Targeting ESE3/EHF With Nifurtimox Inhibits CXCR2+ Neutrophil Infiltration and Overcomes Pancreatic Cancer Resistance to Chemotherapy and Immunotherapy.

BACKGROUND & AIMS: Because pancreatic cancer responds poorly to chemotherapy and immunotherapy, it is necessary to identify novel targets and compounds to overcome resistance to treatment. METHODS: This study analyzed genomic single nucleotide polymorphism sequencing, single-cell RNA sequencing, and spatial transcriptomics. Ehf-knockout mice, KPC (LSL-KrasG12D/+, LSL-Trp53R172H/+ and Pdx1-Cre) mice, CD45.1+ BALB/C nude mice, and CD34+ humanized mice were also used as subjects. Multiplexed immunohistochemistry and flow cytometry were performed to investigate the proportion of tumor-infiltrated C-X-C motif chemokine receptor 2 (CXCR2)+ neutrophils. In addition, multiplexed cytokines assays and chromatin immunoprecipitation assays were used to examine the mechanism. RESULTS: The TP53 mutation-mediated loss of tumoral EHF increased the recruitment of CXCR2+ neutrophils, modulated their spatial distribution, and further induced chemo- and immunotherapy resistance in clinical cohorts and preclinical syngeneic mice models. Mechanistically, EHF deficiency induced C-X-C motif chemokine ligand 1 (CXCL1) transcription to enhance in vitro and in vivo CXCR2+ neutrophils migration. Moreover, CXCL1 or CXCR2 blockade completely abolished the effect, indicating that EHF regulated CXCR2+ neutrophils migration in a CXCL1-CXCR2-dependent manner. The depletion of CXCR2+ neutrophils also blocked the in vivo effects of EHF deficiency on chemotherapy and immunotherapy resistance. The single-cell RNA-sequencing results of PDAC treated with Nifurtimox highlighted the therapeutic significance of Nifurtimox by elevating the expression of tumoral EHF and decreasing the weightage of CXCL1-CXCR2 pathway within the microenvironment. Importantly, by simultaneously inhibiting the JAK1/STAT1 pathway, it could significantly suppress the recruitment and function of CXCR2+ neutrophils, further sensitizing PDAC to chemotherapy and immunotherapies. CONCLUSIONS: The study demonstrated the role of EHF in the recruitment of CXCR2+ neutrophils and the promising role of Nifurtimox in sensitizing pancreatic cancer to chemotherapy and immunotherapy.

Triple-negative breast cancer survival prediction using artificial intelligence through integrated analysis of tertiary lymphoid structures and tumor budding.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly heterogeneous and clinically aggressive disease. Accumulating evidence indicates that tertiary lymphoid structures (TLSs) and tumor budding (TB) are significantly correlated with the outcomes of patients who have TNBC, but no integrated TLS-TB profile has been established to predict their survival. The objective of this study was to investigate the relationship between the TLS/TB ratio and clinical outcomes of patients with TNBC using artificial intelligence (AI)-based analysis. METHODS: The infiltration levels of TLSs and TB were evaluated using hematoxylin and eosin staining, immunohistochemistry staining, and AI-based analysis. Various cellular subtypes within TLS were determined by multiplex immunofluorescence. Subsequently, the authors established a nomogram model, conducted calibration curve analyses, and performed decision curve analyses using R software. RESULTS: In both the training and validation cohorts, the antitumor/protumor model established by the authors demonstrated a positive correlation between the TLS/TB index and the overall survival (OS) and relapse-free survival (RFS) of patients with TNBC. Notably, patients who had a high percentage of CD8-positive T cells, CD45RO-positive T cells, or CD20-positive B cells within the TLSs experienced improved OS and RFS. Furthermore, the authors developed a comprehensive TLS-TB profile nomogram based on the TLS/TB index. This novel model outperformed the classical tumor-lymph node-metastasis staging system in predicting the OS and RFS of patients with TNBC. CONCLUSIONS: A novel strategy for predicting the prognosis of patients with TNBC was established through integrated AI-based analysis and a machine-learning workflow. The TLS/TB index was identified as an independent prognostic factor for TNBC. This nomogram-based TLS-TB profile would help improve the accuracy of predicting the prognosis of patients who have TNBC.

