Single-Cell RNA Sequencing Identifies Crucial Genes Influencing the Polarization of Tumor-Associated Macrophages in Liver Cancer.

Background: In the context of hepatocellular carcinoma (HCC), tumor-associated macrophages (TAMs) are pivotal for the immunosuppressive nature of the tumor microenvironment (TME). This investigation delves into the functional transformations of TAMs within the TME by leveraging single-cell transcriptomics to pinpoint critical genes influencing TAM subset polarization. Methods: We procured single-cell and bulk transcriptomic data from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA), implementing quality assurance, dimensional reduction, clustering, and annotation on the single-cell sequencing data. To examine cellular interactions, CellChat was utilized, while single-cell regulatory network inference and clustering (SCENIC) was applied to deduce transcription factors (TFs) and their associated targets. Through gene enrichment, survival, and immune infiltration correlation analyses, we sought to pinpoint and validate influential genes. A TAM model under HCC conditions was then established to confirm the expression levels of these key genes. Results: Our analysis encompassed 74,742 cells and 23,110 genes. Through postdimensional reduction and clustering, we identified seven distinct cell types and nine TAM subtypes. Analysis via CellChat highlighted a predominance of M2-phenotype-inclined TAM subsets within the tumor's core. SCENIC pinpointed the transcription factor PRDM1 and its target genes as pivotal in this region. Further analysis indicated these genes' involvement in macrophage polarization. Employing trajectory analysis, survival analysis, and immune infiltration correlation, we scrutinized and validated genes likely directing M2 polarization. Experimental validation confirmed PRDM1's heightened expression in TAMs conditioned by HCC. Conclusions: Our findings suggest the PRDM1 gene is a key regulator of M2 macrophage polarization, contributing to the immunosuppressive TME in HCC.

Small Molecule-Inducible and Photoactivatable Cellular RNA N1-Methyladenosine Editing.

N1-methyladenosine (m1A) modification is one of the most prevalent epigenetic modifications on RNA. Given the vital role of m1A modification in RNA processing such as splicing, stability and translation, developing a precise and controllable m1A editing tool is pivotal for in-depth investigating the biological functions of m1A. In this study, we developed an abscisic acid (ABA)-inducible and reversible m1A demethylation tool (termed AI-dm1A), which targets specific transcripts by combining the chemical proximity-induction techniques with the CRISPR/dCas13b system and ALKBH3. We successfully employed AI-dm1A to selectively demethylate the m1A modifications at A8422 of MALAT1 RNA, and this demethylation process could be reversed by removing ABA. Furthermore, we validated its demethylation function on various types of cellular RNAs including mRNA, rRNA and lncRNA. Additionally, we used AI-dm1A to specifically demethylate m1A on ATP5D mRNA, which promoted ATP5D expression and enhanced the glycolysis activity of tumor cells. Conversely, by replacing the demethylase ALKBH3 with methyltransferase TRMT61A, we also developed a controllable m1A methylation tool, namely AI-m1A. Finally, we caged ABA by 4,5-dimethoxy-2-nitrobenzyl (DMNB) to achieve light-inducible m1A methylation or demethylation on specific transcripts. Collectively, our m1A editing tool enables us to flexibly study how m1A modifications on specific transcript influence biological functions and phenotypes.

Clinical characteristics of postoperative necrotizing enterocolitis in patients with congenital jejunoileal atresia and its risk factors.

Objective: To review postoperative necrotizing enterocolitis (NEC) in patients with jejunoileal atresia (JIA) and to explore the potential risk factors related to the concurrence of NEC. Methods: Patients diagnosed with JIA who received surgical treatment from January 2016 to June 2021 were enrolled. Demographics, viral infection of the fetus, transfusion within 48 hours before NEC, sepsis before JIA repair, pathological and anatomical classification of JIA, combined malformation, occurrence time of NEC after the operation, treatment, and prognosis of patients were analyzed. Patients were divided into NEC group and non-NEC group, and all patients were followed up for 3-6 months to observe for complications. Results: A total of 180 patients with JIA were included, of whom 12 were diagnosed with NEC after surgery and 1 patient with NEC died during follow-up. The average age, birth weight, gestational age, proportion of premature infants, proportion of preoperative infections, and pathological classification of JIA did not significantly differ between the two groups. The probability of patients with proximal jejunal atresia (PJA) in the NEC group (58.3%) was higher than that in the non-NEC group (22.6%) (p=0.011), and patients with PJA had longer parenteral nutrition time than patients without PJA (26.64±9.21 days vs 15.11±6.58 days, p<0.001). Conclusion: PJA was more likely to be associated with concurrent NEC after surgery, which is a highly NEC-related risk factor inherent in JIA.

