Cholesterol metabolism disorders are a major contributor to type 2 diabetes mellitus (T2DM). Cholesterol accumulation in the liver exacerbates insulin resistance, while cholesterol overload in the pancreatic islets impairs insulin secretion. Peroxisome proliferator activated receptors (PPARs) are considered potential therapeutic targets for cholesterol regulation. Herein, we report a series of novel aromatic amide derivatives as pan-PPAR agonists. Among them, compound 15a exhibited potent and well balanced pan-PPAR agonistic activity and significantly upregulated the expression of ATP binding cassette transporter A1 (ABCA1), a PPAR target gene involved in cholesterol efflux. In a high fat and high sucrose diet induced KKAy diabetic mouse model, 15a markedly reduced plasma cholesterol levels, hepatic cholesterol accumulation, and islet cholesterol deposition. It also demonstrated favorable regulation of glucolipid metabolism, leading to pronounced alleviation of hepatic steatosis and islet dysfunction, while avoiding the weight gain and adiposity side effects associated with PPARγ agonists. SPR experiments demonstrated that 15a interacts with the ligand binding domains (LBDs) of all three PPAR subtypes. Molecular docking and single point mutation assays of key residues confirmed that 15a likely exerts its pan PPAR agonist activity by stabilizing helix 3 of the three PPAR subtypes. Furthermore, 15a exhibits improved pharmacokinetic properties and a favorable safety profile compared to the lead compound. In summary, this study demonstrates that compound 15a significantly ameliorates hepatic steatosis and islet dysfunction associated with T2DM by regulating cholesterol metabolism disorders along the liver-islet axis, highlighting its strong potential for treating hepatic lipid accumulation and T2DM related islet dysfunction.

C-type lectins (CTLs) are crucial pattern recognition receptors in invertebrate innate immunity. Previous studies revealed that the silkworm (Bombyx mori) C-type lectin gene BmIML-2 suppresses Nucleopolyhedrovirus (BmNPV) proliferation through apoptotic signaling pathways. However, whether BmIML-2 regulates oxidative stress and glycolysis during viral infection remains unknown. In this study, we found that BmIML-2 was significantly upregulated in resistant strains upon BmNPV infection. Gain- and loss-of-function assays indicated that BmIML-2 suppressed viral replication by enhancing oxidative stress and inhibiting glycolytic metabolism. Mechanistically, BmIML-2 promotes reactive oxygen species (ROS) accumulation, modulates oxidative stress-related gene expression, disrupts mitochondrial membrane potential, downregulates glycolytic pathway genes, and reduces ATP production. These findings uncover a novel antiviral mechanism of C-type lectins through coordinated regulation of oxidative stress and metabolic reprogramming, providing insights into insect antiviral immunity.

Two novel diketopiperazine dimers (1 and 2) and two new 4-hydroxyphenylacetates (5 and 6), along with two previously known diketopiperazine dimers were isolated from the culture of the endolichenic fungus Aspergillus sp. CPCC 400810. Their structures were determined through comprehensive spectroscopic analysis, including high-resolution electrospray ionization mass spectrometry (HRESIMS) and 1D and 2D nuclear magnetic resonance (NMR) data. The absolute configurations of the new compounds were confirmed using Marfey's method and chemical synthesis.

Infectious diseases caused by drug-resistant bacteria represent one of the most significant global public challenges of this century. There is an urgent need for the treatment of drug-resistant Gram-negative bacterial infections. A series of 3,4-dihydro-2H-[1,3]oxazino[5,6-h]quinoline derivatives were synthesized and evaluated for their antibacterial activity against Gram-negative bacteria including strains from ATCC and clinical isolates, initially revealing the structure-activity relationship. Among them, 22 compounds demonstrated inhibitory activity (MICs: 3.125-12.5 μg/mL) against Escherichia coli (E. coli) ATCC 25922 and Acinetobacter baumannii (A. baumannii) ATCC 19606. Among these, 7 compounds exhibited good inhibitory activity against MDR A. baumannii clinical isolates, with MICs ranging from 3.125 to 12.5 μg/mL. Most of these compounds also showed lower cytotoxicity than IMB-881. Notably, 2 compounds, 4n1 and 4b3, significantly extended the survival of Galleria mellonella larvae infected with E. coli. Mechanism studies have revealed that compounds 4n1 and 4b3 might disrupt the interaction between LptA and LptC, showing moderate affinity for LptA protein. These compounds also induce abnormal bacterial morphology and cause outer membrane damage. This finding provides a novel class of antibiotic sensitizers with the potential to effectively fight against E. coli and A. baumannii.

