Cell Culture-based Assays Research Articles

Evaluation of the Survivability of SARS-CoV-2 on Cardboard and Plastic Surfaces and the Transferability of Virus from Surface to Skin

Objective: To 1) characterize the decay curve of infective SARS-CoV-2 over time on the surface of cardboard packaging and plastic mailer packaging; 2) characterize the transferability over time of virus-inoculated cardboard packaging and plastic mailer packaging to skin. Methods: We inoculated samples of plastic and cardboard packaging with a titer of SARS-CoV-2 > 106 TCID50/ mL to evaluate the survivability and transferability to the skin (pig skin) over time. A cell culture-based infectivity assay (TCID50) was used to determine viral titers. Regression analysis was used to characterize decay curves. Results: The time that SARS-CoV-2 remained transferable to skin was reduced on both packaging substrates compared to the total time of survivability, though cardboard demonstrated a substantially larger reduction. Virus inoculated plastic substrates continued to transfer the virus to the skin after 7 hours of holding time and regression analysis predicts this transferability would remain detectable up to 9.5 hours of holding time. Inoculated cardboard substrates demonstrated detectable transfer at 15 minutes of holding time, but no viable virus could be detected on the skin after 30 minutes of holding time. Conclusions: The type of material used as a packaging substrate substantially modifies the potential for SARS-CoV-2 fomite transmission. The use of materials that limit fomite transmission from packaging should be considered among strategies to reduce the transmission of SARS-CoV-2. Future research should investigate the generalizability of these findings for other viral pathogens that potentially transmit via fomite.

Development of a Novel Lateral Flow Biosensor Combined With Aptamer-Based Isolation: Application for Rapid Detection of Grouper Nervous Necrosis Virus.

Nervous necrosis virus (NNV) has infected more than 50 fish species worldwide, and has caused serious economic losses in the aquaculture industries. However, there is no effective antiviral therapy. The development of a rapid and accurate point-of-care diagnostic method for the prevention and control of NNV infection is urgently required. Commonly used methods for NNV detection include the cell culture-based assay, antibody-based assay and polymerase chain reaction (PCR)-based assay. However, these methods have disadvantages as they are time-consuming and complex. In the present study, we developed a simple and sensitive aptamer-based lateral flow biosensor (LFB) method for the rapid detection of red-spotted grouper nervous necrosis virus (RGNNV). An aptamer is a single-stranded nucleotide, which can specifically bind to the target and has many advantages. Based on a previously selected aptamer, which specifically bound to the coat protein of RGNNV (RGNNV-CP), two modified aptamers were used in this study. One aptamer was used for magnetic bead enrichment and the other was used for isothermal strand displacement amplification (SDA). After amplification, the product was further tested by the LFB, and the detection results were observed by the naked eye within 5 min with high specificity and sensitivity. The LFB method could detect RGNNV-CP protein as low as 5 ng/mL or 5 × 103 RGNNV-infected GB (grouper brain) cells. Overall, it is the first application of a LFB combined with aptamer in the rapid diagnosis of virus from aquatic animals, which provides a new option for virus detection in aquaculture.

5' splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity.

In the human genome, most 5' splice sites (~99%) employ the canonical GT dinucleotide whereas a small minority (~1%) use the noncanonical GC dinucleotide. The functionality and pathogenicity of 5' splice site GT>GC (+2T>C) variants have been extensively studied but we know very little about 5' splice site GC>GT (+2C>T) variants. Herein, we have addressed this deficiency by performing a meta-analysis of reported +2C>T "pathogenic" variants together with a functional analysis of engineered +2C>T substitutions using a cell culture-based full-length gene splicing assay. Our results establish proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and suggest that, in sharp contrast to +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants and understanding the evolutionary switching between GT and GC 5' splice sites in mammalian genomes.

