Cholesteric liquid crystal biosensor platform with image analysis for rapid detection of COVID-19.

Rapid and low-cost diagnosis of coronavirus disease 2019 (COVID-19) is essential to identify infected subjects, particularly asymptomatic cases, primarily to arrest the spread of the disease through local transmission. Antibody-based chromatographic serological tests, as an alternative to the RT-PCR technique, offer only limited help due to high false positives. We propose to exploit our cholesteric liquid crystal biosensor platform for one-step, wash-free, rapid detection of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus directly with minimal sample pre-processing. As mentioned above, cholesteric liquid crystals are an effective and innovative approach to healthcare as a rapid test for the diagnosis of COVID-19 and other diseases.

Label-Free, Smartphone-Based, and Sensitive Nano-Structural Liquid Crystal Aligned by Ceramic Silicon Compound-Constructed DMOAP-Based Biosensor for the Detection of Urine Albumin.

INTRODUCTION: The sensitive interfacial interaction of liquid crystals (LC) holds potential for precision biosensors. In the past, the developments of LC biosensors were limited by the complicated manufacturing process, which hinders commercialization and wider applications of such devices. In this report, we demonstrate the first nano-structural polymeric stabilized-cholesteric LC (PSCLC) thin films to be a new label-free biosensing technology. METHODS: The transmission spectra of PSCLC devices were measured by the fiber-optic spectrometer with high-resolution. In addition, a smartphone was set on the stage, and the camera of smartphone was placed and aligned with a set of lenses embedded in the designed stage. To decrease the chromatic and spherical aberrations, an achromatic lens set composition, consisting of both dual-convex lens and concave-plane lens, was applied for measuring and imaging the PSCLC texture. The average and the estimated standard deviation (SD) were used to present quantitative experimental results. The test BSA was immobilized and fulfilled by the ceramic silicon-constructed DMOAP-coated glass in aqueous BSA solutions at 1 mg/mL, 1 µg/mL, and 1 ng/mL. RESULTS: The fabrication process of PSCLC is much simplified compared to previous LC biosensors. The color of PSCLC biosensor altered with the BSA concentration, making detection result easy to read. The detection limit of 1 ng/mL is achieved for label-free PSCLC biosensor. The PSCLC biosensor was able to successfully detect due to the albumin concentration's alteration, with a linear range of 1 ng/mL-2 mg/mL. Thus, the label-free-proposed design-integrated nanoscale PSCLCs smartphone-based biosensor could successfully detect BSA in a preclinical urine sample. CONCLUSION: Finally, we propose a design to integrate the PSCLC biosensor with a smartphone. The PSCLC owns potential for high performance, low cost for detecting various disease biomarkers in home use. Owing to its great potential for high performance and low cost, the PSCLC biosensors can be used as a label-free point-of-care for detecting various disease biomarkers for patients in care homes.

Dielectric Thermal Smart Glass Based on Tunable Helical Polymer-Based Superstructure for Biosensor with Antibacterial Property.

A dielectric thermal smart glass (DTSG) based on the dielectric heating optical (DHO) effect in tunable helical polymer-based superstructures-cholesteric liquid crystals (CLCs)-was exhibited in this study. Field-induced dielectric heating can strongly affect the orientation of liquid crystals and change its optical properties. The purpose of this research focuses on dual-frequency CLC materials characterized by their specific properties on dielectric relaxation and demonstrates their potential for antibacterial biosensor applications. The developed DTSG is driven by voltages with modulated frequencies. The principal of DTSG in transparent states are a planar (P) state and a heated planar (HP) state reflecting infrared light, operated with the voltage at low and high frequencies, respectively. The scattering states are a focal conic (FC) state and a heated FC (HFC) state, with an applied frequency near the crossover frequency. The biomolecule detection of the antibacterial property was also demonstrated. The detection limitation of the DTSG biosensor was found to be about 0.5 µg/mL. The DTSG material has many potential industrial applications, such as in buildings, photonic devices, and biosensor applications.

