Overexpression of wild-type HRAS drives non-alcoholic steatohepatitis to hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC), a prevalent solid carcinoma of significant concern, is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes. The etiology and pathological progression of non-alcoholic steatohepatitis (NASH)-related HCC is multifactorial and multistage. However, no single animal model can accurately mimic the full NASH-related HCC pathological progression, posing considerable challenges to transition and mechanistic studies. Herein, a novel conditional inducible wild-type human HRAS overexpressed mouse model (HRAS-HCC) was established, demonstrating 100% morbidity and mortality within approximately one month under normal dietary and lifestyle conditions. Advanced symptoms of HCC such as ascites, thrombus, internal hemorrhage, jaundice, and lung metastasis were successfully replicated in mice. In-depth pathological features of NASH- related HCC were demonstrated by pathological staining, biochemical analyses, and typical marker gene detections. Combined murine anti-PD-1 and sorafenib treatment effectively prolonged mouse survival, further confirming the accuracy and reliability of the model. Based on protein-protein interaction (PPI) network and RNA sequencing analyses, we speculated that overexpression of HRAS may initiate the THBS1-COL4A3 axis to induce NASH with severe fibrosis, with subsequent progression to HCC. Collectively, our study successfully duplicated natural sequential progression in a single murine model over a very short period, providing an accurate and reliable preclinical tool for therapeutic evaluations targeting the NASH to HCC continuum.

Upregulation of LRRC8A by m5C modification-mediated mRNA stability suppresses apoptosis and facilitates tumorigenesis in cervical cancer.

Cervical cancer (CC) is one of the most common gynecological malignancies with poor prognosis for advanced CC patients. LRRC8A is a volume-regulated anion channel protein involved in cellular homeostasis, but its role in CC remains largely unknown. In this study, we found that LRRC8A is elevated in CC and associated with poor prognosis. LRRC8A maintains cell survivals under the hypotonic condition, and promotes tumorigenesis through apoptosis suppression in vitro and in vivo. Notably, LRRC8A is upregulated by NSUN2-mediated m5C modification. m5C modified-LRRC8A mRNA is bound by the RNA binding protein YBX1 followed by the increased RNA stability. Moreover, loss of NSUN2 suppresses the proliferation and metastasis of CC cells, and NSUN2 expression is positively correlated with LRRC8A expression in CC. Altogether, our study demonstrates that the NSUN2-m5C-LRRC8A axis is crucial and would be a potential therapeutic target for CC.

Phosphorylation of enteroviral 2Apro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

Enteroviral 2A proteinase (2Apro ), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential for enterovirus A71 (EV-A71) and other viruses replication. We recently showed that ERK1/2 is required for the proteolytic activity of viral 2Apro ; however, the mechanism underlying the regulation of 2Apro remains unknown. Here, we demonstrated that the 125th residue Ser125 of EV-A71 2Apro or Thr125 of coxsackievirus B3 2Apro , which is highly conserved in the Enterovirus, was phosphorylated by ERK1/2. Importantly, 2Apro with phosphor-Ser/Thr125 had much stronger proteolytic activity toward eukaryotic initiation factor 4GI and rendered the virus more efficient for multiplication and pathogenesis in hSCARB2 knock-in mice than that in nonphospho-Ser/Thr125A (S/T125A) mutants. Notably, phosphorylation-mimic mutations caused deleterious changes in 2Apro catalytic function (S/T125D/E) and in viral propagation (S125D). Crystal structure simulation analysis showed that Ser125 phosphorylation in EV-A71 2Apro enabled catalytic Cys to adopt an optimal conformation in the catalytic triad His-Asp-Cys, which enhances 2Apro proteolysis. Therefore, we are the first to report Ser/Thr125 phosphorylation of 2Apro increases enteroviral adaptation to the host to ensure enteroviral multiplication, causing pathogenicity. Additionally, weakened viruses containing a S/T125A mutation could be a general strategy to develop attenuated Enterovirus vaccines.

Immune Dysfunction Mediated by the ceRNA Regulatory Network in Human Placenta Tissue of Intrahepatic Cholestasis Pregnancy.

