Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

In vivo assessment of dual-function submicron textured nitric oxide releasing catheters in a 7-day rabbit model.

Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. Herein, a dualfunction submicron textured nitric oxide (NO)-releasing catheter was developed. The hemocompatibility and antithrombotic activity of vascular catheters were evaluated in both 20 h in vitro blood loop and 7 d in vivo rabbit model. Surface characterization assessments via atomic force microscopy show the durability of the submicron pattern after incorporation of NO donor S-nitroso-N-acetylpenicillamine (SNAP). The SNAP-doped catheters exhibited prolonged and controlled NO release mimicking the levels released by endothelium. Fabricated catheters showed cytocompatibility when evaluated against BJ human fibroblast cell lines. After 20h in vitro evaluation of catheters in a blood loop, textured-NO catheters exhibited a 13-times reduction in surface thrombus formation compared to the control catheters, which had 83% of the total area covered by clots. After the 7 d in vivo rabbit model, analysis on the catheter surface was examined via scanning electron microscopy, where significant reduction of platelet adhesion, fibrin mesh, and thrombi can be observed on the NO-releasing textured surfaces. Moreover, compared to relative controls, a 63% reduction in the degree of thrombus formation within the jugular vein was observed. Decreased levels of fibrotic tissue decomposition on the jugular vein and reduced platelet adhesion and thrombus formation on the texture of the NO-releasing catheter surface are indications of mitigated foreign body response. This study demonstrated a biocompatible and robust dual-functioning textured NO PU catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. STATEMENT OF SIGNIFICANCE: Catheter-induced thrombosis is a major contributor to infectious and mechanical complications of biomaterials that lead to device failure. This study demonstrated a robust, biocompatible, dual-functioning textured nitric oxide (NO) polyurethane catheter in limiting fouling-induced complications for longer-term blood-contacting device applications. The fabricated catheters exhibited prolonged and controlled NO release that mimics endothelium levels. After the 7 d in vivo model, a significant reduction in platelet adhesion, fibrin mesh, and thrombi was observed on the NO-releasing textured catheters, along with decreased levels of fibrotic tissue decomposition on the jugular vein. Results illustrate that NO-textured catheter surface mitigates foreign body response.

Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

Challenging applicability of ISO 10993-5 for calcium phosphate biomaterials evaluation: Towards more accurate in vitro cytotoxicity assessment.

Research on biomaterials typically starts with cytocompatibility evaluation, using the ISO 10993-5 standard as a reference that relies on extract tests to determine whether the material is safe (cell metabolic activity should exceed 70 %). However, the generalized approach within the standard may not accurately reflect the material's behavior in direct contact with cells, raising concerns about its effectiveness. Calcium phosphates (CaPs) are a group of materials that, despite being highly biocompatible and promoting bone formation, still exhibit inconsistencies in basic cytotoxicity evaluations. Hence, in order to test the cytocompatibility dependence on different experimental setups and material-cell interactions, we used amorphous calcium phosphate, α-tricalcium phosphate, hydroxyapatite, and octacalcium phosphate (0.1 mg/mL to 5 mg/mL) with core cell lines of bone microenvironment: mesenchymal stem cells, osteoblast-like and endothelial cells. All materials have been characterized for their physicochemical properties before and after cellular contact and once in vitro assays were finalized, groups identified as 'cytotoxic' were further analyzed using a modified Annexin V apoptosis assay to accurately determine cell death. The obtained results showed that indirect contact following ISO standards had no sensitivity of tested cells to the materials, but direct contact tests at physiological concentrations revealed decreased metabolic activity and viability. In summary, our findings offer valuable guidelines for handling biomaterials, especially in powder form, to better evaluate their biological properties and avoid false negatives commonly associated with the traditional standard approach.

Adapting the in vitro micronucleus assay (OECD Test Guideline No. 487) for testing of manufactured nanomaterials: recommendations for best practices.