Noteworthy Consensus Effects of D/E Residues in Umami Peptides Used for Designing the Novel Umami Peptides.

Aspartic acid (D) and glutamic acid (E) play vital roles in the umami peptides. To understand their exact mechanism of action, umami peptides were collected and cut into 1/2/3/4 fragments. Connecting D/E to the N/C-termini of the fragments formed D/E consensus effect groups (DEEGs), and all fragments containing DEEG were summarized according to the ratio and ranking obtained in the above four situations. The interaction patterns between peptides in DEEG and T1R1/T1R3-VFD were compared by statistical analysis and molecular docking, and the most conservative contacts were found to be HdB_277_ARG and HdB_148_SER. The molecular docking score of the effector peptides significantly dropped compared to that of their original peptides (-1.076 ± 0.658 kcal/mol, p value < 0.05). Six types of consensus fingerprints were set according to the Top7 contacts. The exponential of relative umami was linearly correlated with ΔGbind (R2 = 0.961). Under the D/E consensus effect, the electrostatic effect of the umami peptide was improved, and the energy gap between the highest occupied molecular orbital-the least unoccupied molecular orbital (HOMO-LUMO) was decreased. The shortest path map showed that the peptides had similar T1R1-T1R3 recognition pathways. This study helps to reveal umami perception rules and provides support for the efficient screening of umami peptides based on the material richness in D/E sequences.

TastePeptides-EEG: An Ensemble Model for Umami Taste Evaluation Based on Electroencephalogram and Machine Learning.

In the field of food, the sensory evaluation of food still relies on the results of manual sensory evaluation, but the results of human sensory evaluation are not universal, and there is a problem of speech fraud. This work proposed an electroencephalography (EEG)-based analysis method that effectively enables the identification of umami/non-umami substances. First, the key features were extracted using percentage conversion, standardization, and significance screening, and based on these features, the top four models were selected from 19 common binary classification algorithms as submodels. Then, the support vector machine (SVM) algorithm was used to fit the outputs of these four submodels to establish TastePeptides-EEG. The validation set of the model achieved a judgment accuracy of 90.2%, and the test set achieved a judgment accuracy of 77.8%. This study discovered the frequency change of α wave in umami taste perception and found the frequency response delay phenomenon of the F/RT/C area under umami taste stimulation for the first time. The model is published at www.tastepeptides-meta.com/TastePeptides-EEG, which is convenient for relevant researchers to speed up the analysis of umami perception and provide help for the development of the next generation of brain-computer interfaces for flavor perception.

Irbesartan overcomes gemcitabine resistance in pancreatic cancer by suppressing stemness and iron metabolism via inhibition of the Hippo/YAP1/c-Jun axis.