Pregnancy-related intrahepatic cholestasis (ICP) is a serious complication with adverse perinatal outcomes of preterm labor, fetal distress, or stillbirth. As a result, it is important to investigate and identify the potential critical pathogenic mechanisms of ICP. First, we collected the placental tissues from the ICP with placental weight and fetal birth weight loss for the whole transcriptome sequencing. Then we analyzed the differentially expressed (DE) circRNAs (DEcircRNAs) by SRPBM, DElncRNAs by FRKM, DEmiRNAs by TPM, and DEmRNAs by TPM and RSEM. Based on differential expression of term pregnancy placental tissues from pregnancies impacted by ICP (n=7) as compared to gestational aged matched control tissues (n=5), the circ/lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) regulatory networks were constructed. The ceRNA regulatory networks covered 3,714 events, including 21 DEmiRNAs, 36 DEcircRNAs, 146 DElncRNAs, and 169 DEmRNAs. According to the functional analysis, ICP complications were linked to the immune system, signal transduction, endocrine system, cell growth and death, and transport and catabolism. Further evidence suggested that the expression of immune-related genes KLRD1, BRAF, and NFATC4 might have a potential ceRNA mechanism by individual lncRNA sponging miR372-3p, miR-371a-3p, miR-7851-3p, and miR-449a to control downstream the level of TNF-α, IFN-γ, and IL-10, thereby regulating the pathophysiology of ICP. Furthermore, our results were validated by the qRT-PCR, western blotting and ELISA assays. In conclusion, this study is the first to evaluate placental ceRNA networks in pregnancies affected by ICP, showing alterations in immune regulatory networks which may impact fetal and placental growth. Overall our these data suggest that the ceRNA regulatory network may refine biomarker predictions for developing novel therapeutic approaches in ICP.

Evaluation of the Effect of Food on the Pharmacokinetics of SHR6390, An Oral CDK4/6 Inhibitor, in Healthy Volunteers.

BACKGROUND AND INTRODUCTION: SHR6390 is a new developed highly effective and selective small-molecule oral CDK4/6 inhibitor. We aimed to evaluate the effect of food on the pharmacokinetics of SHR6390 tablets. METHODS: In an open-label two-way crossover study, 24 healthy Chinese volunteers were randomly divided into Group A and Group B, and 12 volunteers in each group received a single oral dose of a SHR6390 150-mg tablet under fasting and high-fat conditions. Blood samples were collected and determined for pharmacokinetic analyses. A liquid chromatography-tandem mass spectrometry method was developed and validated for determining the SHR6390 concentration. RESULTS: The time to maximum plasma concentration was not significantly affected by a high-fat diet. Compared with the fasting group, maximum plasma concentration, i.e., the area under the concentration-time curve (AUC0-t and AUC0-∞) was altered significantly, as evidenced by an increase of 56.9%, 38.6%, and 37.5% respectively. We identified seven metabolites of SHR6390 from the plasma samples, and we found no sex differences in metabolic pathways. All treatment-emergent adverse events were Grade 1 or 2. CONCLUSIONS: Food intake increased the maximum plasma concentration, AUC0-t, and AUC0-∞ significantly compared with the fasting condition. Meanwhile, single-dose SHR6390 for two treatment cycles is safe. SHR6390 was administered in a fasting status in the pivotal phase III study (NCT03927456) and chosen for the final drug label.

RNA 5-Methylcytosine Regulators Contribute to Metabolism Heterogeneity and Predict Prognosis in Ovarian Cancer.