Novel purification method and antibiotic activity of recombinant Momordica charantia MAP30.

Ribosome-inactivating proteins (RIPs) are a group of enzymes originally isolated from plants that possess the ability to damage ribosomes in an irreversible manner, leading to inhibition of protein synthesis in eukaryotic cells. In this study, we aimed to purify recombinant RIPs, investigate their function in the treatment of bacterial infection, and determine their toxicity in mice. We employed a pMAL protein fusion and purification system using E. coli transformed with a plasmid containing MBP-tagged MAP30 cDNA. MBP-tagged MAP30 was purified using a modified novel protocol to effectively produce highly active MAP30 of high purity. In an acute toxicity study in mice, no mortality occurred at doses lower than 1.25 mg/kg. MAP30 at both 0.42 and 0.14 mg/kg induced anti-MAP30 IgG, which reached a maximum titer at week 3. In conclusion, recombinant MAP30 prepared using our purification method possesses bioactivity, and has a synergistic bacteria-killing effect that can significantly reduce the required dosages of chloramphenicol and erythromycin. Therefore, when MAP30 is used in combination with chloramphenicol or erythromycin, it may of benefit in terms of reducing the side effects of the antibiotics, as lower concentrations of antibiotics are required.

Ursolic Acid Suppresses Hepatitis B Virus X Protein-mediated Autophagy and Chemotherapeutic Drug Resistance.

Hepatitis B virus X (HBx) protein is a multifunctional oncoprotein that affects diverse cell activities via regulation of various host cell signaling pathways. The current investigation demonstrated that ursolic acid (UA), a pentacyclic triterpenoid, protected hepatoma cells and reduced HBx-mediated autophagy through modulation of Ras homolog gene family member A (RhoA). Low-level ectopic HBx expression in Huh7 cells induced more significant autophagosome formation than high-level HBx expression. HBx activated beclin-1 promoter and enhanced the beclin-1 protein expression under low HBx expression. Transcription factor AP-1 played an essential function in HBx-mediated beclin-1 promoter activation. Inhibition of RhoA and its downstream effector Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) alleviated HBx-mediated autophagy significantly. Transiently-expressed HBx elicited an increased RhoA-GTP level, as well as phospho-ROCK1 transient accumulation. Utilization of transactivation-deficient HBx demonstrated that the transactivation activity of HBx is required for autophagy induction. Furthermore, UA suppressed HBx-mediated RhoA activation, beclin-1 promoter activation and subsequent autophagy induction, while, most importantly, reversed HBx-induced anti-cancer drug resistance.

Potentiation of Acute Promyelocytic Leukemia Cell Differentiation and Prevention of Leukemia Development in Mice by Oleanolic Acid.

Although differentiation therapy with all-trans retinoic acid (ATRA) induces complete remission in most acute promyelocytic leukemia (APL) patients, it is associated with organ toxicity. The present study focused on investigating the effects of the natural compounds oleanolic acid (OA) and ursolic acid (UA) on proliferation and differentiation of human APL HL-60 cells in vitro and murine APL WEHI-3 cells in vivo. Results demonstrated that OA and UA significantly inhibited cellular proliferation of HL-60 in a concentration- and time-dependent manner. Non-cytotoxic concentration of OA exhibited a marked differentiation-inducing effect on HL-60 and enhanced ATRA-induced HL-60 differentiation. In contrast, UA showed only a moderate effect. Activation of MAPK/NF-κB signaling pathway was likely found to be involved in the mechanism. Moreover, OA increased survival duration of WEHI-3 transplanted BALB/c mice, and decreased leukemia cells infiltration in the liver and spleen. Thus, these results may provide new insight for developing alternative therapy in APL patients.

RhoA/ROCK1 regulates Avian Reovirus S1133-induced switch from autophagy to apoptosis.