Development of an In Vitro Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release

Gene therapy product release requires reliable and consistent demonstration of biopotency. In hemophilia B vectors, this is usually determined in vivo by measuring the plasma levels of the expressed human factor IX (FIX) transgene product in FIX knockout mice. To circumvent this laborious assay, we developed an in vitro method in which the HepG2 human liver cell line was infected with the vector, and the resulting FIX activity was determined in the conditioned medium using a chromogenic assay. The initial low sensitivity of the assay, particularly toward adeno-associated viral serotype 8 (AAV8), increased approximately 100-fold and allowed linear measurement in a broad range of multiplicities of infection. Statistical parameters indicated high assay repeatability (relative standard deviation (RSD) < 5%) and intra-assay reproducibility (RSD < 20%). To compare the performance of the in vitro and in vivo biopotency assay, we applied statistical analyses including regression techniques and variation decomposition to the results obtained for 25 AAV8-FIX vector lots (BAX 335). These showed a highly significant correlation, with the cell culture-based assay demonstrating less variation than the in vivo test. The in vitro assay thus constitutes a viable alternative to using animals for lot release testing.

Abstract P5-05-08: Comparative evaluation of a trastuzumab biosimilar or originator trastuzumab in association with pertuzumab: Binding and biological activities in cell culture-based assays

Abstract Background: trastuzumab (TZB) is approved for the treatment of HER2-positive (HER2+) breast cancer in different indications and schemes. The association of pertuzumab (PTZ) in some regimens has shown to improve the clinical benefit. Recently, biosimilars (BS) of trastuzumab were approved after extensive exercises of comparison with the originator, proving that these new biologicals are highly similar in structure and biological activity, and inherent microheterogeneity is not clinically relevant, since efficacy, safety and immunogenicity were found to be equivalent in pivotal phase 3 trials. In this study, we aimed to compare binding and biological activities of biosimilar trastuzumab (BS-TZB) or trastuzumab originator in association with PTZ in cell culture-based assays. Methods: two cancer cell lines were used to perform all the tests: BT474 (HER2+/ progesterone (PR) and estrogen receptor (ER)- positive) and HCC1954 (HER2+/PR and ER-negative); fluorescent labeling of trastuzumab originator (Herceptin®- Produtos Roche Químicos e Farmacêuticos S.A.) with Alexa 488, biosimilar of trastuzumab (trastuzumab-dkst, Biocon Limited/ Zedora- Libbs Farmacêutica Ltda.) with Alexa 488 and pertuzumab (Perjeta®- Produtos Roche Químicos e Farmacêuticos S.A.) with Alexa 647; FACS-based antigen binding evaluations and live cell imaging were performed (TZB or BS-TZB, and PTZ at concentrations of: 0; 10; 100; 1,000 ng/mL in every combinations. Predefined acceptance criteria for ligand-binding assay (LBA) was ±20% (25% at lower limit of quantification - LLOQ). Proliferation and CDC (complement-dependent cytotoxicity) assays: TZB or BS-TZB were tested at concentrations of: 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 100 μg/mL combined with PTZ at concentrations 1; 10; 100 μg/mL; the number of viable cells were estimated by quantitation of the ATP present using the CellTiter-Glo® kit. Equivalence was stablished with 95% confidence if 90% confidence interval (CI) of difference between means (DBM) lies within the zone of indifference (±20%). Results: tests were done using different concentrations and combinations of antibodies. For LBA, results are shown with concentrations in which the effect was neither minimal nor maximal (for both cell lines: 100 ng/mL for PTZ; 1,000 ng/mL for TZB or BS-TZB) (Table 1). For proliferation and CDC analysis, results at concentrations 100 μg/mL for PTZ, and TZB or BS-TZB (for both cell lines) are shown as a ratio to control. Table 1: Ligand-binding, proliferation and CDC assays. SD: standard deviation.Cell lineAnti-bodiesLBA (% cells) mean ± SDDBM 90% CIProliferation (ratio) mean ±SDDBM 90% CICDC (% cells) mean ± SDDBM 90% CIBT474BS-TZB + PTZ69.6 ±0.3-4.5 to -3.465.3 ±2.5-7.2 to -0.358.2 ±0.1-5.8 to -3.2TZB + PTZ73.5 ±0.369.6 ±1.262.8 ±1.1HCC1954BS-TZB + PTZ94.2 ±0.35.8 to 6.480.5 ±1.9-14.3 to -0.349.8 ±0.1-4.3 to -1.9TZB + PTZ88.1 ±0.187.8 ±5.452.9 ±1.0 Conclusions: trastuzumab and pertuzumab bind to HER2 receptor at different subdomains acting synergistically and, ultimately, improving the clinical benefit. In this study we comparatively evaluated the ligand and biological activities of the biosimilar BS-TZB and the TZB originator in the presence of PTZ. In addition to the previous findings of highly similar structure and biological activities of biosimilar to its originator, this study showed that not only PTZ binding to HER2 domain II but also biological activity showed the same pattern in the presence of BS-TZB as well as in the presence of TZB. Herein, in evaluated mechanisms, we have showed that the combination of BS-TZB and PTZ are highly similar to the combination of TZB and PTZ. Citation Format: Franklin F Pimentel, Juliano S Toledo, João Gonçalves, Leandro L Alves, Mayara I de Paula, Jurandyr M de Andrade, Daniel G Tiezzi, Roger Chammas. Comparative evaluation of a trastuzumab biosimilar or originator trastuzumab in association with pertuzumab: Binding and biological activities in cell culture-based assays [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-08.