HMGB1 promotes mitochondrial transfer between hepatocellular carcinoma cells through RHOT1 and RAC1 under hypoxia.

Mitochondrial transfer plays an important role in various diseases, and many mitochondrial biological functions can be regulated by HMGB1. To explore the role of mitochondrial transfer in hepatocellular carcinoma (HCC) and its relationship with HMGB1, field emission scanning electron microscopy, immunofluorescence, and flow cytometry were used to detect the mitochondrial transfer between HCC cells. We found that mitochondrial transfer between HCC cells was confirmed using tunnel nanotubes (TNTs). The transfer of mitochondria from the highly invasive HCC cells to the less invasive HCC cells could enhance the migration and invasion ability of the latter. The hypoxic conditions increased the mitochondrial transfer between HCC cells. Then the mechanism was identified using co-immunoprecipitation, luciferase reporter assay, and chromatin immunoprecipitation. We found that RHOT1, a mitochondrial transport protein, promoted mitochondrial transfer and the migration and metastasis of HCC cells during this process. Under hypoxia, HMGB1 further regulated RHOT1 expression by increasing the expression of NFYA and NFYC subunits of the NF-Y complex. RAC1, a protein associated with TNTs formation, promoted mitochondrial transfer and HCC development. Besides, HMGB1 regulated RAC1 aggregation to the cell membrane under hypoxia. Finally, the changes and significance of related molecules in clinical samples of HCC were analyzed using bioinformatics and tissue microarray analyses. We found that HCC patients with high HMGB1, RHOT1, or RAC1 expression exhibited a relatively shorter overall survival period. In conclusion, under hypoxic conditions, HMGB1 promoted mitochondrial transfer and migration and invasion of HCC cells by increasing the expression of mitochondrial transport protein RHOT1 and TNTs formation-related protein RAC1.

Discovery of the First-in-Class RORγ Covalent Inhibitors for Treatment of Castration-Resistant Prostate Cancer.

Nuclear receptor receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor and has been established as a key player in castration-resistant prostate cancers (CRPC) by driving androgen receptor (AR) overexpression, representing a potential therapeutical target for advanced prostate cancers. Here, we report the identification of the first-in-class RORγ covalent inhibitor 29 via the structure-based drug design approach following structure-activity relationship (SAR) exploration. Mass spectrometry assay validated its covalent inhibition mechanism. Compound 29 significantly inhibited RORγ transcriptional activity and remarkably suppressed the expression levels of AR and AR-targeted genes. Compound 29 also exhibited much superior activity in inhibiting the proliferation and colony formation and inducing apoptosis of the CRPC cell lines relative to the positive control 2 and noncovalent control 33. Importantly, it markedly suppressed the tumor growth in a 22Rv1 mouse tumor xenograft model with good safety. These results clearly demonstrate that 29 is a highly potent and selective RORγ covalent inhibitor.

Design, synthesis and evaluation of quinazoline derivatives as Gαq/11 proteins inhibitors against uveal melanoma.