Cathepsin L (CTSL), a lysosomal cysteine protease belonging to the papain-like protease family, is primarily involved in intracellular protein degradation, antigen processing, and extracellular matrix remodeling. It plays critical roles in pathological conditions, including cancer metastasis, neurodegenerative disorders, and viral infection, due to dysregulated activity or overexpression. Thus, inhibitors targeting CTSL are under investigation for therapeutic applications. Current approaches for identifying CTSL inhibitors predominantly rely on fluorescence-labeled substrates, fluorescence resonance energy transfer (FRET), and cell-based screening assays. Here, we applied the principle of fluorescence polarization (FP) to the detection of substrate cleavage activity by CTSL through changes in millipolarization unit (mp) values and established a cost-effective, quantitative, reagent- and time-saving inhibitor high-throughput screening (HTS) assay. We also provide detailed steps for the expression and purification of highly active CTSL from eukaryotic cells, which lays a solid foundation for the FP-based assay. A key advantage of this assay lies in its reduced susceptibility to fluorescence interference, as the fluorescein isothiocyanate (FITC) fluorophore exhibits high quantum efficiency with an emission peak at 535 nm-a wavelength range distinct from most naturally occurring fluorescent molecules. The assay's adaptability to reaction time, temperature, and dimethyl sulfoxide (DMSO) concentration minimizes false-positive or false-negative results caused by minor experimental inconsistencies, streamlining the screening process. Furthermore, the protocol requires fewer operational steps, reduced incubation time, and lower quantities of CTSL and substrates compared to conventional methods. This rapid, cost-effective, and scalable approach aligns well with the demands of HTS platforms. Key features • Detailed procedures for eukaryotic expression, purification, and identification of active recombinant CTSL and determination of its biological activity. • Full description for application of fluorescence polarization (FP)-based high-throughput screening (HTS) assay targeting CTSL. • Elaboration for the application of the FP-based assay in CTSL activity evaluation or drug discovery. • Protocol is readily adaptable to other proteases with a similar catalytic mechanism.

IFN-γ and TNF-α are two vital inflammatory factors elevated aberrantly in many diseases. Such an inflammatory microenvironment is detrimental to residual cells such as mesenchymal stem cells (MSCs), yet the precise mechanisms are not fully understood. IFN-γ and TNF-α have distinct effects on the immunoregulatory properties of MSCs, and they have been proposed as optimal priming factors to enhance the immunosuppressive capacity of engineered MSCs. Thus, the overall effects of IFN-γ and/or TNF-α exposure on MSCs needs to be elucidated. Here, it is found that IFN-γ and TNF-α synergistically induce cell death of MSCs via necroptosis. When MSCs are exposed to both IFN-γ and TNF-α, their morphological features and biological functions are impaired. Mechanistically revealed by RNA-Sequencing, the injured MSCs undergo a unique cell death process, namely necroptosis. Compared with controls, IFN-γ synergized with TNF-α to increase the expression of RIPK1, RIPK3, MLKL, and all other genes associated with necroptosis. Rescue experiments further demonstrate that this process can be reversed by RIPK3 and MLKL inhibitors but not by the RIPK1 inhibitor, suggesting a RIPK1-independent pathway. Collectively, this study discloses an inflammatory injury mechanism of MSCs, which may shed new light on revealing the MSCs deficits in many inflammatory diseases with expectations to inspire potential targeted therapies. In addition, inflammatory impairment should be taken into consideration when delivering cell therapy based on MSCs primed with IFN-γ and TNF-α.