SRNA/L1 retrotransposition: using siRNAs and miRNAs to expand the applications of the cell culture-based LINE-1 retrotransposition assay.

The cell culture-based retrotransposition reporter assay has been (and is) an essential tool for the study of vertebrate Long INterspersed Elements (LINEs). Developed more than 20 years ago, this assay has been instrumental in characterizing the role of LINE-encoded proteins in retrotransposition, understanding how ribonucleoprotein particles are formed, how host factors regulate LINE mobilization, etc. Moreover, variations of the conventional assay have been developed to investigate the biology of other currently active human retrotransposons, such as Alu and SVA. Here, we describe a protocol that allows combination of the conventional cell culture-based LINE-1 retrotransposition reporter assay with short interfering RNAs (siRNAs) and microRNA (miRNAs) mimics or inhibitors, which has allowed us to uncover specific miRNAs and host factors that regulate retrotransposition. The protocol described here is highly reproducible, quantitative, robust and flexible, and allows the study of several small RNA classes and various retrotransposons. To illustrate its utility, here we show that siRNAs to Fanconi anaemia proteins (FANC-A and FANC-C) and an inhibitor of miRNA-20 upregulate and downregulate human L1 retrotransposition, respectively. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Assessment of Gene Variant Amenability for Pharmacological Chaperone Therapy with 1-Deoxygalactonojirimycin in Fabry Disease.

Fabry disease is one of the most common lysosomal storage disorders caused by mutations in the gene encoding lysosomal α-galactosidase A (α-Gal A) and resultant accumulation of glycosphingolipids. The sugar mimetic 1-deoxygalactonojirimycin (DGJ), an orally available pharmacological chaperone, was clinically approved as an alternative to intravenous enzyme replacement therapy. The decision as to whether a patient should be treated with DGJ depends on the genetic variant within the α-galactosidase A encoding gene (GLA). A good laboratory practice (GLP)-validated cell culture-based assay to investigate the biochemical responsiveness of the variants is currently the only source available to obtain pivotal information about susceptibility to treatment. Herein, variants were defined amenable when an absolute increase in enzyme activity of ≥3% of wild type enzyme activity and a relative increase in enzyme activity of ≥1.2-fold was achieved following DGJ treatment. Efficacy testing was carried out for over 1000 identified GLA variants in cell culture. Recent data suggest that about one-third of the variants comply with the amenability criteria. A recent study highlighted the impact of inter-assay variability on DGJ amenability, thereby reducing the power of the assay to predict eligible patients. This prompted us to compare our own α-galactosidase A enzyme activity data in a very similar in-house developed assay with those from the GLP assay. In an essentially retrospective approach, we reviewed 148 GLA gene variants from our former studies for which enzyme data from the GLP study were available and added novel data for 30 variants. We also present data for 18 GLA gene variants for which no data from the GLP assay are currently available. We found that both differences in experimental biochemical data and the criteria for the classification of amenability cause inter-assay discrepancy. We conclude that low baseline activity, borderline biochemical responsiveness, and inter-assay discrepancy are alarm signals for misclassifying a variant that must not be ignored. Furthermore, there is no solid basis for setting a minimum response threshold on which a clinical indication with DGJ can be justified.