5-Methylcytosine (m5C) is an abundant and highly conserved modification in RNAs. The dysregulation of RNA m5C methylation has been reported in cancers, but the regulatory network in ovarian cancer of RNA m5C methylation-related genes and its implication in metabolic regulation remain largely unexplored. In this study, RNA-sequencing data and clinical information of 374 ovarian cancer patients were downloaded from The Cancer Genome Atlas database, and a total of 14 RNA m5C regulators were included. Through unsupervised consensus clustering, two clusters with different m5C modification patterns were identified with distinct survivals. According to enrichment analyses, glycosaminoglycan and collagen metabolism-related pathways were specifically activated in cluster 1, whereas fatty acid metabolism-related pathways were enriched in cluster 2, which had better overall survival (OS). Besides the metabolism heterogeneity, the higher sensitivity to platinum and paclitaxel in cluster 2 can further explain the improved OS. Ultimately, a least absolute shrinkage and selection operator prediction model formed by ALYREF, NOP2, and TET2 toward OS was constructed. In conclusion, distinct m5C modification pattern exhibited metabolism heterogeneity, different chemotherapy sensitivity, and consequently survival difference, providing evidence for risk stratification.

Metabolite profiles and mass balance of fuzuloparib, a novel poly (ADP-ribose) polymerase inhibitor, in subjects with advanced solid cancers.

AIM: This trial (NCT04013048) investigated the metabolite profiles, mass balance and pharmacokinetics of fuzuloparib, a novel poly (ADP-ribose) polymerase inhibitor, in subjects with advanced solid cancers. METHODS: A single dose of 150 mg [14 C]fuzuloparib was administered to five subjects with advanced solid cancers. Blood, urine and faecal samples were collected, analysed for radioactivity and unchanged fuzuloparib, and profiled for metabolites. The safety of the medicine was assessed during the study. RESULTS: The maximum concentrations (Cmax ) of the total radioactivity (TRA) and unchanged fuzuloparib in plasma were 5.39 µg eq./mL and 4.19 µg/mL, respectively, at approximately 4 hours post dose. The exposure (AUC0-t ) of fuzuloparib accounted for 70.7% of the TRA in plasma, and no single metabolite was observed accounting for more than 10% of the plasma TRA. The recovery of TRA in excreta was 103.3 ± 3.8% in 288 hours, including 59.1 ± 9.9% in urine and 44.2 ± 10.8% in faeces. Sixteen metabolites of fuzuloparib were identified, including mono-oxidation (M1), hydrogenation (M2), di-oxidation (M3), trioxidation (M4), glucuronidation (M5, M7, M8) and de-ethylation (M6) products, and there was no specific binding between these metabolites and blood cells. Aliphatic hydroxylated fuzuloparib (M1-1) was the primary metabolite in the excreta, accounting for more than 40% of the dose for subjects. There were no serious adverse events observed in the study. CONCLUSION: Fuzuloparib was widely metabolized and excreted completely through urine and faeces in subjects with advanced solid cancer. Unchanged fuzuloparib was indicated to be the primary drug-related compound in circulation. [14 C]fuzuloparib was well-tolerated at the study dose.

Metabolic modulation of immune checkpoints and novel therapeutic strategies in cancer.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) or programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1)-based immune checkpoint inhibitors (ICIs) have led to significant improvements in the overall survival of patients with certain cancers and are expected to benefit patients by achieving complete, long-lasting remissions and cure. However, some patients who receive ICIs either fail treatment or eventually develop immunotherapy resistance. The existence of such patients necessitates a deeper understanding of cancer progression, specifically nutrient regulation in the tumor microenvironment (TME), which includes both metabolic cross-talk between metabolites and tumor cells, and intracellular metabolism in immune and cancer cells. Here we review the features and behaviors of the TME and discuss the recently identified major immune checkpoints. We comprehensively and systematically summarize the metabolic modulation of tumor immunity and immune checkpoints in the TME, including glycolysis, amino acid metabolism, lipid metabolism, and other metabolic pathways, and further discuss the potential metabolism-based therapeutic strategies tested in preclinical and clinical settings. These findings will help to determine the existence of a link or crosstalk between tumor metabolism and immunotherapy, which will provide an important insight into cancer treatment and cancer research.

The Role of Prophylactic Application of Granulocyte Colony Stimulating Factor in Adjuvant Chemotherapy for Epithelial Ovarian Cancer.