BACKGROUND: Chemoresistance is the main reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Thus, there is an urgent need to screen out new targets and compounds to reverse chemotherapeutic resistance. METHODS: We established a bio-bank of human PDAC organoid models, covering a representative range of PDAC tumor subtypes. We screened a library of 1304 FDA-approved compounds to identify candidates efficiently overcoming chemotherapy resistance. The effects of the compounds were evaluated with a CellTiter-Glo-3D assay, organoid apoptosis assay and in vivo patient-derived xenograft (PDX), patient-derived organoid (PDO) and LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) genetically engineered mouse models. RNA-sequencing, genome editing, sphere formation assays, iron assays and luciferase assays were conducted to elucidate the mechanism. RESULTS: High-throughput drug screening of chemotherapy-resistant PDOs identified irbesartan, an angiotensin ‖ type 1 (AT1) receptor antagonist, which could synergistically enhance the ability of chemotherapy to kill PDAC cells. In vitro and in vivo validation using PDO, PDX and KPC mouse models showed that irbesartan efficiently sensitized PDAC tumors to chemotherapy. Mechanistically, we found that irbesartan decreased c-Jun expression by inhibiting the Hippo/YAP1 pathway and further overcame chemotherapy resistance in PDAC. We also explored c-Jun, a potential target of irbesartan, which can transcriptionally upregulate the expression of key genes involved in stemness maintenance (SOX9/SOX2/OCT4) and iron metabolism (FTH1/FTL/TFRC). More importantly, we observed that PDAC patients with high levels of c-Jun expression demonstrated poor responses to the current standard chemotherapy regimen (gemcitabine plus nab-paclitaxel). Moreover, patients with PDAC had significant survival benefits from treatment with irbesartan plus a standard chemotherapy regimen in two-center retrospective clinical cohorts and patients with high c-Jun expression exhibited a better response to combination chemotherapy. CONCLUSIONS: Irbesartan could be used in combination with chemotherapy to improve the therapeutic efficacy in PDAC patients with high levels of c-Jun expression. Irbesartan effectively inhibited chemotherapy resistance by suppressing the Hippo/YAP1/c-Jun/stemness/iron metabolism axis. Based on our findings, we are designing an investigator-initiated phase II clinical trial on the efficacy and safety of irbesartan plus a standard gemcitabine/nab-paclitaxel regimen in the treatment of patients with advanced III/IV staged PDAC and are hopeful that we will observe patient benefits.

Survival outcomes of neoadjuvant therapy followed by radical resection versus upfront surgery for stage I-III pancreatic ductal adenocarcinoma: a retrospective cohort study.

BACKGROUND: Neoadjuvant therapy remains controversial in treating resectable pancreatic ductal adenocarcinoma (PDAC) patients. This study aims to assess the impact of neoadjuvant therapy on survival in patients with PDAC according to their clinical stage. METHODS: Patients with resected clinical Stage I-III PDAC from 2010 to 2019 were identified in the surveillance, epidemiology, and end results database. A propensity score matching method was utilized within each stage to reduce potential selection bias between patients who underwent neoadjuvant chemotherapy followed by surgery and patients who underwent upfront surgery. An overall survival (OS) analysis was performed using the Kaplan-Meier method and a multivariate Cox proportional hazards model. RESULTS: A total of 13 674 patients were included in the study. The majority of the patients ( N =10 715, 78.4%) underwent upfront surgery. Patients receiving neoadjuvant therapy followed by surgery had significantly longer OS than those with upfront surgery. Subgroup analysis revealed that the neoadjuvant chemoradiotherapy group's OS is comparable to neoadjuvant chemotherapy. In clinical Stage IA PDAC, there was no difference in survival between the neoadjuvant treatment and upfront surgery groups before or after matching. In stage IB-III patients, neoadjuvant therapy followed by surgery improved OS before and after matching compared to upfront surgery. The results revealed the same OS benefits using the multivariate Cox proportional hazards model. CONCLUSION: Neoadjuvant therapy followed by surgery could improve OS over upfront surgery in Stage IB-III PDAC but did not provide a significant survival advantage in Stage IA PDAC.

Conserved Sites and Recognition Mechanisms of T1R1 and T2R14 Receptors Revealed by Ensemble Docking and Molecular Descriptors and Fingerprints Combined with Machine Learning.