Prion protein transcription is auto-regulated through dynamic interactions with G-quadruplex motifs in its own promoter

Cellular prion protein (PrP) misfolds into an aberrant and infectious scrapie form (PrPSc) that lead to fatal transmissible spongiform encephalopathies (TSEs). Association of prions with G-quadruplex (GQ) forming nucleic acid motifs has been reported, but implications of these interactions remain elusive. Herein, we show that the promoter region of the human prion gene (PRNP) contains two putative GQ motifs (Q1 and Q2) that assume stable, hybrid, intra-molecular quadruplex structures and bind with high affinity to PrP. Here, we investigate the ability of PrP to bind to the quadruplexes in its own promoter. We used a battery of techniques including SPR, NMR, CD, MD simulations and cell culture-based reporter assays. Our results show that PrP auto-regulates its expression by binding and resolving the GQs present in its own promoter. Furthermore, we map this resolvase-like activity to the N-terminal region (residues 23–89) of PrP. Our findings highlight a positive transcriptional-translational feedback regulation of the PRNP gene by PrP through dynamic unwinding of GQs in its promoter. Taken together, our results shed light on a yet unknown mechanism of regulation of the PRNP gene. This work provides the necessary framework for a plethora of studies on understanding the regulation of PrP levels and its implications in prion pathogenesis.

ADP-Ribosylation Levels and Patterns Correlate with Gene Expression and Clinical Outcomes in Ovarian Cancers.

Inhibitors of nuclear PARP enzymes (e.g., PARP-1) have improved clinical outcomes in ovarian cancer, especially in patients with BRCA1/2 gene mutations or additional homologous recombination (HR) DNA repair pathway deficiencies. These defects serve as biomarkers for response to PARP inhibitors (PARPi). We sought to identify an additional biomarker that could predict responses to both conventional chemotherapy and PARPi in ovarian cancers. We focused on cellular ADP-ribosylation (ADPRylation), which is catalyzed by PARP enzymes and detected by detection reagents we developed previously. We determined molecular phenotypes of 34 high-grade serous ovarian cancers and associated them with clinical outcomes. We used the levels and patterns of ADPRylation and PARP-1 to distribute ovarian cancers into distinct molecular phenotypes, which exhibit dramatically different gene expression profiles. In addition, the levels and patterns of ADPRylation, PARP-1 protein, and gene expression correlated with clinical outcomes in response to platinum-based chemotherapy, with cancers exhibiting the highest levels of ADPRylation having the best outcomes independent of BRCA1/2 status. Finally, in cell culture-based assays using patient-derived ovarian cancer cell lines, ADPRylation levels correlated with sensitivity to the PARPi, Olaparib, with cell lines exhibiting high levels of ADPRylation having greater sensitivity to Olaparib. Collectively, our study demonstrates that ovarian cancers exhibit a wide range of ADPRylation levels, which correlate with therapeutic responses and clinical outcomes. These results suggest ADPRylation may be a useful biomarker for PARPi sensitivity in ovarian cancers, independent of BRCA1/2 or homologous recombination deficiency status.

WZ66, a novel acetyl-CoA carboxylase inhibitor, alleviates nonalcoholic steatohepatitis (NASH) in mice.

The global prevalence of nonalcoholic steatohepatitis (NASH) increases incredibly. NASH ends up to advanced liver disease, which is highly threatening to human health. Currently, treatment of NASH is very limited. Acetyl-CoA carboxylases (ACC1/ACC2) are proved as effective drug targets for NASH. We aimed to develop novel ACC inhibitors and evaluate their therapeutic value for NASH prevention. ACC inhibitors were obtained through structure-based drug design, synthesized, screened from ACC enzymatic measurement platform and elucidated in cell culture-based assays and animal models. The lipidome and microbiome analysis were integrated to assess the effects of WZ66 on lipids profiles in liver and plasma as well as gut microbiota in the intestine. WZ66 was identified as a novel ACC1/2 inhibitor. It entered systemic circulation rapidly and could accumulate in liver. WZ66 alleviated NASH-related liver features including steatosis, Kupffer cells and hepatic stellate cells activation in diet-induced obese mice. The triglycerides (TGs) and other lipids including diglycerides (DGs), phosphatidylcholine (PC) and sphingomyelin (SM) were decreased in WZ66-treated mice as evidenced by lipidome analysis in livers. The lipids profiles in plasma were also altered with WZ66 treatment. Plasma TG were moderately increased, while the activation of SREBP1c was not detected. WZ66 also downregulated the abundance of Allobaculum, Mucispirillum and Prevotella genera as well as Mucispirillum schaedleri species in gut microbiota. WZ66 is an ideal lead compound and a potential drug candidate deserving further investigation in the therapeutics of NASH.