The current Organisation for Economic Co-Operation and Development test guideline number 487 (OECD TG No. 487) provides instruction on how to conduct the in vitro micronucleus assay. This assay is one of the gold standard approaches for measuring the mutagenicity of test items; however, it is directed at testing low molecular weight molecules and may not be appropriate for particulate materials (e.g. engineered nanoparticles [ENPs]). This study aimed to adapt the in vitro micronucleus assay for ENP testing and underpins the development of an OECD guidance document. A harmonized, nano-specific protocol was generated and evaluated by two independent laboratories. Cell lines utilized were human lymphoblastoid (TK6) cells, human liver hepatocytes (HepG2) cells, Chinese hamster lung fibroblast (V79) cells, whole blood, and buffy coat cells from healthy human volunteers. These cells were exposed to reference ENPs from the Joint Research Council (JRC): SiO2 (RLS-0102), Au5nm and Au30nm (RLS-03, RLS-010), CeO2 (NM212), and BaSO4 (NM220). Tungsten carbide-cobalt (WC/Co) was used as a trial particulate positive control. The chemical controls were positive in all cell cultures, but WC/Co was only positive in TK6 and buffy coat cells. In TK6 cells, mutagenicity was observed for SiO2- and both Au types. In HepG2 cells, Au5nm and SiO2 showed sub-two-fold increases in micronuclei. In V79 cells, whole blood, and buffy coat cells, no genotoxicity was detected with the test materials. The data confirmed that ENPs could be tested with the harmonized protocol, additionally, concordant data were observed across the two laboratories with V79 cells. WC/Co may be a suitable particulate positive control in the in vitro micronucleus assay when using TK6 and buffy coat cells. Detailed recommendations are therefore provided to adapt OECD TG No. 487 for testing ENP.

The application of a self-designed microfluidic lung chip in the assessment of different inhalable aerosols.

Microfluidic-based assessment platforms have recently attracted considerable attention and have been widely used for evaluating in vitro toxic effects. In the present study, we developed an original real-time aerosol exposure system, which focused on a self-designed microfluidic chip, in order to evaluate the toxicological effects following exposure to inhalable aerosols. The three-layer structured microfluidic chip enables real-time aerosol exposure at the gas-liquid interface. The comprehensive detection of toxic effect biomarkers based on this assessment platform encompasses transcriptomics, in situ fluorescence detection, and the identification of extracellular secretagogues. Correspondingly, the effects of selected inhalable aerosols such as cigarette smoke (CS), heated tobacco product smoke (HS), and electronic cigarette smoke (ES) on gene expression profiles, cell viability, intracellular biomarkers (reactive oxygen species and nitric oxide), apoptosis (caspase-3/7 activity), and extracellular biomarkers (IL-8, IL-1ß, TNF-α, and malondialdehyde) in the BEAS-2B cells present on the chip were investigated. Following exposure to aerosols derived from CS, HS, and ES, the transcriptome analysis revealed differential expression in these cells. In addition, the overlapping DEGs from the different treatment groups were found to be primarily associated with stimuli and inflammatory responses. Correspondingly, each of the three categories of selected inhalable aerosols was confirmed to induce significant changes in biomarkers that were associated with toxic effects. These results suggest that the original real-time aerosol exposure system centered around a self-designed chip can be applied to the toxic effect evaluation of inhalable aerosol exposure.

Assessment of Mitochondrial Function in the AmE-711 Honey Bee Cell Line: Boscalid and Pyraclostrobin Effects.

There is a growing concern that chronic exposure to fungicides contributes to negative effects on honey bee development, life span, and behavior. Field and caged-bee studies have helped to characterize the adverse outcomes (AOs) of environmentally relevant exposures, but linking AOs to molecular/cellular mechanisms of toxicity would benefit from the use of readily controllable, simplified host platforms like cell lines. Our objective was to develop and optimize an in vitro-based mitochondrial toxicity assay suite using the honey bee as a model pollinator, and the electron transport chain (ETC) modulators boscalid and pyraclostrobin as model fungicides. We measured the effects of short (~30 min) and extended exposures (16-24 h) to boscalid and pyraclostrobin on AmE-711 honey bee cell viability and mitochondrial function. Short exposure to pyraclostrobin did not affect cell viability, but extended exposure reduced viability in a concentration-dependent manner (median lethal concentration = 4175 µg/L; ppb). Mitochondrial membrane potential (MMP) was affected by pyraclostrobin in both short (median effect concentration [EC50] = 515 µg/L) and extended exposure (EC50 = 982 µg/L) scenarios. Short exposure to 10 and 1000 µg/L pyraclostrobin resulted in a rapid decrease in the oxygen consumption rate (OCR), approximately 24% reduction by 10 µg/L relative to the baseline OCR, and 64% by 1000 µg/L. Extended exposure to 1000 µg/L pyraclostrobin reduced all respiratory parameters (e.g., spare capacity, coupling efficiency), whereas 1- and 10-µg/L treatments had no significant effects. The viability of AmE-711 cells, as well as the MMP and cellular respiration were unaffected by short and extended exposures to boscalid. The present study demonstrates that the AmE-711-based assessment of viability, MMP, and ETC functionality can provide a time- and cost-effective platform for mitochondrial toxicity screening relevant to bees. Environ Toxicol Chem 2024;43:976-987. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Green-Synthesized Silver and Selenium Nanoparticles Using Berberine: A Comparative Assessment of In Vitro Anticancer Potential on Human Hepatocellular Carcinoma Cell Line (HepG2).