Uveal melanoma (UM) represents the predominant ocular malignancy among adults, exhibiting high malignancy and proclivity for liver metastasis. GNAQ and GNA11 encoding Gαq and Gα11 proteins are key genes to drive UM, making the selective inhibition of Gαq/11 proteins to be a potential therapeutic approach for combating UM. In this study, forty-six quinazoline derivatives were designed, synthesized, and assessed for their ability to inhibit Gαq/11 proteins and UM cells. Compound F33 emerged as the most favorable candidate, and displayed moderate inhibitory activity against Gαq/11 proteins (IC50 = 9.4 µM) and two UM cell lines MP41 (IC50 = 6.7 µM) and 92.1 (IC50 = 3.7 µM). Being a small molecule inhibitor of Gαq/11 proteins, F33 could effectively suppress the activation of downstream signaling pathways in a dose-dependent manner, and significantly inhibits UM in vitro.F33 represents a promising lead compound for developing therapeutics for UM by targeting Gαq/11 proteins.

Discovery of novel indazole derivatives as SOS1 agonists that activate KRAS signaling.

KRAS serves as a vital regulator for cellular signaling and drives tumor pathogenesis after mutation. Despite extensive research efforts spanning several decades, targeting KRAS is still challenging due to the multiple KRAS mutations and the emergence of drug resistance. Interfering the interactions between KRAS and SOS1 is one of the promising approaches for modulating KRAS functions. Herein, we discovered small-molecule SOS1 agonists with novel indazole scaffold. Through structure-based optimization, compound 11 was identified with high SOS1 activation potency (p-ERK EC50 = 1.53 µM). In HeLa cells, compound 11 enhances cellular RAS-GTP levels and exhibits biphasic modulation of ERK1/2 phosphorylation through an on-target mechanism and presents the therapeutic potential to modulate RAS signaling by activating SOS1.

Identification of PANoptosis-Based Prognostic Signature for Predicting Efficacy of Immunotherapy and Chemotherapy in Hepatocellular Carcinoma.

Background: PANoptosis has been a research hotspot, but the role of PANoptosis in hepatocellular carcinoma (HCC) remains widely unknown. Drug resistance and low response rate are the main limitations of chemotherapy and immunotherapy in HCC. Thus, construction of a prognostic signature to predict prognosis and recognize ideal patients for corresponding chemotherapy and immunotherapy is necessary. Method: The mRNA expression data of HCC patients was collected from TCGA database. Through LASSO and Cox regression, we developed a prognostic signature based on PANoptosis-related genes. KM analysis and ROC curve were implemented to evaluate the prognostic efficacy of this signature, and ICGC and GEO database were used as external validation cohorts. The immune cell infiltration, immune status, and IC50 of chemotherapeutic drugs were compared among different risk subgroups. The relationships between the signature and the efficacy of ICI therapy, sorafenib treatment, and transcatheter arterial chemoembolization (TACE) therapy were investigated. Result: A 3-gene prognostic signature was constructed which divided the patients into low- and high-risk subgroups. Low-risk patients had better prognosis, and the risk score was proved to be an independent predictor of overall survival (OS), which had a well predictive effect. Patients in high-risk population had more immunosuppressive cells (Tregs, M0 macrophages, and MDSCs), higher TIDE score and TP53 mutation rate, and elevated activity of base excision repair (BER) pathways. Patients with low risk benefited more from ICI, TACE, and sorafenib therapy. The predictive value of the risk score was comparable with TIDE and MSI for OS under ICI therapy. The risk score could be a biomarker to predict the response to ICI, TACE, and sorafenib therapy. Conclusion: The novel signature based on PANoptosis is a promising biomarker to distinguish the prognosis predict the benefit of ICI, TACE, and sorafenib therapy, and forecast the response to them.

Instillation of Amphotericin B by bronchoscopy combined with systemic voriconazole in advanced non-small cell lung cancer patients with chronic cavitary pulmonary aspergillosis: A case series and literature review.