The continuous emergence of SARS-CoV-2 variants as well as other potential future coronavirus has challenged the effectiveness of current COVID-19 vaccines. Therefore, there remains a need for alternative antivirals that target processes less susceptible to mutations, such as the formation of six-helix bundle (6-HB) during the viral fusion step of host cell entry. In this study, a novel high-throughput screening (HTS) assay employing a yeast-two-hybrid (Y2H) system was established to identify inhibitors of HR1/HR2 interaction. The compound IMB-9C, which achieved single-digit micromolar inhibition of SARS-CoV-2 and its Omicron variants with low cytotoxicity, was selected. IMB-9C effectively blocks the HR1/HR2 interaction in vitro and inhibits SARS-CoV-2-S-mediated cell-cell fusion. It binds to both HR1 and HR2 through non-covalent interaction and influences the secondary structure of HR1/HR2 complex. In addition, virtual docking and site-mutagenesis results suggest that amino acid residues A930, I931, K933, T941, and L945 are critical for IMB-9C binding to HR1. Collectively, in this study, we have developed a novel screening method for HR1/HR2 interaction inhibitors and identified IMB-9C as a potential antiviral small molecule against COVID-19 and its variants.

The recent coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has exerted considerable impact on global health. To prepare for rapidly mutating viruses and for the forthcoming pandemic, effective therapies targeting the critical stages of the viral life cycle need to be developed. Viruses are dependent on the interaction between the receptor-binding domain (RBD) of the viral Spike (S) protein (S-RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor to efficiently establish infection and the following replicate. Targeting this interaction provides a promising strategy to inhibit the entry process of the virus, which in turn has both preventive and therapeutic effects. In this study, we developed a robust and straightforward assay based on the Yeast-Two Hybrid system (Y2H) for identifying inhibitors targeting the S-RBD-ACE2 interaction of SARS-CoV-2. Through high-throughput screening, two compounds were identified as potential entry inhibitors. Among them, IMB-1C was superior in terms of pseudovirus entry inhibition and toxicity. It could bind to both ACE2 and S-RBD and induce conformational change in the S-RBD+ACE2 complex. This is the first study to verify the feasibility of utilizing the Y2H system to discover potent SARS-CoV-2 inhibitors targeting the receptor recognition stage. This approach may also be applied in the discovery of other virus receptor recognition inhibitors.

The probability of cardiovascular events has been reported lower in rheumatoid arthritis (RA) patients treated with leflunomide. However, the anti-atherosclerotic and cardiovascular protective effects and metabolism of leflunomide are not explored. In this study, we assessed the potential benefits of leflunomide on atherosclerosis and revealed the underlying mechanism. ApoE-/- mice were fed a western diet (WD) alone or supplemented with leflunomide (20 mg/kg, oral gavage, once per day) for 12 weeks. Samples of the aorta, heart, liver, serum, and macrophages were collected. We found that leflunomide significantly reduced lesion size in both en-face aortas and aortic root in WD-fed ApoE-/- mice. Leflunomide also obviously improved dyslipidemia, reduced hepatic lipid content, and improved disorders of glucose and lipid metabolism in vivo. RNA-Seq results showed that leflunomide effectively regulated the genes' expression involved in the lipid metabolism pathway. Importantly, leflunomide significantly increased the phosphorylation levels of AMPKα and acetyl-CoA carboxylase (ACC) in vivo. Furthermore, leflunomide and its active metabolite teriflunomide suppressed lipid accumulation in free fatty acid (FFA)-induced AML12 cells and improved endothelial dysfunction in palmitic acid (PA)-induced HUVECs through activating AMPK signaling and inhibiting dihydroorotate dehydrogenase (DHODH) signaling pathway. We present evidence that leflunomide and teriflunomide ameliorate atherosclerosis by regulating lipid metabolism and endothelial dysfunction. Our findings suggest a promising use of antirheumatic small-molecule drugs leflunomide and teriflunomide for the treatment of atherosclerosis and related cardiovascular diseases (CVDs).