BACKGROUND: The goal of this study was to investigate the relative merits of granulocyte colony stimulating factor (G-CSF) prophylaxis for patients with epithelial ovarian cancer (EOC). METHODS: The Institutional Review Board of the Women's Hospital, Zhejiang University School of Medicine has approved this study (No. IRB-20200132-R) with a waiver of informed consent. Patients with EOC who received the combination of a platinum drug and paclitaxel (TP) chemotherapy regimens in the hospital from January 1, 2016, to November 30, 2020, were included in this retrospective cohort study. To assess clinical effectiveness, patients were categorized into groups who received either long-acting G-CSF or short-acting G-CSF prophylaxis with and without prophylaxis. The incidence of neutropenia and adverse events were compared between groups. All results of chemotherapy were pooled for analysis. RESULTS: Of the identified cases, 128 patients were evaluated. Long-acting G-CSF and short-acting G-CSF were applied in 51 and 41 patients, respectively. The absolute neutrophil count at the nadir was significantly lower in patients with G-CSF prophylaxis than those without G-CSF (p = 0.001). The duration of ANC levels < 2.0 x 109/L in cycles using short-acting G-CSF was longer than that in those receiving long-acting G-CSF (p = 0.045). There were no serious adverse events observed in patients with G-CSF. No significant differences in the incidence of febrile neutropenia (FN) and duration of grade 2 - 4 neutropenia were observed between groups receiving G-CSF prophylaxis and those without. CONCLUSIONS: Primary prophylaxis with G-CSF in chemotherapy for epithelial ovarian cancer appears to be of low value in terms of its relationship to the incidence of FN and prognosis.

The impact of rifampicin on the pharmacokinetics of fuzuloparib in healthy Chinese male volunteers.

AIMS: This clinical study was conducted to evaluate the impact of rifampicin on the pharmacokinetics of fuzuloparib. METHODS: In this single-centre, single-arm, open-label, fixed-sequence study, healthy male subjects took a single 50 mg dose of fuzuloparib on two separate occasions: the first was on Day 1 as monotherapy, and the second was on Day 12 after oral administration of rifampicin 600 mg once daily for 8 days. Series of blood samples were obtained before and after fuzuloparib administration at different time points: pre-dose, and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 36, 48 and 72 hours post-dose. All samples were examined using liquid chromatography with tandem mass spectrometry. PK parameters were estimated by using a non-compartmental method with Phoenix WinNonlin software. Safety was assessed by monitoring for changes in vital signs and laboratory tests, physical examinations, and incidences of adverse events (AEs). RESULTS: A total of 16 Chinese male subjects were enrolled. Of these, 16 and 15 cases were evaluable for PK analysis following administration with fuzuloparib alone and pretreatment with rifampicin, respectively. Pretreatment with rifampicin resulted in a statistically significant reduction in the systemic exposure to fuzuloparib. The treatment ratio and 90% confidence intervals (CIs) for AUC0-∞ and Cmax were 0.10 (0.095-0.115) and 0.32 (0.281-0.365), respectively. A single administration of fuzuloparib after multiple oral dosing of rifampicin was well-tolerated, without severe AEs. CONCLUSION: The exposure of fuzuloparib was dramatically decreased when pretreated with rifampicin. Strong CYP3A4 inducers should be avoided during fuzuloparib treatment.

Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway.

Introduction: Venlafaxine is one of the most commonly used anti-depressant and antineoplastic drug. Previous studies have predicted venlafaxine as an anti-cancer compound, but the therapeutic effects of venlafaxine in melanoma have not yet been demonstrated. Nur77 is an orphan nuclear receptor that highly expressed in melanoma cells and can interact with Bcl-2 to convert Bcl-2 from an antiapoptotic to a pro-apoptotic protein. Method: We examined the effects of venlafaxine in MV3 cells in vitro and MV3 xenograft tumor in nude mice. Western-blot, PCR, TUNEL assay and immunofluorescence were used to reveal the growth of melanoma cells. Results: Here, our data revealed that venlafaxine could reduce the growth, and induce apoptosis of melanoma cells through a Nur77-dependent way. Our results also showed that treatment with venlafaxine (20 mg/kg, i.p.) potently inhibited the growth of melanoma cells in nude mice. Mechanistically, venlafaxine activated JNK1/2 signaling, induced Nur77 expressions and mitochondrial localization, thereby promoting apoptosis of melanoma cells. Knockdown of Nur77 and JNK1/2, or inhibition of JNK1/2 signaling with its inhibitor SP600125 attenuated the anti-cancer effects of venlafaxine. Conclusion: In summary, our results suggested venlafaxine as a potential therapy for melanoma.

