New Advances in Rapid Pretreatment for Small Dense LDL Cholesterol Measurement Using Shear Horizontal Surface Acoustic Wave (SH-SAW) Technology.

Atherosclerosis is an inflammatory disease of the arteries associated with alterations in lipid and other metabolism and is a major cause of cardiovascular disease (CVD). LDL consists of several subclasses with different sizes, densities, and physicochemical compositions. Small dense LDL (sd-LDL) is a subclass of LDL. There is growing evidence that sd-LDL-C is associated with CVD risk, metabolic dysregulation, and several pathophysiological processes. In this study, we present a straightforward membrane device filtration method that can be performed with simple laboratory methods to directly determine sd-LDL in serum without the need for specialized equipment. The method consists of three steps: first, the precipitation of lipoproteins with magnesium harpin; second, the collection of effluent from a 100 nm filter; and third, the quantification of sd-LDL-ApoB in the effluent with an SH-SAW biosensor. There was a good correlation between ApoB values obtained using the centrifugation (y = 1.0411x + 12.96, r = 0.82, n = 20) and filtration (y = 1.0633x + 15.13, r = 0.88, n = 20) methods and commercially available sd-LDL-C assay values. In addition to the filtrate method, there was also a close correlation between sd-LDL-C and ELISA assay values (y = 1.0483x - 4489, r = 0.88, n = 20). The filtration treatment method also showed a high correlation with LDL subfractions and NMR spectra ApoB measurements (y = 2.4846x + 4.637, r = 0.89, n = 20). The presence of sd-LDL-ApoB in the effluent was also confirmed by ELISA assay. These results suggest that this filtration method is a simple and promising pretreatment for use with the SH-SAW biosensor as a rapid in vitro diagnostic (IVD) method for predicting sd-LDL concentrations. Overall, we propose a very sensitive and specific SH-SAW biosensor with the ApoB antibody in its sensitive region to monitor sd-LDL levels by employing a simple delay-time phase shifted SH-SAW device. In conclusion, based on the demonstration of our study, the SH-SAW biosensor could be a strong candidate for the future measurement of sd-LDL.

Augmenting Neutrophil Extracellular Traps with Carbonized Polymer Dots: A Potential Treatment for Bacterial Sepsis.

Sepsis is a life-threatening condition that can progress to septic shock as the body's extreme response to pathogenesis damages its own vital organs. Staphylococcus aureus (S. aureus) accounts for 50% of nosocomial infections, which are clinically treated with antibiotics. However, methicillin-resistant strains (MRSA) have emerged and can withstand harsh antibiotic treatment. To address this problem, curcumin (CCM) is employed to prepare carbonized polymer dots (CPDs) through mild pyrolysis. Contrary to curcumin, the as-formed CCM-CPDs are highly biocompatible and soluble in aqueous solution. Most importantly, the CCM-CPDs induce the release of neutrophil extracellular traps (NETs) from the neutrophils, which entrap and eliminate microbes. In an MRSA-induced septic mouse model, it is observed that CCM-CPDs efficiently suppress bacterial colonization. Moreover, the intrinsic antioxidative, anti-inflammatory, and anticoagulation activities resulting from the preserved functional groups of the precursor molecule on the CCM-CPDs prevent progression to severe sepsis. As a result, infected mice treated with CCM-CPDs show a significant decrease in mortality even through oral administration. Histological staining indicates negligible organ damage in the MRSA-infected mice treated with CCM-CPDs. It is believed that the in vivo studies presented herein demonstrate that multifunctional therapeutic CPDs hold great potential against life-threatening infectious diseases.

Potential relevance of salivary legumain for the clinical diagnostic of hand, foot, and mouth disease.

The fight against hand, foot, and mouth disease (HFMD) remains an arduous challenge without existing point-of-care (POC) diagnostic platforms for accurate diagnosis and prompt case quarantine. Hence, the purpose of this salivary biomarker discovery study is to set the fundamentals for the realization of POC diagnostics for HFMD. Whole salivary proteome profiling was performed on the saliva obtained from children with HFMD and healthy children, using a reductive dimethylation chemical labeling method coupled with high-resolution mass spectrometry-based quantitative proteomics technology. We identified 19 upregulated (fold change = 1.5-5.8) and 51 downregulated proteins (fold change = 0.1-0.6) in the saliva samples of HFMD patients in comparison to that of healthy volunteers. Four upregulated protein candidates were selected for dot blot-based validation assay, based on novelty as biomarkers and exclusions in oral diseases and cancers. Salivary legumain was validated in the Singapore (n = 43 healthy, 28 HFMD cases) and Taiwan (n = 60 healthy, 47 HFMD cases) cohorts with an area under the receiver operating characteristic curve of 0.7583 and 0.8028, respectively. This study demonstrates the feasibility of a broad-spectrum HFMD POC diagnostic test based on legumain, a virus-specific host systemic signature, in saliva.