BACKGROUND:Atherosclerosis is the most common cause of cardiovascular diseases. Clinical studies indicate that loss-of-function ASGR1 (asialoglycoprotein receptor 1) is significantly associated with lower plasma cholesterol levels and reduces cardiovascular disease risk. However, the effect of ASGR1 on atherosclerosis remains incompletely understood; whether inhibition of ASGR1 causes liver injury remains controversial. Here, we comprehensively investigated the effects and the underlying molecular mechanisms of ASGR1 deficiency and overexpression on atherosclerosis and liver injury in mice. METHODS:We engineered Asgr1 knockout mice (Asgr1-/-), Asgr1 and ApoE double-knockout mice (Asgr1-/-ApoE-/-), and ASGR1-overexpressing mice on an ApoE-/- background and then fed them different diets to assess the role of ASGR1 in atherosclerosis and liver injury. RESULTS:After being fed a Western diet for 12 weeks, Asgr1-/-ApoE-/- mice exhibited significantly decreased atherosclerotic lesion areas in the aorta and aortic root sections, reduced plasma VLDL (very-low-density lipoprotein) cholesterol and LDL (low-density lipoprotein) cholesterol levels, decreased VLDL production, and increased fecal cholesterol contents. Conversely, ASGR1 overexpression in ApoE-/- mice increased atherosclerotic lesions in the aorta and aortic root sections, augmented plasma VLDL cholesterol and LDL cholesterol levels and VLDL production, and decreased fecal cholesterol contents. Mechanistically, ASGR1 deficiency reduced VLDL production by inhibiting the expression of MTTP (microsomal triglyceride transfer protein) and ANGPTL3 (angiopoietin-like protein 3)/ANGPTL8 (angiopoietin-like protein 8) but increasing LPL (lipoprotein lipase) activity, increased LDL uptake by increasing LDLR (LDL receptor) expression, and promoted cholesterol efflux through increasing expression of LXRα (liver X receptor-α), ABCA1 (ATP-binding cassette subfamily A member 1), ABCG5 (ATP-binding cassette subfamily G member 5), and CYP7A1 (cytochrome P450 family 7 subfamily A member 1). These underlying alterations were confirmed in ASGR1-overexpressing ApoE-/- mice. In addition, ASGR1 deficiency exacerbates liver injury in Western diet-induced Asgr1-/-ApoE-/- mice and high-fat diet-induced but not normal laboratory diet-induced and high-fat and high-cholesterol diet-induced Asgr1-/- mice, while its overexpression mitigates liver injury in Western diet-induced ASGR1-overexpressing ApoE-/- mice. CONCLUSIONS:Inhibition of ASGR1 inhibits atherosclerosis in Western diet-fed ApoE-/- mice, suggesting that inhibiting ASGR1 may serve as a novel therapeutic strategy to treat atherosclerosis and cardiovascular diseases.