Although the treatment of aspergillosis has been studied for years, the optimal nonsurgical treatment of chronic cavitary pulmonary aspergillosis (CCPA) remains unsatisfactory, especially in lung cancer. We report two advanced non-small cell lung cancer (NSCLC) patients who recovered from CCPA following instillation of Amphotericin B (AmB) by bronchoscopy combined with systemic voriconazole. The first patient was diagnosed with lung adenocarcinoma after right upper lobe resection and was treated with anaplastic lymphoma kinase-targeted therapy. Chest computed tomography (CT) revealed a right pulmonary cavity containing solid materials. The second patient was diagnosed with squamous cell carcinoma and received immunotherapy following surgery, chemotherapy, and radiotherapy. Chest CT tomography revealed a mass in the right lung cavity. Both patients' cultures and next-generation sequencing of their bronchoalveolar lavage (BAL) samples revealed presence of Aspergillus fumigatus. In addition, the galactomannan test of both patients BAL samples was positive. Systemic voriconazole was prescribed based on in vitro susceptibility testing. The chest images and clinical symptoms of both patients did not improve after one month of voriconazole therapy within the therapeutic blood concentration. Considering the low local concentrations of antifungals against CCPA, AmB instillation by bronchoscopy combined with systemic voriconazole was utilized. The chest CT images and clinical symptoms of both patients markedly improved in the following third month. Instillation of AmB combined with systemic voriconazole may be a promising treatment option for NSCLC patients with CCPA who fail voriconazole monotherapy.

Histone H3 activates caspase-1 and promotes proliferation and metastasis in hepatocellular carcinoma.

Background: As a component of nucleosomes, histone H3 plays an important role in chromosome structure and gene expression. Current studies have mostly focused on the role of histones in epigenetics, but in addition to this, the role of histones themselves in tumor development and microenvironment have been less explored. Methods: Western blot and immunofluorescence were carried out to detect the content and localization of histone H3 in hepatocellular carcinoma. The changes of histone H3 were observed in hypoxia treatment cells, the specific action mechanism of histone H3 was studied by CoIP and other methods. Cell Counting Kit-8 assay, plate cloning assay and transwell assay were used to exam the effect of histone H3 on cell proliferation and metastasis, which were verified by subcutaneous tumors in mice and lung metastasis by tail vein injection in mice. Results: We found that histone H3 was overexpressed in hepatocellular carcinoma tumor tissues compared to adjacent non-tumor tissues, and there was concomitant translocation of histone H3 from the nucleus to the cytoplasm. We found that hypoxia could contribute to this phenomenon of histone H3 translocation from the nucleus to the cytoplasm in hepatocellular carcinoma cells and increased binding levels to TLR9. At the same time, hypoxia induced downstream activation of TLR9 and caspase-1, as well as cleavage and release of the pro-inflammatory cytokines IL-1ß and IL-18. We further demonstrated that histone H3 could also promote proliferation and metastasis of hepatocellular carcinoma through TLR9 activation of NLRP3 inflammasome. In addition, overexpression of histone H3 was also confirmed to promote hepatocellular carcinoma proliferation and metastasis in mouse models of hepatocellular carcinoma growth assay and lung metastasis. Conclusions: In hypoxic hepatocellular carcinoma cells, histone H3 can translocate to the cytoplasm and activate caspase-1 via TLR9, thereby producing pro-inflammatory cytokines that promote tumor proliferation and metastasis.

Identification of novel Pyrrolo[2,3-d]Pyrimidine-based KRAS G12C inhibitors with anticancer effects.

Oncogene KRAS plays predominant roles in human cancers by regulating cell proliferation, differentiation, and migration. Recent progress revealed that directly target KRAS G12C with allosteric inhibitors that covalently bind to the switch Ⅱ pocket is feasible. Herein, series of pyrrolo[2,3-d]pyrimidine derivatives were designed and synthesized through systematic structural optimization, leading to the discovery of compound 2-((S)-1-acryloyl-4-(2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-methyl-6-(8-methylnaphthalen-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-2-yl)acetonitrile (50) with high KRAS/SOS1 inhibitory potency (IC50 = 0.21 µM) and strong anti-proliferation activities on cancer cells harboring KRAS p.G12C. Compound 50 also exhibited satisfactory selectivity, moderate pharmacokinetic characters, and good anticancer effects in vivo. Meaningfully, the identification of these compounds highlights the necessity of an appropriate conformational constraint for acquiring the applicable binding pose in the cryptic pocket of KRAS, and the results support efforts toward design of KRAS inhibitors with novel skeleton and binding mechanism could be beneficial for targeting the acquired drug resistance.

