Grape seed proanthocyanidin extract alleviates inflammation in experimental colitis mice by inhibiting NF-κB signaling pathway.

Ulcerative colitis (UC) is a complex inflammatory disease of colorectum that induces abnormal immune responses and severely affects the quality of life of the patients. Grape seed proanthocyanidin extract (GSPE) exerts anti-inflammatory and antioxidant functions in many inflammatory diseases. The objective of this study was to investigate the potential therapeutic effects and underlying mechanisms of GSPE in UC using a dextran sodium sulfate (DSS)-induced mouse UC model and a lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage model. In this study, we found that the GSPE markedly prevented DSS-induced weight loss and colon length shortening in UC mice. Further investigations showed that GSPE significantly attenuated the expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß, and elevated the expression of anti-inflammatory cytokine IL-10 in the colon tissues and serum of DSS-induced colitis mice by suppressing NF-κB signaling pathway. Furthermore, LPS-induced inflammation in RAW264.7 cells was also reversed by GSPE. Taken together, our results confirm that GSPE can ameliorate inflammatory response in experimental colitis via inhibiting NF-κB signaling pathway. This study advances the research progress on a potentially effective therapeutic strategy for inflammatory bowel diseases.

Red-light is an environmental effector for mutualism between begomovirus and its vector whitefly.

Environments such as light condition influence the spread of infectious diseases by affecting insect vector behavior. However, whether and how light affects the host defense which further affects insect preference and performance, remains unclear, nor has been demonstrated how pathogens co-adapt light condition to facilitate vector transmission. We previously showed that begomoviral ßC1 inhibits MYC2-mediated jasmonate signaling to establish plant-dependent mutualism with its insect vector. Here we show red-light as an environmental catalyzer to promote mutualism of whitefly-begomovirus by stabilizing ßC1, which interacts with PHYTOCHROME-INTERACTING FACTORS (PIFs) transcription factors. PIFs positively control plant defenses against whitefly by directly binding to the promoter of terpene synthase genes and promoting their transcription. Moreover, PIFs interact with MYC2 to integrate light and jasmonate signaling and regulate the transcription of terpene synthase genes. However, begomovirus encoded ßC1 inhibits PIFs' and MYC2' transcriptional activity via disturbing their dimerization, thereby impairing plant defenses against whitefly-transmitted begomoviruses. Our results thus describe how a viral pathogen hijacks host external and internal signaling to enhance the mutualistic relationship with its insect vector.

[Protective effect of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress].

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P<0.05 or P<0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P<0.05 or P<0.01), while Bcl-2 was significantly up-regulated(P<0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P<0.01), whereas Bcl-2 was significantly up-regulated(P<0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.

Hybridization Liquid Chromatography-Tandem Mass Spectrometry: An Alternative Bioanalytical Method for Antisense Oligonucleotide Quantitation in Plasma and Tissue Samples.

Quantitative bioanalysis in plasma and tissues samples is required to study the pharmacokinetic and pharmacodynamic properties of antisense oligonucleotides (ASOs). To overcome intrinsic drawbacks in specificity, sensitivity, and throughput of traditional ligand-binding assay (LBA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, an alternative bioanalytical method was developed by combining oligonucleotide hybridization and LC-MS/MS technologies. Target ASOs were extracted from biological samples by hybridization with biotinylated sense-strand oligonucleotides coupled to streptavidin magnetic beads. Using ion-pairing chromatography and tandem mass spectrometry, this method demonstrated high sensitivity (0.5 ng/mL using 100 µL of plasma), high specificity, wide linear range, complete automation, and generic applications in tests with multiple ASOs. The typical challenge of sensitivity drop in traditional ion-pairing LC-MS/MS was for the first time overcome by the introduction of a ternary pump system. Due to the high specificity, quantitation in various biological matrixes was achieved using calibration standards in plasma, largely improving efficiency and consistency. Another major advantage was the capability of simultaneous quantitation of ASO metabolites. The hybridization LC-MS/MS was considered an improved alternative for quantitation of ASOs and metabolites in plasma and tissue samples, showing a great potential to replace traditional LBA and LC-MS/MS methods.

KLF15-activating Twist2 ameliorated hepatic steatosis by inhibiting inflammation and improving mitochondrial dysfunction via NF-κB-FGF21 or SREBP1c-FGF21 pathway.