Acquired aplastic anemia (AAA) is the most prevalent bone marrow failure disease, characterized by an autoimmune disorder that damages hematopoietic stem/progenitor cells, which is pivotal in the disease's progression. Nearly 70% of AA patients show hematologic responses to antithymocyte globulin (ATG) and ciclosporin within 6 months. Although massive laboratory data support the immune pathophysiology of AA, the mechanisms underlying abnormal immune response remain unclear. Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous cells characterized by their immature state and ability to inhibit T cell-mediated immune responses. Our previous studies have shown that the number and inhibitory function of MDSCs are impaired in patients with AA compared to healthy donors (HD). However, the biological alterations of MDSCs from patients with severe AA (SAA) after treatment of ATG are largely unknown. In this study, a total of 62 HD and 118 SAA patients were included, with a median age of 38 years; 97 were male and 83 were female. Among the 118 SAA patients, 66 were newly diagnosed and 52 had been treated with ATG previously. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation, followed by the evaluation of MDSCs via flow cytometry. The proportion of MDSCs (CD33+CD11b+HLA-DR-), especially polymorphonuclear MDSCs (CD33+CD11b+HLA-DR-CD14-CD15+) and early-stage MDSCs (CD33+CD11b+HLA-DR-Lin-), in circulation was significantly lower in SAA patients than in HD. Following ATG treatment, there was a significant increase in the percentage of MDSCs in PBMCs from SAA patients and HD. Furthermore, MDSC-mediated suppression on T-cell responses correlated with the expression of Arginase 1(Arg1) and interleukin 10 (IL-10). Arg1 and IL-10 expression in circulating MDSCs were analyzed by flow cytometry. Compared to HD, SAA patients had significantly lower Arg1 expression level in MDSCs. After ATG treatment, Arg1 expression was significantly elevated. PBMCs from SAA patients and HD were cultured with IgG or ATG in vitro and harvested on day 6. ATG could significantly increased the percentage and absolute number of MDSCs in both SAA patients and HD. Furthermore, ATG markedly augmented the expression level of Arg1 and IL-10 in MDSCs from both groups. To further confirm the effect of ATG on MDSCs' immunosuppressive function, MDSCs from both SAA patients and HD were treated with IgG or ATG and then cultured with CFSE-labeled CD3+T cells. The proliferation and activation rate of CD3+T cells and CD4+T cells were lower when cultured with ATG-treated MDSCs, indicating that the immunosuppressive function of MDSCs was significantly enhanced by ATG. To further investigate the molecular mechanisms by which ATG modulates MDSCs in SAA patients, we performed genome-wide RNA-seq of MDSCs cultured with IgG or ATG. The hierarchical clustering of differentially expressed transcripts showed 1048 upregulated and 1248 downregulated transcripts in ATG-treated MDSCs compared with IgG-treated MDSCs (fold change >2 and P value <0.05). PCA of the transcriptome revealed that MDSCs treated with IgG and ATG clustered separately, representing significant differences in overall gene expression. GO analysis revealed that enriched genes were related to immunoregulation (e.g., inhibitory MHC class I receptor activity) and cellular process (e.g., translation and apoptotic process). Immunologically relevant signaling pathways (T cell receptor signaling pathway, Th17 cell differentiation, and NF-kappa B signaling pathway) and biological processes (ferroptosis and apoptosis) were also enriched, as revealed by KEGG pathway analysis. In conclusion, our study indicates that MDSCs play a role in the immunopathogenesis of SAA, and that ATG treatment could enhance their expansion and immunosuppressive capabilities.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases. Silencing information regulator 1 (SIRT1) was demonstrated to modulate cholesterol and lipid metabolism in NAFLD. Here, a novel SIRT1 activator, E1231, was studied for its potential improvement effects on NAFLD. C57BL/6J mice were fed a high-fat and high-cholesterol diet (HFHC) for 40 weeks to create a NAFLD mouse model, and E1231 was administered by oral gavage (50 mg/kg body weight, once/day) for 4 weeks. Liver-related plasma biochemistry parameter tests, Oil Red O staining, and hematoxylin-eosin staining results showed that E1231 treatment ameliorated plasma dyslipidemia, plasma marker levels of liver damage (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)), liver total cholesterol (TC) and triglycerides (TG) contents, and obviously decreased hepatic steatosis score and NAFLD Activity Score (NAS) in the NAFLD mouse model. Western blot results showed that E1231 treatment significantly regulated lipid-metabolism-related protein expression. In particular, E1231 treatment increased SIRT1, PGC-1α, and p-AMPKα protein expression but decreased ACC and SCD-1 protein expression. Additionally, in vitro studies demonstrated that E1231 inhibited lipid accumulation and improved mitochondrial function in free-fatty-acid-challenged hepatocytes, and required SIRT1 activation. In conclusion, this study illustrated that the SIRT1 activator E1231 alleviated HFHC-induced NAFLD development and improved liver injury by regulating the SIRT1-AMPKα pathway, and might be a promising candidate compound for NAFLD treatment.