A well-known natural ingredient found in several medicinal plants, berberine (Ber), has been shown to have anticancer properties against a range of malignancies. The limited solubility and bioavailability of berberine can be addressed using Ber-loaded nanoparticles. In this study, we compared the in vitro cytotoxic effects of both Ber-loaded silver nanoparticles (Ber-AgNPs) and Ber-loaded selenium nanoparticles (Ber-SeNPs) in the human liver cancer cell line (HepG2) and mouse normal liver cells (BNL). The IC50 values in HepG2 for berberine, Ber-AgNPs, Ber-SeNPs, and cisplatin were 26.69, 1.16, 0.04, and 0.33 µg/mL, respectively. Our results show that Ber and its Ag and Se nanoparticles exerted a good antitumor effect against HepG2 cells by inducing apoptosis via upregulating p53, Bax, cytosolic cytochrome C levels, and caspase-3 activity, and the down-regulation of Bcl-2 levels. Similarly, incubation with Ber and both Ber-NPs (Ag and Se) led to a significant dose-dependent elevation in inflammatory markers' (TNF-α, NF-κB, and COX-2) levels compared to the control group. In addition, it led to the arrest of the G1 cell cycle by depleting the expression of cyclin D1 and CDK-2 mRNA. Furthermore, Ber and both Ber-NPs (Ag and Se) caused a significant dose-dependent increase in LDH activity in HepG2 cells. Furthermore, our findings offer evidence that Ber and its nanoparticles intensified oxidative stress in HepG2 cells. Furthermore, the migration rate of cells subjected to berberine and its nanoforms was notably decreased compared to that of control cells. It can be inferred that Ber nanoparticles exhibited superior anticancer efficacy against HepG2 compared to unprocessed Ber, perhaps due to their improved solubility and bioavailability. Furthermore, Ber-SeNPs exhibited greater efficacy than Ber-AgNPs, possibly as a result of the inherent anticancer characteristics of selenium.

Calculation of biological effectiveness of SOBP proton beams: a TOPAS Monte Carlo study.

Objective.This study aims to investigate the biological effectiveness of Spread-Out Bragg-Peak (SOBP) proton beams with initial kinetic energies 50-250 MeV at different depths in water using TOPAS Monte Carlo code.Approach.The study modelled SOBP proton beams using TOPAS time feature. Various LET-based models and Repair-Misrepair-Fixation model were employed to calculate Relative Biological Effectiveness (RBE) for V79 cell lines at different on-axis depths based on TOPAS. Microdosimetric Kinetic Model and biological weighting function-based models, which utilize microdosimetric distributions, were also used to estimate the RBE. A phase-space-based method was adopted for calculating microdosimetric distributions.Main results.The trend of variation of RBE with depth is similar in all the RBE models, but the absolute RBE values vary based on the calculation models. RBE sharply increases at the distal edge of SOBP proton beams. In the entrance region of all the proton beams, RBE values at 4 Gy i.e. RBE(4 Gy) resulting from different models are in the range of 1.04-1.07, comparable to clinically used generic RBE of 1.1. Moving from the proximal to distal end of the SOBP, RBE(4 Gy) is in the range of 1.15-1.33, 1.13-1.21, 1.11-1.17, 1.13-1.18 and 1.17-1.21, respectively for 50, 100, 150, 200 and 250 MeV SOBP beams, whereas at the distal dose fall-off region, these values are 1.68, 1.53, 1.44, 1.42 and 1.40, respectively.Significance.The study emphasises application of depth-, dose- and energy- dependent RBE values in clinical application of proton beams.

