, we observed that Cyp3a11, Cyp2f2, Cyp2d9, Cyp2d10, and Cyp2d26 were significantly decreased in Hep2 and Hep3 of the TGT group. Interestingly, Cyp2e1 and Cyp26b1 were significantly reduced only in Hep2 and Cyp2a12 only in Hep3  after activating ER stress in the cells. Due to the regulatory effects of LCM cells on cytokines, we analyzed the differentially expressed cytokines of all LCM cell subtypes. In the TGT group, LCM1 cells had a higher expression of CCL2, CCR1, IL-1b, CCR2, CCL9, and CXCL2, but lower expression of CCR7 and IGF-1; LCM2 cells had a higher expression of CCL2, CCR1, CCL7, Inhbb, and Tgfb1, but lower expression of CXCL10, CXCL9, CX3CR1, CCL24, CCL5, and Pf4; LCM3 cells had a higher expression of CCR1, but lower expression of IL-1b, CXCL10, Tnfsf9, CCL6, IL-6, and CX3CR1; LCM4 cells had a higher expression of CCL2 and CCR1, but lower expression of CXCL10, IGF-1, Ifnar2, IL-10RA, and CX3CR1 , alternatively known as macrophage inflammatory protein 2, has the major function of chemokine leukocytes at the inflammatory sites. Interestingly, TGT treatment elevated the expression of CXCL2 in all the liver lymphocyte subtypes, reflecting immune regulation  belong to IFITM proteins that are the cofactors for efficient virus infection in human cell types, including SARS-CoV-2 infection [Ifitm2 and Ifitm3 in all the subtypes of lymphocytes. In summary, under TGT-induced acute liver injury conditions, the liver lymphocytes demonstrated immune dysfunction.Lymphocytes play a critical role in immune regulation in the liver. In the current study, we delineated the subtypes of liver lymphocytes, including T cells, NK cells, and B cells, based on key gene markers. As shown in he liver  and S6E.he liver  for threhe liver  for NK cgulation J. IFN-innfection  and acutnfection . Thus, t3.8CXCL and CCL signaling pathways showed enhanced interactions compared to the control group (Tgfb1-(Tgfbr1 + Tgfbr2), Mif-(Cd74 + Cd44), CCL5-CCR5, and CCL5-CCR1, indicating that T cells communicated with other cell types to regulate the inflammation and immune response (Mif-(Cd74 + CXCR4), Mif-(Cd74 + Cd44), and CXCL2-CXCR2 indicating its inhibitory effects on regulation, as described in CXCL2-CXCR2 in the TGT treatment group  pairs analysis between sender and receiver cells . First, ol group . Next, wol group C. Thus, response D. Similaresponse E. In addnt group H. In addup , CCL5-CCR5, and CCL5-CCR1, are closely involved in the innate immune response and the activation and amplification of liver injury [Finally, according to the cell-cell communication crosstalk analysis, several cell types, such as T cells, B cells, KCs, HSCs, and pDCs play a central role in association with other cell types. Notably, in the liver tissues of TGT-treated mice, T cells showed maximal interactions with other cell types when acting as either source or target by ligand-receptor pairing. This included proinflammatory cytokines and the corresponding receptors, such as r injury . Reporter injury . In thisr injury .in vitro investigation. Secondly, we analyzed the recruitment and activation of immune cells and found that both the clonal correlation and the environmental location of immune cells will require other techniques  to decipher the intricate activities of TGT in the liver fully. Finally, due to the complexity of the pathogenesis of TGT-induced acute liver injury, additional experiments are needed at the cell levels with single stimuli to validate the single-cell transcriptomic results.Nevertheless, the present study has several limitations. Firstly, the molecular mechanisms underlying the regulation of different liver cell types under TGT-induced acute injury conditions need further In summary, this study integrated the in vivo experiments and single-cell RNA-seq, uncovering specific responses in different cell types and the microenvironment alterations in the liver tissues under TGT-induced acute liver injury conditions. These novel findings provided insights into the nature of TGT-induced acute liver injury and suggested potential pathways and targets for future therapeutic interventions to improve the safe and rational clinical applications of TGT.5In conclusion, we generated a comprehensive single-cell landscape for TGT-induced acute liver injury to identify the marked alterations and heterogeneity in 15 specific subtypes of liver cells under injury conditions. Importantly, our analysis expands our understanding of the mechanism of liver injury initiated by TGT, which would be valuable in identifying new biomarkers and therapeutic targets for liver protection. The present study also provided novel insights into the safe and rational usage of TGT in other therapeutic applications against rheumatoid arthritis, nephrotic syndrome, and other diseases.Qiuyan Guo: Conceptualization, Methodology, Resources, Visualization, Writing - Original draft preparation, Reviewing and Editing, Funding acquisition; Jiangpeng Wu: Software, Formal analysis, Investigation, Data Curation, Visualization, Writing - Original draft preparation, Reviewing and Editing; Qixin Wang: Validation, Formal analysis, Investigation, Visualization, Writing - Original draft preparation, Reviewing and Editing; Yuwen Huang: Investigation, Methodology, Software; Lin Chen: Investigation, Validation, Methodology; Jie Gong: Investigation, Writing - Reviewing and Editing; Maobo Du: Investigation, Writing - Reviewing and Editing; Guangqing Cheng: Investigation, Validation, Methodology; Tianming Lu: Investigation, Visualization, Validation; Minghong Zhao: Investigation, Visualization, Validation; Yuan Zhao: Software, Investigation, Resources; Chong Qiu: Investigation, Validation, Resources; Fei Xia: Investigation, Validation, Resources; Junzhe Zhang: Investigation, Validation, Resources; Jiayun Chen: Software, Methodology, Investigation; Feng Qiu: Supervision, Writing - Reviewing and Editing; Jigang Wang: Conceptualization, Resources, Supervision, Writing - Reviewing and Editing, Project administration, Funding acquisition.The authors declare that there are no conflicts of interest.


Malignant T lymphocyte proliferation in mycosis fungoides (MF) is largely restricted to the skin, implying that malignant cells are dependent on their specific cutaneous tumor microenvironment (TME), including interactions with non-malignant immune and stromal cells, cytokines, and other immunomodulatory factors. To explore these interactions, we performed a comprehensive transcriptome analysis of the TME in advanced-stage MF skin tumors by single-cell RNA sequencing. Our analysis identified cell-type compositions, cellular functions, and cell-to-cell interactions in the MF TME that were distinct from those from healthy skin and benign dermatoses. While patterns of gene expression were common among patient samples, high transcriptional diversity was also observed in immune and stromal cells, with dynamic interactions and crosstalk between these cells and malignant T lymphocytes. This heterogeneity mapped to processes such as cell trafficking, matrix interactions, angiogenesis, immune functions, and metabolism that affect cancer cell growth, migration, and invasion, as well as antitumor immunity. By comprehensively characterizing the transcriptomes of immune and stromal cells within the cutaneous microenvironment of individual MF tumors, we have identified patterns of dysfunction common to all tumors that represent a resource for identifying candidates with therapeutic potential as well as patient-specific heterogeneity that has important implications for personalized disease management.  PatiA striking feature of MF is the restriction of lymphocyte proliferation to the skin, which implies that the malignant cells are dependent on their specific cutaneous tumor microenvironment (TME), including cell-to-cell interactions, spatial distribution, and secreted factors , 6. Mali+ and CD8+ lineages were characterized by a memory phenotype and the activation of antitumor Th1 and cytotoxic pathways but, conversely, upregulation of checkpoint receptor expression as well as several anti-inflammatory and immunosuppressive mechanisms. We also detected patient-specific immune responses mediated by CXCL13+ T follicular helper (TFH) cells, TNFR2 signaling, NRF2-mediated oxidative stress, and ferroptosis. However, these studies did not analyze all immune and stromal cells in the TME of advanced-stage MF patients.Advances in single-cell RNA sequencing (scRNA-Seq)  allow trHere we report a comprehensive study including transcriptome analyses of all cell types from skin lesions of patients with advanced-stage MF. Our analysis revealed a distinct MF-specific TME in comparison with skin from healthy controls and benign dermatoses but with extensive interpatient heterogeneity. Major diversity was observed in both the proportion of specific cell types and their gene expression. Focusing on the cell types in the MF TME that exhibit the greatest changes, such as myeloid cells, B cells, keratinocytes, fibroblasts, and endothelial cells, we identified alterations in cellular transcriptomes leading to changes associated with pathways affecting cell trafficking, matrix interactions, angiogenesis, immune functions, and metabolism in patient samples, with dynamic interactions and crosstalk between these cell types and malignant T lymphocytes. We also detected substantial differences in MF TME between patients, indicating that differences in individual MF TMEs may also affect T cell clonal expansions. This work provides a comprehensive view of immune and stromal cell heterogeneity within the skin microenvironment of individual MF tumors and offers important implications for personalized disease management.https://doi.org/10.1172/jci.insight.170015DS1) that were previously studied to identify T cell heterogeneity and clonality  and dendritic cells (DCs)   . The tra LILRA4) ; however LILRA4) .Seurat analysis of combined MF and HC samples identified 9 Louvain clusters , and we EMP1, CXCL12, EGR1, CXCL14) and 3  corresponded to tissue-resident macrophages  contained cells from most MF samples  and upregulated several processes including leukocyte motility and extravasation, IFN signaling, cachexia, and production of reactive oxygen species  comprised cells deriving from MF6, MF12, MF18, and MF24 and identified a subset of plasmacytoid dendritic cells (pDCs)  identified M2-like macrophages (The MF-specific macrophage cluster 2 ( species . Cells f species  such as s (pDCs) , upreguls (pDCs) . Cells frophages  and actirophages .CD1C, LY6C, FCER1A, S100A7), as well as CD1A, which characterizes Langerhans cells; however, we found no expression of langerin (CD207)   (DCs from cluster 1 expressed markers of cDC2 ( (CD207) , thus id (CD207) . Pathway (CD207) . Cluster, BATF3)  and a LA, CD200) . They ac, CD200) .