Twist-related protein 2 (TWIST2) is identified as a basic helix-loop-helix (b-HLH) transcription repressor by dimerizing with other b-HLH proteins. The significance of TWIST2 has been emphasized in various tumors; however, few studies report its functions in metabolism and metabolic diseases. Here we aimed to explore the novel role and regulation mechanism of TWIST2 in hepatic steatosis. Our results showed that Twist2 knockdown caused mice obesity, insulin resistance, and hepatic steatosis, which were accompanied with inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction. In vitro, TWIST2 overexpression ameliorated hepatocellular steatosis, inhibited inflammation, and improved mitochondrial content and function with a fibroblast growth factor 21 (FGF21)-dependent pattern. NF-κB negatively regulated FGF21 transcription by directly binding to FGF21 promoter DNA, which was eliminated by TWIST2 overexpression by inhibiting NF-κB expression and translocation to nucleus. TWIST2 overexpression decreased intracellular reactive oxygen species level, increased mitochondrial DNA and biogenesis, and enhanced ATP production and antioxidation ability. Additionally, TWIST2 expression was repressed by insulin-targeting sterol regulatory element-binding protein 1c (SREBP1c) and forkhead box protein O1 and was enhanced by dexamethasone targeting Krüppel-like factor 15, which directly interacted with Twist2 promoter DNA. Together, our studies identify an important role and regulation mechanism of TWIST2 in maintaining hepatic homeostasis by ameliorating steatosis, inflammation, and oxidative stress via the NF-κB-FGF21 or SREBP1c-FGF21 pathway, which may provide a new therapeutic scheme for nonalcoholic fatty liver disease.-Zhou, L., Li, Q., Chen, A., Liu, N., Chen, N., Chen, X., Zhu, L., Xia, B., Gong, Y., Chen, X. KLF15-activating Twist2 ameliorated hepatic steatosis by inhibiting inflammation and improving mitochondrial dysfunction via NF-κB-FGF21 or SREBP1c-FGF21 pathway.

Influence of Ethanol on Darunavir Hepatic Clearance and Intracellular PK/PD in HIV-Infected Monocytes, and CYP3A4-Darunavir Interactions Using Inhibition and in Silico Binding Studies.

PURPOSE: Although the prevalence of alcohol consumption is higher in HIV+ people than general public, limited information is available on how alcohol affects the metabolism and bioavailability of darunavir (DRV). METHODS: DRV was quantified by using LC-MS/MS method. All in vitro experiments were performed using human liver microsomes and HIV-infected monocytic cells. CYP3A4 and DRV/Ritonavir (RTV) docking was performed using GOLD suite 5.8. RESULTS: Ethanol (20 mM) significantly decreased apparent half-life and increased degradation rate constant of RTV-boosted DRV but not for DRV alone. Similarly, ethanol exposure increased hepatic intrinsic clearance for RTV-boosted DRV with no significant influence on DRV alone. Ethanol showed a limited influence on intracellular total DRV exposure in the presence of RTV without altering maximum concentration (Cmax) values in HIV-infected monocytic cells. Ethanol alone elevated HIV replication but this effect was nullified with the addition of DRV or DRV + RTV. Additionally, inhibitory potency of DRV was significantly reduced in the presence of ethanol. Our docking results projected that ethanol increases the average distance between DRV and CYP3A4 heme, and alter the orientation of DRV-CYP3A4 binding. CONCLUSIONS: Collectively these findings suggest that DRV metabolism is primarily influenced by ethanol in the liver, but has minor effect in HIV-residing monocytes.

Leveraging Modeling and Simulation to Enhance the Efficiency of Bioequivalence Approaches for Generic Drugs: Highlights from the 2023 Generic Drug Science and Research Initiatives Public Workshop.

The 2023 Generic Drug Science and Research Initiative Public Workshop organized by the U.S. Food and Drug Administration (FDA) discussed the research needs to improve and enhance bioequivalence (BE) approaches for generic drug development. FDA takes such research needs and panel discussions into account to develop its Generic Drug User Fee Amendments III (GDUFA III) Science and Research Initiatives specific to generics. During the five workshop sessions, presentations and panel discussions focused on identifying and addressing scientific gaps and research needs related to nitrosamine impurity issues, BE assessment for oral products, innovative BE approaches for long-acting injectable products, alternative BE approaches for orally inhaled products, and advanced BE methods for topical products. Specifically, this report highlights the discussions on how to improve BE assessment for developing generic drug products based on research priorities for leveraging quantitative methods and modeling, as well as artificial intelligence/machine learning (AI/ML).

Pharmacokinetic Models for Inhaled Fluticasone Propionate and Salmeterol Xinafoate to Quantify Batch-to-Batch Variability.