The Upf1 protein restricts EV-A71 viral replication.

Enterovirus A71 (EV-A71) is transmitted through the respiratory tract, gastrointestinal system, and fecal-oral routes. The main symptoms caused by EV-A71 are hand, foot, and mouth disease (HFMD) or vesicular sore throat. Upf1 (Up-frameshift protein 1) was reported to degrade mRNA containing early stop codons, known as nonsense-mediated decay (NMD). Upf1 is also involved in the NMD mechanism as a host factor detrimental to viral replication. In this study, we dissected the potential roles of Upf1 in the EV-A71-infected cells. Upf1 was virulently down-regulated in three different EV-A71-infected cells, RD, Hela, and 293T, implying that Upf1 is a host protein unfavorable for EV-A71 replication. Knockdown of Upf1 protein resulted in increased viral RNA expression and production of progeny virus, and conversely, overexpression of Upf1 protein resulted in decreased viral RNA expression and production of progeny virus. Importantly, we observed increased RNA levels of asparagine synthetase (ASNS), one of the indicator substrates for the NMD mechanism, which indirectly suggests that EV-A71 infection of cells suppresses NMD activity in the host. The results shown in this study are useful for subsequent analysis of the relationship between the NMD/Upf1 mechanism and other picornaviruses, which may lead to the development of anti-picornavirus drugs.

Application of Shear Horizontal Surface Acoustic Wave (SH-SAW) Immunosensor in Point-of-Care Diagnosis.

Point-of-care testing (POCT), also known as on-site or near-patient testing, has been exploding in the last 20 years. A favorable POCT device requires minimal sample handling (e.g., finger-prick samples, but plasma for analysis), minimal sample volume (e.g., one drop of blood), and very fast results. Shear horizontal surface acoustic wave (SH-SAW) biosensors have attracted a lot of attention as one of the effective solutions to complete whole blood measurements in less than 3 min, while providing a low-cost and small-sized device. This review provides an overview of the SH-SAW biosensor system that has been successfully commercialized for medical use. Three unique features of the system are a disposable test cartridge with an SH-SAW sensor chip, a mass-produced bio-coating, and a palm-sized reader. This paper first discusses the characteristics and performance of the SH-SAW sensor system. Subsequently, the method of cross-linking biomaterials and the analysis of SH-SAW real-time signals are investigated, and the detection range and detection limit are presented.

Measurements of Anti-SARS-CoV-2 Antibody Levels after Vaccination Using a SH-SAW Biosensor.

To prevent the COVID-19 pandemic that threatens human health, vaccination has become a useful and necessary tool in the response to the pandemic. The vaccine not only induces antibodies in the body, but may also cause adverse effects such as fatigue, muscle pain, blood clots, and myocarditis, especially in patients with chronic disease. To reduce unnecessary vaccinations, it is becoming increasingly important to monitor the amount of anti-SARS-CoV-2 S protein antibodies prior to vaccination. A novel SH-SAW biosensor, coated with SARS-CoV-2 spike protein, can help quantify the amount of anti-SARS-CoV-2 S protein antibodies with 5 µL of finger blood within 40 s. The LoD of the spike-protein-coated SAW biosensor was determined to be 41.91 BAU/mL, and the cut-off point was determined to be 50 BAU/mL (Youden's J statistic = 0.94733). By using the SH-SAW biosensor, we found that the total anti-SARS-CoV-2 S protein antibody concentrations spiked 10−14 days after the first vaccination (p = 0.0002) and 7−9 days after the second vaccination (p = 0.0116). Furthermore, mRNA vaccines, such as Moderna or BNT, could achieve higher concentrations of total anti-SARS-CoV-2 S protein antibodies compared with adenovirus vaccine, AZ (p < 0.0001). SH-SAW sensors in vitro diagnostic systems are a simple and powerful technology to investigate the local prevalence of COVID-19.

Development of antiviral carbon quantum dots that target the Japanese encephalitis virus envelope protein.