Evaluation of the proinflammatory effects of contaminated bathing water

ABSTRACTContaminated marine bathing water has been reported to adversely affect human health. Our data demonstrated a correlation between total endotoxin (lipopolysaccharide; LPS) levels and degree of contamination of marine bathing waters. To assess the potential health implications of LPS present in marine bathing waters, the inflammation-inducing potency of water samples collected at different time points at multiple sampling sites were assessed using a cell culture-based assay. The numbers of fecal indicator bacteria (FIB) were also examined in the same samples. Water samples were used to stimulate two cell culture models: (1) a novel non-transformed continuously growing murine cell line Max Plank Institute (MPI) characteristic of alveolar macrophages and (2) human MonoMac 6 monocyte cell line. The inflammatory potential of the samples was assessed by measuring the release of inflammatory cytokines. The presence of high levels of LPS in contaminated bathing water led to induction of inflammatory response from our in vitro cell-based bioassays suggesting its potential health impact. This finding introduces an in vitro culture assay that reflects the level of LPS in water samples. These observations further promote previous finding that LPS is a reliable surrogate biomarker for fecal contamination of bathing water.

Folic acid conjugated polymeric drug delivery vehicle for targeted cancer detection in hepatocellular carcinoma.

Targeted therapies provide increased efficiency for the detection and treatment of cancer with reduced side effects. Folate receptor (alpha subunit) is overexpressed in multiple tumors including liver cancer. In this study, we evaluated the specificity and toxicity of a folic acid-containing drug delivery vehicle (DDV) in a hepatocellular carcinoma (HCC) model. The DDV was prepared with two units each of folic acid (FA) and fluorescein isothiocyanate (FITC) molecules and conjugated to a central poly (ethylene glycol) (PEG) core via a modified chemo-enzymatic synthetic process. Rat hepatoma (N1S1) and human monocytic (U937) cell lines were used for cell culture-based assays and tested for DDV uptake and toxicity. Folate receptor expressions in liver tissues and cell lines were verified using standard immunohistochemistry techniques. Rat HCC model was used for in vivo assessment. The DDV was injected via intra-arterial or intravenous methods and imaged with IVIS spectrum in vivo imaging system. Strong signals of FITC in the liver tumor region correlated to targeted DDV uptake. The use of PEG enhanced water-solubility and provided flexibility for the interaction of FA ligands with multiple cell surface folate receptors that resulted in increased specific uptake. Our study suggested that PEG incorporation and folate targeting via intra-arterial approach is an efficient strategy for targeted delivery in HCC therapy.

First estimate of the scale of canonical 5' splice site GT>GC variants capable of generating wild-type transcripts.

It has long been known that canonical 5' splice site (5'SS) GT>GC variants may be compatible with normal splicing. However, to date, the actual scale of canonical 5'SSs capable of generating wild-type transcripts in the case of GT>GC substitutions remains unknown. Herein, combining data derived from a meta-analysis of 45 human disease-causing 5'SS GT>GC variants and a cell culture-based full-length gene splicing assay of 103 5'SS GT>GC substitutions, we estimate that ~15-18% of canonical GT 5'SSs retain their capacity to generate between 1% and 84% normal transcripts when GT is substituted by GC. We further demonstrate that the canonical 5'SSs in which substitution of GT by GC-generated normal transcripts exhibit stronger complementarity to the 5' end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were capable of reliably distinguishing 5'SS GT>GC variants that generated wild-type transcripts from those that did not. Our findings imply that 5'SS GT>GC variants in human disease genes may not invariably be pathogenic.

Microgels Sopping Up Toxins-GM1a-Functionalized Microgels as Scavengers for Cholera Toxin.

