Inhibition of PTPRE suppresses tumor progression and improves sorafenib response in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis, and the efficacy of current therapeutic strategies is extremely limited in advanced diseases. Our previous study reported that protein tyrosine phosphatase receptor epsilon (PTPRE) is a promoting factor in HCC progression. In this study, our objective was to evaluate the treatment effect of PTPRE inhibitors in different HCC preclinical models. Our results indicated that the PTPRE inhibitory compound 63 (Cpd-63) inhibited tumor cell proliferation, migration, and HCC organoid growth. Mechanism research revealed that Cpd-63 could inhibit the expression of MYC and MYC targets by inhibiting the activation of SRC. Additionally, we found that Cpd-63 could improve the response of sorafenib in HCC cells. In conclusion, we demonstrated that the PTPRE inhibitors represented a potential therapeutic agent for HCC management.

Kinome-wide siRNA screen identifies a DCLK2-TBK1 oncogenic signaling axis in clear cell renal cell carcinoma.

TANK-binding kinase 1 (TBK1) is a potential therapeutic target in multiple cancers, including clear cell renal cell carcinoma (ccRCC). However, targeting TBK1 in clinical practice is challenging. One approach to overcome this challenge would be to identify an upstream TBK1 regulator that could be targeted therapeutically in cancer specifically. In this study, we perform a kinome-wide small interfering RNA (siRNA) screen and identify doublecortin-like kinase 2 (DCLK2) as a TBK1 regulator in ccRCC. DCLK2 binds to and directly phosphorylates TBK1 on Ser172. Depletion of DCLK2 inhibits anchorage-independent colony growth and kidney tumorigenesis in orthotopic xenograft models. Conversely, overexpression of DCLK2203, a short isoform that predominates in ccRCC, promotes ccRCC cell growth and tumorigenesis in vivo. Mechanistically, DCLK2203 elicits its oncogenic signaling via TBK1 phosphorylation and activation. Taken together, these results suggest that DCLK2 is a TBK1 activator and potential therapeutic target for ccRCC.

Applications of cryo-EM in drug development for STING.

STING is a critical adaptor protein in the cGAS-mediated DNA-sensing innate immune pathway. Binding of the second messenger cGAMP generated by cGAS to STING induces the high-order oligomerization and activation of the STING dimer. STING is a promising target for diseases associated with the cGAS/STING pathway such as cancer and autoimmune diseases. Recent applications of cryo-EM to STING have led to exciting progress in the understanding of its regulatory mechanism. Cryo-EM structures of STING bound to either cGAMP mimetics or novel small molecule ligands not only revealed the action mechanisms of these ligands but also suggested new ways to modulate the activity of STING for therapeutic purposes. Some of these recent studies are highlighted here.

Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly.

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We use electron cryomicroscopy to determine a 3.2 Å helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a distinct protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism shows that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.

Activation of human STING by a molecular glue-like compound.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

The intracellular monoamine oxidase-A inhibitory activity and the protective effect of small hairtail-related peptides in nerve cells (SH-SY5Y).

This study was to investigate the inhibitory activity of small hairtail-related peptides (VFEVFW, LPNSLYQQ, LPNSLYQK, and FADAME) on intracellular monoamine oxidase-A (MAO-A) and their protective effects in a cell model. Specifically, the inhibition activity in SH-SY5Y cells indicated that VFEVFW and LPNSLYQK reduced ∼50% of MAO-A activity in cells, at 0.5 m m. The survival experiment demonstrated that the toxic effect of dexamethasone (DEX) on cells can be significantly alleviated in the presence of peptides, and these peptides can restore (>20%) the mitochondrial membrane potential of SH-SY5Y cells reduced by DEX. Circular dichroism displayed that peptides affected the secondary structure of MAO-A in a concentration-dependent manner. Finally, the real-time quantitative polymerase chain reaction assay revealed that the MAO-A inhibitory activity of the peptides was associated with the upregulation of brain derived neurotrophic factor/cAMP (Cyclic adenosine monophosphate) response element binding protein)/B-cell lymphoma-2 mRNA levels.

Structural basis of insulin fibrillation.