Taste peptides, as an important component of protein-rich foodstuffs, potentiate the nutrition and taste of food. Thereinto, umami- and bitter-taste peptides have been ex tensively reported, while their taste mechanisms remain unclear. Meanwhile, the identification of taste peptides is still a time-consuming and costly task. In this study, 489 peptides with umami/bitter taste from TPDB (http://tastepeptides-meta.com/) were collected and used to train the classification models based on docking analysis, molecular descriptors (MDs), and molecular fingerprints (FPs). A consensus model, taste peptide docking machine (TPDM), was generated based on five learning algorithms (linear regression, random forest, gaussian naive bayes, gradient boosting tree, and stochastic gradient descent) and four molecular representation schemes. Model interpretive analysis showed that MDs (VSA_EState, MinEstateIndex, MolLogP) and FPs (598, 322, 952) had the greatest impact on the umami/bitter prediction of peptides. Based on the consensus docking results, we obtained the key recognition modes of umami/bitter receptors (T1Rs/T2Rs): (1) residues 107S-109S, 148S-154T, 247F-249A mainly form hydrogen bonding contacts and (2) residues 153A-158L, 163L, 181Q, 218D, 247F-249A in T1R1 and 56D, 106P, 107V, 152V-156F, 173K-180F in T2R14 constituted their hydrogen bond pockets. The model is available at http://www.tastepeptides-meta.com/yyds.

Artificial intelligence-based comprehensive analysis of immune-stemness-tumor budding profile to predict survival of patients with pancreatic adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy. CD8+ T cells, cancer stem cells (CSCs), and tumor budding (TB) have been significantly correlated with the outcome of patients with PDAC, but the correlations have been independently reported. In addition, no integrated immune-CSC-TB profile for predicting survival in patients with PDAC has been established. METHODS: Multiplexed immunofluorescence and artificial intelligence (AI)-based comprehensive analyses were used for quantification and spatial distribution analysis of CD8+ T cells, CD133+ CSCs, and TB. In vivo humanized patient-derived xenograft (PDX) models were established. Nomogram analysis, calibration curve, time-dependent receiver operating characteristic curve, and decision curve analyses were performed using R software. RESULTS: The established 'anti-/pro-tumor' models showed that the CD8+ T cell/TB, CD8+ T cell/CD133+ CSC, TB-adjacent CD8+ T cell, and CD133+ CSC-adjacent CD8+ T cell indices were positively associated with survival of patients with PDAC. These findings were validated using PDX-transplanted humanized mouse models. An integrated nomogram-based immune-CSC-TB profile that included the CD8+ T cell/TB and CD8+ T cell/CD133+ CSC indices was established and shown to be superior to the tumor-node-metastasis stage model in predicting survival of patients with PDAC. CONCLUSIONS: 'Anti-/pro-tumor' models and the spatial relationship among CD8+ T cells, CSCs, and TB within the tumor microenvironment were investigated. Novel strategies to predict the prognosis of patients with PDAC were established using AI-based comprehensive analysis and machine learning workflow. The nomogram-based immune-CSC-TB profile can provide accurate prognosis prediction for patients with PDAC.

SDCBP promotes pancreatic cancer progression by preventing YAP1 from ß-TrCP-mediated proteasomal degradation.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal tumour with limited treatment options. Here, we identified syndecan binding protein (SDCBP), also known as syntenin1, as a novel targetable factor in promoting PDAC tumour progression. We also explored a therapeutic strategy for suppressing SDCBP expression. DESIGN: We used samples from patients with PDAC, human organoid models, LSL-KrasG12D/+mice, LSL-Trp53R172H/+ and Pdx1-Cre (KPC) mouse models, and PDX mouse models. Immunostaining, colony formation assay, ethynyl-2-deoxyuridine incorporation assay, real-time cell analysis, cell apoptosis assay, automated cell tracking, invadopodia detection and gelatin degradation assays, coimmunoprecipitation, and pull-down assays were performed in this study. RESULTS: The median overall survival and recurrence-free survival rates in the high-SDCBP group were significantly shorter than those in the low-SDCBP group. In vitro and in vivo studies have demonstrated that SDCBP promotes PDAC proliferation and metastasis. Mechanically, SDCBP inhibits CK1δ/ε-mediated YAP-S384/S387 phosphorylation, which further suppresses ß-TrCP-mediated YAP1 ubiquitination and proteasome degradation by directly interacting with YAP1. SDCBP interacts with the TAD domain of YAP1, mainly through its PDZ1 domain. Preclinical KPC mouse cohorts demonstrated that zinc pyrithione (ZnPT) suppresses PDAC tumour progression by suppressing SDCBP. CONCLUSIONS: SDCBP promotes the proliferation and metastasis of PDAC by preventing YAP1 from ß-TrCP-mediated proteasomal degradation. Therefore, ZnPT could be a promising therapeutic strategy to inhibit PDAC progression by suppressing SDCBP.