Advair Diskus is an essential treatment for asthma and chronic obstructive pulmonary disease. It is a dry powder inhaler with a combination of fluticasone propionate (FP) and salmeterol xinafoate (SX). However, the pharmacokinetics (PK) batch-to-batch variability of the reference-listed drug (RLD) hindered its generic product development. This work developed the PK models for inhaled FP and SX that could represent potential batch variability. Two batches each of the reference and the test product (R1, R2, T1, T2) of Advair Diskus (100 µg FP/50 µg SX inhalation) were administered to 60 healthy subjects in a 4-period, 4-sequence crossover study. The failure of the bioequivalence (BE) between R1 and R2 confirmed the high between-batch variability of the RLD. Non-linear mixed effect modeling was used to estimate the population mean PK parameters for each batch. For FP, a 2-compartment model with a sequential dual zero-order absorption best described the PK profile. For SX, a 2-compartment model with a first-order absorption model best fit the data. Both models were able to capture the plasma concentration, the maximum concentration, and the total exposure (AUCinf) adequately for each batch, which could be used to simulate the BE study in the future. In vitro properties were also measured for each batch, and the batch with a higher fraction of the fine particle (diameter < 1 µm, < 2 µm) had a higher AUCinf. This positive correlation for both FP and SX could potentially assist the batch selection for the PK BE study.

Development of Quantitative Comparative Approaches to Support Complex Generic Drug Development.

On October 27-28, 2022, the US Food and Drug Administration (FDA) and the Center for Research on Complex Generics (CRCG) hosted a virtual public workshop titled "Best Practices for Utilizing Modeling Approaches to Support Generic Product Development." This report summarizes the presentations and panel discussions for a session titled "Development of Quantitative Comparative Approaches to Support Complex Generic Drug Development." This session featured speakers and panelists from both the generic industry and the FDA who described applications of advanced quantitative approaches for generic drug development and regulatory assessment within three main topics of interest: (1) API sameness assessment for complex generics, (2) particle size distribution assessment, and (3) dissolution profile similarity comparison. The key takeaways were that the analysis of complex data poses significant challenges to the application of conventional statistical bioequivalence methods, and there are various opportunities for using data analytics approaches for developing and applying suitable equivalence assessment method.

The Role of Model Master Files for Sharing, Acceptance, and Communication with FDA.

With the evolving role of Model Integrated Evidence (MIE) in generic drug development and regulatory applications, the need for improving Model Sharing, Acceptance, and Communication with the FDA is warranted. Model Master File (MMF) refers to a quantitative model or a modeling platform that has undergone sufficient model Verification & Validation to be recognized as sharable intellectual property that is acceptable for regulatory purposes. MMF provides a framework for regulatorily acceptable modeling practice, which can be used with confidence to support MIE by both the industry and the U.S. Food and Drug Administration (FDA). In 2022, the FDA and the Center for Research on Complex Generics (CRCG) hosted a virtual public workshop to discuss the best practices for utilizing modeling approaches to support generic product development. This report summarizes the presentations and panel discussions of the workshop symposium entitled "Model Sharing, Acceptance, and Communication with the FDA". The symposium and this report serve as a kick-off discussion for further utilities of MMF and best practices of utilizing MMF in drug development and regulatory submissions. The potential advantages of MMFs have garnered acknowledgment from model developers, industries, and the FDA throughout the workshop. To foster a unified comprehension of MMFs and establish best practices for their application, further dialogue and cooperation among stakeholders are imperative. To this end, a subsequent workshop is scheduled for May 2-3, 2024, in Rockville, Maryland, aiming to delve into the practical facets and best practices of MMFs pertinent to regulatory submissions involving modeling and simulation methodologies.

Tumor suppressor FRMD3 controls mammary epithelial cell fate determination via notch signaling pathway.

The luminal-to-basal transition in mammary epithelial cells (MECs) is accompanied by changes in epithelial cell lineage plasticity; however, the underlying mechanism remains elusive. Here, we report that deficiency of Frmd3 inhibits mammary gland lineage development and induces stemness of MECs, subsequently leading to the occurrence of triple-negative breast cancer. Loss of Frmd3 in PyMT mice results in a luminal-to-basal transition phenotype. Single-cell RNA sequencing of MECs indicated that knockout of Frmd3 inhibits the Notch signaling pathway. Mechanistically, FERM domain-containing protein 3 (FRMD3) promotes the degradation of Disheveled-2 by disrupting its interaction with deubiquitinase USP9x. FRMD3 also interrupts the interaction of Disheveled-2 with CK1, FOXK1/2, and NICD and decreases Disheveled-2 phosphorylation and nuclear localization, thereby impairing Notch-dependent luminal epithelial lineage plasticity in MECs. A low level of FRMD3 predicts poor outcomes for breast cancer patients. Together, we demonstrated that FRMD3 is a tumor suppressor that functions as an endogenous activator of the Notch signaling pathway, facilitating the basal-to-luminal transformation in MECs.