Translocating lipopolysaccharide correlates with the severity of enterovirus A71-induced HFMD by promoting pro-inflammation and viral IRES activity.

BACKGROUND: The increase of inflammation-inducing enterobacteria was recently observed in severe hand, foot, and mouth disease (HFMD) caused by Enterovirus A71 (EV-A71). This study aimed to verify the occurrence of bacterial translocation (BT) and further explore the contributory role of BT to severity of EV-A71-mediated HFMD cases. METHODS: Serum specimens from 65 mild and 65 severe EV-A71-associated HFMD cases and 65 healthy children were collected. EV-A71 VP1 in serum, inflammatory mediators including C-reactive protein, IL-1ß, IL-6, interferon-γ and tumor necrosis factor-α, BT related biomarkers including Claudin-3, intestinal fatty acid binding protein, lipopolysaccharide (LPS), soluble CD14 (sCD14) and endotoxin core antibody were measured by ELISA. Bacterial DNA (BactDNA) fragments were quantified by quantified PCR (qPCR). Rhabdomyosarcoma (RD) or SH-SY5Y cells, infected with LPS-pre-incubated EV-A71 or transfected with plasmid containing viral 2Apro or mRNA containing viral internal ribosomal entry site (IRES), were post-treated with or without LPS in vitro. EV-A71 RNA and viral or cellular proteins were determined by qPCR and western blot, respectively. RESULTS: Compared to mild HFMD patients, remarkably higher inflammatory mediators as well as BT-related biomarkers except BactDNA were observed in severe HFMD cases (all P < 0.05). In severe HFMD group, circulating concentrations of LPS and sCD14 showed statistical correlations with inflammation indices (all P < 0.05), serum levels of EV-A71 VP1 were found to be positively correlated with serum LPS (r = 0.341, P = 0.005) and serum sCD14 (r = 0.458, P < 0.001). In vitro, EV-A71 attachment and internalization were only slightly promoted by LPS pre-incubation; however, EV-A71 proliferation and viral 2Apro-mediated IRES activity were significantly accelerated by LPS post-treatment. CONCLUSIONS: Our results collectively indicate that gut-derived translocating LPS contributes to the severity of EV-A71-induced HFMD by driving inflammatory response and viral proliferation via viral 2Apro-mediated IRES.

The RNA binding protein QKI5 suppresses ovarian cancer via downregulating transcriptional coactivator TAZ.

RNA-binding proteins (RBPs) are a set of proteins involved in many steps of post-transcriptional regulation to maintain cellular homeostasis. Ovarian cancer (OC) is the most deadly gynecological cancer, but the roles of RBPs in OC are not fully understood. Here, we reported that the RBP QKI5 was significantly negatively correlated with aggressive tumor stage and worse prognosis in serous OC patients. QKI5 could suppress the growth and metastasis of OC cells both in vitro and in vivo. Transcriptome analysis showed that QKI5 negatively regulated the expression of the transcriptional coactivator TAZ and its downstream targets (e.g., CTGF and CYR61). Mechanistically, QKI5 bound to TAZ mRNA and recruited EDC4, thus decreasing the stability of TAZ mRNA. Functionally, TAZ was involved in the QKI5-mediated tumor suppression of OC cells, and QKI5 expression was inversely correlated with TAZ, CTGF, and CYR61 expression in OC patients. Together, our study indicates that QKI5 plays a tumor-suppressive role and negatively regulates TAZ expression in OC.

Letrozole combined with oral contraceptives versus oral contraceptives alone in the treatment of endometriosis-related pain symptoms: a pilot study.