Insulin is a hormone responsible for maintaining normal glucose levels by activating insulin receptor (IR) and is the primary treatment for diabetes. However, insulin is prone to unfolding and forming cross-ß fibers. Fibrillation complicates insulin storage and therapeutic application. Molecular details of insulin fibrillation remain unclear, hindering efforts to prevent fibrillation process. Here, we characterized insulin fibrils using cryo-electron microscopy (cryo-EM), showing multiple forms that contain one or more of the protofilaments containing both the A and B chains of insulin linked by disulfide bonds. We solved the cryo-EM structure of one of the fibril forms composed of two protofilaments at 3.2-Å resolution, which reveals both the ß sheet conformation of the protofilament and the packing interaction between them that underlie the fibrillation. On the basis of this structure, we designed several insulin mutants that display reduced fibrillation while maintaining native IR signaling activity. These designed insulin analogs may be developed into more effective therapeutics for type 1 diabetes.

Anticancer potential of Bacillus coagulans MZY531 on mouse H22 hepatocellular carcinoma cells via anti-proliferation and apoptosis induction.

Bacillus coagulans have recently revealed its anticancer effects, but few investigations are available on their effects on liver cancer proliferation, and the precise mechanism to mark its impact on apoptosis-related signaling pathways has yet to be elucidated. The aim of this study was to evaluate the anti-proliferative effect of B. coagulans MZY531 and apoptosis induction in the mouse H22 hepatocellular carcinoma cell line. The anti-proliferative activity of B. coagulans MZY531 was evaluated by Cell Counting Kit-8 (CCK-8) assay, and cell apoptosis was revealed with Terminal Deoxynucleotidyl Transferase (TDT)-mediated dUTP Nick-End Labeling (TUNEL) staining and flow cytometric analysis. The expressions of apoptosis-related protein were determined by western blot analysis. The CCK-8 assay revealed that B. coagulans MZY531 inhibited the H22 cells proliferation in a concentration-dependent manner. TUNEL staining revealed an increased apoptosis rate in H22 cells following intervention with B. coagulans MZY531. Furthermore, flow cytometric analysis showed that B. coagulans MZY531 treatment (MOI = 50 and 100) significantly alleviated the H22 cells apoptosis compared with the control group. Western blot analysis found B. coagulans MZY531 significantly decreased level of phospho-PI3K (p-PI3K), phospho-AKT (p-AKT), and phospho-mTOR (p-mTOR) compared with the control group. Furthermore, H22 cells treatment with B. coagulans MZY531 enhanced the expression of caspase-3 and Bax and jeopardized the expression of Bcl-2. Taken together, apoptosis induction and cell proliferation inhibition via PI3K/AKT/mTOR and Bax/Bcl-2/Caspase-3 pathway are promising evidence to support B. coagulans MZY531 as a potential therapeutic agent for cancer.

Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly.

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We determined a 3.2 Å electron cryomicroscopy helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a unique protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism revealed that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.

Bacillus coagulans MZY531 alleviates intestinal mucosal injury in immunosuppressive mice via modulating intestinal barrier, inflammatory response, and gut microbiota.

Bacillus coagulans has a potential role in improving intestinal injury. However, the specific mechanism is still unclear. In this study, the protective effect of B. coagulans MZY531 on intestinal mucosa injury in cyclophosphamide (CYP)-induced immunosuppressed mice were investigated. The results indicated that the immune organ (thymus and spleen) indices of B. coagulans MZY531 treatment groups were significantly increased compared to the CYP group. B. coagulans MZY531 administration promotes the expression of immune proteins (IgA, IgE, IgG, and IgM). B. coagulans MZY531 could upregulate the ileum levels of IFN-γ, IL-2, IL-4, and IL-10 in immunosuppressed mice. Moreover, B. coagulans MZY531 restores the villus height and crypt depth of the jejunum and alleviates injury of intestinal endothelial cells caused by CYP. Furthermore, the western blotting results showed that B. coagulans MZY531 ameliorated CYP-induced intestinal mucosal injury and inflammatory via up-regulates the ZO-1 pathway and down-regulates the expression of the TLR4/MyD88/NF-κB pathway. After treatment with B. coagulans MZY531, the relative abundance of Firmicutes phylum was dramatically increased, as well as the genera of Prevotella and Bifidobacterium, and reducing harmful bacteria. These findings suggested that B. coagulans MZY531 has a potential immunomodulatory activity on chemotherapy-induced immunosuppression.

Tubular cell transcriptional intermediary factor 1γ deficiency exacerbates kidney injury-induced tubular cell polyploidy and fibrosis.