Japanese encephalitis is a mosquito-borne disease caused by the Japanese encephalitis virus (JEV) that is prevalent in Asia and the Western Pacific. Currently, there is no effective treatment for Japanese encephalitis. Curcumin (Cur) is a compound extracted from the roots of Curcuma longa, and many studies have reported its antiviral and anti-inflammatory activities. However, the high cytotoxicity and very low solubility of Cur limit its biomedical applications. In this study, Cur carbon quantum dots (Cur-CQDs) were synthesized by mild pyrolysis-induced polymerization and carbonization, leading to higher water solubility and lower cytotoxicity, as well as superior antiviral activity against JEV infection. We found that Cur-CQDs effectively bound to the E protein of JEV, preventing viral entry into the host cells. In addition, after continued treatment of JEV with Cur-CQDs, a mutant strain of JEV was evolved that did not support binding of Cur-CQDs to the JEV envelope. Using transmission electron microscopy, biolayer interferometry, and molecular docking analysis, we revealed that the S123R and K312R mutations in the E protein play a key role in binding Cur-CQDs. The S123 and K312 residues are located in structural domains II and III of the E protein, respectively, and are responsible for binding to receptors on and fusing with the cell membrane. Taken together, our results suggest that the E protein of flaviviruses represents a potential target for the development of CQD-based inhibitors to prevent or treat viral infections.

A Novel Rapid Test to Detect Anti-SARS-CoV-2 N Protein IgG Based on Shear Horizontal Surface Acoustic Wave (SH-SAW).

Since the Coronavirus disease 2019 (COVID-19) pandemic outbreak, many methods have been used to detect antigens or antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including viral culture, nucleic acid test, and immunoassay. The shear-horizontal surface acoustic wave (SH-SAW) biosensor is a novel pathogen detection platform with the advantages of high sensitivity and short detection time. The objective of this study is to develop a SH-SAW biosensor to detect the anti-SARS-CoV-2 nucleocapsid antibody. The rabbit sera collected from rabbits on different days after SARS-CoV-2 N protein injection were evaluated by SH-SAW biosensor and enzyme-linked immunosorbent assay (ELISA). The results showed that the SH-SAW biosensor achieved a high correlation coefficient (R = 0.9997) with different concentrations (34.375-1100 ng/mL) of the "spike-in" anti-N protein antibodies. Compared to ELISA, the SH-SAW biosensor has better sensitivity and can detect anti-N protein IgG signals earlier than ELISA on day 6 (p < 0.05). Overall, in this study, we demonstrated that the SH-SAW biosensor is a promising platform for rapid in vitro diagnostic (IVD) testing, especially for antigen or antibody testing.

High Incidence and Early Onset of Urinary Tract Cancers in Patients with BK Polyomavirus Associated Nephropathy.

Over-immunosuppressed kidney transplant recipients are susceptible to malignancies and BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN). This study aimed to verify the association between BKPyV infection and urinary tract cancers (UTC). A total of 244 kidney transplant recipients were enrolled at Chang Gung Memorial Hospital from June 2000 to February 2020. Biopsy-proven BKPyVAN patients (n = 17) had worse kidney function (eGFR: 26 ± 13.7 vs. 47.8 ± 31.0 mL/min/1.73 m2). The 5-year allograft survival rates for patients with and without BKPyVAN were 67% and 93%, respectively (p = 0.0002), while the 10-year patient survival was not different between the two groups. BKPyVAN patients had a significantly higher incidence of UTC compared to the non-BKPyVAN group (29.4% vs. 6.6%). Kaplan-Meier analysis showed that the UTC-free survival rate was significantly lower in BKPyVAN patients, and the onset of UTC was significantly shorter in BKPyVAN patients (53.4 vs. 108.9 months). The multivariate logistic regression analysis demonstrated that age (RR = 1.062) and BKVAN (RR = 6.459) were the most significant risk factors for the development of UTC. Our study demonstrates that BKPyVAN patients have greater allograft losses, higher incidence, a lower cancer-free survival rate, and an earlier onset with a higher relative risk of developing UTC compared to non-BKPyVAN patients.

Evaluation of ViroTrack Sero Zika IgG/IgM, a New Rapid and Quantitative Zika Serological Diagnostic Assay.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the flavivirus genus and are antigenically closely related. They also share the same mosquito vector and can cause similar symptoms upon infection. However, DENV and ZIKV infections lead to different clinical sequelae and treatments; therefore, clinicians need rapid and accurate diagnostics capable of distinguishing between the two diseases. METHODS: We employed the immuno-magnetic assay technology on a microfluidic cartridge (ViroTrack Sero Zika IgG/IgM) for diagnosis of ZIKV infection based on the aggregation of magnetic nanoparticles. We carried out three serological studies including samples from the Dominican Republic, USA, and Nicaragua, aimed at detecting ZIKV-specific IgG and IgM using the ViroTrack Sero Zika IgG/IgM test. RESULTS: The seroconversion results were comparable with ZIKV IgG and IgM reactivity measured by the commercial ZIKV ELISA kit. The sensitivity and specificity for both ZIKV IgG and IgM tested by the ViroTrack Sero Zika IgG/IgM was approximately 98% and 93%, respectively. CONCLUSION: Serological detection of ZIKV infection by the new ViroTrack Sero Zika IgG/IgM test shows promising performance and limited cross-reactivity with DENV.