Vibrio cholerae is a Gram-negative bacterium that causes secretory diarrhea and constitutes a major health threat in the industrialized world and even more in developing countries. Its main virulence factor is the cholera toxin, which is internalized by intestinal epithelial cells after binding to the glycosphingolipid receptor GM1a on their apical surface. A potential future solution to dampen complications of cholera infection is by scavenging the cholera toxin by presenting competitive binding motifs to diminish the in vivo toxicity of V. cholerae. Here, we generate GM1a-functionalized and biocompatible microgels with diameters of 20 μm using drop-based microfluidics. The microgels are designed to exhibit a mesoporous and widely meshed network structure, allowing diffusion of the toxin protein deep into the microgel scavengers. Flow cytometry demonstrates strong and multivalent binding at high capacity of these microgels to the binding domain of the cholera toxin. Cell culture-based assays reveal the ability of these microgels to scavenge and retain the cholera toxin in direct binding competition to colorectal cells. This ability is evidenced by suppressed cyclic adenosine monophosphate production as well as reduced vacuole formation in mucus-forming colorectal HT-29 cells. Therefore, glycan-functionalized microgels show great potential as a non-antibiotic treatment for toxin-mediated infectious disorders.

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

The Polyphenolic Composition of Extracts Derived from Different Greek Extra Virgin Olive Oils Is Correlated with Their Antioxidant Potency.

Olive oil possesses a predominant role in the diet of countries around the Mediterranean basin, whereas it is a known constituent of several sectors of human culture. The polyphenolic composition of olive oil seems to be a key factor in its beneficial biological properties. Based on the above, the aim of this study was to correlate the polyphenolic composition of five extracts derived from a Greek olive oil variety with their antioxidant potency and antimutagenic activities in vitro with chemical-based techniques and cell culture-based assays. According to the results obtained, the polyphenol samples with higher concentration of hydroxytyrosol (HT) were more potent in antioxidant and antimutagenic activity in vitro, as indicated by their ability to scavenge ABTS·+ radical and to protect the strand of plasmid DNA from free radical-induced breaking compared to the corresponding samples with higher levels of tyrosol (T) and its derivatives. However, this observation was not evident in the cell culture model (i.e., the HeLa cervical cancer cell line) to which the tested extracts were administered. Specifically, the T-rich extracts more effectively increased endogenous GSH levels measured by flow cytometry than did the HT-rich compounds. Also, olive oil compounds contributed variously to the expression of genes implicated in the cell antioxidant machinery, as indicated by quantitative PCR. Therefore, the relationship between structure and function in redox regulation is complex and merits the combination of tests. Given that factors like the production and storage regimen of the plants are major determinants of the composition of the generated extracts, we propose that specific conditions should be adopted in order to achieve their maximum biological activity. These results followed by others in the same direction could provide a solid basis for the production of functional foods enriched in olive oil extracts with potential antioxidant action in vivo.

Baloxavir marboxil susceptibility of influenza viruses from the Asia-Pacific, 2012–2018

Baloxavir Marboxil (BXM) is an influenza polymerase inhibitor antiviral that binds to the endonuclease region in the PA subunit of influenza A and B viruses. To establish the baseline susceptibility of viruses circulating prior to licensure of BXM and to monitor for susceptibility post-BXM use, a cell culture-based focus reduction assay was developed to determine the susceptibility of 286 circulating seasonal influenza viruses, A(H1N1)pdm09, A(H3N2), B (Yamagata/Victoria) lineage viruses, including neuraminidase inhibitor (NAI) resistant viruses, to Baloxavir Acid (BXA), the active metabolic form of BXM. BXA was effective against all influenza subtypes tested with mean EC50 values (minimum-maximum) of 0.7 ± 0.5 nM (0.1–2.1 nM), 1.2 ± 0.6 nM (0.1–2.4), 7.2 ± 3.5 nM (0.7–14.8), and 5.8 ± 4.5 nM (1.8–15.5) obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses, respectively. Using reverse genetics, amino acid substitutions known to alter BXA susceptibility were introduced into the PA protein resulting in EC50 fold change increases that ranged from 2 to 65. Our study demonstrates that currently circulating viruses are susceptible to BXA and that the newly developed focus reduction assay is well suited to susceptibility monitoring in reference laboratories.