BACKGROUND: To compare the efficacy and the tolerability of letrozole combined with oral contraceptives versus oral contraceptives alone in treating endometriosis-related pain. METHODS: A total of 820 women with endometriosis presented with endometriosis-related pain were enrolled with this study. Patients were randomly treated either with letrozole (2.5 mg/day) combined with oral contraceptives (Desogestrel and Ethinylestradiol Tablets) or oral contraceptives (Desogestrel and Ethinylestradiol Tablets) alone for 6 months. Changes in pain symptoms during treatment and in 1 months after treatment, 6-month follow-up and 12-month follow-up were evaluated. Adverse effects of each treatment protocol were recorded. RESULTS: At completion of treatment, the intensity of chronic pelvic pain continued to decrease during treatment and at 1-month after treatment it was significantly lower than at 6-month follow-up and baseline level both in LE + oral contraceptives group (Mean ± SD,1.5 ± 1.4) and in oral contraceptives alone group(Mean ± SD,2.9 ± 1.2).The intensity of chronic pelvic pain and deep dyspareunia was significantly decrease at both 1-month after treatment and 6-month follow-up. CONCLUSIONS: This treatment for endometriosis is a promising new modality that warrants further investigation.

Population pharmacokinetics of the anti-PD-1 antibody camrelizumab in patients with multiple tumor types and model-informed dosing strategy.

Camrelizumab, a programmed cell death 1 (PD-1) inhibitor, has been approved for the treatment of patients with relapsed or refractory classical Hodgkin lymphoma, nasopharyngeal cancer and non-small cell lung cancer. The aim of this study was to perform a population pharmacokinetic (PK) analysis of camrelizumab to quantify the impact of patient characteristics and to investigate the appropriateness of a flat dose in the dosing regimen. A total of 3092 camrelizumab concentrations from 133 patients in four clinical trials with advanced melanoma, relapsed or refractory classical Hodgkin lymphoma and other solid tumor types were analyzed using nonlinear mixed effects modeling. The PKs of camrelizumab were properly described using a two-compartment model with parallel linear and nonlinear clearance. Then, covariate model building was conducted using stepwise forward addition and backward elimination. The results showed that baseline albumin had significant effects on linear clearance, while actual body weight affected intercompartmental clearance. However, their impacts were limited, and no dose adjustments were required. The final model was further evaluated by goodness-of-fit plots, bootstrap procedures, and visual predictive checks and showed satisfactory model performance. Moreover, dosing regimens of 200 mg every 2 weeks and 3 mg/kg every 2 weeks provided similar exposure distributions by model-based Monte Carlo simulation. The population analyses demonstrated that patient characteristics have no clinically meaningful impact on the PKs of camrelizumab and present evidence for no advantage of either the flat dose or weight-based dose regimen for most patients with advanced solid tumors.

Different polymorphisms in HIF-1α may exhibit different effects on cancer risk in Asians: evidence from nearly forty thousand participants.

The effect of different SNPs in HIF-1α and cancer susceptibility remain indistinct. Here, we evaluated the association between all identified SNPs (rs11549465, rs11549467 and rs2057482) in HIF-1α and the overall risk of cancer in all case-control studies published before April 2020. A total of 54 articles including 56 case-control studies were included in this analysis. We found that variant genotypes of rs11549465 and rs11549467 were associated with a significantly increased overall cancer risk. In contrast, the variant T allele of rs2057482 showed a significantly reduced risk of overall cancer. In addition, variant genotypes of the three studied SNPs exhibited a significant association with cancer risk in Asians and specific cancer types. Meanwhile, HIF-1α was significantly highly expressed in head and neck squamous cell carcinoma and pancreatic cancer tissues. More importantly, survival analysis indicated that the high expression of HIF-1α was associated with a poor survival in patients with lung cancer. These findings further provided evidence that different SNPs in HIF-1α may exhibit different effects on overall cancer risk; these effects were ethnicity and type-specific. Further studies with functional evaluations are required to confirm the biological mechanisms underlying the role of HIF-1α SNPs in cancer development and progression.

IMI - Oral biopharmaceutics tools project - Evaluation of bottom-up PBPK prediction success part 4: Prediction accuracy and software comparisons with improved data and modelling strategies.

