Pluchea indica is a shrub plant found in mangrove forests. The leaves are consumed as food and herbal tea and exhibit various biological effects, such as antioxidant and anti-inflammatory activities, in macrophages and animal models. However, the inhibitory activity of P. indica leaf extract on vascular inflammation remains unknown. Therefore, this study investigated the effect of an ethanol extract from P. indica herbal tea leaves (PIE) on tumor necrosis factor-α (TNF-α)-induced human vascular endothelial EA.hy926 cells. The cytotoxic effect of PIE was determined by thiazolyl blue tetrazolium bromide assays. PIE at concentrations of 12.5 - 50 µg/mL did not show significant cytotoxicity, whereas PIE at concentrations ≥ 100 µg/mL decreased cell viability. PIE inhibited the production of reactive oxygen species (ROS) in TNF-α-stimulated endothelial cells. To evaluate the PIE’s anti-vascular inflammatory activity, the protein expression of cell adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), was determined by western blot. PIE significantly decreased TNF-α-induced ICAM-1 and VCAM-1 expression in a concentration-dependent manner. Furthermore, PIE upregulated heme oxygenase-1 (HO-1) in a concentration- and time-dependent manner. Inhibiting the activity of HO-1 by tin protoporphyrin IX significantly blocked the suppressive effect of PIE on ICAM-1 but not VCAM-1 expression. Therefore, PIE exerts anti-inflammatory activity on vascular endothelial cells, at least in part, by suppressing ROS production and the induction of HO-1. The obtained data suggest that PIE is a promising substance for developing therapeutic agents or as an ingredient of functional food. HIGHLIGHTS Pluchea indica leaf extract (PIE) at non-toxic doses inhibited ICAM-1 and VCAM-1 in TNF-α-induced human vascular endothelial cells PIE suppressed the production of ROS in TNF-α-stimulated endothelial cells PIE exerts anti-inflammatory activity on vascular endothelial cells mediated partly through the upregulation of HO-1

Viral contamination may occur at any stage of food processing. The study aim was to develop a two-step reverse transcription (RT)-recombinase polymerase amplification (RPA) assay and evaluate for in-field duplex detection of hepatitis A virus (HAV) and norovirus in oysters. The RNA expression plasmids were generated by amplifying a fragment of the VP1 gene of HAV and norovirus through PCR and cloning it into an expression vector. The RNAs were transcribed in vitro from the plasmids and further used for reverse transcription. The resulting single-stranded cDNAs were used as the purified or spiking templates to determine the sensitivities of simplex and duplex RT-RPA compared with RT-PCR, and RT-qPCR assays. The reproducibility and application of duplex RT-RPA in the field were also evaluated. Our results showed that simplex RT-RPA was at least 100 times more sensitive than RT-PCR and RT-qPCR and even more than duplex detection using purified targets. Unlike RT-PCR, the RT-RPA reaction was unaffected by inhibitors found in food, allowing simple sample preparation methods for detection within a fraction of the time. The duplex assay detected HAV, norovirus, or both in 12/30 (40%) oyster samples tested. Duplex RT-RPA proved to be a rapid, accurate, and reproducible method in a field test for detecting HAV and norovirus. Thus, duplex RT-RPA should be suitable for use in minimally equipped laboratories and field settings. If in-field RT-RPA services are provided to oyster farmers, the technique can minimize the risk of infection to consumers, thereby improving food safety. Keywords: Food safety, Hepatitis A virus, Direct extraction, Norovirus, Nucleic acid amplification

Monitoring foodborne viruses via nucleic acid amplification tests rely on stable RNA standards to obtain reliable testing. This study aimed to produce RNA-based standard reagents for hepatitis virus (HAV) or norovirus detections which relies on viral-like particle (VLP) technology. Using a plasmid packaging system, plasmids containing DNA encoding Qβcoat protein (CP) monomer and the VP1 gene of viruses were co-transformed into E. coli host cells. In cell lysates, expressed CP was characterized by western blot and the whole icosahedral formation of VLPs was proved by electron microscope analysis. Encapsidated RNAs were measured and assessed as a standard by a two-step reverse transcription recombinase polymerase amplification (RT-RPA). Our results showed that CP has a distinguished protein band with a molecular weight of 14.5 kDa but a few variabilities of particle size were visualized. When adjusting the pH of the lysate to lower than 6, a more intense protein band and substantial particles with homogenous particle size were observed. These VLPs were found to enclose HAV and norovirus RNA contents to 1.2×107 copies/ng and 1.9×107 copies/ng, respectively. When analyzed by RT-RPA, linear regression analysis confirmed the alternative application of RNAs enclosed in VLPs to naked RNA synthesized from in vitro transcription. Using the E. coli expression system to produce Qβ VLPs allows cost-effective production and, therefore, can be implemented in laboratories with basic equipment. These encapsidated RNAs may become an ideal “standard” for detecting foodborne viruses via a molecular test in food and clinical samples. Keywords: Molecular testing, Nanoparticles, Nucleic amplification, RNA standards, Viral-like particles