+FCN1+ and CLEC4C+LILRA4+ cells in MF skin lesions that are absent in HC skin.Finally, cluster 8 represented a subset of proliferating macrophages and DCs . To valiThus, our transcriptome analysis details phenotypic shifts of myeloid cells in the TME of advanced-stage MF, including the prominent expansion of TAMs, M2-like macrophages, and pDC populations.MS4A1, CD19, PAX5, CD27)  (+ (MS4A1) B cells and CD138+ (SDC1) plasma cells in MF skin lesions, whereas no B memory and few plasma cells were detected in HC skin  derived from MF24 (78%) and MF18 (15.3%) and upregulated B cell development as well as the IL-4 and PI3K signaling cascades  comprised cells from MF6 (83%), MF8 (4.4%), MF12 (9.7%), and MF18 (3%) and was enriched in antigen presentation, B cell development, and estrogen receptor signaling pathways  derived from most MF samples and activated B cell receptor signaling as well as endoplasmic reticulum stress and NAD pathways  were from MF6 (62%), MF12 (19.3%), MF18 (11.1%), and MF24 (6%) and upregulated signaling by Rho GTPases and IL-4 as well as antigen presentation  . Transcr HC skin . Louvain HC skin . We founcascades . Clusterpathways . Plasma pathways . The majentation .However, B cells from individual patient samples also exhibited distinct gene expression . While BKRT1, KRT10, and KRT14  were specifically enriched in pathways such as necroptosis, eicosanoid signaling, and IL-17 signaling in psoriasis, in line with the MF8 histological pattern of psoriasiform epidermal hyperplasia, whereas cells from cluster 4  upregulated the IFN, estrogen, and integrin signaling cascades  identified proliferating keratinocytes  of keratinocytes among MF samples, we identified 237 genes in common, of which we show a selection of 20 genes that exhibited little or no expression in HC samples . These i HC skin . Thus, wIFI27, IFI30, STAT1, EPSTI1), ECM production , metabolism , and inflammation . Notably, all samples upregulated several processes in common, including wound healing and IL-6, IL-8, IL-13, TGF-β, VEGFA, PDGF, FGF, IGF-1, and IGF-2 signaling cascades   that showed increased PLVAP expression by all MF samples except MF6 compared with individual HC samples. However, most of the latter exhibited increased expression of SELE, important for neutrophil rolling in postcapillary venules. Markers for venule  and lymphatic  ECs were mostly detected in control samples. Notably, ECs from MF samples exhibited increased expression of angiogenic and immunomodulatory gene signatures. The angiogenic phenotype was more prominent in MF17, MF18, and MF21 and included the expression of genes encoding pro-angiogenic factors, endothelial tip cell markers, VEGF and NOTCH signaling molecules, and vascular membrane remodeling factors  as well as factors contributing to EC homeostasis , microvascular permeability, and leukocyte recirculation  (+SOX18+) in microvascular areas of the TME in several MF patient samples, whereas ECs from HC samples were negative for SOX18 expression  were also proportionally decreased in patient samples compared with HCs and exhibited a non-overlapping transcriptional profile . ECs wer, CLDN5)  and show, CLDN5)   . These i factors . We also factors . ECs fro ACVRL1) . Notably ACVRL1) . All MF pression . Thus, MTo further investigate the MF cell types analyzed above, we integrated our MF data sets with publicly available scRNA-Seq data from benign inflammatory skin diseases including atopic dermatitis (AD)  and psorWe first aimed to profile differences in the phenotype and effector functions of myeloid cells between MF and AD/PS samples. Although we noticed a considerable overlap between the transcriptional profiles of MF and AD myeloid cells, we also found MF-specific gene expression that included signatures also identified in the comparison with HC skin . These iAs observed in the MF comparison with HC skin samples, integration of MF and AD/PS keratinocyte data sets revealed distinct MF8 and MF21 transcriptional profiles . SimilarComparison of the MF and AD/PS fibroblast data sets showed MF differential expression of multiple genes associated with wound healing and fibrosis, including collagens, TNC, LOXL, and MMP enzymes, and signaling through various cytokines and growth factors such as IL-6, IL-8, IL-13, IL-17, IFN, and PDGF . In furtLastly, integration of the EC data sets showed an extensive overlap between the MF comparisons with AD/PS and with HC skin . In commTogether, integration of our data sets with those from benign dermatoses validates the results of our comparison with HC skin and further emphasizes the role of the advanced MF TME in promoting dysfunctional cell trafficking, antitumor immunity, matrix interactions, and angiogenesis.+TOX+ITGB1+ cells were detected in HC or in AD/PS skin samples   on highly suppressive TNFR2+ Tregs  exhibited dichotomous functional phenotypes characterized by the expression of proinflammatory and pro-tumorigenic pathways. Interestingly, cells from this cluster also expressed markers of MDSCs (+ TAMs) identified M2-like TAMs  signaling  and signaling pathways fueling necroptosis. In contrast, a previous study showed a decreased expression of alarmins that correlated with predisposition to Staphylococcus aureus infection in CTCL skin , which favors the influx of Tregs and TAMs, downregulation of VCAM1 and ICAM1, or secretion of soluble adhesion molecules  that inhibit the recruitment of effector CD4+ and CD8+ T cells. Although MF ECs could function in antigen presentation, they lack expression of CD80 and CD86 (not shown), thereby promoting CD4+ T cell unresponsiveness (CD274 (PDL1), HAVCR2 (TIM3), and IDO1, to directly inhibit T cell activation and suppress antitumor immunity. However, several studies have reported expression of inhibitory checkpoint molecules by malignant MF T lymphocytes (Increased microvessel density (MVD) in CTCL lesional skin correlates with aggressive disease subtypes and disease progression . Our anativation , 72. TGFeability . Vessel eability , 72. Whieability . Examplesiveness . FurtherMycosis fungoides is a rare cancer, which limits sample availability and homogeneity. Although all samples analyzed were derived from patients with advanced-stage MF and most of them were not receiving any treatment at the time of biopsy, all patients underwent prior and diverse treatments, which may affect the heterogeneity of the TME. Nonetheless, compared with HC skin or benign dermatoses, we robustly detected common patterns of abnormal transcription in MF that may be used as a basis for functional predictions. Thus, our characterization of the TME and its interactions with malignant lymphocytes offers critical insights for developing treatments targeted to TME components that may be further personalized to specific patients.n = 9) obtained from age- and sex-matched donors at the Health Sciences Tissue Bank, University of Pittsburgh.Skin samples (4 mm punch biopsies) were obtained from 10 patients with a confirmed diagnosis of advanced MF (stage IIB–IVA) at the Comprehensive Skin Cancer Center, Columbia University Medical Center. Patients were well characterized in terms of demographic, disease type, clinical features, and therapy as described in P value less than 0.05 for significance. For gene differential tests, we used the FindMarkers function, which uses Wilcoxon’s rank sum test to show differential genes with a minimum percentage of cells of 10% per identity requested, log fold change >0.1, and P value less than 0.05 for significance  for significant upregulated pathways. Pathways were selected by enrichment scores  and absolute z scores over 2  as previously described . The antP value of 0.05 was used throughout to determine significance. Statistical differences of cell-type proportions between MF and HC samples within the skin microenvironment were estimated by unpaired 2-tailed Student’s t test.For analysis of single-cell data, relevant statistical analyses are indicated in the respective sections of Methods. A Clinical information and biological specimens were deidentified and coded. Research protocols involving humans were approved by the Institutional Review Board of Columbia University. All participants gave written informed consent in accordance with the Declaration of Helsinki.All scRNA-Seq data generated in this study were deposited in the Gene Expression Omnibus (GEO) database under accession numbers GSE206123 and GSE182861. The AD and PS data sets analyzed in this study are available in the GEO database under accession numbers GSE153760 and GSE151177. Data used in the Figures and AMG and TT performed experiments and analyzed data. LJG and CJS acquired samples and collected clinical descriptions. RL analyzed data and prepared the manuscript. PF developed the project, analyzed data, and prepared the manuscript.