Toward a clinical diagnostic pipeline for SPINK1 intronic variants

BackgroundThe clinical significance of SPINK1 intronic variants in chronic pancreatitis has been previously assessed by various approaches including a cell culture-based full-length gene assay. A close correlation between the results of this assay and in silico splicing prediction was apparent. However, until now, a clinical diagnostic pipeline specifically designed to classify SPINK1 intronic variants accurately and efficiently has been lacking. Herein, we present just such a pipeline and explore its efficacy and potential utility in potentiating the classification of newly described SPINK1 intronic variants.ResultsWe confirm a close correlation between in silico splicing prediction and results from the cell culture-based full-length gene assay in the context of three recently reported pathogenic SPINK1 intronic variants. We then integrated in silico splicing prediction and the full-length gene assay into a stepwise approach and tested its utility in the classification of two novel datasets of SPINK1 intronic variants. The first dataset comprised 16 deep intronic variants identified in 52 genetically unexplained Chinese chronic pancreatitis patients by sequencing the entire intronic sequence of the SPINK1 gene. The second dataset comprised five novel rare proximal intronic variants identified through the routine analysis of the SPINK1 gene in French pancreatitis patients. Employing a minor allele frequency of > 5% as a population frequency filter, 6 of the 16 deep intronic variants were immediately classified as benign. In silico prediction of the remaining ten deep intronic variants and the five rare proximal intronic variants with respect to their likely impact on splice site selection suggested that only one proximal intronic variant, c.194 + 5G > A, was likely to be of functional significance. Employing the cell culture-based full-length gene assay, we functionally analyzed c.194 + 5G > A, together with seven predicted non-functional variants, thereby validating their predicted effects on splicing in all cases.ConclusionsWe demonstrated the accuracy and efficiency of in silico prediction in combination with the cell culture-based full-length gene assay for the classification of SPINK1 intronic variants. Based upon these findings, we propose an operational pipeline for classifying SPINK1 intronic variants in the clinical diagnostic setting.

In vitro anti-inflammatory activity of terpenes via suppression of superoxide and nitric oxide generation and the NF-κB signalling pathway.

Terpenes are considered the main components of essential oils and an important source for the identification of novel lead molecules. This study aimed to investigate the in vitro anti-inflammatory activity of L-carveol, L-carvone, and m-cimene (monoterpenes) and of valencene and guaiene (sesquiterpenes). The influence on intracellular nitric oxide (NO) and pro- and anti-inflammatory cytokine (TNF-α, IL-1α and IL-10) production and on nuclear factor kappa B (NF-κB) activity was determined using Griess reagent, immunoenzymatic assay kits (ELISA) and chemiluminescence measurements in cell-based assays, respectively. Antioxidant activity was assayed through the protective effect against cellular oxidative damage produced by superoxide anion production (O 2 ·- ) and hydrogen peroxide on macrophages and by the quenching activity of the NO radical. Terpenes reduced the pro-inflammatory cytokines TNF-α and IL-1α and increased the production of IL-10. In addition, the terpenes, especially guaiene (53.3 ± 2.4%) and m-cymene (38.1 ± 0.6%), significantly inhibited NO production in a macrophage cell culture-based assay, whereas no effect was observed in the scavenging activity of this radical. L-carveol and m-cymene significantly inhibited O 2 ·- production with reductions of approximately 68.6 ± 2.2% and 48.2 ± 4.2%, respectively, at a concentration of 10μM. Moreover, these terpenes were verified to suppress NF-κB activity. The results indicate that these terpenes have therapeutic potential and may be used to suppress inflammatory diseases or as a leading compounds.

Second-Generation Nitazoxanide Derivatives: Thiazolides are Effective Inhibitors of the Influenza a Virus

The only small molecule drugs currently available for treatment of influenza A virus (IAV) are M2 ion channel blockers and sialidase inhibitors. The prototype thiazolide, nitazoxanide, has successfully completed Phase III clinical trials against acute uncomplicated influenza. We report the activity of seventeen thiazolide analogs against A/PuertoRico/8/1934(H1N1), a laboratory-adapted strain of the H1N1 subtype of IAV, in a cell culture-based assay. A total of eight analogs showed IC50s in the range of 0.14-5.0μM. Additionally a quantitative structure-property relationship study showed high correlation between experimental and predicted activity based on a molecular descriptor set. A range of thiazolides show useful activity against an H1N1 strain of IAV. Further evaluation of these molecules as potential new small molecule therapies is justified.