Tubulointerstitial fibrosis is considered the final convergent pathway of progressive chronic kidney diseases (CKD) regardless of etiology. However, mechanisms underlying kidney injury-induced fibrosis largely remain unknown. Recent studies have indicated that transcriptional intermediary factor 1γ (TIF1γ) inhibits the progression of fibrosis in other organs. Here, we found that TIF1γ was highly expressed in the cytoplasm and nucleus of the kidney proximal tubule. Interestingly, we found tubular TIF1γ expression was decreased in patients with CKD, including those with diabetes, hypertension, and IgA nephropathy, and in mouse models with experimental kidney fibrosis (unilateral ureteral obstruction [UUO], folic acid nephropathy [FAN], and aristolochic acid-induced nephrotoxicity). Tubule-specific knock out of TIF1γ in mice exacerbated UUO- and FAN-induced tubular cell polyploidy and subsequent fibrosis, whereas overexpression of kidney TIF1γ protected mice against kidney fibrosis. Mechanistically, in tubular epithelial cells, TIF1γ exerted an antifibrotic role via transforming growth factor-ß (TGF-ß)-dependent and -independent signaling. TIF1γ hindered TGF-ß signaling directly by inhibiting the formation and activity of the transcription factor Smad complex in tubular cells, and we discovered that TIF1γ suppressed epidermal growth factor receptor (EGFR) signaling upstream of TGF-ß signaling in tubular cells by ubiquitylating EGFR at its lysine 851/905 sites thereby promoting EGFR internalization and lysosomal degradation. Pharmacological inhibition of EGFR signaling attenuated exacerbated polyploidization and the fibrotic phenotype in mice with tubule deletion of TIF1γ. Thus, tubular TIF1γ plays an important role in kidney fibrosis by suppressing profibrotic EGFR and TGF-ß signaling. Hence, our findings suggest that maintaining homeostasis of tubular TIF1γ may be a new therapeutic option for treating tubulointerstitial fibrosis and subsequent CKD.

Trimethoprim-sulfamethoxazole prevents interstitial pneumonitis in B-cell lymphoma patients receiving chemotherapy: a propensity score matching analysis.

B-cell lymphoma is the most prevalent type of non-Hodgkin lymphoma, for which the standard treatment regimen includes rituximab combined with CHOP. However, some patients may develop interstitial pneumonitis (IP), which can be caused by various factors; one of the most important factors is Pneumocystis jirovecii. It is crucial to investigate the pathophysiology of IP and implement preventive measures since IP can be fatal for some people. The data were collected from the First Affiliated Hospital, Zhejiang University School of Medicine, where patients with B-cell lymphoma received the R-CHOP/R-CDOP regimen with or without prophylactic use of trimethoprim-sulfamethoxazole (TMP-SMX). Multivariable logistic regression and propensity score matching (PSM) were used to investigate any potential association. Eight hundred thirty-one patients with B-cell lymphoma were classified into two groups: the non-prophylaxis group without TMP-SMX (n=699) and the prophylaxis group with TMP-SMX (n = 132). IP occurred in 66 patients (9.4%, all in the non-prophylaxis group), with an onset median of three cycles of chemotherapy. Multiple logistic regression analysis demonstrated that IP incidence was associated with pegylated liposome doxorubicin (OR=3.29, 95% CI 1.84-5.90, P<0.001). After utilizing a 1:1 matching algorithm for PSM, 90 patients from each group were obtained. There was a statistical difference between the two cohorts in the IP incidence (non-prophylaxis 12.2% vs prophylaxis 0.0%, P <0.001). The prophylactic use of TMP-SMX could prevent the occurrence of IP whose risk factor was pegylated liposome doxorubicin after chemotherapy for B-cell lymphoma.

1H NMR-based urinary metabolic analysis of high-dose cyclophosphamide-induced toxicity in mice.