Gold nanobipyramid@copper sulfide nanotheranostics for image-guided NIR-II photo/chemodynamic cancer therapy with enhanced immune response.

Photothermal therapy (PTT), photodynamic therapy (PDT), and chemodynamic therapy (CDT) can cause cancer cell death through an immunogenic process. However, the study of second near-infrared window (NIR-II)-triggered PTT and PDT combined with CDT to induce an immune response has not been recently reported. Here, we integrated gold nanobipyramids and copper sulfide in a core/shell architecture (AuNBP@CuS). The material displays both photodynamic and photothermal properties under irradiation with a NIR-II laser. The released Cu2+ from CuS under an acidic tumor microenvironment can be converted to Cu+ by glutathione following a Fenton-like reaction with hydrogen peroxide to generate highly toxic hydroxyl radicals in the tumor region. Both in vitro and in vivo results demonstrated that such multifunctional nanoplatforms could achieve enhanced efficiency for image-guided tumor suppression based on the NIR-II photo/chemodynamic therapy. We found that damage-associated molecular pattern molecules such as adenosine triphosphate, pre-apoptotic calreticulin, and high mobility group box-1 in dying cells induced by the NIR-II photo/chemodynamic therapy could simultaneously trigger adaptive immune responses. This is the first report revealing that NIR-II photo/chemodynamic therapy based on AuNBP@CuS had promising performance on tumor suppressor with an effective immunogenic cell death process. STATEMENT OF SIGNIFICANCE: 1. AuNBP@CuS displays both NIR-II photodynamic and photothermal properties. 2. Cu+ following a Fenton-like reaction to generate highly toxic hydroxyl radicals. 3. The NIR-II photo/chemodynamic therapy can trigger adaptive immune responses. 4. Such multifunctional nanoplatforms could achieve enhanced efficiency for tumor suppression.

A TastePeptides-Meta system including an umami/bitter classification model Umami_YYDS, a TastePeptidesDB database and an open-source package Auto_Taste_ML.

Taste peptides with umami/bitterness play a role in food attributes. However, the taste mechanisms of peptides are not fully understood, and the identification of these peptides is time-consuming. Here, we created a taste peptide database by collecting the reported taste peptide information. Eight key molecular descriptors from di/tri-peptides were selected and obtained by modeling screening. A gradient boosting decision tree model named Umami_YYDS (89.6% accuracy) was established by data enhancement, comparison algorithm and model optimization. Our model showed a great prediction performance compared to other models, and its outstanding ability was verified by sensory experiments. To provide a convenient approach, we deployed a prediction website based on Umami_YYDS and uploaded the Auto_Taste_ML machine learning package. In summary, we established the system TastePeptides-Meta, containing a taste peptide database TastePeptidesDB an umami/bitter taste prediction model Umami_YYDS and an open-source machine learning package Auto_Taste_ML, which were helpful for rapid screening of umami peptides.

All-In-One Second Near-Infrared Light-Responsive Drug Delivery System for Synergistic Chemo-Photothermal Therapy.