Role of cholinergic innervation in biliary remnants of patients with biliary atresia.

Objective: Biliary innervation is considered important in regulating the function of bile ducts, whereas the role of innervation in the hepatobiliary system of patients with biliary atresia (BA) remains unknown. This current study aims to investigate the role of innervation in biliary remnants and analyze the relationship between the innervation and prognosis of BA after surgery. Methods: Eighty-seven patients with type III BA who underwent the Kasai procedure were consecutively enrolled from January 2017 to September 2020. Innervation and ductules in remnants were examined by pathologists. Liver function, onset of cholangitis, jaundice clearance, and survival with the native liver were recorded. Patients were followed up for 24 months. The relationship between innervation and prognosis was analyzed. Results: In total, 67 patients had bile drainage postoperatively, and 21 biliary remnants contained neuronal plexuses where there was no neuron but nerve fiber bundles. Acetylcholinesterase staining was positive in all plexuses. In patients with bile drainage, those with plexuses had improved postoperative liver function, significantly better jaundice clearance 3 or 6 months postoperatively (50.0% vs. 19.1%, or 90.0% vs. 63.8%, respectively), fewer episodes of early cholangitis (10.0% vs. 34.0%), and better survival (80.0% vs. 61.7%) compared to those without. In addition, a larger area of plexuses was associated with a larger area of ductules (R2 = 0.786, p = 0.000), less frequent (p = 0.000) and later cholangitis onset (p = 0.012), and better jaundice clearance (p = 0.063). Conclusions: Increased cholinergic innervation in biliary remnants may help reduce the onset of cholangitis and lead to better and earlier jaundice clearance. Thus, it improves the postoperative prognosis of patients with BA.

Discovery of small molecule Gαq/11 protein inhibitors against uveal melanoma.

Constitutively activated G proteins caused by specific mutations mediate the development of multiple malignancies. The mutated Gαq/11 are perceived as oncogenic drivers in the vast majority of uveal melanoma (UM) cases, making directly targeting Gαq/11 to be a promising strategy for combating UM. Herein, we report the optimization of imidazopiperazine derivatives as Gαq/11 inhibitors, and identified GQ262 with improved Gαq/11 inhibitory activity and drug-like properties. GQ262 efficiently blocked UM cell proliferation and migration in vitro. Analysis of the apoptosis-related proteins, extracellular signal-regulated kinase (ERK), and yes-associated protein (YAP) demonstrated that GQ262 distinctly induced UM cells apoptosis and disrupted the downstream effectors by targeting Gαq/11 directly. Significantly, GQ262 showed outstanding antitumor efficacy in vivo with good safety at the testing dose. Collectively, our findings along with the favorable pharmacokinetics of GQ262 revealed that directly targeting Gαq/11 may be an efficient strategy against uveal melanoma.

Immunophenotype in acute exacerbation of chronic obstructive pulmonary disease: a cross-sectional study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease, and the immune inflammatory response is thought to play an important role in pathogenesis. However, the immunophenotype of patients with COPD is unknown. Herein, we evaluated the immunophenotype of patients with acute exacerbation of COPD (AECOPD). METHODS: A cross-sectional study was conducted in West China Hospital from September 2018 to October 2019. The proportion of CD4 + T lymphocyte subtypes (Th1, Th2, Th17 and Treg) and levels of serum cytokines in the peripheral blood of patients with AECOPD, stable COPD (SCOPD), healthy smokers (HSs)and healthy controls (HCs) were evaluated. RESULTS: A total of 15 HCs, 19 HSs, 42 patients with SCOPD, and 55 patients with AECOPD were included. Compared to patients with SCOPD, Th1 cells, Th17 cells, Treg cell ratio, Th1/Th2 cell ratio, and the levels of C-reactive protein, interleukin (IL)-6, and IL-10 were significantly increased in patients with AECOPD (P < 0.001), while the proportion of Th2 cells was significantly reduced (P < 0.01). The proportion of Th17 cells was positively correlated with COPD Assessment Test score (r = 0.266, P = 0.009), modified Medical Research Council dyspnea score (r = 0.858, P < 0.0001), and Th1 cell ratio (r = 0.403, P < 0.0001) and negatively correlated with forced vital capacity (r = - 0.367, P = 0.009) and proportion of Th2 cells (r = - 0.655, P < 0.0001). CONCLUSIONS: The immunophenotype of patients with AECOPD shows abnormal activation of Th1, Th17, and Treg cells. There is a correlation between the proportion of Th17 cells and the severity of COPD; therefore, this may represent a novel index for the evaluation of COPD severity. TRIAL REGISTRATION: China Clinical Trials Registry, ChiCTR1800018452, registered 19 September 2018, https://www.chictr.org.cn/index.aspx .