IFI27, ISG15, and CYBRD1 as potential suitable biomarkers of disease severity. Thus, this study characterizes the landscape of the interferome signature in DENV infection, indicating that interferome dynamics are a crucial and central part of the anti-dengue immune response.Dengue virus (DENV) infection manifests as a febrile illness with three distinct phases: early acute, late acute, and convalescent. Dengue can result in clinical manifestations with different degrees of severity, dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Interferons (IFNs) are antiviral cytokines central to the anti-DENV immune response. Notably, the distinct global signature of type I, II, and III interferon-regulated genes (the interferome) remains uncharacterized in dengue patients to date. Therefore, we performed an in-depth cross-study for the integrative analysis of transcriptome data related to DENV infection. Our systems biology analysis shows that the anti-dengue immune response is characterized by the modulation of numerous interferon-regulated genes (IRGs) enriching, for instance, cytokine-mediated signaling  and chemotaxis, which is then followed by a transcriptional wave of genes associated with cell cycle, also regulated by the IFN cascade. The adjunct analysis of disease stratification potential, followed by a transcriptional meta-analysis of the interferome, indicated genes such as   Dengue Aedes aegypti and Aedes albopictus mosquitoes (Four different DENV serotypes DENV1-4) are transmitted by the vector squitoes . The cousquitoes , 4. are tranThe acute phase can be classified into two stages: 1) Early acute (day 0 to 3 from symptom onset) and 2) Late acute or defervescence (day 4 to end of acute phase) . The criMoreover, dengue can be clinically classified by disease severity: 1) dengue fever (DF), characterized by fever and two or more dengue symptoms; 2) dengue hemorrhagic fever (DHF), marked by increased vascular permeability, plasma leakage, bleeding, and thrombocytopenia , 4; and The DENV infection typically presents as a self-limiting febrile disease, indicating a central role of the immune system in controlling disease . AntigenOne key mechanism of the anti-viral immune response is the production of interferons (IFNs) by various cell types. IFNs are rapidly induced cytokines that respond strongly to DENV , 11. TheFor instance, interferon-alpha/beta (IFN-α/β)-binding to their receptor (IFNAR) triggers the activation of multiple downstream signaling pathways, including the canonical STAT1–STAT2–IFN-regulatory factor 9 (IRF9) signaling complex , which tIndividual transcriptome studies of DENV-infected patients revealed that different IFN-associated genes are elicited in response to DENV infection , 13–15 aA comprehensive analysis of the IFN signature in the context of dengue infection has not been conducted yet. Here, we performed an integrative analysis of multiple studies of dengue patients’ transcriptomes, considering the disease phases and severities, to characterize the dengue interferome  Figure 122.1https://www.ncbi.nlm.nih.gov/gds). Our search query (“dengue virus”[MeSH Terms] OR “dengue virus”[All Fields] OR “DENV”[All Fields] OR “dengue”[MeSH Terms] OR “dengue”[All Fields]) AND “Homo sapiens”[Organism] AND (Expression profiling by high throughput sequencing[DataSet Type] OR Expression profiling by array[DataSet Type]) resulted in 93 series as of 17th of August of 2021  natural dengue infection, (3) blood or PBMC samples, (4) availability of convalescent-phase samples, and (5) at least one group with ten or more samples. The exclusion criteria included (1) in vitro samples, (2) cohorts with only infants and/or children, and (3) results from vaccine or drug trials. We included in our analysis seven datasets: GSE25001 (The inclusion criteria included: (1) GSE25001 , 21, GSEGSE25001 , 22, GSEGSE25001 , GSE2899GSE25001 , GSE4377GSE25001 , 26, andGSE25001 , 27.The samples were categorized into three groups according to time after symptom onset: 1) Early acute (0-3 days), 2) Late acute (4-8 days), and 3) Convalescent (≥14 days). For dataset GSE43777, the late acute phase comprised days 4-10 from disease onset. When applicable, samples were also classified according to disease severity as: 1) Dengue fever (DF), 2) Dengue hemorrhagic fever (DHF), and 3) Dengue shock syndrome (DSS), as determined by the authors when samples were collected. Information about the included series is provided in 2.2https://www.ncbi.nlm.nih.gov/geo/geo2r/)  was performed with the GEO2R web tool (/geo2r/) , applyin package . All gro2.3http://www.interferome.org/interferome/home.jspx)  , which h2.4http://circos.ca/) (https://asntech.shinyapps.io/intervene/) (https://software.broadinstitute.org/morpheus/) (The overlap of the IFN-regulated DEGs between datasets both by IFN type and disease phase were retrieved and represented with Circos (cos.ca/)  and UpSeervene/) . The datervene/) Table S7rpheus/) . The hie2.5https://maayanlab.cloud/Enrichr/) (http://revigo.irb.hr/)  . The resirb.hr/) . The mosirb.hr/) . The funirb.hr/)  R packag2.6https://www.expressanalyst.ca/) , were selected to analyze the power of the interferome to discriminate the disease severities. The expression values were processed on the ExpressAnalyst web tool (yst.ca/) , and logyst.ca/) , 40 wereyst.ca/) , ggplot2yst.ca/) , and ggEyst.ca/) .2.7The samples from datasets A and E were grouped by disease severity for each phase. The log2-transformed expression values of the commonalities used in the respective PCAs were applied in the random forest (RF), a machine learning algorithm, which uses the combination of multiple tree classifiers  to ident2.8https://www.expressanalyst.ca/)  genes from dataset G (GSE51808) (data available in https://www.ncbi.nlm.nih.gov/gene/) (https://www.uniprot.org/)   (resultsyst.ca/)  with R 4yst.ca/) . A box pv/gene/)  and UniPot.org/)   natural dengue infection, (3) blood or PBMC samples, (4) availability of convalescent-phase samples, and (5) at least one group with ten or more samples; and exclusion criteria of (1) in vitro samples, (2) cohorts with only infants and/or children, and (3) results from vaccine or drug trials. All these datasets were generated through microarray technology  GSE25001 , 21 (A),GSE25001 , 22 (B),GSE25001  (C), GSEGSE25001  (D), GSEGSE25001 , 26 (F),GSE25001 , 27 (G) GSE25001 Table 1.We compared the gene expression profiles of the different disease phases  and severities  through differential expression analyses (DEAs). Following, we identified the interferon-regulated genes (IRGs), as summarized in 3.2We next analyzed the overlap between dengue’s early and late acute phases, identifying the transcriptional intersections between studies A to E. The investigation of common IRGs found 173 common DEGs between the datasets during early and late acute phases Table S5We found a distinct interferome signature across the datasets during early and late acute phases (3.3We characterized the interferome expression patterns across the transcriptome datasets from dengue patients at acute phases (datasets A-E) with an unsupervised hierarchical clustering analysis Table S8Next, we characterized the clusters through gene ontology (GO) analysis to identify distinct biological processes (BPs) that are enriched by the differential expression signatures found in our gene sets , considering disease phases and severities. The PCA of IRGs indicated that they stratify DF, DHF, non-DSS, and DSS dengue patients at early and late acute phases from their counterparts in convalescence , a machine learning algorithm, to identify classifiers of dengue severity . The acute and convalescent phases were significantly different, while there was no difference between the convalescent and healthy control samples  , which pOur current study emphasizes the predominance of IFN-regulated genes among DEGs, characterizing the interferome signature as an evident hallmark of the acute response to DENV. This finding agrees with the observation of high levels of type I IFN during acute dengue infection with concomitant CD4+ and CD8+ T cell activation at symptom onset . It is aClinical investigations of dengue patients have assessed varying timeframes of IFN kinetics and levels during infection, e.g., higher IFN-α levels were identified in the period comprising the early acute phase (0 to 3 days after fever onset) , 60, butThe observed cell-cycle-related effects of the distinct anti-DENV interferome signature should be investigated further. They may be explained by the fact that IFNs have been reported to negatively regulate the proliferation and differentiation of cell types, such as innate lymphoid type 2 cells and dendritic cells , as meanIFI27, SULF2, ISG15, NME1, CYBRD1, GINS2, GAS6, UBE2T, RNASE1, MKI67) for differentiation of DHF and DF in the late acute phase. Among them, IFI27, ISG15, and CYBRD1 were also meta-significant genes. We confirmed in an independent dataset that these putative biomarkers are differentially expressed during acute infection for both DF and DHF and return to baseline expression upon disease resolution. Further, IFI27 and ISG15 were significantly upregulated in the DHF late acute phase and have previously been associated with dengue severity by Zanini et al.  results demonstrate effective discrimination between disease severities based on the interferome signature only in the late acute phase, indicating that the interferome has a more prominent role at this stage. The late acute phase is critical, wherein the disease course is defined as severe or non-severe dengue . So far,i et al. .IFI27  is involved in type-I interferon-induced apoptosis , accession numbers GSE25001, GSE28405, GSE28988, GSE28991, GSE43777, GSE40628, GSE51808. Codes utilized in this article are available at the following link: https://github.com/JNUsuda/dengue_interferome.Publicly available datasets were analyzed in this study. This data can be found here: GEO Datasets (Ethical approval was not required for the study involving human samples in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because publicly available datasets were used in this study.JU, DP, and OC-M conceived this study. JU performed the data analysis. DF, AM, IF, VC, AA, AT-C, MH, PF, RC, GM, GC, LS, DP, and OC-M provided scientific insights. JU, RC, GM, DP, and OC-M wrote and revised the final version of the manuscript. DP and OC-M supervised the work. All authors contributed to the article and approved the submitted version.