Light-responsive nanocarrier-based drug delivery systems (NDDSs), due to their unique advantages such as safety, minimal cross-reaction, and spatiotemporal precision, have received wide attention. Notably, second near-infrared (NIR-II) light, which has a high penetration depth for manipulating NDDSs to release drugs, is in high demand. Herein, polyethylene glycol (PEG)-modified hollow CuxS nanoparticles (NPs) are developed as an all-in-one NIR-II light-responsive NDDS for synergistic chemo-photothermal therapy. First, CuxS-PEG NPs were prepared under mild conditions by using Cu2O NPs as sacrificial templates. The morphology, photothermal effect, drug loading/releasing abilities, and synergistic chemo-photothermal therapy of CuxS-PEG NPs have been investigated. The CuxS-PEG NPs with hollow structures showed a high drug loading capacity (∼255 µg Dox per mg of CuxS NPs) and stimuli-responsive drug release triggered by NIR-II laser irradiation. The synergistic chemo-photothermal therapy based on the Dox/CuxS-PEG NPs showed 98.5% tumor elimination. Our study emphasizes the great potential of CuxS-PEG NPs as an all-in-one NIR-II light-responsive NDDS for applications in biomedicine.

In-silico investigation of umami peptides with receptor T1R1/T1R3 for the discovering potential targets: A combined modeling approach.

Umami, providing amino acids/peptides for animal growth, represents one of the major attractive taste modalities. The biochemical and umami properties of peptide are both important for scientific research and food industry. In this study, we did the sequence analysis of 205 umami peptides with 2-18 amino acids, sought the active sites of umami peptides by quantum chemical simulations and investigated their recognition residues with receptor T1R1/T1R3 by molecular docking. The results showed the peptides with 2-3 amino acids accounting for 44% of the total umami peptides. Residues D and E are the key active sites no matter where they are in the peptides (N-terminal, C-terminal or middle), when umami peptides contain D/E residues. N69, D147, R151, A170, S172, S276 and R277 residues in T1R1 receptor were deemed to be the key residues binding umami peptides. Finally, a powerful decision rule for umami peptides was proposed to predict potential umami peptides, which was convenient and efficient.

ESE3/EHF, a promising target of rosiglitazone, suppresses pancreatic cancer stemness by downregulating CXCR4.

BACKGROUND AND AIMS: The crosstalk between cancer stem cells (CSCs) and their niche is required for the maintenance of stem cell-like phenotypes of CSCs. Here, we identified E26 transformation-specific homologous factor (EHF) as a key molecule in decreasing the sensitivity of pancreatic cancer (PC) cells to CSCs' niche stimulus. We also explored a therapeutic strategy to restore the expression of EHF. DESIGN: We used a LSL-KrasG12D/+mice, LSL-Trp53R172H/+ and Pdx1-Cre (KPC) mouse model and samples from patients with PC. Immunostaining, flow cytometry, sphere formation assays, anchorage-independent growth assay, in vivo tumourigenicity, reverse transcription PCR, chromatin immunoprecipitation (ChIP) and luciferase analyses were conducted in this study. RESULTS: CXCL12 derived from pancreatic stellate cells (PSCs) mediates the crosstalk between PC cells and PSCs to promote PC stemness. Tumorous EHF suppressed CSC stemness by decreasing the sensitivity of PC to CXCL12 stimulus and inhibiting the crosstalk between PC and CSC-supportive niches. Mechanically, EHF suppressed the transcription of the CXCL12 receptor CXCR4. EHF had a cell autonomous role in suppressing cancer stemness by inhibiting the transcription of Sox9, Sox2, Oct4 and Nanog. Rosiglitazone suppressed PC stemness and inhibited the crosstalk between PC and PSCs by upregulating EHF. Preclinical KPC mouse cohorts demonstrated that rosiglitazone sensitised PDAC to gemcitabine therapy. CONCLUSIONS: EHF decreased the sensitivity of PC to the stimulus from PSC-derived CSC-supportive niche by negatively regulating tumorous CXCR4. Rosiglitazone could be used to target PC stem cells and the crosstalk between CSCs and their niche by upregulating EHF.

A Virulent PEDV Strain FJzz1 with Genomic Mutations and Deletions at the High Passage Level Was Attenuated in Piglets via Serial Passage In Vitro.