Cyclophosphamide (CP) is widely used in clinical fields. Beside its therapeutic effects, CP shows toxicity depending on dose and administration schedule. In this study, the urinary metabolic profiles were investigated in mice intraperitoneally injected with high-dose CP (150 mg/kg body weight) once a week over four weeks using nuclear magnetic resonance (NMR)-based metabolomics. Twenty-six metabolites were identified as potential biomarkers by multivariate statistical analysis. A decrease in isoleucine, alanine, N-acetylglutamic acid, proline, methionine, valine, phenylacetylglulamine, dimethylamine, hippurate, acetic acid, lactate, α-oxoglutarate, citrate, malonic acid, creatinine, niacin, ß-hydroxybutyrate, and betaine, whereas an increase in leucine, glutamate, glycine, taurine, phenylacetylglycine, glucose, creatine, and choline were observed in the urine of high-dose CP-treated mice. Metabolites related to amino acid metabolism, energy metabolism, and gut microbial metabolism were changed markedly in the urine. Further metabolic pathway analysis suggested that seven metabolic pathways, including alanine, aspartate, and glutamate metabolism, arginine biosynthesis, glyoxylate, and dicarboxylate metabolism, glycine, serine and threonine metabolism, d-glutamine and d-glutamate metabolism, arginine, and proline metabolism, citrate cycle, as well as the gut microbiota metabolism, were significantly involved in response to high-dose CP treatment. These findings help to predict the toxicity of CP and understand the biological mechanism of the toxicity of CP.

Inhibition of FAM46/TENT5 activity by BCCIPα adopting a unique fold.

The FAM46 (also known as TENT5) proteins are noncanonical poly(A) polymerases (PAPs) implicated in regulating RNA stability. The regulatory mechanisms of FAM46 are poorly understood. Here, we report that the nuclear protein BCCIPα, but not the alternatively spliced isoform BCCIPß, binds FAM46 and inhibits their PAP activity. Unexpectedly, our structures of the FAM46A/BCCIPα and FAM46C/BCCIPα complexes show that, despite sharing most of the sequence and differing only at the C-terminal portion, BCCIPα adopts a unique structure completely different from BCCIPß. The distinct C-terminal segment of BCCIPα supports the adoption of the unique fold but does not directly interact with FAM46. The ß sheets in BCCIPα and FAM46 pack side by side to form an extended ß sheet. A helix-loop-helix segment in BCCIPα inserts into the active site cleft of FAM46, thereby inhibiting the PAP activity. Our results together show that the unique fold of BCCIPα underlies its interaction with and functional regulation of FAM46.

Case Report: Parvovirus B19 infection complicated by hemophagocytic lymphohistiocytosis in a heart-lung transplant patient.

Immunosuppressed patients can contract parvovirus B19, and some may experience hemophagocytic lymphohistiocytosis (HLH). Herein, we describe the first report of hemophagocytic lymphohistiocytosis in a heart-lung transplant patient with concomitant parvovirus B19 infection. The patient was treated with intravenous immune globulin (IVIG) and the features of HLH were remission. This instance emphasizes the significance of parvovirus B19 monitoring in transplant patients with anemia; if HLH complicates the situation, IVIG may be an adequate remedy. Finally, a summary of the development in diagnosing and managing parvovirus B19 infection complicated by HLH is provided.

A nomogram based on LI-RADS features, clinical indicators and quantitative contrast-enhanced MRI parameters for predicting glypican-3 expression in hepatocellular carcinoma.

Purpose: Noninvasively assessing the tumor biology and microenvironment before treatment is greatly important, and glypican-3 (GPC-3) is a new-generation immunotherapy target for hepatocellular carcinoma (HCC). This study investigated the application value of a nomogram based on LI-RADS features, quantitative contrast-enhanced MRI parameters and clinical indicators in the noninvasive preoperative prediction of GPC-3 expression in HCC. Methods and materials: We retrospectively reviewed 127 patients with pathologically confirmed solitary HCC who underwent Gd-EOB-DTPA MRI examinations and related laboratory tests. Quantitative contrast-enhanced MRI parameters and clinical indicators were collected by an abdominal radiologist, and LI-RADS features were independently assessed and recorded by three trained intermediate- and senior-level radiologists. The pathological and immunohistochemical results of HCC were determined by two senior pathologists. All patients were divided into a training cohort (88 cases) and validation cohort (39 cases). Univariate analysis and multivariate logistic regression were performed to identify independent predictors of GPC-3 expression in HCC, and a nomogram model was established in the training cohort. The performance of the nomogram was assessed by the area under the receiver operating characteristic curve (AUC) and the calibration curve in the training cohort and validation cohort, respectively. Results: Blood products in mass, nodule-in-nodule architecture, mosaic architecture, contrast enhancement ratio (CER), transition phase lesion-liver parenchyma signal ratio (TP-LNR), and serum ferritin (Fer) were independent predictors of GPC-3 expression, with odds ratios (ORs) of 5.437, 10.682, 5.477, 11.788, 0.028, and 1.005, respectively. Nomogram based on LI-RADS features (blood products in mass, nodule-in-nodule architecture and mosaic architecture), quantitative contrast-enhanced MRI parameters (CER and TP-LNR) and clinical indicators (Fer) for predicting GPC-3 expression in HCC was established successfully. The nomogram showed good discrimination (AUC of 0.925 in the training cohort and 0.908 in the validation cohort) and favorable calibration. The diagnostic sensitivity and specificity were 76.9% and 92.3% in the training cohort, 76.8% and 93.8% in the validation cohort respectively. Conclusion: The nomogram constructed from LI-RADS features, quantitative contrast-enhanced MRI parameters and clinical indicators has high application value, can accurately predict GPC-3 expression in HCC and may help noninvasively identify potential patients for GPC-3 immunotherapy.