Unlocking the Power of Transcriptomic Biomarkers in Qualitative and Quantitative Genotoxicity Assessment of Chemicals.

To modernize genotoxicity assessment and reduce reliance on experimental animals, new approach methodologies (NAMs) that provide human-relevant dose-response data are needed. Two transcriptomic biomarkers, GENOMARK and TGx-DDI, have shown a high classification accuracy for genotoxicity. As these biomarkers were extracted from different training sets, we investigated whether combining the two biomarkers in a human-derived metabolically competent cell line (i.e., HepaRG) provides complementary information for the classification of genotoxic hazard identification and potency ranking. First, the applicability of GENOMARK to TempO-Seq, a high-throughput transcriptomic technology, was evaluated. HepaRG cells were exposed for 72 h to increasing concentrations of 10 chemicals (i.e., eight known in vivo genotoxicants and two in vivo nongenotoxicants). Gene expression data were generated using the TempO-Seq technology. We found a prediction performance of 100%, confirming the applicability of GENOMARK to TempO-Seq. Classification using TGx-DDI was then compared to GENOMARK. For the chemicals identified as genotoxic, benchmark concentration modeling was conducted to perform potency ranking. The high concordance observed for both hazard classification and potency ranking by GENOMARK and TGx-DDI highlights the value of integrating these NAMs in a weight of evidence evaluation of genotoxicity.

Assessment of possible biomedical applications of green synthesized TiO2NPs-an in-vitro approach.

Biomedical applications for various types of nanoparticles are emerging on a daily basis. Hence this research was performed to evaluate the antifungal (Aspergillus sp., Alternaria sp., Trichophyton sp., Candida sp., and Penicillium sp.), cytotoxicity (MCF10A cell lines), and antioxidant (DPPH) potential of Coleus aromaticus mediated and pre-characterized TiO2NPs were studied with respective standard methodology. Interestingly, the TiO2NPs exhibited significant antifungal activity on pathogenic fungal strains like Alternaria sp., Aspergillus sp. (31 ± 1.4), Penicillium sp. (31 ± 1.9) Trichophyton sp. (27 ± 2.1), and Candida sp. (26 ± 2.3) at high concentration (250 µg mL-1). However, the considerable levels of zone of inhibitions on fungal pathogens were recorded at 100 µg mL-1 of TiO2NPs as well as it was considerably greater than positive control. It also demonstrated dose based anti-inflammatory and antidiabetic activities. The plant-mediated TiO2NPs demonstrated a maximum DPPH scavenging efficiency of 91% at a dosage of 250 µg mL-1, comparable to the positive control's 94%. Furthermore, TiO2NPs at 100 µg mL-1 concentration did not cause cytotoxicity in MCF10A cell lines. At higher concentrations (250 µg mL-1), the nanoparticles showed the lowest cytotoxicity (17%). These findings suggest that C. aromaticus-mediated TiO2NPs have significant biomedical applications. However, in-vivo studies are needed to learn more about their (C. aromaticus-mediated TiO2NPs) potential biomedical applications.

Bioconcentration Assessment of Three Cationic Surfactants in Permanent Fish Cell Lines.

Cationic surfactants are used in many industrial processes and in consumer products with concurrent release into the aquatic environment, where they may accumulate in aquatic organisms to regulatoryly relevant thresholds. Here, we aimed to better understand the bioconcentration behavior of three selected cationic surfactants, namely N,N-dimethyldecylamine (T10), N-methyldodecylamine (S12), and N,N,N-trimethyltetradecylammonium cation (Q14), in the cells of fish liver (RTL-W1) and gill (RTgill-W1) cell lines. We conducted full mass balances for bioconcentration tests with the cell cultures, in which the medium, the cell surface, the cells themselves, and the plastic compartment were sampled and quantified for each surfactant by HPLC MS/MS. Accumulation in/to cells correlated with the surfactants' alkyl chain lengths and their membrane lipid-water partitioning coefficient, DMLW. Cell-derived bioconcentration factors (BCF) of T10 and S12 were within a factor of 3.5 to in vivo BCF obtained from the literature, while the cell-derived BCF values for Q14 were >100 times higher than the in vivo BCF. From our experiments, rainbow trout cell lines appear as a suitable conservative in vitro screening method for bioconcentration assessment of cationic surfactants and are promising for further testing.