ZmSSL genes in maize growth and development and its response to stresses is unknown. Herein, we undertook the systematic identification and analysis of maize SSL genes. Twenty SSL genes were identified in the maize genome. Except for chromosomes 3, 5, 6, and 10, they were unevenly distributed on the remaining 6 chromosomes. A total of 105 SSL genes from maize, sorghum, rice, Aegilops tauschii, and Arabidopsis were divided into five evolutionary groups, and ZmSSL gene structures and conserved protein motifs in the same group were similar. A collinearity analysis showed that tandem duplication plays an important role in the evolution of the SSL family in maize, and ZmSSL genes share more collinear genes in crops  than in Arabidopsis. Cis-element analysis in the ZmSSL gene promoter region revealed that most genes contained many development and stress response elements. We evaluated the expression levels of ZmSSL genes under normal conditions and stress treatments. ZmSSL4–9 were widely expressed in different tissues and were positively or negatively regulated by heat, cold, and infection stress from Colletotrichum graminicola and Cercospora zeina. Moreover, ZmSSL4 and ZmSSL5 were localized in the chloroplast. Taken together, we provide insight into the evolutionary relationships of the ZmSSL genes, which would be useful to further identify the potential functions of ZmSSLs in maize.Maize is often subjected to various environmental stresses. The strictosidine synthase-like (SSL) family is thought to catalyze the key step in the monoterpene alkaloids synthesis pathway in response to environmental stresses. However, the role of  This, in turn, activates the expression of relevant defense genes to enhance plant stress tolerance [STR and other genes, thereby improving the drought tolerance and yield of plants [Maize is one of the most widely distributed food crops in the world, with the total area under cultivation only less than wheat and rice. In addition to being eaten, it can also be used to produce feed and ethanol. Crops (as for example maize) are often subjected to various natural disasters (abiotic and biotic stress) during their growth and development stages, which have a great impact on their quality and yield . Becauseolerance ,4,5,6. Aolerance ,8. Stricolerance . Previouf plants ,11,12.SSL gene family have two conservative motifs: strictosidine synthase (SS) and the SS-like N-terminal domain [Drosophila immune-related protein  [The protein sequences of the plant l domain . The SS l domain . The seqeceptor) ,9.SSL gene family has been systematically characterized only in Populus trichocarpa (13 members) [Arabidopsis thaliana (15 members) [Salix purpurea (22 members) [SSL members are biotic/abiotic-stress-responsive [AtSSL4–7 displays the enzyme activity necessary to catalyze the synthesis of STR, and the AtSSL4–7 gene is upregulated by treatments of SA, JA, and ethylene [PtrSSL2, 6, 8, 10, and 12 genes is elevated by drought and high salt levels, and the PtrSSL2, 7, 8, and 10 genes respond to leaf blight  stress [PtrSSL family members were highly expressed in stems or leaves, respectively [SSL genes play an important role in normal growth and response to biotic/abiotic stresses in Poplar trees. There are a few reports about the numbers and function of SSL genes in other species. In Catharanthus roseus, the SSL gene is highly induced by drought stress, thus conferring plant drought tolerance [STR gene mRNA and negatively regulates its accumulation; the over-expression of miR1916 in tomato plants significantly decreased the drought tolerance of transgenic lines [miR1916 in Solanum lycopersicum increased the expression level of STR-2, some immune-response-related proteins, and MYB transcription factor genes, which, in turn, significantly elevated the tolerance of tomato plant leaves to two common fungi (Phytophthora infestans and Botrytis cinerea) [SSL gene family is related to plant stress responses, the role of ZmSSL genes in maize growth and development or response to biotic/abiotic stresses is far from known.Until now, the members) , Arabidochocarpa  members members) . All Arasponsive ,13. Furtic lines . Conversthaliana  members SSL gene family members. With the help of homology comparisons using conserved protein motifs, 20 ZmSSL genes were identified; chromosomal localization was also undertaken along with the clarification of phylogenetics and gene structure. Cis-elements located in promoter analysis were also carried out. Moreover, we analyzed the expression level of ZmSSL genes under normal growth and various biotic/abiotic conditions using published transcriptome data and the RT-qPCR method. This work provides new information about the evolutionary relationships of the ZmSSL gene family, which provides a foundation for further detecting their possible biological functions.In the present study, we systematically identified and analyzed the maize SSL gene family members in maize (Zea mays L.), sorghum (Sorghum bicolor L.), rice (Oryza sativa L.), and one of the parents of common wheat, Aegilops tauschii (Aegilops tauschii Coss.), a homology analysis was conducted in a recent version of the genomic database of each species using AtSSLs protein sequences [SSL genes were evaluated in maize, sorghum, rice, and Ae. tauschii, respectively. All of these identified genes were named in the order of the position in their chromosomes (SSL gene family is unevenly distributed on 6 of the 10 chromosomes with the highest number (10 members) on chromosome 8 and the lowest on chromosomes 4 and 9  in length (average: 340 aa), with isoelectric points (PI) ranging from 5.81 to 8.92 (average: 6.57), and protein molecular weights (Mw) of 20.81–46.54 kDa (average: 36.52). Among the 21 SbSSL proteins, the average aa, PI, and Mw were 353, 7.23, and 38.23, respectively. Ae. tauschii contains 33 SSL members; the average aa, PI, and Mw were 337, 7.61, and 36.63, respectively. The number of OsSSLs was almost identical to Arabidopsis; the average aa, PI, and Mw were 384, 8.00, and 41.66, respectively. Based on the predicted subcellular localization, ZmSSL genes were located in the chloroplast/thylakoid (14 members), extracellular/cell wall (4 members), plasma membrane (1 member), and endoplasmic reticulum (1 member), respectively , a maximum likelihood phylogenetic tree was constructed using the protein sequences of the SSL genes listed in SSL members from five species could be divided into five groups (I–V), each group including 32, 11, 26, 29, and 7 SSL genes, respectively. SSL genes were present in Group I and Group II from all five plants, implying that members in these two groups may undergo a similar evolutionary pattern (AtSSLs were only represented in Groups I and II (14 members in I and 1 member in V) (SSL genes of four crops were mostly represented in Groups III–V (from the same ancestor) (SSL genes were predominantly in Group IV (13 members), and all branches of ZmSSL genes were more adjacent to SbSSL gene branches than to the other two crops (To examine the evolutionary relationships of  pattern . Meanwhier in V) ; the SSLncestor) , which mwo crops . This inZmSSL family members, we evaluated the gene structure and conserve motifs of ZmSSL genes. The gene exon–intron structure/distribution analysis indicated that, except for ZmSSL4/5/6/20 (Group II), where the number of exons and introns varies from each other, the same group displays structural similarities; ZmSSL7 (Group I) contained 2 exons and 1 intron; four Group II members contained 3, 4, or 6 exons and 2, 3, or 5 introns, respectively; two Group III members (ZmSSL8 and ZmSSL9) included 3 exons and 2 introns; and all of Group IV (13 members) contained only 1 exon and no introns  were obtained   . This inZmSLL genes, we identified a large number of cis-elements involved in the development and hormones/abiotic/biotic stress responses in the promoter regions of most ZmSSL genes  (ZmSSL8 and 9) showed the highest expression in the leaf-tip and SAM  showed almost no expression levels in any tissues, indicating that these genes may be involved in stress responses.To explore the expression patterns of ing data  (Supplem tissues . ZmSSL4– embryo) , suggest and SAM , revealiZmSSL genes in maize tissues under abiotic and biotic stresses, published transcriptome sequencing data (Colletotrichum graminicola and Cercospora zeina) [ZmSSL genes were induced during 1 h of dehydration stress  ,20 were n stress , even thn stress . ZmSSL5 nfection , indicatZmSSL7–9 . ZmSSL4 fections . Similarfections , none ofZmSSL genes under abiotic/biotic stresses, we treated maize B73 to heat, cold, C. graminicola, and C. zeina stresses, and performed an RT-qPCR to explore the transcript level of ZmSSL4–9. The expression of four genes  was notably downregulated at 42 °C (ZmSSL8 and ZmSSL9 both positively responded to cold stress (C. graminicola infection indicated that ZmSSL5 and ZmSSL8 were upregulated after 24 and 48 h post-inoculation (hpi), suggesting they may be involved in the response process . In the current study, using AtSSLs and the conserved HMM motif of SSL protein as a query to blast genome data, a total of 20, 21, 16, and 33 SSL genes were identified in maize, sorghum, rice, and Ae. tauschii, respectively  to build a phylogenetic tree. The results showed that these SSL genes are divided into five groups . TBtools were used to analyze the structure of the ZmSSL genes and the conserved motif/domain of amino acid sequences. As shown in ZmSSL genes in the same evolutionary group contained similar numbers of exons, introns, and protein motifs, which showed high correlations with the evolutionary analysis results. A recent study indicated that the SSL family members of Populus trichocarpa (13 members), Arabidopsis thaliana, and Salix purpurea (22 members) are divided into four groups based on the neighbor-joining (NJ) method of constructing a phylogenetic tree, and each group includes both SSL genes from three species, indicating that those from herbaceous and woody SSL genes might undergo relatively consistent evolutionary progress [AtSSL genes were mainly included in Group I  with the brush method [6 conidia per mL) leaf material (two leaves per plant) was harvested at four time points  from three biological replicates. The fungus was prepared as described [The maize B73 inbred line was used in this research. Surface-sterilized B73 seeds were planted in nutrient soil and grown under a 16 h/8 h day/night photoperiod at 25 °C in a greenhouse. Two-week-old (V3 stage) maize seedlings were used to assess abiotic stress. For either heat or cold stress, the seedlings were put in a 42 °C or 4 °C growth chamber for 0, 2, 4, or 8 h. There were three replicates for each treatment and three pots of seedlings for each replicate. Two fungal pathogens, h method ,20. Inocescribed ,20. LeavZea mays L. , Sorghum bicolor L. , Oryza sativa L. , and Aegilops tauschii  genome annotate documents using BLASTP . The non-redundant protein sequences obtained from these two methods were further analyzed. The physical parameters and subcellular localization of SSL proteins were envaulted using the online ExPASy  and WoLF PSORT .Arabidopsis thaliana SSL protein sequences (download in TAIR10)  and two Ae. tauschii, and Arabidopsis were aligned using Clustal X (Version 1.83) [ZmSSL genes were evaluated with MEME (Version 5.5.0) [ZmSSL genes were obtained and visualized with TBtools v1.120.The identified SSL protein sequences from maize, sorghum, rice, on 1.83) . The alin 5.5.0)  and visun 5.5.0) . The exoSSL gene to its corresponding chromosomal location using MapGene2Chrom  and TBtools v1.120. [ZmSSL genes or SSL genes from different species; the results were visualized using TBtools v1.120 [Genomic data downloaded from NCBI  [The 2000 bp upstream of the transcription start site (TSS) sequence of each st 2023)  and visust 2023) .ZmSSL genes under normal conditions were obtained in the published research [The RNA-seq data used for the tissue-specific expression analysis of research . The pubresearch , droughtresearch , and funresearch ,20 were research . DiffereZmActin1 (GRMZM2G126010) was used as the internal control. All primers used in the RT-qPCR are listed in Total leaf RNA extraction was performed following a published protocol . Total RZmSSL4 and ZmSSL5, the CDS of ZmSSL4 and ZmSSL5 were amplified without the stop codon using primers ZmSSL4-F/R and ZmSSL5-F/R (ZmSSL4/5-GFP plasmid DNA (20 μg) was transformed into maize mesophyll protoplasts following a published protocol [https://andor.oxinst.com/).For the subcellular localization of SSL5-F/R . Amplicoprotocol ,37. The p < 0.05; p < 0.01) was assessed by using the Student’s t test.Statistical analysis was performed using SPSS 19.0. Significance . Furthermore, six members of the ZmSSL genes, ZmSSL4–9, displayed wide expression in different tissues under normal conditions and were positively or negatively modulated by abiotic (heat and cold) and biotic (C. graminicola and C.zeina infection) stresses. This work may provide useful information to further evaluate the biological functions of ZmSSL genes.In this study, 20