Targeting of GSDMD sensitizes HCC to anti-PD-1 by activating cGAS pathway and downregulating PD-L1 expression.

BACKGROUND: Gasdermin D (GSDMD) is well known as a downstream of inflammasomes. However, the roles of GSDMD itself in hepatocellular carcinoma (HCC) remain unclear. METHODS: Two independent cohorts of patients with HCC were analyzed to evaluate the pathological relevance of GSDMD/pTBK1/PD-L1. GSDMD knockout (GSDMD-/-) mice, Alb-Cre mice administered with an adeno-associated virus (AAV) vector that expressed the gasdermin-N domain (AAV9-FLEX-GSDMD-N) and their wild-type littermates were used to induce hepatocarcinogenesis or metastatic HCC. Combination of anti-programmed cell death protein-1 (PD-1) and GSDMD inhibitor dimethyl fumarate (DMF) was used to test for improved therapeutic efficacy. RNA sequencing was used to explore the mechanisms how GSDMD promoted HCC progression. RESULTS: The expression of GSDMD and GSDMD-N was upregulated in HCC tissues or metastatic HCC tissues and positive GSDMD expression indicated grim prognosis. Diethylnitrosamine/carbon tetrachloride or thioacetamide-treated GSDMD-/- mice exhibited decreased liver tumors. In contrast, AAV9-FLEX-GSDMD-N promoted hepatocarcinogenesis. RNA sequencing manifested that knockout of GSDMD impacted the cyclic GMP-AMP synthase (cGAS) pathway and immune-associated pathway. GSDMD damped cGAS activation by promoting autophagy via outputting potassium (K+) and promoted programmed death ligand-1 (PD-L1) expression by histone deacetylases/signal transducer and activator of transcription 1 (STAT1)-induced transactivation of PD-L1 via importing calcium (Ca2+). High Mobility Group Box 1/toll-like receptor 4 (TLR4)/caspase-1 pathway contributed to the overexpression and cleavage of GSDMD. Anti-PD-1 combining with DMF largely impaired HCC progression and metastasis. CONCLUSIONS: Targeting GSDMD could promote expression of interferons through inactivation of cGAS pathway and downregulated the PD-L1 expression. Therefore, combined anti-PD-1 and GSDMD inhibitor might serve as an effective treatment option for patients with HCC with GSDMD upregulation.

Structure-based optimization of Toddacoumalone as highly potent and selective PDE4 inhibitors with anti-inflammatory effects.

Phosphodiesterase-4 (PDE4) is an important drug target for inflammatory diseases. Previously, we identified a series of novel PDE4 inhibitors derived from the natural Toddacoumalone, among which the hit compound 2 with a naphthyridine scaffold showed moderate potency with the IC50 value of 400 nM. Based on the co-crystal structure of PDE4D-2, further structural optimizations and structure-activity relationship studies led to a highly potent PDE4 inhibitor 23a with the IC50 value of 0.25 nM and excellent selectivity profiles over other PDEs (>4000-fold). The co-crystal structure of PDE4D-23a elucidated that 23a has strong interactions with the M and Q pocket of PDE4D. Importantly, compound 23a significantly inhibits the release of inflammatory cytokines TNF-α and IL-6 in lipopolysaccharide-stimulated RAW264.7 cells. Thus, compound 23a with a naphthyridine scaffold is a promising PDE4 inhibitor for the treatment of inflammatory diseases.