BACKGROUND: Autophagy is an essential process in the control of cellular homeostasis. It enables cells under certain stress conditions to survive by removing toxic cellular components, and may protect cells from apoptosis. In the present study, the signaling pathways involved in ARV S1133 regulated switch from autophagy to apoptosis were investigated. RESULTS: ARV S1133 infection caused autophagy in the early to middle infectious stages in Vero and DF1 cells, and apoptosis in the middle to late stages. Conversion of the autophagy marker LC3-I to LC3-II occurred earlier than cleavage of the apoptotic marker caspase-3. ARV S1133 also activated the Beclin-1 promoter in the early to middle stages of infection. Levels of RhoA-GTP and ROCK1 activity were elevated upon ARV S1133 infection, while inhibition of RhoA and ROCK1 reduced autophagy and subsequent apoptosis. Conversely, inhibition of caspase-3 did not affect the level of autophagy. Beclin-1 knockdown and treatment with autophagy inhibitors, 3-MA and Bafilomycin A1, suppressed ARV S1133-induced autophagy and apoptosis simultaneously, suggesting the shift from autophagy to apoptosis. A co-immunoprecipitation assay demonstrated that the formation of a RhoA, ROCK1 and Beclin-1 complex coincided with the induction of autophagy. CONCLUSION: Our results demonstrate that RhoA/ROCK1 signaling play critical roles in the transition of cell activity from autophagy to apoptosis in ARV S1133-infected cells.

Avian reovirus S1133-induced apoptosis is associated with Bip/GRP79-mediated Bim translocation to the endoplasmic reticulum.

In this study the mechanism of avian reovirus (ARV) S1133-induced pathogenesis was investigated, with a focus on the contribution of ER stress to apoptosis. Our results showed that upregulation of the ER stress response protein, as well as caspase-3 activation, occurred in ARV S1133-infected cultured cells and in SPF White Leghorn chicks organs. Upon infection, Bim was translocated specifically to the ER, but not mitochondria, in the middle to late infectious stages. In addition, ARV S1133 induced JNK phosphorylation and promoted JNK-Bim complex formation, which correlated with the Bim translocation and apoptosis induction that was observed at the same time point. Knockdown of BiP/GRP78 by siRNA and inhibition of BiP/GRP78 using EGCG both abolished the formation of the JNK-Bim complex, caspase-3 activation, and subsequent apoptosis induction by ARV S1133 efficiently. These results suggest that BiP/GRP78 played critical roles and works upstream of JNK-Bim in response to the ARV S1133-mediated apoptosis process.

Suppression of apoptosis by pseudorabies virus Us3 protein kinase through the activation of PI3-K/Akt and NF-κB pathways.

The pseudorabies virus (PRV) is a major viral disease that causes huge economic loss in the pig industry globally. Most viruses have been found to generate anti-apoptotic factors that facilitate cell survival in the early stages of infection. This study aimed to investigate the anti-apoptotic effects of PRV and study the underlying mechanisms in the early stage of infection. We investigated and compared whether the two PRV Us3 isoforms, Us3a and Us3b, could block apoptosis induced by virus infection, and further identified molecules involved in the signaling pathways. Our results demonstrated that PRV elicits 3-phosphoinositide dependent protein kinase-1/phosphatidylinositide 3-kinases/Akt (PDK-1/PI3-K/Akt)- and nuclear factor-κB (NF-κB)-dependent signaling in the early stage of infection. Inhibition of the PI3-K/Akt or NF-κB pathway enhanced cell death but no effect was observed on virus replication or PRV gene expression. Transiently-expressed GFP- or His-tagged PRV Us3a and Us3b cDNA protect cells against PRV-, avian reovirus- or bovine ephemeral fever virus-induced apoptosis in the cell lines. Us3a and Us3b transient over-expression upregulated several anti-apopototic signaling events, and the anti-apoptosis activity of Us3a is greater than that of Us3b. Kinase activity-deficient point or double point mutated Us3a lost the kinase activity of Us3a, which showed that kinase activity is required for the anti-apoptosis effect of Us3. Akt and NF-κB activation still occurred in UV-inactivated PRV- and cycloheximide-treated cells. In vivo study showed that PRV-infected trigeminal ganglion increases the expression of anti-apoptosis signaling molecules, including Akt, PDK-1 and IκBα, which is a similar result to that seen in the in vitro experiments. Our study suggests that signaling mechanisms may play important roles in PRV pathogenesis.