At present, the activated T cell-mediated destruction of hematopoietic stem/progenitor cells is the widely-recognized pathogenesis of aplastic anemia (AA). As the most important antigen-presenting cells in humans, dendritic cells (DCs) play an important role in the upstream link of immune pathogenesis in AA patients, so it may be the reason for the breakdown of immune tolerance in AA patients. In addition, abatacept, a cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) fusion protein, can block T cell activation and function by binding CD80 and CD86 and blocking their interaction with CD28. Thus may be a potential direction for targeting DCs to treat AA. However, relevant reports have not been seen so far. To investigate the potential role of DCs in the pathogenesis of AA, we collected peripheral blood (PB) and bone marrow (BM) and measured the proportion of DCs. Results revealed that compared with healthy donors (HD), the proportion of myeloid DC in PB and BM in AA patients was significantly higher. In contrast, the proportion of plasmacytoid DC decreased significantly. Besides, the expression of CD80 and CD86 on PB DC surface of AA patients was higher than that of HD patients. Next, we cultured monocyte-derived DC in vitro and detected its phenotype and function. We confirmed that compared with HD, the expression rates of the co-stimulatory molecule CD80 and mature molecule CD83 of DC in AA patients were significantly increased, the apoptosis level was significantly decreased, and the ability to promote proliferation of CD3 +, CD4 + and CD8 + T lymphocytes was enhanced. At the same time, AA-DC significantly enhanced the ability of CD4 + T and CD8 + T cells to express CD25 and CD69. In addition, the ability of AA-DC to promote the differentiation of Th0 cells to Th1 and Th17 cells was stronger than HD. The ability to promote the differentiation of Th0 to Treg cells was weaker than HD. To investigate the feasibility of abatacept targeting DC in the treatment of AA, we compared the proportions of CTLA-4 and found the expression of CTLA-4 on CD4 + T cells and Treg cells in PB of AA patients was significantly decreased. Then, our in vitro tests confirmed that abatacept can bind to CD80 on the surface of DC and significantly down-regulate the expression of CD80. Adding abatacept into the co-culture system of DC and CD3 + T lymphocytes significantly inhibited the proliferation of CD3 +, CD4 + and CD8 + T lymphocytes, and significantly inhibited the expression of CD25 and CD69 in CD4 + T and CD8 + T cells. In addition, the ability of DC to promote the secretion of interferon-γ by CD4 + and CD8 + T cells in AA patients was significantly weakened after the addition of abatacept, while the ability to promote the secretion of interleukin-4 by CD4 + and CD8 + T cells was significantly enhanced. Finally, we performed RNA-Seq analysis using DCs cultured in vitro from HD and AA patients and using DC from AA patients added with abatacept to uncover the possible molecular mechanism. Result showed that compared with HD, there were significant differences in the expression levels of 58 genes of DC in patients with AA, including 22 up-regulated genes and 36 down-regulated genes. Go analysis inferred that the differentially expressed genes were mainly concentrated in cell migration, cell chemotaxis, regulation of cytokine production, and immune response. Besides, KEGG pathway analysis implied that the signaling pathway of significant enrichment of differentially expressed genes was cytokine-cytokine receptor interaction, NF-κB signaling pathway, chemokine signaling pathway and IL-17 signaling pathway. Meanwhile, compared with AA patients, after adding abatacept, 1058 genes showed significant differences in expression levels. Go analysis inferred that the differentially expressed genes were mainly concentrated in immune response, cytokine secretion, regulation of hemopoiesis, cell activation, differentiation, proliferation, and adhesion. Moreover, KEGG pathway analysis implied that the signaling pathway enriched in cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, NF-κB signaling pathway and Th1, Th2 and Th17 cell differentiation. Our finding revealed the increased number and hyperfunction of DC in AA patients may be involved in the immune pathogenesis of AA. Abatacept can block T cell activation by blocking the DC surface co-stimulatory molecule CD80 and is a promising strategy for AA treatment.

OBJECTIVE:To evaluate the expression level of melatonin and its effects on immune function in aplastic anemia (AA) patients.METHODS:The enzyme-linked immunosorbent assay (ELISA) was used to detect the plasma levels of melatonin in AA patients, and the correlation between melatonin levels and laboratory indexs was analyzed. The activation, proliferation, and apoptosis of T cells from AA patients were analyzed by flow cytometry with or without melatonin in vitro.RESULTS:The plasma levels of melatonin in AA patients were significantly lower compared with healthy controls (HC) (12.23 pg/ml vs 20.04 pg/ml, P < 0.01), while the plasma melatonin levels of AA patients in remission group after immunosuppressive therapy (IST) were significantly higher than those in non-remission group (29.16 pg/ml vs 11.73 pg/ml, P =0.04). Moreover, the melatonin levels were positively correlated with platelets (r =0.49), the absolute reticulocyte count (r =0.45), and the percentage of neutrophils (r =0.43). Meanwhile, there was a negative correlation between melatonin levels and the percentages of lymphocytes (r =-0.45). The expressions of CD25 and CD69 in both CD4+ and CD8+ T cells from AA patients were remarkably inhibited by melatonin in vitro (all P < 0.05). When cultured with melatonin, the proliferation rates of both CD4+ and CD8+ T cells from AA patients were markedly suppressed (P =0.01 andP < 0.01).CONCLUSION:The plasma levels of melatonin were decreased in AA patients, which might play an important role in the mechanism of immunological abnormalities. The hyperimmune status of AA patients could be partially ameliorated by melatonin in vitro.