Isolation and identification of dipeptidyl peptidase-IV inhibitory peptides from Sacha inchi meal.

BACKGROUND: Sacha inchi meal (SIM) is a by-product of oil processing. Our previous studies showed that SIM hydrolysates exhibited dipeptidyl peptidase-IV (DPP-IV) inhibition activity. The objective of the present work was to identify and characterize the bioactive peptides from protein hydrolysates of SIM; enzyme kinetics and peptide-enzyme interaction were also investigated. RESULTS: From SIM hydrolysates, ten peptides responsible for the activity were identified: GPSRGF (GF-6), FPILSPDPA (FA-9), APYRRGGKI (AI-9), WPYH (WH-4), DPATWLALPT (DT-10), NPEDEFRQQ (NQ-9), APESKPVGV (AV-9), LEWRDR (LR-6), APVYWVQ (AQ-7) and LLMWPY (LY-6). The IC50 values of five peptides (GF-6, WH-4, AQ-7, AV-9 and LY-6) with better inhibitory activity on DPP-IV were within the range of 23.43-128.40 µmol L-1 . AQ-7 had the best activity, with an IC50 value of 23.43 µmol L-1 . Enzyme kinetics indicated the presence of various inhibition types (mixed, non-competitive and competitive). Isothermal titration microcalorimetry showed that the main forces of the binding sites between peptide (GF-6 or AQ-7) and DPP-IV were hydrogen bond, hydrophobic interaction and van der Waals force. The key residues involved in peptide-enzyme interaction were determined by molecular docking. Furthermore, at a concentration of 800 µmol L-1 , GF-6 was found to significantly increase the glucose consumption in insulin-resistant HepG2 cells (P < 0.05) compared with the model group. CONCLUSION: Sacha inchi meal-derived peptides displayed potent DPP-IV inhibition activity and could be used in the health food industry and as lead compounds for diabetes therapy. © 2023 Society of Chemical Industry.

Combined therapy of prednisone and mTOR inhibitor sirolimus for treating retroperitoneal fibrosis.

OBJECTIVES: Retroperitoneal fibrosis (RPF) is a rare autoimmune disease with fibrous tissue growth and inflammation in retroperitoneum. Its current treatments involve long-term uptake of glucocorticoids (e.g., prednisone) for controlling inflammation; however, side effects are common. We strived for an improved therapy for fibrosis remission while reducing side effects. METHODS: We surveyed gene-disease-drug databases and discovered that mammalian target of rapamycin (mTOR) was a key signalling protein in RPF and the mTOR inhibitor compound sirolimus affected many RPF pathways. We designed a therapy combining a gradual reduction of prednisone with a long-term, stable dosage of sirolimus. We then implemented a single-arm clinical trial and assessed the effects in eight RPF patients at 0, 12 and 48 weeks of treatment by measuring fibrous tissue mass by CT, markers of inflammation and kidney functions by lab tests, immune cell profiles by flow cytometry and plasma inflammatory proteins by Olink proteomics. RESULTS: With the combined therapy, fibrous tissue shrunk about by half, markers of acute inflammation reduced by 70% and most patients with abnormal kidney functions had them restored to normal range. Molecularly, fibrosis-related T cell subsets, including TH2, TH17 and circulating TFH cells, were reduced and tumour necrosis factor and related cytokines restored to healthy levels. No severe long-term side effects were observed. CONCLUSIONS: Our combined therapy resulted in significant fibrosis remission and an overall regression of the immune system towards healthy states, while achieving good tolerance. We concluded that this new therapy had the potential to replace the steroid monotherapy for treating RPF.