Suppression of AMF/PGI-mediated tumorigenic activities by ursolic acid in cultured hepatoma cells and in a mouse model.

Our previous studies demonstrated that autocrine motility factor/phosphoglucose isomerase (AMF/PGI) possesses tumorigenic activities through the modulation of intracellular signaling. We then investigated the effects of ursolic acid (UA), oleanolic acid (OA), tangeretin, and nobiletin against AMF/PGI-mediated oncogenesis in cultured stable Huh7 and Hep3B cells expressing wild-type or mutated AMF/PGI and in a mouse model in this study. The working concentrations of the tested compounds were lower than their IC10 , which was determined by Brdu incorporation and colony formation assay. Only UA efficiently suppressed the AMF/PGI-induced Huh7 cell migration and MMP-3 secretion. Additionally, UA inhibited the AMF/PGI-mediated protection against TGF-ß-induced apoptosis in Hep3B cells, whereas OA, tangeretin, and nobiletin had no effect. In Huh7 cells and tumor tissues, UA disrupted the Src/RhoA/PI 3-kinase signaling and complex formation induced by AMF/PGI. In the Hep3B system, UA dramatically suppressed AMF/PGI-induced anti-apoptotic signaling transmission, including Akt, p85, Bad, and Stat3 phosphorylation. AMF/PGI enhances tumor growth, angiogenesis, and pulmonary metastasis in mice, which is correlated with its enzymatic activity, and critically, UA intraperitoneal injection reduces the tumorigenesis in vivo, enhances apoptosis in tumor tissues and also prolongs mouse survival. Combination of sub-optimal dose of UA and cisplatin, a synergistic tumor cell-killing effects was found. Thus, UA modulates intracellular signaling and might serve as a functional natural compound for preventing or alleviating hepatocellular carcinoma.

Avian reovirus S1133-induced DNA damage signaling and subsequent apoptosis in cultured cells and in chickens.

In this study, intracellular signaling in ARV S1133-mediated apoptosis was investigated. A microarray was used to examine the gene expression profiles of cells upon ARV S1133 infection and ARV-encoded pro-apoptotic protein σC overexpression. The analysis indicated that in the set of DNA-damage-responsive genes, DDIT-3 and GADD45α were both upregulated by viral infection and σC overexpression. Further investigation demonstrated that both treatments caused DNA breaks, which increased the expression and/or phosphorylation of DNA damage response proteins. ROS and lipid peroxidation levels were increased, and ARV S1133 and σC caused apoptosis mediated by DNA damage signaling. ROS scavenger NAC, caffeine and an ATM-specific inhibitor significantly reduced ARV S1133- and σC-induced DNA breaks, DDIT-3 and GADD45α expression, H2AX phosphorylation, and apoptosis. Overexpression of DDIT-3 and GADD45α enhanced the oxidative stress and apoptosis induced by ARV S1133 and σC. In conclusion, our results demonstrate the involvement of the DNA-damage-signaling pathway in ARV S1133- and σC-induced apoptosis.

Suppression of hepatitis B virus x protein-mediated tumorigenic effects by ursolic Acid.

This study investigated the potential effects of natural products ursolic acid (UA) and oleanolic acid (OA) against HBx-mediated tumorigenic activities in vitro and in vivo. HBx transactivated Sp-1 and Smad 3/4 in Huh7 and FL83B hepatocytes and induced cell migration of Huh7 and HepG2. HBx also induced MMP-3 secretion in Huh7 and acted against TGF-ß-induced apoptosis in Hep3B. UA almost completely blocked the HBx-mediated effects, while OA had a partial inhibitive effect. Utilization of specific MAPK inhibitors and immunoblotting demonstrated that UA selectively activated MAPK signaling in certain tested cells. Preintraperitoneal injection of UA fully prevented the tumor growth of HBV-containing 2.2.15 cells, while OA-treated mice had smaller tumors than the control group. Our results suggested that UA possesses a hepatoprotective ability and illustrated the evident effects against HBx-mediated tumorigenic activities without toxicity in a mouse model.