Oral drug absorption is a complex process depending on many factors, including the physicochemical properties of the drug, formulation characteristics and their interplay with gastrointestinal physiology and biology. Physiological-based pharmacokinetic (PBPK) models integrate all available information on gastro-intestinal system with drug and formulation data to predict oral drug absorption. The latter together with in vitro-in vivo extrapolation and other preclinical data on drug disposition can be used to predict plasma concentration-time profiles in silico. Despite recent successes of PBPK in many areas of drug development, an improvement in their utility for evaluating oral absorption is much needed. Current status of predictive performance, within the confinement of commonly available in vitro data on drugs and formulations alongside systems information, were tested using 3 PBPK software packages (GI-Sim (ver.4.1), Simcyp® Simulator (ver.15.0.86.0), and GastroPlus™ (ver.9.0.00xx)). This was part of the Innovative Medicines Initiative (IMI) Oral Biopharmaceutics Tools (OrBiTo) project. Fifty eight active pharmaceutical ingredients (APIs) were qualified from the OrBiTo database to be part of the investigation based on a priori set criteria on availability of minimum necessary information to allow modelling exercise. The set entailed over 200 human clinical studies with over 700 study arms. These were simulated using input parameters which had been harmonised by a panel of experts across different software packages prior to conduct of any simulation. Overall prediction performance and software packages comparison were evaluated based on performance indicators (Fold error (FE), Average fold error (AFE) and absolute average fold error (AAFE)) of pharmacokinetic (PK) parameters. On average, PK parameters (Area Under the Concentration-time curve (AUC0-tlast), Maximal concentration (Cmax), half-life (t1/2)) were predicted with AFE values between 1.11 and 1.97. Variability in FEs of these PK parameters was relatively high with AAFE values ranging from 2.08 to 2.74. Around half of the simulations were within the 2-fold error for AUC0-tlast and around 90% of the simulations were within 10-fold error for AUC0-tlast. Oral bioavailability (Foral) predictions, which were limited to 19 APIs having intravenous (i.v.) human data, showed AFE and AAFE of values 1.37 and 1.75 respectively. Across different APIs, AFE of AUC0-tlast predictions were between 0.22 and 22.76 with 70% of the APIs showing an AFE > 1. When compared across different formulations and routes of administration, AUC0-tlast for oral controlled release and i.v. administration were better predicted than that for oral immediate release formulations. Average predictive performance did not clearly differ between software packages but some APIs showed a high level of variability in predictive performance across different software packages. This variability could be related to several factors such as compound specific properties, the quality and availability of information, and errors in scaling from in vitro and preclinical in vivo data to human in vivo behaviour which will be explored further. Results were compared with previous similar exercise when the input data selection was carried by the modeller rather than a panel of experts on each in vitro test. Overall, average predictive performance was increased as reflected in smaller AAFE value of 2.8 as compared to AAFE value of 3.8 in case of previous exercise.

Effects of gibberellic acid (GA3 ) application before anthesis on rachis elongation and berry quality and aroma and flavour compounds in Vitis vinifera L. 'Cabernet Franc' and 'Cabernet Sauvignon' grapes.

BACKGROUND: Gibberellic acid (GA3 ), a plant-growth regulator, is often used to obtain enlarged table grape berries and induce seedlessness in them. However, the effects of GA3 on rachis elongation and bunch compactness have seldom been reported in wine-grape production. We assessed the effects of GA3 spraying on wine-grape inflorescences and bunches and their practical implications for viticulture in the Jiaodong Peninsula, China. RESULTS: Various GA3 concentrations were sprayed on field-grown Vitis vinifera L. 'Cabernet Franc' (CF) and 'Cabernet Sauvignon' (CS) grapevines before anthesis in the Jiaodong Peninsula, China, in 2015 and 2016. Inflorescence length during berry development was measured, and flavonoids and aroma compounds in the fruit were detected by high-performance liquid chromatography - mass spectrometry (HPLC-MS) and gas chromatography - mass spectrometry (GC-MS), respectively. For both cultivars, 50 and 100 mg L-1 GA3 caused significant elongation of the rachis, whereas there was no significant effect on inflorescence growth and berry seed number. Anthocyanin, flavonol, and flavan-3-ol levels in mature berries were not significantly influenced by GA3 spraying, whereas C13 -norisoprenoids were modified. CONCLUSION: The application of 50-100 mg L-1 GA3 prior to grapevine anthesis caused elongation of inflorescences and bunches, and eased cluster compactness in CF and CS, and no negative effects were observed on the yield and seed numbers. The concentration and composition of flavonoids and most aroma compounds were not influenced, except that the norisoprenoids were increased by 50 mg L-1 GA3 applications. © 2020 Society of Chemical Industry.