FRMD8 targets both CDK4 activation and RB degradation to suppress colon cancer growth.

Cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (RB) are both important cell-cycle regulators that function in different scenarios. Here, we report that FERM domain-containing 8 (FRMD8) inhibits CDK4 activation and stabilizes RB, thereby causing cell-cycle arrest and inhibiting colorectal cancer (CRC) cell growth. FRMD8 interacts separately with CDK7 and CDK4, and it disrupts the interaction of CDK7 with CDK4, subsequently inhibiting CDK4 activation. FRMD8 competes with MDM2 to bind RB and attenuates MDM2-mediated RB degradation. Frmd8 deficiency in mice accelerates azoxymethane/dextran-sodium-sulfate-induced colorectal adenoma formation. The FRMD8 promoter is hypermethylated, and low expression of FRMD8 predicts poor prognosis in CRC patients. Further, we identify an LKCHE-containing FRMD8 peptide that blocks MDM2 binding to RB and stabilizes RB. Combined application of the CDK4 inhibitor and FRMD8 peptide leads to marked suppression of CRC cell growth. Therefore, using an LKCHE-containing peptide to interfere with the MDM2-RB interaction may have therapeutic value in CDK4/6 inhibitor-resistant patients.

Establishing the suitability of model-integrated evidence to demonstrate bioequivalence for long-acting injectable and implantable drug products: Summary of workshop.

On November 30, 2021, the US Food and Drug administration (FDA) and the Center for Research on Complex Generics (CRCG) hosted a virtual public workshop titled "Establishing the Suitability of Model-Integrated Evidence (MIE) to Demonstrate Bioequivalence for Long-Acting Injectable and Implantable (LAI) Drug Products." This workshop brought relevant parties from the industry, academia, and the FDA in the field of modeling and simulation to explore, identify, and recommend best practices on utilizing MIE for bioequivalence (BE) assessment of LAI products. This report summerized presentations and panel discussions for topics including challenges and opportunities in development and assessment of generic LAI products, current status of utilizing MIE, recent research progress of utilizing MIE in generic LAI products, alternative designs for BE studies of LAI products, and model validation/verification strategies associated with different types of MIE approaches.

Life cycle assessment of rice bran oil production: a case study in China.

Environmental problems caused by the food processing industry have always been one of the concerns for the public. Herein, for the first time, a gate-to-gate life cycle assessment (LCA) was employed to evaluate the environmental impact of rice bran oil production. Four subsystems, namely, transportation of the raw rice bran to oil factory, crude oil extraction, oil refining, and oil storage, were established. The product sustainability software GaBi and the method CML 2001-Jan. 2016 were used to calculate and analyze the environmental burdens at each stage of the rice bran oil production chain. The results show the oil refining stage had the greatest environmental impact, followed by the oil extraction stage. High demands for coal and electricity make a critical difference in generating vast majority of environmental impacts. Modifying the electricity source and replacing traditional fuels with cleaner ones will do bring benefits to the sustainable development of the industry.

Usage of Compartmental Models in Predicting COVID-19 Outbreaks.

Accurately predicting the spread of the SARS-CoV-2, the cause of the COVID-19 pandemic, is of great value for global regulatory authorities to overcome a number of challenges including medication shortage, outcome of vaccination, and control strategies planning. Modeling methods that are used to simulate and predict the spread of COVID-19 include compartmental model, structured metapopulations, agent-based networks, deep learning, and complex network, with compartmental modeling as one of the most widely used methods. Compartmental model has two noteworthy features, a flexible framework that allows users to easily customize the model structure and its high adaptivity that allows well-matured approaches (e.g., Bayesian inference and mixed-effects modeling) to improve parameter estimation. We retrospectively evaluated the prediction performances of the compartmental models on the CDC COVID-19 Mathematical Modeling webpage based on data collected between August 2020 and February 2021, and subsequently discussed in detail their corresponding model enhancement. Finally, we presented examples using the compartmental models to assist policymaking. By evaluating all models in parallel, we systemically evaluated the performance and evolution of using compartmental models for COVID-19 pandemic prediction. In summary, as a 100-year-old epidemic approach, the compartmental model presents a powerful tool that is extremely adaptive and can be readily customized and implemented to address new data or emerging needs during a pandemic.