Highly virulent porcine epidemic diarrhea virus (PEDV) strains re-emerged and circulated in China at the end of 2010, causing significant economic losses in the pork industry worldwide. To understand the genetic dynamics of PEDV during its passage in vitro, the PEDV G2 strain FJzz1 was serially propagated in Vero cells for up to 200 passages. The susceptibility and adaptability of the FJzz1 strain increased gradually as it was serially passaged in vitro. Sequence analysis revealed that amino acid (aa) changes were mainly concentrated in the S glycoprotein, which accounted for 72.22%-85.71% of all aa changes. A continuous aa deletion (55I56G57E → 55K56Δ57Δ) occurred in the N-terminal domain of S1 (S1-NTD). To examine how the aa changes affected its virulence, FJzz1-F20 and FJzz1-F200 were selected to simultaneously evaluate their pathogenicity in suckling piglets. All the piglets in the FJzz1-F20-infected group showed typical diarrhea at 24 h postinfection, and the piglets died successively by 48 h postinfection. However, the clinical signs of the piglets in the FJzz1-F200-infected group were significantly weaker, and no deaths occurred. The FJzz1-F200-infected group also showed a lower level of fecal viral shedding and lower viral loads in the intestinal tissues, and no obvious histopathological lesions. Type I and III interferon were induced in the FJzz1-F200 infection group, together with pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-8. These results indicate that the identified genetic changes may contribute to the attenuation of FJzz1 strain, and the attenuated FJzz1-F200 may have the potential for developing PEDV live-attenuated vaccines.

Porcine Epidemic Diarrhoea Virus Induces Cell-cycle Arrest through the DNA Damage-signalling Pathway.

INTRODUCTION: Porcine epidemic diarrhoea virus (PEDV) infection causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, inflicting on them morbidity and mortality potentially reaching 90%-100%. Despite it being known that certain mammalian cell phases are arrested by PEDV, the mechanisms have not been elucidated, and PEDV pathogenesis is poorly understood. This study determined the effect of an epidemic PEDV strain on cell cycle progression. MATERIAL AND METHODS: We observed the effect of the PEDV SHpd/2012 strain on an infected Vero cell cycle through flow cytometry and Western blot, investigating the interrelationships of cell-cycle arrest, the DNA damage-signalling pathway caused by PEDV and the phosphorylation levels of the key molecules Chk.2 and H2A.X involved upstream and downstream in this pathway. RESULTS: PEDV induced Vero cell-cycle arrest at the G1/G0 phase. The phosphorylation levels of Chk.2 and H2A.X increased with the prolongation of PEDV infection, and no significant cell-cycle arrest was observed after treatment with ATM or Chk.2 inhibitors. The proliferation of PEDV was also inhibited by treatment with ATM or Chk.2 inhibitors. CONCLUSION: PEDV-induced cell-cycle arrest is associated with activation of DNA damage-signalling pathways. Our findings elucidate the molecular basis of PEDV replication and provide evidence to support further evaluation of PEDV pathogenesis.

A robust, integrated platform for comprehensive analyses of acyl-coenzyme As and acyl-carnitines revealed chain length-dependent disparity in fatty acyl metabolic fates across Drosophila development.