Additionally, it reduces the homeostatic model assessment levels of insulin resistance , fasting glucose , and fasting insulin . Based on our results, we hypothesize that TRT can improve glycemic control and hormone levels, as well as lower total cholesterol, triglyceride, and LDL cholesterol levels while raising HDL cholesterol in hypogonadal type 2 diabetes patients. To this end, we recommend TRT for these patients in addition to standard diabetes care.Testosterone replacement therapy (TRT) has been used to treat hypogonadal males with type 2 diabetes mellitus (T2DM) for a long time, despite variable results. This meta-analysis examines TRT’s role in hypogonadal males with T2DM. The databases PubMed, Embase, and Google Scholar were searched for relevant RCTs and observational studies. Estimated pooled mean differences (MDs) and relative risks with 95% confidence intervals were used to measure the effects of TRT (CIs). When compared to the placebo, TRT improves glycemic management by significantly reducing glycated hemoglobin (HBA1c) levels (WMD = −0.29   Hypogonadism is a clinical syndrome in which the testis fails to produce physiological testosterone levels or a normal percentage of spermatozoa due to pathology in one or more hormonal concentrations of the hypothalamic–pituitary–testicular axis. Male hypogonadism is characterized by low serum total and free testosterone concentrations, as well as the presence of hypogonadism symptoms . HypogonThe association between TRT and T2DM in male hypogonadism has only been investigated in a few randomized controlled trials and observational studies, with contradictory results. Consequently, we undertook a systematic review and meta-analysis to determine the efficacy profile of TRT in hypogonadal males with type 2 diabetes. This meta-analysis offers the most up-to-date look at how testosterone therapy compares to no treatment or placebo, as far as we are aware.In accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis, the Preferred Reporting Items were used to execute this meta-analysis  .PubMed  and Cochrane  were combed extensively from the study’s inception until 25 February 2023. Gray literature and preprints were discovered by searches on ClinicalTrials.gov , Google Scholar , and Medrxiv . Medical Subject Headings (MESH terms) and keywords were compiled to form a search strategy. These terms and keywords included [‘Testosterone’ OR ‘TRT’ OR Testosterone undecanoate] AND [‘Hypogonadism’ OR ‘Hormonal deficiency’] AND [Diabetes Mellitus’]. Information about the search methodology is provided in The following criteria were used to select the studies: language, study design, patient population, intervention, comparison, outcomes of interest, and definition.English publications.Study design: eligible, completed, randomized clinical trials or observational studies were extracted to perform the meta-analysis.Patient population: all patients with confirmed type 2 diabetes who met the criteria of hypogonadism.Exposure: patients who received testosterone therapy.Comparison: this includes the non-TRT group, which received the usual standard of care or placebo.Primary outcomes: effects on glucose metabolism and post-treatment hormonal levels.Secondary outcomes: cholesterol levels, BMI, waist circumference, body fat, and systolic and diastolic blood pressure.No clear definitions of the diagnosis of late-onset hypogonadism and T2DM, population, dosage and method of testosterone administration, or outcome evaluation.Insufficient data for estimating a mean difference (MD) with a 95% confidence interval (CI).Duplicates of previous publications.To ensure the quality of this meta-analysis, the following significant exclusion criteria were established:Furthermore, all included RCTs were evaluated using the 25-item CONSORT checklists, which emphasized describing how trials were conceived, analyzed, and interpreted . The numTwo researchers (MK and SK) independently read and analyzed the articles in order to determine their inclusion in the review. Uncertain facts were discussed and clarified. From each trial, we extracted the first author’s name, publication year, nation, ethnicity, testosterone cut-off point, diabetes duration, testosterone regimen, medications on comparators, mean age, HBA1c percentage, and total serum testosterone level. These details are condensed in The quality of published RCTs was assessed using the modified Cochrane Collaboration risk of bias tool , whereasp-value of less than 0.05 was considered statistically significant. Funnel plots were formed for primary outcomes to evaluate potential publication bias.Review Manager 5.4 (Cochrane Collaboration) was utilized to perform the aforementioned meta-analysis. Relative risks (RRs) and 95% confidence intervals (CIs) were calculated for binary outcomes. Continuous outcomes were represented by mean and standard deviation values. This meta-analysis presents the combined effect of relative risks (RRs) and weighted mean differences (WMDs) determined with the generic-inverse variance and continuous outcome functions with a random-effects model. A The amount of discordance between research was estimated using I2 statistics. The I2 value of 25% showed low heterogeneity, the range of 25% to 50% indicated moderate heterogeneity, and the range of 50% and above indicated strong heterogeneity. The effect of individual studies on the overall pooled estimate was examined by conducting a sensitivity analysis on outcomes with a high degree of heterogeneity.A total of 659 articles were found in the preliminary literature search. A total of 2 observational ,13 and 1There was a total of 3002 participants who fit the criteria for hypogonadism across the 15 studies; 1484 received testosterone and 1518 received a placebo. Hypogonadism was defined as the presence of at least 3 sexual symptoms and a total testosterone level of 12 nmol/L in 6 studies ,15,19,21The Newcastle–Ottawa scale, a tool used to evaluate the quality of studies, found a minimal likelihood of bias in the observational studies . Trials The effects on glucometabolism .p = 0.08; I2 = 56.3%). Similarly, 14 [p = 0.19; I2 = 84.4%). A total of 8 [p = 0.31; I2 = 49.3%). However, the above-mentioned findings are statistically non-significant. A total of 13 [p = 0.04; I2 = 89.8%). For illustration, the pre-treatment values for the TRT group were 7.21 ± 1.09, while the post-treatment values were 6.60 ± 1.09. In contrast, the pre-treatment values for the placebo group were 7.52 ± 1.08, whereas the post-treatment values were 7.59 ± 1.19.Testosterone’s impact on glucometabolism was evaluated using the HOMA-IR, HBA1c, fasting serum glucose (FSG), and fasting serum insulin (FSI) measures. A total of 9 ,18,22,23arly, 14 ,22,23,24tal of 8 ,18,19,23al of 13 ,22,23,24The effects on hormone levels .p = 0.0001; I2 = 96.3%). Excluding the studies individually from the pooled analysis had no effect on the in-study heterogeneity.In order to assess the effect of testosterone on hormone levels, the following variables were considered: total testosterone, free testosterone, SHBG, and PSA. A total of 9 studies ,22,23,24p = 0.07; I2 = 70%). The SHBG level was included in 5 studies [p = 0.55; I2 = 0%). A total of 7 studies [p = 0.65; I2 = 0%).A total of 3 articles ,15,22 in studies ,22,23,24 studies ,18,22,24Secondary outcomes: .In addition to improving HDL cholesterol and IIEF, testosterone treatment has been shown to reduce total cholesterol, LDL cholesterol, triglycerides, body fat, waist circumference, BMI, systolic blood pressure, diastolic blood pressure, and arterial mean stiffness in a pooled analysis of secondary outcomes.p = 0.03, but had no effect on body weight or waist circumference [New studies have found that a high percentage of men with type 2 diabetes also suffer from hypogonadism. Despite the increasing knowledge of the association between T2D and hypogonadism, no universally accepted standards exist for patients who suffer from both. This systematic review aimed to develop clear, evidence-based recommendations for treating hypogonadism in males with type 2 diabetes mellitus by administering testosterone replacement therapy. There is solid evidence presenting the link between type 2 diabetes and low blood testosterone levels due to an amplified insulin signaling pathway, as evidenced by multiple studies showing a high incidence 30–80%) of hypogonadism in males with diabetes mellitus  . Althou0% of hypmference . MultiplOur meta-analysis evaluated total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglyceride levels as a part of a lipid panel. In thirteen studies, testosterone recipients had lower total cholesterol levels than placebo recipients. Nonetheless, a decrease in triglyceride levels and increased HDL cholesterol were found in 14 trials. However, the two groups had a more negligible difference in LDL cholesterol levels. Similarly, the Si Hyun Kim et al. meta-analysis from 2021 showed that TRT significantly reduced total cholesterol compared to a placebo. Triglyceride levels also improved, albeit the change was not statistically significant. Intriguingly, compared to the placebo, HDL levels significantly decreased after TRT. More contradictory data about TRT’s role in HDL are necessary. High-dose TRT has been shown to lower HDL and lipoprotein A levels.TRT’s potential impact on blood lipid and lipoprotein levels is still debated . The effTotal testosterone, free testosterone, SHBG, and PSA enzyme  concentrations were studied. A marked reduction in SHBG accompanied a significant elevation in both total and free testosterone. Nonetheless, no correlation was seen between PSA levels and this treatment. Several meta-analyses have been conducted to date to explore the effect of TRT on PSA . HoweverOur meta-analysis provides several advantages: (1) our meta-analysis is strengthened by the inclusion of two additional studies, approximately doubling the total sample size. (2) We also compared our meta-analysis with previous meta-analyses by Jianzhong Zhang et al. . It inclAlthough our study generated significant statistical data, its limitations must be acknowledged. (1) The majority of studies had varying follow-up durations, with some indicating longer times. Longitudinal follow-up studies are preferable when evaluating hormonal diseases, such as hypogonadism, because of the importance of maintaining homeostasis in the body. Numerous studies over a wide range of weeks used testosterone in various doses and administration routes. (2) Variations in study designs, interventions, and patient factors  may have contributed to the observed clinical heterogeneity. (3) Only a small number of randomized controlled trials examined the relationship between body fat, AMS and IIEF scores, free testosterone, and death rates; consequently, further research should be conducted in the future, focusing on these outcomes in order to establish a more accurate association. (4) All included RCTs displayed signs of selective reporting bias, except for Groti 2020. Further research was needed to ascertain how testosterone therapy affected libido. (5) Additionally, most studies did not include information on the doses for the control groups, which may have added uncertainty.Our results demonstrate that hypogonadal T2DM patients who underwent long-term testosterone replacement therapy experienced a sustained remission of their diabetes. This therapy improved glycemic control, decreased total cholesterol, HDL levels, and triglycerides, and reduced body mass index and waist circumference. We propose that this treatment be taken in conjunction with anti-diabetes medications for these patients. The intervention’s long-term durability, safety, and cardiovascular effects need to be studied further.