The Biological Activity of 3-O-Acetyl-11-keto-ß-Boswellic Acid in Nervous System Diseases.

Frankincense is a hard gelatinous resin exuded by Boswellia serrata. It contains a complex array of components, of which acetyl-11-keto-beta-boswellic acid (AKBA), a pentacyclic triterpenoid of the resin class, is the main active component. AKBA has a variety of physiological actions, including anti-infection, anti-tumor, and antioxidant effects. The use of AKBA for the treatment of mental diseases has been documented as early as ancient Greece. Recent studies have found that AKBA has anti-aging and other neurological effects, suggesting its potential for the treatment of neurological diseases. This review focuses on nervous system-related diseases, summarizes the functions and mechanisms of AKBA in promoting nerve repair and regeneration after injury, protecting against ischemic brain injury and aging, inhibiting neuroinflammation, ameliorating memory deficits, and alleviating neurotoxicity, as well as having anti-glioma effects and relieving brain edema. The mechanisms by which AKBA functions in different diseases and the relationships between dosage and biological effects are discussed in depth with the aim of increasing understanding of AKBA and guiding its use for the treatment of nervous system diseases.

Quantitative methods and modeling to assess COVID-19-interrupted in vivo pharmacokinetic bioequivalence studies with two reference batches.

The coronavirus disease 2019 (COVID-19) has presented unprecedented challenges to the generic drug development, including interruptions in bioequivalence (BE) studies. Per guidance published by the US Food and Drug Administration (FDA) during the COVID-19 public health emergency, any protocol changes or alternative statistical analysis plan for COVID-19-interrupted BE study should be accompanied with adequate justifications and not lead to biased equivalence determination. In this study, we used a modeling and simulation approach to assess the potential impact of study outcomes when two different batches of a Reference Standard (RS) were to be used in an in vivo pharmacokinetic BE study due to the RS expiration during the COVID-19 pandemic. Simulations were performed with hypothetical drugs under two scenarios: (1) uninterrupted study using a single batch of an RS, and (2) interrupted study using two batches of an RS. The acceptability of BE outcomes was evaluated by comparing the results obtained from interrupted studies with those from uninterrupted studies. The simulation results demonstrated that using a conventional statistical approach to evaluate BE for COVID-19-interrupted studies may be acceptable based on the pooled data from two batches. An alternative statistical method which includes a "batch" effect to the mixed effects model may be used when a significant "batch" effect was found in interrupted four-way crossover studies. However, such alternative method is not applicable for interrupted two-way crossover studies. Overall, the simulated scenarios are only for demonstration purpose, the acceptability of BE outcomes for the COVID19-interrupted studies could be case-specific.

Combined effects of cyclic stretch and TNF-α on the osteogenic differentiation in MC3T3-E1 cells.

OBJECTIVE: The study aimed to investigate the combined effects of cyclic stretch and tumor necrosis factor-alpha (TNF-α) on the osteogenic differentiation of MC3T3-E1 cells and the role of the nuclear factor-kappaB (NF-κB) signaling pathway in this process. DESIGN: MC3T3-E1 cells were treated with TNF-α (0.5 and 10 ng/mL) and cyclically stretched using the Flexcell tension system 4000 with 12 % elongation for 12 h. Furthermore, to explore which cytokines might be regulated by the NF-κB signaling pathway in osteogenic differentiation, the cells were pre-treated with NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), and then cyclically stretched for 12 h in the presence of 10 ng/mL of TNF-α. RT-PCR and western blot were utilized to detect the expression of type Ⅰ collagen (COL1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), NF-κB, and phosphorylated NF-κB (p-NF-κB) at gene and protein levels. RESULTS: Cyclic stretch alone increased the expression of COL1, OCN, Runx2, ALP, and OPG, decreasing the expression of RANKL and the RANKL/OPG ratio. The upregulation or downregulation induced by cyclic stretch were restrained in the presence of TNF-α. The p-NF-κB/NF-κB ratio increased at any stimulation. Inhibition of NF-κB signaling pathway restrained the expression variations of COL1, OCN, ALP, OPG, and RANKL induced by TNF-α combined with cyclic stretch. CONCLUSION: The results indicated that TNF-α inhibited the osteogenic differentiation of MC3T3-E1 cells induced by cyclic stretch and NF-κB signaling pathway might play a role in this process.