Purple-fleshed sweet potato P40 has been shown to prevent colorectal cancer in a murine model. This study is to identify anthocyanins by using HPLC/MS-MS and assess the stability during various cooking conditions. P40 possesses a high content of anthocyanins up to 14 mg/g dry matter. Total 12 acylated anthocyanins are identified. Top three anthocyanins, e.g., cyanidin 3-caffeoyl-p-hydroxybenzoyl sophoroside-5-glucoside, peonidin 3-caffeoyl sophoroside-5-glucoside, and cyanidin 3-(6"-caffeoyl-6"-feruloylsophoroside)-5-glucoside, account for half of the anthocyanin contents. Over 80% of anthocyanins measured by acid hydrolysis were cyanidin derivatives, indicating P40 is unique when compared with other purple-fleshed sweet potatoes that usually contain more peonidin than cyanidin. Steaming, pressure cooking, microwaving, and frying but not baking significantly reduced 8-16% of total anthocyanin contents. Mono-acylated anthocyanins showed a higher resistance against heat than di- and non-acylated. Among of which, cyanidin 3-p-hydroxybenzoylsophoroside-5-glucoside exhibited the best thermal stability. The stable acylated and cyanidin-predominated anthocyanins in P40 may provide extra benefits for cancer prevention.

Intercellular nanotube connections have been identified as an alternative pathway for cellular spreading of certain viruses. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nanotubes were observed connecting two distant cells with contiguous membranes, with the core infectious viral machinery (viral RNA, certain replicases, and certain structural proteins) present in/on the intercellular nanotubes. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2. In MARC-145 cells expressing PRRSV receptors, GFP-nsp2 moved from one cell to another through nanotubes in the presence of virus-neutralizing antibodies. Intercellular transport of viral proteins did not require the PRRSV receptor as it was observed in receptor-negative HEK-293T cells after transfection with an infectious clone of GFP-PRRSV. In addition, GFP-nsp2 was detected in HEK-293T cells cocultured with recombinant PRRSV-infected MARC-145 cells. The intercellular nanotubes contained filamentous actin (F-actin) with myosin-associated motor proteins. The F-actin and myosin IIA were identified as coprecipitates with PRRSV nsp1β, nsp2, nsp2TF, nsp4, nsp7-nsp8, GP5, and N proteins. Drugs inhibiting actin polymerization or myosin IIA activation prevented nanotube formation and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response. IMPORTANCE Extracellular virus particles transmit infection between organisms, but within infected hosts intercellular infection can be spread by additional mechanisms. In this study, we describe an alternative pathway for intercellular transmission of PRRSV in which the virus uses nanotube connections to transport infectious viral RNA, certain replicases, and certain structural proteins to neighboring cells. This process involves interaction of viral proteins with cytoskeletal proteins that form the nanotube connections. Intercellular viral spread through nanotubes allows the virus to escape the neutralizing antibody response and may contribute to the pathogenesis of viral infections. The development of strategies that interfere with this process could be critical in preventing the spread of viral infection.

Traditional vaccination strategies have failed to generate effective vaccines for many infections like tuberculosis and HIV. New approaches are needed for each type of disease. The protective immunity and distinct responses of many successful vaccines come from activating multiple Toll-like receptors (TLRs). Vaccines with multiple TLRs as adjuvants have proven effective in preclinical studies, but current research has not explored two important elements. First, few multi-TLR systems explore spatial organization—a critical feature of whole-cell vaccines. Second, no multi-TLR systems to date provide systematic analysis of the combinatorial space of three TLR agonists. Here, we present the first examination of the combinatorial space of several spatially defined triple-TLR adjuvants, by synthesizing a series of five triple-TLR agonists and testing their innate activity both in vitro and in vivo. The combinations were evaluated by measuring activation of immune stimulatory genes (Nf-κB, ISGs), cytokine profiles (IL12-p70, TNF-α, IL-6, IL-10, CCL2, IFN-α, IFN-β, IFN-γ), and in vivo cytokine serum levels (IL-6, TNF-α, IL12-p40, IFN-α, IFN-β). We demonstrate that linking TLR agonists substantially alters the resulting immune response compared to their unlinked counterparts and that each combination results in a distinct immune response, particularly between linked combinations. We show that combinations containing a TLR9 agonist produce more Th1 biasing immune response profiles, and that the effect is amplified upon conjugation. However, combinations containing TLR2/6 agonist are skewed toward T_H2 biasing profiles despite the presence of TLR9. These results demonstrate the profound effects that conjugation and combinatorial administration of TLR agonists can have on immune responses, a critical element of vaccine development.

Botanical folk medicines have been used throughout human history to treat common disorders such as hypertension, often with unknown underlying mechanisms. Here, we discovered that hypotensive folk medicines from a genetically diverse range of plant species each selectively activated the vascular-expressed KCNQ5 potassium channel, a feature lacking in the modern synthetic pharmacopeia, whereas nonhypotensive plant extracts did not. Analyzing constituents of the hypotensive Sophora flavescens root, we found that the quinolizidine alkaloid aloperine is a KCNQ-dependent vasorelaxant that potently and isoform-selectively activates KCNQ5 by binding near the foot of the channel voltage sensor. Our findings reveal that KCNQ5-selective activation is a defining molecular mechanistic signature of genetically diverse traditional botanical hypotensives, transcending plant genus and human cultural boundaries. Discovery of botanical KCNQ5-selective potassium channel openers may enable future targeted therapies for diseases including hypertension and KCNQ5 loss-of-function encephalopathy. Significance Botanical folk medicines have been used by diverse human populations and cultures for several millennia. Many are still in use today, but the underlying molecular mechanisms often remain elusive. Here we report the discovery of a molecular mechanism linking diverse plant extracts used traditionally to lower blood pressure (hypotensives). All of the hypotensive plants tested activated the KCNQ5 vascular-expressed potassium channel, whereas nonhypotensives did not. For one hypotensive plant, we describe discovery of the active small molecule (aloperine) and demonstrate that it KCNQ-dependently relaxes blood vessels. The discovery opens up a new source of potential therapeutic drugs and explains the mechanism behind folk hypotensive medicines used by diverse populations for thousands of years.

The ubiquitous soilborne and plant-pathogenic basidiomycete Rhizoctonia solani, although classified as a single species, is a complex of anastomosis groups (AGs) that cause disease in a broad range of higher plants. Here, we investigated the persistent co-isolation of bacteria with R. solani from brown patch-infected, cool-season turfgrasses, and report the presence of endo-hyphal bacteria, related to members in the genus Enterobacter, in an isolate of R. solani AG 2-2IIIB. The intracellular localization of the bacteria was corroborated by fluorescence, confocal and electron microscopy, and DNA analysis. Furthermore, the Enterobacter sp., which is rod-shaped in the free-living form, exists as an L-form (spheroid) within the fungus, a phenomenon not previously reported in endosymbionts. Our findings also indicate that the bacterium is required for full virulence of R. solani on creeping bentgrass and production of wild type levels of the toxin phenylacetic acid in fungal cultures. The possible presence of bacterial endosymbionts in R. solani AG 2-2IIIB may portend the presence of bacteria in additional AGs as well as other Rhizoctonia species, and may help resolve some of the complexities of R. solani pathogenicity. A closely associated bacterium could influence aspects of plant host pathology.