Murine cytomegalovirus (MCMV), and, in particular, recombinant virus derived from MCMV-bacmid pSM3fr, is widely used as the small animal infection model for human cytomegalovirus (HCMV). We sequenced the complete genomes of MCMV strains and recombinants for quality control. However, we noticed deviances from the deposited reference sequences of MCMV-bacmid pSM3fr. This prompted us to re-analyze pSM3fr and reannotate the reference sequence, as well as that for the commonly used MCMV-m157luc reporter virus. A correct reference sequence for this frequently used pSM3fr, containing a repaired version of m129 (MCK-2) and the luciferase gene instead of ORF m157, was constructed. The new reference also contains the original bacmid sequence, and it has a hybrid origin from MCMV strains Smith and K181. These play an important role in viral antigen presentation and, therefore, immune evasion  is a widely distributed pathogen, with seroprevalence of the lifelong infection varying from 40% to more than 90%. HCMV is the most frequent congenital pathogen after infection epatitis . Researcepatitis ,3,4, or epatitis . Murine epatitis ,7, showiepatitis . Experimepatitis . Typicaly glands ,11. In gy glands ,13. m150 to m158 genes belonging to the K181 strain within the genome of strain Smith. Thereafter, several groups observed an impaired replication of the pSM3fr-derived viruses in salivary glands; this was finally shown to be attributable to a frameshift mutation in the MCK-2 (MCMV chemokine homologue) gene, encoded in the ORF m129, leading to impaired replication in salivary glands. Repair of the m129 frameshift in pSM3fr-MCK-2fl (full length) restored salivary gland replication [m157 gene with subsequent replacement by a luciferase gene does not impair the fitness of the resulting viruses during infection but sensitizes C57BL6 mice due to reduced NK cell defense. In other mouse strains, which are Ly49H-negative, M157 might act as ligand for different receptors. The original pSM3fr bacmid and derivatives like MCMV-m157luc were used for multiple studies  containing a near-complete sequence of a herpesvirus, MCMV strain Smith by Messerle and colleagues , represelication . Furtherlication . Intereslication ,22. Thers e.g., ,24); MCM; MCMm150ng MCK-2 . In the In the scope of generating modified MCMV bacmids derived from the widely used pSM3fr bacmid, we performed full-genome sequencing of the constructed DNA via next-generation sequencing (NGS) to verify the correct assembly and search for potential mutations. Since we detected a high number of unexpected mismatches in specific regions, we also sequenced the original pSM3fr bacmid ,17.m145 family  and the deposited pSM3fr-MCK-2fl reference sequence (GenBank Acc. No.: KY348373) with average coverages of 160 and 162, respectively. Interestingly, for both mappings, the variant detection showed a heavily mutated region in the open reading frames (ORFs) m150 and m151, which encode type 1 membrane proteins of the 5 family A. In tot5 family B,C. Thes5 family A. Lodha 5 family B. Furtheribed in . m129) [Another derivative of the widely used bacmid pSM3fr derived from MCMV contains a repaired version of the MCK-2 gene (m129)  and a HCm129) ,24. For Furthermore, we sequenced two different stocks of low-passage viruses reconstituted from the MCMV-m157luc bacmid with an average coverage of 330. The resulting reads were then mapped to our newly generated reference sequence for MCMV-m157luc  and reacFurthermore, since HCMV has a similar genome size to MCMV, we would expect a tendency of gene loss in bacmid systems as well and would recommend full-genome sequencing of bacmid-derived HMCV.In conclusion, we provide the CMV research community with new and corrected references for the extensively used pSM3fr bacmid (accession: ERZ20801763) and the MCK-2-repaired version, MCMV-m157luc reporter virus, containing a luciferase instead of ORF m157 (accession: ERZ20801746).Escherichia coli DH10B-derived EL250 [TM HiPure Plasmid Maxiprep Kit of Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA (K210006) according to the manufacturer’s instructions.ed EL250  or GS178ed EL250  carrying® Ultra™ II FS DNA Library Prep Kit from Illumina, San Diego, CA, USA (E7805), according to manufacturer’s instructions, with a fragmentation time of 15 min.Purified bacmid DNA was obtained via standard precipitation protocols. Viral DNA was extracted utilizing a Qiagen  EZ-1 instrument. For the library preparation for next-generation sequencing, 500 ng of bacmid DNA or extracted viral DNA was fragmented and adapters and indices were ligated using the NEBNextSequencing was performed via paired-end sequencing utilizing the MiSeq Reagent Kit v3 (150 cycles) on a MiSeq™ Instrument . Sequence analysis was conducted using CLC Genomics Workbenches 22 and 23 . Raw reads were trimmed for quality , adapters, and ambiguities. The trimmed reads were mapped with different stringencies, testing for the best distribution. Duplicate reads were removed from the mapping. Variants from the reference sequence were detected, only considering regions with coverages above 50 and a frequency of the variant in the reads of more than 80%. 5 virions were added to 25 mL of complete DMEM. After extensive vortexing, the MEF supernatant of each flask was exchanged for 25 mL of complete DMEM containing MCMV virions and the cells were incubated for 1 week under cell culture conditions. Cell culture supernatants were centrifuged  to remove cell debris. The supernatants were ultracentrifuged . The supernatant was discarded; then, the pellets were resuspended in a few microliters of residual media, pooled, and dispersed using a syringe. Following that, 1 mL of concentrated virions was carefully loaded on a 9 mL cushion of 15% sucrose in virus suspension buffer , 12 mM KCl, 5 mM EDTA) and purified via ultracentrifugation . The sucrose cushion was discarded; then, the pellets were washed in 10 mL of PBS and repelleted . PBS was discarded and 500 µL of VSP was added and incubated on ice overnight. The next day, pellets were resuspended, pooled, dispersed using a syringe, and stored at −80 °C in 40 µL aliquots.In order to generate high-titer stocks of MCMV, murine embryonic fibroblast (MEF) cells were seeded 1:2 into twelve T175 cell culture flasks. On the next day, purified MCMV virions were thawed and 1 × 10https://www.ebi.ac.uk/ena/browser/home, accessed on 31 July 2023). The accession number for the original pSM3fr bacmid is ERZ20801763, while the accession number for the MCK-2 repaired version with a luciferase instead of ORF m157 is ERZ20801746.Reference sequences were deposited in the European Nucleotide Archive (


To this end, wecreated a focused virtual library using reaction-based enumerationtools in the Schrödinger suite. These compounds were dockedinto the Mpro active site and subsequently prioritizedfor synthesis based upon relative binding affinity values calculatedby FEP+. Fourteen compounds were selected, synthesized, and evaluatedboth biochemically and in cell culture. Several of the synthesizedcompounds proved to be potent, competitive Mpro inhibitorswith improved metabolic stability profiles.The SARS-CoV-2 main protease (M Moreover, the effectiveness of currently availablevaccines is waning as newer SARS-CoV-2 variants emerge.2 Thus, there is an urgent, ongoing need for effective,orally bioavailable treatment options. Currently, the FDA has approvedthree direct-acting antiviral treatments, namely Veklury (remdesivir),Lagevrio (molnupiravir), and Paxlovid (nirmatrelvir and ritonavir).Unfortunately, all three treatments have shortcomings. The first ofthe viral polymerase substrates, remdesivir, is not orally bioavailableand must be administered intravenously in a hospital setting. Consequently,the drug is mostly used for patients already exhibiting severe COVID-19symptoms and, in this setting, is of limited efficacy.3 Molnupiravir, which is also a substrate for the viral polymerase,should not be administered to pregnant woman, as it may cause fetalharm.4 Finally, nirmatrelvir, the firstapproved inhibitor targeting the viral main protease (Mpro), must be co-administered with ritonavir (a CYP3A4 inhibitor) toimprove its pharmacokinetic properties.5 The incorporation of a CYP3A4 inhibitor is sub-optimal for patientson other chronic medications that are metabolized by CYP3A4. Mentionmust also be made of Xocova ,another SARS-CoV-2 Mpro inhibitor, which has been demonstratedto be highly efficacious,6 further highlightingthe importance of the main protease as a viral target. Unfortunately,drugs targeting viral proteases are often challenged with the developmentof resistance, and it would seem that this is, indeed, the case forthe SARS-CoV-2 Mpro, as strains exhibiting some resistanceto nirmatrelvir and ensitrelvir have recently been reported.7 Thus, there remains an ongoing need to developnext-generation SARS-CoV-2 Mpro inhibitors capable of circumventingresistance-causing mutations.Severe acute respiratory syndromecoronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic,pro inhibitors,we sought to follow the path less traveled, by designing inhibitorsthat were neither peptidic nor covalent modifiers. Peptide-like compoundsare often highly polar and are also susceptible to metabolic degradation,both attributes often leading to poor oral bioavailability. At thestart of our project, the vast majority of inhibitors being publishedemanated from earlier work carried out from SARS-CoV research in theearly 2000s and thus were almost entirely composed of peptidic compounds. However, we were inspired by a seriesof four publications originating from the Jorgensen and Anderson groups,11 as these compounds were far more drug-like, and we also saw potentialfor their further improvement. In this work, these groups, with theirexceptional strengths in molecular modeling techniques and pharmacologicevaluations, initially carried out a virtual screening of around 2000approved drugs in search of a viable starting point.8 Of the results, 17 compounds were chosen for evaluationin a kinetic Mpro inhibition assay, and, remarkably, 14compounds exhibited inhibition, some with IC50 values aslow as 5 μM. Within this set, perampanel 1  of just 7.2 min.In our quest to discover novelSARS-CoV-2 Mmpanel 1 1 was sel12 to calculaterelative binding free energy values (using the Jorgensen–Andersonstructure from PDB 7N44)10 revealed that, of the