Activation of PI 3-kinase/Akt/NF-kappaB and Stat3 signaling by avian reovirus S1133 in the early stages of infection results in an inflammatory response and delayed apoptosis.

Avian reovirus (ARV) strain S1133 causes apoptosis in host cells in the middle to late stages of infection. This study investigated the early-stage biological response and intracellular signaling in ARV S1133-infected Vero and chicken cells. Treatment with conditioned medium from ARV S1133-infected cells increased the chemotactic activity of U937 cells. Neutralizing antibodies against IL-1beta and IL-6 showed that both cytokines contribute to viral-induced inflammation but neither affect cell survival. Inhibition of Akt, NF-kappaB, and Stat3 released the chemotactic activity and anti-apoptotic effect elicited by ARV S1133. ARV S1133 activated PI 3-kinase-dependent Akt/NF-kappaB and p70 S6 kinase, as well as Stat3; however, p70 S6 kinase was not involved in ARV S1133-mediated effects. DF1 cells over-expressing constitutively active PI 3-kinase and Stat3 showed association with enhancement of anti-apoptotic activity. In conclusion, in the early stages of ARV S1133 infection, activation of cell survival signals contributes to virus-induced inflammation and anti-apoptotic response.

PI 3-kinase/Akt and STAT3 are required for the prevention of TGF-beta-induced Hep3B cell apoptosis by autocrine motility factor/phosphoglucose isomerase.

We established Hep3B cells stably-expressing wild-type and mutated AMF/PGI with differing enzymatic activities in order to investigate how AMF/PGI affects TGF-beta-induced apoptosis, and demonstrated that AMF/PGI against TGF-beta-induced apoptosis was correlated with its enzymatic activity. AMF/PGI did not alter TGF-beta-receptor expression nor affect TGF-beta-induced PAI-1 gene promoter or Smad3/4 activity. AMF/PGI induced PI 3-kinase activity, IRS and Akt phosphorylation, which can further regulate BAD phosphorylation. Constitutively-active p110 enhanced AMF/PGI-mediated anti-apoptosis activity, and dominant negative Akt alleviated anti-TGF-beta-induced apoptosis. We also demonstrated that STAT3 is a weak anti-apoptotic agent but has an increased anti-apoptotic effect in cooperation with PI 3-kinase/Akt.

Modulation of p53 by mitogen-activated protein kinase pathways and protein kinase C delta during avian reovirus S1133-induced apoptosis.

ARV S1133 infection caused apoptosis in vivo and in vitro; however, the intracellular signaling pathways have not been fully delineated. We have previously demonstrated that ARV S1133 activates proapoptotic signaling from Src to p53, and further investigated how ARV S1133 modulates p53. We found that ARV S1133 forms syncytia and induces apoptosis in CEF, DF1 and Vero cells with different kinetics. Enhancement of p53 phosphorylation and DNA-binding capacity to bax and bad promoters was found in this study to increase bax and bad expression in ARV S1133-infected cells. ARV S1133 activates PKC delta and p38 and JNK/SAPK pathways, and inhibition of Ras, p38, JNK/SAPK and PKC delta works efficiently against apoptosis. Suppression of p38, JNK/SAPK and PKC delta selectively abolished ARV S1133-mediated p53 phosphorylation; moreover, inhibition of Src did not affect ARV S1133-induced p38 and JNK/SAPK activation, whereas blocking of Ras resulted in a reduction in the activities of p38 and JNK/SAPK.

TNF-alpha mediates pseudorabies virus-induced apoptosis via the activation of p38 MAPK and JNK/SAPK signaling.