Acyl-coenzyme A thioesters (acyl-CoAs) denote a key class of intermediary metabolites that lies at the hub of major metabolic pathways. The great diversity in polarity between short- and long-chain acyl-CoAs makes it technically challenging to cover an inclusive range of acyl-CoAs within a single method. Levels of acyl-carnitines, which function to convey fatty acyls into mitochondria matrix for ß-oxidation, indicate the efficiency of mitochondrial import and utilization of corresponding acyl-CoAs. Herein, we report a robust, integrated platform to allow simultaneous quantitation of endogenous acyl-CoAs and acyl-carnitines. Using this method, we monitored changes in intermediary lipid profiles across Drosophila development under control (ND) and high-fat diet (HFD). We observed specific accumulations of medium-chain (C8-C12) and long-chain (≥C16) acyl-carnitines distinct to L3 larval and pupal stages, respectively. These observations suggested development-specific, chain length-dependent disparity in metabolic fates of acyl-CoAs across Drosophila development, which was validated by deploying the same platform to monitor isotope incorporation introduced from labelled 12:0 and 16:0 fatty acids into extra- and intra-mitochondrial acyl-CoA pools. We found that pupal mitochondria preferentially import and oxidise C12:0-CoAs (accumulated as C12:0-carnitines in L3 stage) over C16:0-CoAs. Preferential oxidation of medium-chain acyl-CoAs limits mitochondrial utilization of long-chain acyl-CoAs (C16-C18), leading to pupal-specific accumulation of long-chain acyl-carnitines mediated by enhanced CPT1-6A activity. HFD skewed C16:0-CoAs towards catabolism over anabolism in pupa, thereby adversely affecting overall development. Our developed platform emphasizes the importance of integrating biological knowledge in the design of pathway-oriented platforms to derive maximal physiological insights from analysis of complex biological systems.

Tumoral EHF predicts the efficacy of anti-PD1 therapy in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly immune-suppressive tumor with a low response rate to single checkpoint blockade therapy. ETS homologous factor (EHF) is a tumor suppressor in PDAC. Here, we report a novel function of EHF in pancreatic cancer immune microenvironment editing and efficacy prediction for anti-PD1 therapy. Our findings support that the deficiency of tumoral EHF induced the accumulation of regulatory T (T reg) cells and myeloid-derived suppressor cells (MDSCs) and a decrease in the number of tumor-infiltrating CD8+ T cells. Mechanistically, EHF deficiency induced the conversion and expansion of T reg cells and MDSCs through inhibiting tumor TGFß1 and GM-CSF secretion. EHF suppressed the transcription of TGFB1 and CSF2 by directly binding to their promoters. Mice bearing EHF overexpression tumors exhibited significantly better response to anti-PD1 therapy than those with control tumors. Our findings delineate the immunosuppressive mechanism of EHF deficiency in PDAC and highlight that EHF overexpression may improve PDAC checkpoint immunotherapy.

Isolation and chatacterization of homoisoflavonoids from Dracaena cochinchinensis and their osteogenic activities in mouse mesenchymal stem cells.

Two new homoisoflavonoids, dracaeconolide A (1) and dracaeconolide B (2), together with ten known compounds, namely (3R)-7,4'-dihydroxy-8-methoxyhomoisoflavane (3), (3R)-7-hydroxy-3-(4-hydroxybenzyl)chromane (4), (3R)-7,4'-dihydroxy-5-methoxy-homoisoflavane (5), (3R)-6,4'-dihydroxy-8-methoxyhomoisoflavan (6), 7,4'-dihydroxy-8-methylflavan (7), (2R)-7,4'-dihydroxy-5-methoxy-8-methylflavan (8), 5,4'-dihydroxy-7-methoxy-6-methylflavane (9), 7,4'-dihydroxy-3'-methoxyflavan (10), 7,4'-dihydroxyflavan (11), 4,4'-dihydroxy-2,6-dimethoxydihydrochlcone (12), were isolated from the red resin of Dracaena cochinchinensis (dragon's blood, DB). All the compounds were then evaluated for their effects on mouse bone marrow-derived mesenchymal stem cells (MSCs) proliferation using CCK8 assay and their abilities in promoting MSCs differentiating into osteoblast through the assay of alkaline phosphatase (ALP) activity in vitro. Compounds 2, 3, 4, 7, 9, and 11, at a concentration of 10µM with no cytotoxicity, significantly promoted MSC osteogenic differentiation by increasing the levels of ALP activity to percents of 159.6±5.9, 167.6±10.9, 162.0±1.4, 151.3±4.0, 171.0±8.2, and 169.9±7.3 in relative to the control, respectively. The results of ALP staining were in accordance to that of ALP activity.