Cellular Caspase-3 Contributes to EV-A71 2Apro-Mediated Down-Regulation of IFNAR1 at the Translation Level.

Enterovirus A71 (EV-A71) is the major pathogen responsible for the severe hand, foot and mouth disease worldwide, for which few effective antiviral drugs are presently available. Interferon-α (IFN-α) has been used in antiviral therapy for decades; it has been reported that EV-A71 antagonizes the antiviral activity of IFN-α based on viral 2Apro-mediated reduction of the interferon-alpha receptor 1 (IFNAR1); however, the mechanism remains unknown. Here, we showed a significant increase in IFNAR1 protein induced by IFN-α in RD cells, whereas EV-A71 infection caused obvious down-regulation of the IFNAR1 protein and blockage of IFN-α signaling. Subsequently, we observed that EV-A71 2Apro inhibited IFNAR1 translation by cleavage of the eukaryotic initiation factor 4GI (eIF4GI), without affecting IFNAR1 mRNA levels induced by IFN-α. The inhibition of IFNAR1 translation also occurred in puromycin-induced apoptotic cells when caspase-3 cleaved eIF4GI. Importantly, we verified that 2Apro could activate cellular caspase-3, which was subsequently involved in eIF4GI cleavage mediated by 2Apro. Furthermore, inhibition of caspase-3 activation resulted in the partial restoration of IFNAR1 in cells transfected with 2A or infected with EV-A71, suggesting the pivotal role of both viral 2Apro and caspase-3 activation in the disturbance of IFN-α signaling. Collectively, we elucidate a novel mechanism by which cellular caspase-3 contributes to viral 2Apro-mediated down-regulation of IFNAR1 at the translation level during EV-A71 infection, indicating that caspase-3 inhibition could be a potential complementary strategy to improve clinical anti-EV-A71 therapy with IFN-α.

Deficient IL-2 Produced by Activated CD56+ T Cells Contributes to Impaired NK Cell-Mediated ADCC Function in Chronic HIV-1 Infection.

Background: Antibody-dependent cellular cytotoxicity (ADCC), which mainly mediated by natural killer (NK) cells, may play a critical role in human immunodeficiency virus type-1 (HIV-1) disease progression. However, the potential mechanisms that affecting NK-mediated ADCC response are still not well-elucidated. Methods: Antigen-antibody complex model of Ab-opsonized P815 cells was adopted to induce a typical non-specific ADCC response. The capacities of HIV-1 specific NK-ADCC were measured by using the combination model of gp120 protein and plasma of HIV-1 elite controllers. The levels of plasma cytokine were measured by ELISA. Anti-IL-2 blocking antibody was used to analyze the impact of activated CD56+ T cells on NK-ADCC response. Results: IL-2, IL-15, IFN-α, and IFN-ß could effectively enhance the non-specific and HIV-1-specific NK-ADCC responses. Compared with healthy controls, HIV-1-infected patients showed decreased plasma IL-2 levels, while no differences of plasma IFN-α, IL-15, and IFN-ß were presented. IL-2 production was detected from CD56+ T cells activated through antibody-dependent manner. The capability of NK-ADCC could be weakened by blocking IL-2 secretion from activated CD56+ T cells. Although no difference of frequencies of CD56+ T cells was found between HIV-1-infected patients and healthy controls, deficient IL-2 secretion from activated CD56+ T were found in chronic HIV-1 infection. Conclusions: The impaired ability of activated CD56+ T cells to secreting IL-2 might contribute to the attenuated NK cell-mediated ADCC function in HIV-1 infection.