Comparative Studies of Extracts Obtained from Brassica oleracea L. Plants at Different Stages of Growth by Isolation and Determination of Isothiocyanates: An Assessment of Chemopreventive Properties of Broccoli.

The main goal of this work was to develop analytical procedures for the isolation and determination of selected isothiocyanates. As an example, particularly sulforaphane from plants of the Brassicaceae Burnett or Cruciferae Juss family. The applied methodology was mainly based on classical extraction methods and high-performance liquid chromatography coupled with tandem mass spectrometry. Moreover, the effect of temperature on the release of isothiocyanates from plant cells was considered. The cytotoxic activity of the obtained plant extracts against a selected cancer cell line has also been included. The results allow evaluating the usefulness of obtained plant extracts and raw sprouts regarding their content of isothiocyanates-bioactive compounds with chemopreventive properties.

Cell Survival Computation via the Generalized Stochastic Microdosimetric Model (GSM2); Part II: Numerical Results.

In the present paper we numerically investigate, using Monte Carlo simulation, the theoretical results predicted by the Generalized Stochastic Microdosimetric Model (GSM2), as shown in the published companion paper. Taking advantage of the particle irradiation data ensemble (PIDE) dataset, we calculated GSM2 biological parameters of human salivary gland (HSG) and V79 cell lines. Further, exploiting the TOPAS-microdosimetric extension, we simulated the microdosimetric spectra of different radiation fields of therapeutic interest generated by four different ions (protons, helium-4, carbon-12 and oxygen-16) each at three different residual ranges. We investigated the properties of the initial damage distributions as well as the cell survival curve predicted by GSM2, focusing especially on the non-Poissonian effects naturally included in the model. GSM2 successfully computed cell survival curves, accurately describing experimental behavior even under challenging LET and dose conditions.

Preliminary data on cytotoxicity and functional group assessment of a herb-mineral combination against colorectal carcinoma cell line.

OBJECTIVES: The invasive screening methods and the late stage diagnosis of colorectal carcinoma (CRC) are contributing for the devastative prognosis. The gradual shift of the disease pattern among younger generations requires the implementation of phytochemicals and traditional medicines. Arkeshwara rasa (AR) is a herb-mineral combination of Tamra bhasma/incinerated copper ashes and Dwigun Kajjali/mercury sulphide levigated with Calotropis procera leaf juice, Plumbago zeylanica root decoction and the decoction of three myrobalans (Terminalia chebula, Terminalia bellerica, Emblica Officinalis decoction)/Triphala decoction. METHODS: The SW-480 cell line was checked for the cytotoxicity and the cell viability criteria with MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The acridine orange/ethidium bromide (AO/EtBr) assay revealed the depth of apoptosis affected cells in the fluorescent images. The FTIR analysis exhibited the graphical spectrum of functional groups within the compound AR. RESULTS: The IC50 from the 10-7 to 10-3 concentrations against SW-480 cells was 40.4 µg/mL. The staining of AO/EtBr was performed to visualize live and dead cells and it is evident from the result that number of apoptotic cells increases at increasing concentration of AR. The single bond with stretch vibrations of O-H and N-H are more concentrated in the 2,500-3,200 cm-1 and 3,700-4,000 cm-1 of the spectra whereas, the finger print region carries the O-H and S=O type peaks. CONCLUSIONS: The AR shows strong cyto-toxicity against the SW-480 cells by inducing apoptosis. It also modulates cellular metabolism with the involvement of functional groups which antagonizes the strong acids. Moreover, these effects need to be analyzed further based in the in vivo and various in vitro models.

Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

Naringenin Improves Innate Immune Suppression after PRRSV Infection by Reactivating the RIG-I-MAVS Signaling Pathway, Promoting the Production of IFN-I.