Abstract Background Previous studies have verified the dysfunction of mesenchymal stem cells (MSCs) for immunoregulation in acquired aplastic anemia (AA) patients. Exosomes derived from MSCs can partially substitute MSCs acting as immune regulator. Dysfunction of exosomes (Exos) derived from AA-MSC (AA-Exos) may play a key role in immunologic dissonance. Method In this study, CD3 + T cells were collected and cocultured with AA-Exos and exosomes derived from HD-MSC (HD-Exos). The proliferation, differentiation and activation of CD3 + T cells were detected to compare the immunosuppressive effects between AA-Exos and HD-Exos. An immune-mediated murine model of AA was structured to compare the therapeutic effect of AA-Exos and HD-Exos. Furthermore, total RNA including miRNA from exosomes we purified and total RNA of CD3 + T cells were extracted for RNA-seq in order to construct the miRNA–mRNA network for interactions and functional analysis. Results AA-Exos had impaired inhibition effects on CD3 + T cells in terms of cell proliferation, activation and differentiation compared with exosomes from HD-Exos. HD-Exos showed a more effective rescue of AA mice compared to AA-Exos. Importantly, we found some differentially expressed miRNA involved in immune response, such as miR-199, miR-128 and miR-486. The Gene Ontology analysis of differentially expressed genes (DEGs) revealed involvement of various cellular processes, such as lymphocyte chemotaxis, lymphocyte migration and response to interferon-gamma. The Kyoto Encyclopedia of Genes and Genomes analysis illustrated upregulation of critical pathways associated with T cell function after coculturing with AA-Exos compared with HD-Exos, such as graft-versus-host disease, Th17 cell differentiation and JAK-STAT signaling pathway. A miRNA–mRNA network was established to visualize the interaction between them. Conclusion In summary, AA-Exos had impaired immunosuppressive effect on T cells, less ability to rescue AA mice and differently expressed miRNA profile, which might partly account for the pathogenesis of AA as well as provide a new target of AA treatment.

Background: Pin1 is a member of the evolutionarily conserved peptidyl-prolyl isomerase (PPIase) family of proteins. Following phosphorylation, Pin1-catalyzed prolyl-isomerization induces conformational changes, which serve to regulate the function of many phosphorylated proteins that play important roles during oncogenesis. Thus, the inhibition of Pin1 provides a unique means of disrupting oncogenic pathways and therefore represents an appealing target for novel anticancer therapies.Methods: As Pin1 is conserved between yeast and humans, we employed budding yeast to establish a high-throughput screening method for the primary screening of Pin1 inhibitors. This effort culminated in the identification of the compounds HWH8-33 and HWH8-36. Multifaceted approaches were taken to determine the inhibition profiles of these compounds against Pin1 activity in vitro and in vivo, including an isomerization assay, surface plasmon resonance (SPR) technology, virtual docking, MTT proliferation assay, western blotting, cell cycle analysis, apoptosis analysis, immunofluorescence analysis, wound healing, migration assay, and nude mouse assay.Results:In vitro, HWH8-33 and HWH8-36 could bind to purified Pin1 and inhibited its enzyme activity; showed inhibitory effects on cancer cell proliferation; led to G2/M phase arrest, dysregulated downstream protein expression, and apoptosis; and suppressed cancer cell migration. In vivo, HWH8-33 suppressed tumor growth in the xenograft mice after oral administration for 4 weeks, with no noticeable toxicity. Together, these results show the anticancer activity of HWH8-33 and HWH8-36 against Pin1 for the first time.Conclusion: In summary, we identified two hit compounds HWH8-33 and HWH8-36, which after further structure optimization have the potential to be developed as antitumor drugs.