PRV infection causes apoptosis in vitro and in vivo. However, the significance of PRV-induced apoptosis and its signaling pathways is still unknown. This work investigates the role of MAPK pathways in mediating PRV-induced apoptosis. Flow cytometry, apoptosis ELISA and western blotting using antibodies against cleaved caspase-3, -6 and PARP demonstrated that PRV induces apoptosis in a time- and dose-dependent manner. p38 and JNK/SAPK inhibitors significantly protected cells from PRV-induced apoptosis. Inhibitor treatment did not affect Us3a gene transcription and progeny virus production. Western blotting revealed that PRV activates p38 and JNK/SAPK signaling. Inhibition of NF-kappaB had no effect on PRV-mediated apoptosis. Non-replicative PRV failed to activate p38 and JNK/SAPK or induce apoptosis. PRV infection increases TNF-alpha transcription, translation and secretion, as well as TNF-alpha receptor expression. Inhibition of p38 and JNK/SAPK reduced PRV-induced TNF-alpha up-regulation. Neutralization assay confirmed that TNF-alpha is a key mediator involved in PRV-induced apoptosis.

Activation of small GTPases RhoA and Rac1 is required for avian reovirus p10-induced syncytium formation.

The first ORF of the ARV S1133 S1 segment encodes the nonstructural protein p10, which is responsible for the induction of cell syncytium formation. However, p10-dependent signaling during syncytium formation is fully unknown. Here, we show that dominant negative RhoA, Rho inhibitor C3 exoenzyme, ROCK/Rho-kinase inhibitor Y-27632 and Rac1 inhibitor NSC23766 inhibit p10-mediated cell fusion. p10 over-expression is concomitant with activation and membrane translocation of RhoA and Rac1, but not cdc42. RhoA and Rac1 downstream events, including JNK phosphorylation and transcription factor AP-1 and NF-kappaB activation, as well as MLC expression and phosphorylation are simultaneously activated by p10. p10 point mutant T13M possessed 20% fusion-inducing ability and four p10 fusion-deficient mutants V15M, V19M, C21S and L32A reduced or lost their ability to activate RhoA and Rac1 signaling. We conclude that p10-mediated syncytium formation proceeds by utilizing RhoA and Rac1-dependent signaling.

AMF/PGI transactivates the MMP-3 gene through the activation of Src-RhoA-phosphatidylinositol 3-kinase signaling to induce hepatoma cell migration.

We have previously shown that AMF/PGI induces hepatoma cell migration through the induction of MMP-3. This work investigates how AMF/PGI activates the MMP-3 gene. We demonstrated that AMF/PGI transactivates the MMP-3 gene promoter through AP-1. The transactivation and induction of cell migration effect of AMF/PGI directly correlates with its enzymatic activity. Various analyses showed that AMF/PGI stimulated the Src-RhoA-PI3-kinase signaling pathway, and these three signaling molecules could form a complex. Our results demonstrate a new mechanism of AMF/PGI-induced cell migration and a link between Src-RhoA-PI3-kinase, AP-1, MMP-3 and hepatoma cell migration.

Retardation of cell growth by avian reovirus p17 through the activation of p53 pathway.

The second open reading frame of avian reovirus S1 gene segment encodes a 17 kDa non-structural protein, named p17. The biological role of p17 is fully unknown so far. Using trypan blue dye exclusion and MTT assay, we demonstrated that the ectopic expression of p17 results in the reduction of viable cell number and cell proliferation rate of Vero, BHK, 293, and HeLa cells. Measurement of LDH activity and DNA fragmentation analysis revealed that p17 expression did not cause cell death or apoptosis. These data indicated that the p17 possessed the growth retardation function. Semi-quantitative RT-PCR and Western blotting revealed that p17-expressing cells induced the expression of CDK inhibitor p21cip1/waf1 in a time- and dose-dependent manner, but the transcripts of CDK inhibitor p15INK4b, p16INK4a, or p27kip were not altered. In the presence of p17, the p53 protein level and p53-driven reporter activity were elevated significantly. Dominant negative p53 alleviated the p21 accumulation, p53 activation, and growth inhibition effect induced by p17. Taken together, these studies revealed a possible intrinsic function of p17 in growth regulation through the activation of p53 and p21cip1/waf1.