Porcine reproductive and respiratory syndrome (PRRS) has been prevalent for nearly forty years since it was first reported. It has been one of the major diseases jeopardizing the healthy development of the world swine industry, as well as causing great economic losses to the industry's economic development. Furthermore, no way has been found to combat the disease due to the immunosuppressive properties of its pathogen porcine reproductive and respiratory syndrome virus (PRRSV) infection. We previously examined the mRNA expression of IFN-I in PRRSV-infected Marc-145 cells at different time periods using qRT-PCR, and found that the mRNA expression of IFN-I in the late stage of PRRSV infection showed suppression. Naringenin is a flavonoid found in citrus fruits and has a very wide range of pharmacological activities. Therefore, the aim of the present study was to investigate the modulatory effect of naringenin on the suppressed innate immune response after PRRSV infection. The expression of IFN-I, IL-10, and ISGs in the late stage of PRRSV infection was examined using qRT-PCR, and the results showed that naringenin improved the expression of antiviral cytokines suppressed by PRRSV infection. Further results showed that naringenin treatment significantly up-regulated the expression of proteins related to the RIG-I-MAV immune signaling pathway, and that naringenin could not significantly activate the RIG-I-MAVS signaling pathway after the addition of the RIG-I inhibitor Cyclo. Overall, these data demonstrated that naringenin could improve the innate immune response suppressed by PRRSV infection by modulating the RIG-I-MAVS signaling pathway. Therefore, our study will provide a theoretical basis for the development of naringenin as a drug against immunosuppressive viral infectious disease infections.

Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines.

The interaction manner and biological function of Rab7 and its effector, Rab-interacting lysosomal protein (RILP), remain unclear in invertebrates. We provide a protocol for detecting the effects of Rab7 and RILP terminals on lysosome and autophagy in Spodoptera frugiperda Sf9 cells with overexpression and RNA interference. We describe steps for overexpressing plasmids, generating long double-stranded RNA, and transfecting them into Sf9 cells. We then detail procedures for cell immunofluorescence imaging with harmine treatment and fluorescence analysis. For complete details on the use and execution of this protocol, please refer to Cui et al. (2023).1.

Immunoinformatic Risk Assessment of Host Cell Proteins During Process Development for Biologic Therapeutics.

The identification and removal of host cell proteins (HCPs) from biologic products is a critical step in drug development. Despite recent improvements to purification processes, biologics such as monoclonal antibodies, enzyme replacement therapies, and vaccines that are manufactured in a range of cell lines and purified using diverse processes may contain HCP impurities, making it necessary for developers to identify and quantify impurities during process development for each drug product. HCPs that contain sequences that are less conserved with human homologs may be more immunogenic than those that are more conserved. We have developed a computational tool, ISPRI-HCP, that estimates the immunogenic potential of HCP sequences by evaluating and quantifying T cell epitope density and relative conservation with similar T cell epitopes in the human proteome. Here we describe several case studies that support the use of this method for classifying candidate HCP impurities according to their immunogenicity risk.

Long-circulating thiolated chitosan nanoparticles of nintedanib with N-acetyl cysteine for treating idiopathic pulmonary fibrosis: In vitro assessment of cytotoxicity, antioxidant, and antifibrotic potential.

Nintedanib (NIN) is one of the FDA-approved tyrosine kinase inhibitor drugs used to treat idiopathic pulmonary fibrosis (IPF). This study aimed to formulate a long-circulating injection of Nintedanib to treat bedridden patients with IPF. Nintedanib was incorporated into chitosan nanoparticles (NIN-NP) via the ionic gelation method, and N-acetyl cysteine (NAC), a known antioxidant and mucolytic agent, was added to the NIN-NP (NAC-NIN-NP). The lyophilized formulation had a particle size of 174 nm, a polydispersity index of 0.511, and a zeta potential of 18.6 mV. The spherical nanoparticles were observed in transmission electron microscopy, whereas field emission scanning electron microscopy showed irregular clusters of NP. The thiolation of the chitosan in NAC-NIN-NP was confirmed by ATR-FTIR and NMR, which improved drug release profiles showing >90 % drug release that was 2.42-folds greater than NIN-NP lasting for five days. The DPPH assay showed that adding NAC increased the % inhibition of oxidation in blank-NP (from 54.59 % to 87.17 %) and NIN-NP (58.65 %-89.19 %). The MTT assay on A549 cells showed 67.57 % cell viability by NAC-NIN-NP with an IC50 value of 28 µg/mL. The NAC formulation reduced hydroxyproline content (56.77 µg/mL) compared to NIN-NP (69.48 µg/mL) in WI-38 cell lines. Meanwhile, the healthy cells count with NAC-NIN-NP was higher (5.104 × 103) than with NIN-NP (4.878 × 103). In Hoechst staining, no significant damage to DNA was observed by the drug or formulation. Therefore, NAC-NIN-NP could be a promising treatment option for IPF patients and can be studied further clinically.