EDITORIAL article Front. Immunol., 07 February 2023Sec. Viral Immunology Volume 14 - 2023 | https://doi.org/10.3389/fimmu.2023.1139520

Relapsing fever due to Borrelia hermsii is characterized by recurrent bacteremia episodes. However, infection of B. hermsii, if not treated early, can spread to various organs including the central nervous system (CNS). CNS disease manifestations are commonly referred to as relapsing fever neuroborreliosis (RFNB). In the mouse model of B. hermsii infection, we have previously shown that the development of RFNB requires innate immune cells as well as T cells. Here, we found that prior to the onset of RFNB, an increase in the systemic proinflammatory cytokine response followed by sustained levels of IP-10 concurrent with the CNS disease phase. RNA sequencing analysis of the spinal cord tissue during the disease phase revealed an association of the interleukin (IL)-17 signaling pathway in RFNB. To test a possible role for IL-17 in RFNB, we compared B. hermsii infection in wild-type and IL-17A-/- mice. Although the onset of bacteremia and protective anti-B. hermsii antibody responses occurred similarly, the blood-brain barrier permeability, proinflammatory cytokine levels, immune cell infiltration in the spinal cord, and RFNB manifestations were significantly diminished in IL-17A-/- mice compared to wild-type mice. Treatment of B. hermsii-infected wild-type mice with anti-IL-17A antibody ameliorated the severity of spinal cord inflammation, microglial cell activation, and RFNB. These data suggest that the IL-17 signaling pathway plays a major role in the pathogenesis of RFNB, and IL-17A blockade may be a therapeutic modality for controlling neuroborreliosis.

Abstract Because of the emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different regions of the world, the battle with infectious coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been seesawing. Therefore, the identification of antiviral drugs is of particular importance. In order to rapidly identify inhibitors for SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), an enzyme essential for viral replication, we combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) and developed a novel sandwich-like FP screening assay. Through high-throughput screening, two hits of 3CLpro inhibitors, ginkgolic acid (GA) and anacardic acid (AA) were identified, which showed IC50 values of 11.29 ± 0.48 and 12.19 ± 0.50 μM, respectively. Their binding modes were evaluated by HPLC-Q-TOF–MS. There was no mass increase detected for SARS-CoV-2 3CLpro incubated with either GA or AA, indicating the absence of covalent adducts. The kinetic analysis clearly demonstrated that both GA and AA inhibit SARS-CoV-2 3CLpro via reversible and mixed-inhibition manner. Our results argue against conclusion that GA and AA act as irreversible and covalent inhibitors against SARS-CoV-2 3CLpro, which is based on the studies by Chen et al.

目的:比较使用重组人血小板生成素(rhTPO)与重组人白细胞介素-11(rhIL-11)预防卵巢癌化疗相关血小板减少症(CIT)的疗效和安全性.方法:入组Ⅱ度以上卵巢癌CIT患者68例,随机分为预防性注射rhTPO治疗组(34例)和预防性注射rhIL-11治疗组(34例),化疗后第一天、第二天用药.剂量:rhTPO 300 U/kg,rhIL-11 25～50 μg/kg.观察两组患者血小板下降情况、血小板开始恢复时间、血小板输注次数、治疗延迟及不良反应情况.结果:两组患者血小板下降程度均较本组预防性用药前有所改善,rhTPO组患者Ⅲ-Ⅳ度血小板下降比例较rhIL-11组患者明显减少,使用rhTPO的促血小板增殖效果明显高于rhIL-11.rhTPO治疗组血小板开始恢复时间与血小板恢复至100×109/L以上所需要时间较rhIL-11治疗组明显缩短,统计学差异显著.rhIL-11治疗组有1例患者化疗后输入外源性血小板,rhTPO治疗组无患者输注血小板.应用rhTPO及rhIL-11对患者凝血功能无明显影响.与rhIL-11相比,rhTPO较少发生不良反应,患者耐受性较好.结论:既往化疗出现Ⅱ度以上CIT的患者,预防性应用rhTPO和rhIL-11对CIT有积极的预防作用,rhTPO的疗效优于rhIL-11,并且具有良好的耐受性.