two possiblepyridones we could synthesize for the S1′ pocket, the 2-pyridone,we adopted a slightly different design strategy. We envisioned thatit could be replaced by a pyridone, albeit with the loss of one ofthe three electrostatic interactions that occur with Thr26 in theS1′ pocket and the catalytic thiol of Cys145 2A. A docpyridone2B shouldpyridone2C.10 In order to optimize our synthetic approach to accommodatea late-stage diversification strategy, we planned on arriving at phenol 5, which would allow us to incorporate a wide range of S4pocket moieties while minimizing synthetic efforts. With this in mind,we embarked upon a focused virtual screening campaign to identifysuitable small fragments which could be purchased (to speed up ourprogram) and were amenable to phenol attachment by SN2substitution of a suitable halide or a Mitsunobu reaction, which broadenedour options to now also include suitable alcohol-containing buildingblocks. In this exercise, over 1500 small, commercially availablebuilding blocks were identified, and a library of final compoundswas created in silico using the Schrödingerreaction-based enumeration tool. These compounds were then docked(Glide-SP) and ranked by docking score as well as by visual inspection,leading to a list of approximately 100 compounds which were then rankedmore rigorously by determining relative binding free energy valuesusing free energy perturbation methods .13 Finally, relative binding freeenergy values were determined once again for a final shortlist of20 compounds using Schrödinger’s FEP+, leading to theidentification of 14 compounds for synthesis, which included 2 compoundswith the predicated unfavorable 4-pyridone system to validate ourmodeling hypothesis . Fortunately, a survey of the literature revealedjust one single article which rescued our planned synthetic route.Facing a similar chemoselectivity problem, Barnes et al. developedmethodology based on the copper-catalyzed Ulmann–Goldberg reaction,employing N-(2-cyanophenyl)picolinamideas the ligand in a copper-mediated cross-coupling reaction.15 Under these conditions, they achieved a highselectivity for coupling at N1. With this promising new strategy inmind, we switched the order of our planned coupling reactions andnow opted to install the dichlorophenyl moiety first, at N1. Indeed,under the conditions developed by Barnes et al., we achieved couplingexclusively at N1  when reacting 12 with 11, albeit in modest yield, thereby arrivingat 13. With only N3 now available for the reaction, wewere able to successfully install the pyridyl moiety using 14, forming 15. Finally, the pyridone for the S1′pocket was revealed after demethylation using TMSCl and NaI, affordingthe target compound 6.Synthesis of ntyield 1. Atthi7 was similarlysynthesized by starting with 5-bromo-2-methoxypyridine 16  was carriedout regioselectively according to the procedure developed byBarnes et al.15 as described earlier, providingexclusively 22, though often in modest yields. At thispoint, another copper-mediated coupling of 3-pyridyl boronic acidunder Chan–Lam conditions provided 23 in goodyields. Debenzylation of 23 under standard hydrogenationconditions afforded the key phenol 5, ready for attachmentof our assortment of S4 pocket moieties, as prioritized by the FEP+modeling described earlier. Thus, the corresponding halides or alcoholswere reacted with phenol 5 in the presence of potassiumcarbonate or under Mitsunobu conditions, respectively, leading todesired penultimate compounds 24–34 in moderate to excellent yields. It should be mentioned that allof these derivatives were commercially available, with the exceptionof the R3, R10, and R11 derivatives(Supporting Information). Finally, the crucial S1′ pocket pyridones were revealedby treating each compound with TMSCl and NaI, affording target compounds 35–45 in generally moderate yields.Having confirmed thepreferred pyridone for the S1′ pocket,we next set about synthesizing the series containing groups extendinginto the S4 pocket. Thus, starting from 11 3, instalivatives3, which 39, namely 51  was synthesized relativelyuneventfully, starting from 18.In parallel withthis effort, we also synthesized the alternate4-pyridone version of amely 51 4, in ord35–45 and 51 in hand, we were ableto assess their efficacy in a SARS-CoV-2recombinant protease enzymatic assay (50). From the respective IC50 values, the Ki values couldbe determined, allowing us to compare the calculated Gibbs free energyof binding (FEP+) with the experimentally derived values. Pleasingly,an excellent correlation was found between the calculated and experimentalresults , revealing compounds 43, 45, and 6 (somewhat surprisingly since this compound doesnot contain an S4 pocket moiety) as potent compounds exhibiting sub-micromolarEC50 values, comparable to that of the promising new ShionogiSARS-CoV-2 Mpro inhibitor, S-217622 (ensitrelvir).16The compounds were then evaluated in a cellularantiviral assay were significantly less metabolicallystable than our compound 36, which contains an aliphaticS4 pocket moiety. This observation prompted us to re-evaluate ourhypothesis regarding the observed liver microsomal instability ofJorgensen–Anderson compound 4. Indeed, metaboliteidentification studies (not available at the start of our project)revealed that loss of the uracil N1 methyl was, in fact, not the majormetabolite, but rather it was loss of the ortho-chlorobenzyl moiety. This same phenomenonwas observed when we similarly identified the microsomal metabolitesfor compound 43. Furthermore, the inferior stabilityof the Jorgensen–Anderson compound 4 comparedto 43 (even though they both contain the same S4 pocketmoiety) is attributed to the fact that, although the loss of the benzylicgroup is the major metabolite for 4, some loss of themethyl at N1 is also observed. Unfortunately, in our series, compoundswith an aromatic S4 pocket moiety were generally found to be morepotent than their aliphatic counterparts . Theresults of this study indicated a peak plasma concentration at 1.67h, suggesting rapid absorption. Unfortunately, a modest oral bioavailability(41%), despite low plasma clearance (2.49 mL/min/kg) and rapid uptake,was indicative of poor compound solubility. This problem was furtherhighlighted in a dose escalation study (10 mg/kg and 100 mg/kg PO),where it was observed that the 10-fold increase in dose resulted ina only 2-fold increase in AUC .Having established that pro inhibitorsdeveloped by the Jorgensen and Anderson groups, we set out to improveupon their design by changing the uracil moiety occupying the S1′pocket to a pyridone. Analysis of the new design by FEP+ suggestedthat our compounds would be as effective, and indeed, this turnedout to be the case. Furthermore, FEP+ proved to be an extremely valuabletool in prioritizing compounds for synthesis, and an excellent correlationwas obtained between the predicted binding free energy values andthose later calculated from measured IC50 results. Althoughthese new compounds did prove to be metabolically more stable thanthe highly potent Jorgensen–Anderson compound 4, they still present some challenges. In particular, aqueous solubilityremains a problem, despite the polar nature of these compounds. Nevertheless,the design and development of novel, potent SARS-CoV-2 Mpro inhibitorsremain an important priority. The shortcomings of the only FDA-approvedMpro inhibitor (nirmatrelvir) have been mentioned above,and the promising new Mpro inhibitor being developed byShionogi (ensitrelvir) is a strong CYP3A inhibitor that may presentserious adverse drug–drug interactions for patients on otherchronic medication.17 Furthermore, anothernewly emerging opportunity in this area lies in the development ofSARS-CoV-2 Mpro inhibitors capable of overcoming issuespertaining to resistant viral variants, which are just coming to thefore for both ensitrelvir and nirmatrelvir.18In summary, inspired by the potent SARS-CoV-2 M


PITX2 and FOXC1 variants explain the majority of individuals with Axenfeld–Rieger syndrome (ARS) but leave ~30% unsolved. Here, we present pathogenic/likely pathogenic variants in nine families with ARA/ARS or similar phenotypes affecting five different genes/regions. USP9X and JAG1 explained three families each. USP9X was recently linked with syndromic cognitive impairment that includes hearing loss, dental defects, ventriculomegaly, Dandy–Walker malformation, skeletal anomalies (hip dysplasia), and other features showing a significant overlap with FOXC1-ARS. Anterior segment anomalies are not currently associated with USP9X, yet our cases demonstrate ARA, congenital glaucoma, corneal neovascularization, and cataracts. The identification of JAG1 variants, linked with Alagille syndrome, in three separate families with a clinical diagnosis of ARA/ARS highlights the overlapping features and high variability of these two phenotypes. Finally, intragenic variants in CDK13, BCOR, and an X chromosome deletion encompassing HCCS and AMELX  were identified in three additional cases with ARS. Accurate diagnosis has important implications for clinical management. We suggest that broad testing such as exome sequencing be applied as a second-tier test for individuals with ARS with normal results for PITX2/FOXC1 sequencing and copy number analysis, with attention to the described genes/regions.Axenfeld–Rieger anomaly (ARA) is a specific ocular disorder that is frequently associated with other systemic abnormalities.  