Synthesis and evaluation of imidazo[1,2-a]pyrazine derivatives as small molecule Gαq/11 inhibitors against uveal melanoma.

Uveal melanoma (UM) is an aggressive malignancy with high mortality in adults and lacks effective systemic therapies. Activating gene mutations related to the Gαq/11 signaling pathway are prevalent in UM, and Gαq/11 inhibitors have shown anti-UM activity in vitro and in vivo. In this study, we designed and synthesized a series of imidazo[1,2-a]pyrazine derivatives as Gαq/11 inhibitors, and discovered GQ352 with the selective antiproliferative activity against UM cells. Importantly, GQ352 directly binds to the Gαq and inhibits the dissociation of Gαßγ heterotrimers with the IC50 value of 8.9 µM. GQ352 inhibits UM tumorigenesis by suppressing Gαq/11 downstream ERK phosphorylation and YAP dephosphorylation, as shown in Western blot analysis. In addition, GQ352 displayed reasonable physiochemical properties and human liver microsome stability, indicating the potential application in UM treatment.

On-Resin Synthesis of Linear Aryl Thioether Containing Peptides and in-Solution Cyclization via Cysteine SNAr Reaction.

Cyclic peptides represent one of the most promising therapeutic agents in drug discovery due to their good affinity and selectivity. Herein, an on-resin synthesis of aryl thioether containing peptides and a concise cyclization strategy via chemoselective cysteine SNAr reaction was developed. The arylation group could be incorporated into a series of amino acids and used for standard SPPS and peptides cyclization. Constructed cyclic peptides showed increased cellular uptakes compared to their linear peptides.

The m6A demethylase FTO promotes the osteogenesis of mesenchymal stem cells by downregulating PPARG.

N6-methyladenosine (m6A) is the most abundant posttranscriptional methylation modification that occurs in mRNA and modulates the fine-tuning of various biological processes in mammalian development and human diseases. In this study we investigated the role of m6A modification in the osteogenesis of mesenchymal stem cells (MSCs), and the possible mechanisms by which m6A modification regulated the processes of osteoporosis and bone necrosis. We performed systematic analysis of the differential gene signatures in patients with osteoporosis and bone necrosis and conducted m6A-RNA immunoprecipitation (m6A-RIP) sequencing to identify the potential regulatory genes involved in osteogenesis. We showed that fat mass and obesity (FTO), a primary m6A demethylase, was significantly downregulated in patients with osteoporosis and osteonecrosis. During the differentiation of human MSCs into osteoblasts, FTO was markedly upregulated. Both depletion of FTO and application of the FTO inhibitor FB23 or FB23-2 impaired osteogenic differentiation of human MSCs. Knockout of FTO in mice resulted in decreased bone mineral density and impaired bone formation. PPARG, a biomarker for osteoporosis, was identified as a critical downstream target of FTO. We further revealed that FTO mediated m6A demethylation in the 3'UTR of PPARG mRNA, and reduced PPARG mRNA stability in an YTHDF1-dependent manner. Overexpression of PPARG alleviated FTO-mediated osteogenic differentiation of MSCs, whereas knockdown of PPARG promoted FTO-induced expression of the osteoblast biomarkers ALPL and OPN during osteogenic differentiation. Taken together, this study demonstrates the functional significance of the FTO-PPARG axis in promoting the osteogenesis of human MSCs and sheds light on the role of m6A modification in mediating osteoporosis and osteonecrosis.

Demethylative Alkylation of Methionine Residue by Employing the Sulfonium as the Key Intermediate.

Methionine (Met) offers a valuable handle to achieve peptide chemical modification owing to its unique thioether functional group. In contrast with cysteine, the site-selective functionalization of the hydrophobic and redox-sensitive thioether motif on peptides is still challenging, and strategies for diversification on the Met residue are rarely disclosed. Herein we report a transition-metal-free and redox-neutral approach for Met diversification with substrate diversity, which could be applied to synthesize cyclic peptides.