PITX2-assocated ARS (type 1), the ocular anomalies are almost universally associated with dental  and umbilical  features [FOXC1-associated ARS (type 3) causes much more variable nonocular features, with some individuals having isolated ocular features and others affected with a variety of additional anomalies including hearing loss, congenital heart defects, enamel hypoplasia/dental crowding, hip dysplasia and other skeletal anomalies, hypotonia/early delay, and white matter hyperintensities [FOXC1 disruption is De Hauwere syndrome, characterized by anterior segment anomalies, hypertelorism, short stature, hearing loss, hydrocephalus, joint hyperlaxity, and skeletal anomalies including hip dysplasia [FOXC1 [PITX2 or FOXC1, indicating that additional genetic causes remain to be elucidated [Axenfeld–Rieger anomaly/syndrome (ARA/ARS) is a specific type of anterior segment disorder characterized by the triad of posterior embryotoxon (prominent annular white line near the limbus at the level of the Descemet membrane), iridocorneal adhesions, and iris anomalies including iris hypoplasia, corectopia, and/or polycoria ,2. The ofeatures . FOXC1-aARS type  causes mARS type  causes mysplasia ,5. WhileARS type  causes mARS type  causes mCOL4A1 in multiple families [CPAMD8, ADAMTS17, and CYP1B1 reported in one family each [SETD1A and KMT2F in one family each [PAX6, typically associated with aniridia, can present with overlapping anterior segment ocular phenotypes but is not a cause of ARA specifically [Other genetic variants have been occasionally reported in individuals with a clinical diagnosis of ARS or isolated ARA, including dominant variants in families ,7, recesily each , and domily each . Large dily each ,12,13. Pifically .PITX2 or FOXC1 [® caller. Pathogenicity was determined using ACMG/AMP criteria [This human study was approved by the Institutional Review Boards of the Medical College of Wisconsin and University of Iowa. We reviewed our cohort of unexplained individuals with ARA/ARS or similar phenotypes without sequence or copy number variants in or FOXC1 . Exome sor FOXC1 . We revicriteria ,16. VariUPS9X and JAG1, explained multiple cases within our cohort. The most novel of the two was USP9X, with three unrelated individuals carrying causative variants in this gene, which has not been recognized as a cause of anterior segment phenotypes.Pathogenic/likely pathogenic variants were identified in nine families affecting five genes . All varUSP9X, NM_001039590.2:c.1314+2T>C. Parental samples were not available. In silico analysis revealed a strong prediction of loss of the donor splice site (0.73 by SpliceAI). There is a weak prediction (0.04) of donor gain which would result in 91bp deletion from exon 10. Exon skipping would result in 153bp deletion of exon 10, c.1162_1315del p.(Glu388_Gln438del). Exon 10 is highly conserved with a very low rate of benign missense variants in gnomAD, so this change is likely to be deleterious.Individual 1 is a 14-year-old White (U.S.) female diagnosed with ARS by her ophthalmologist. Ocular anomalies include ARA, early signs of glaucoma, and myopia. Nonocular anomalies include choanal atresia with deviated nasal septum, high arched palate with bilateral membranous palate anomaly, chronic sinusitis and otitis media , dysmorphic facial features , abnormal skin pigmentation along lines of Blaschko, and precocious puberty (menses at 9 years old). She has a history of early delays, especially gross motor, but transitioned out of intervention by first grade and is now on the honor roll with adjusted workload, and a history of depression. Height, weight, and head circumference are all normal (50th centile). Exome sequencing identified a likely pathogenic variant in USP9X, c.121G>T; p.(Glu41*), which was not present in the mother (father not available).Individual 2 A is a 4-de novo likely pathogenic variant in USP9X, c.1603dupA p.(Ile535Asnfs*11); she was reported in a previous cohort at 2 years of age [Individual 3 is a 9-year-old White (U.S.) female with features overlapping ARS including enamel hypoplasia and dental crowding, bilateral hip developmental dysplasia, congenital heart defects , and history of feeding difficulties in infancy. Ocular anomalies include phlyctenular keratoconjunctivitis and superficial punctate keratitis, subtle scalloped scarring of the cornea with neovascularization, juvenile cataract , mild myopia , amblyopia, and blepharokeratitis. Additional nonocular anomalies include high arched palate, low-set posteriorly rotated ears with overfolded helices, frontal bossing, anteverted nares, scoliosis with tethered cord, vesicoureteral reflux, left vocal cord paralysis, hypopigmentation of skin with cutis marmorata, eczema, and gross/fine motor delays but normal cognition. Height and weight are within the normal range . Exome sequencing identified a s of age .JAG1, NM_000214.3:c.59dupT p.(Leu21Alafs*52). Parental samples were not available.Individual 4 is a 20-year-old White  female referred with diagnosis of ARS based on ARA, bilateral sensorineural hearing loss, midface hypoplasia, hypertelorism, and dental crowding. Specific ocular anomalies consisted of posterior embryotoxon, iridocorneal adhesions, and iris hypoplasia along with hyperopia. Additional nonocular features included prominent forehead, short philtrum, arachnodactyly, marfanoid habitus, postural orthostatic tachycardia syndrome (POTS), and auditory/visual processing deficit with average cognition. Echocardiogram was normal. Exome sequencing identified a likely pathogenic variant in BTBD3, ISM1, JAG1, MKKS, SLX4IP, SNAP25, and SPTLC3), of which JAG1 is pathogenic. Parental samples were not available.Individual 5 B is a 39JAG1, c.2419G>T p.(Glu807*) in the proband and her son.Individual 6 C,D is a CDK13, BCOR) were identified in a single family each, one of which (CDK13) had not been previously linked with anterior segment anomalies. Individual 7 is an 11-year-old White (U.S.) female with a clinical diagnosis of ARS including ARA and widely spaced eyes. Specific ocular features include bilateral posterior embryotoxon and mild iris hypoplasia with transillumination defects, megalocornea with normal intraocular pressure, and mild myopia (−1.00 OU). Additional nonocular anomalies include broad nasal tip, small mouth, history of recurrent otitis media, nasal speech, developmental delay, moderate hypotonia, and cognitive impairment. Height and weight were normal (75th–90th centile). Her 14-year-old brother, affected with cognitive impairment, mild hypotonia, hypospadias, cryptorchidism, history of feeding difficulties, nasal speech, bipolar disorder, behavior problems, and mild dysmorphic features  was thought to have a different genetic syndrome from the proband. Both parents have a history of developmental delay with special education services in school but were unavailable for testing. The mother also has schizophrenia and an extensive family history of learning disabilities and psychiatric illness. Exome sequencing identified a likely pathogenic variant in the proband and her brother within the protein kinase domain of CDK13, NM_003718.5:c.2252G>A p.(Arg751Gln) with high CADD (31) and REVEL (0.697) scores. The same variant was reported previously in an unrelated individual within the Deciphering Developmental Disorders cohort [Variants in two other genes (BCOR NM_001123385.2: c.3350_3360dupCAGACCAGGTG p.(Ala1121GlnfsTer42). Parental samples were not available.Individual 8 is a 20-year-old White (U.S.) female referred with a clinical diagnosis of ARS based on ocular anomalies, peg-shaped and small teeth with dental crowding, maxillary hypoplasia, hearing loss, and redundant periumbilical skin. Specific ocular anomalies include bilateral congenital cataract, glaucoma diagnosed at 3 years of age, optic nerve hypoplasia, mild microphthalmia , and amblyopia of the left eye. Additional nonocular anomalies include patent foramen ovale, nasal deviation, asymmetric and posteriorly rotated ears, narrow palate with thickened ridges, and fifth finger clinodactyly. Height and weight are normal (10–25th centile), as is cognition. Exome sequencing identified a likely pathogenic variant in AMELX, ANOS1, ARHGAP6, CLCN4, CLDN34, FAM9A, FAM9B, GPR143, HCCS, MID1, PNPLA4, SHROOM2, TBL1X, VCX, VCX2, VCX3B, WWC3), of which HCCS and AMELX are pathogenic. Parental samples were not available.The final case was explained by a combination of two disrupted genes. Individual 9 is a 57-year-old White (U.S.) female who was diagnosed with ARS in early childhood based on ocular features and dental anomalies including small, mispositioned teeth, enamel hypoplasia, and extra teeth. Specific ocular features include bilateral congenital glaucoma with surgery in infancy, polycoria, microphthalmia , corneal opacity with failed corneal transplant in adulthood, and small congenital cataract which did not require removal until 30 years of age. Additional nonocular anomalies include hearing loss (40s), rheumatoid arthritis, irritable bowel syndrome, connective tissue disease, and migraines. Height/weight and cognitive development are normal. Exome sequencing identified a pathogenic 4.08 Mb deletion of X:7370404-11445756 (hg19) including 17 genes  [Dominant loss-of-function and missense variants in pairment ,20. Bothpairment . While aher girl ,20. Featsruption . In supphe brain . USP9X h pathway ,24, whichis gene . Addition (Cdh2) .JAG1 or NOTCH2, is a well-recognized phenotype characterized by highly penetrant posterior embryotoxon and liver dysfunction along with cardiac and vertebral anomalies [JAG1 variants [Alagille syndrome, caused by variants in nomalies . While inomalies ,29. The variants .UPS9X, disruption of CDK13 is associated with syndromic cognitive impairment [Usp9x, expression of Cdk13 in the mouse eye is seen at a similar level to the brain [Similar to pairment ,31. The he brain .BCOR is well recognized with loss-of-function variants linked to X-linked dominant Oculofaciocardiodental (OFCD) syndrome in females, while missense variants are occasionally reported in males, typically with a more severe syndromic microphthalmia phenotype [PITX2) and hearing loss and congenital heart defects (FOXC1); while noted to be rare, umbilical hernia has occasionally been reported as well [BCOR variants [BCOR include 2–3 toe syndactyly, extra teeth, or radiculomegaly, and developmental delay/cognitive impairment is more common with BCOR but highly variable [henotype ,33. OFCD as well ,35. CongHCCS was first recognized as a cause of X-linked dominant linear skin defects with multiple congenital anomalies, with microphthalmia, sclerocornea, heart defects, structural brain anomalies, and linear skin defects on the face and neck [AMELX within the deleted region resulted in amelogenesis imperfecta, type 1E [The final case is an interesting example of a contiguous gene syndrome whose combined features resulted in a phenotype overlapping ARS. and neck . Subsequand neck . The add type 1E , with siPITX2/FOXC1.All of the genetic diagnoses identified here showed substantial overlap with the ARS phenotypic spectrum. Accurate diagnosis has important implications for clinical management and family planning. We suggest that broad testing such as exome sequencing be applied as a second-tier test for individuals with ARS with normal sequencing/copy number results for