Risk of Alzheimer's Disease in Obstructive Sleep Apnea Patients With or Without Treatment: Real-World Evidence.

OBJECTIVE: To assess the risk of Alzheimer's disease (AD) in patients with obstructive sleep apnea (OSA) with or without treatment based on real-world evidence. STUDY DESIGN: Retrospective cohort study. METHODS: Patients newly diagnosed with OSA during 1997-2012 were identified using the National Health Insurance Research Database of Taiwan. Patients without OSA were randomly selected and matched in a 1:4 ratio by age, sex, urbanization level, and income. All patients were followed up until death or the end of 2013. The primary outcome was AD occurrence. RESULTS: This study included 3,978 OSA patients and 15,912 non-OSA patients. OSA was independently and significantly associated with a higher incidence of AD in an adjusted Cox proportional hazard model (adjusted hazard ratio: 2.12; 95% confidence interval [CI], 1.27-3.56). The average period of AD detection from the time of OSA occurrence was 5.44 years (standard deviation: 2.96). Subgroup analyses revealed that the effect of OSA remained significant in patients aged ≥60 years, male subgroups, patients without CPAP or surgical treatment, and patients without pharmacological therapies. Patients with OSA who received treatment (continuous positive airway pressure or surgery) exhibited a significantly reduced risk of AD compared with those without treatment (incidence rate ratio 0.23, 95% CI, 0.06-0.98). CONCLUSION: OSA is independently associated with an increased risk of AD. Treatment for OSA reduces the AD risk in OSA patients. AD irreversibility renders OSA as a potential modifiable target for slowing or preventing the process of AD development. LEVEL OF EVIDENCE: IV Laryngoscope, 130:2292-2298, 2020.

MicroRNA 876-5p modulates EV-A71 replication through downregulation of host antiviral factors.

BACKGROUND: Human enterovirus 71 (EV-A71) is a non-enveloped virus that has a single stranded positive sense RNA genome. In a previous study, we showed that miR-876-5p upregulation was observed in the serum of patients with severe EV-A71 infection. Micro-876-5p (miR-876-5p) is a circulating miRNA that can be identified to modulate EV-A71 infections through both in vitro and in vivo studies. However, the regulatory mechanisms that involve miR-876-5p in the EV-A71 infection cycle remain unclear. METHODS: We demonstrated that miR-876-5p facilitated EV-A71 replication and expression by overexpression and knocking-down of miR-876-5p through the transfection of miR-876-5p plasmid and miR-876-5p inhibitor. Although miR-876-5p suppressed CREB5 expression, luciferase reporter assay confirmed this. We also evaluated the role of miR-876-5p in the EV-A71 infection cycle by CREB5 mediated by transfection with an anti-miR-876-5P inhibitor or in combination with an si-CREB5 plasmid. RESULTS: MicroR-876-5p was upregulated in EV-A71-infected neuroblastoma cells. Overexpression of miR-876-5p or knockdown of cyclic-AMP responsive element binding protein 5 (CREB5) promoted EV-A71 replication. The downregulation of miR-876-5p inhibited the accumulation of viral RNA and the production of viral proteins. Interestingly, CREB5 overexpression also suppressed EV-A71 replication. Our in vitro studies reveal that miR-876-5p directly targets CREB5. Finally, downregulation of CREB5 protein abated the inhibitory effect of anti-miR-876-5p and induced inhibitory effect of EV-A71 replication. CONCLUSIONS: Our results suggest that intracellular miR-876-5p promotes EV-A71 replication indirectly by targeting the host CREB5 protein.

Translation control of Enterovirus A71 gene expression.

Upon EV-A71 infection of a host cell, EV-A71 RNA is translated into a viral polyprotein. Although EV-A71 can use the cellular translation machinery to produce viral proteins, unlike cellular translation, which is cap-dependent, the viral RNA genome of EV-A71 does not contain a 5' cap and the translation of EV-A71 protein is cap-independent, which is mediated by the internal ribosomal entry site (IRES) located in the 5' UTR of EV-A71 mRNA. Like many other eukaryotic viruses, EV-A71 manipulates the host cell translation devices, using an elegant RNA-centric strategy in infected cells. During viral translation, viral RNA plays an important role in controlling the stage of protein synthesis. In addition, due to the cellular defense mechanism, viral replication is limited by down-regulating translation. EV-A71 also utilizes protein factors in the host to overcome antiviral responses or even use them to promote viral translation rather than host cell translation. In this review, we provide an introduction to the known strategies for EV-A71 to exploit cellular translation mechanisms.

Alterations in Alzheimer's Disease-Associated Gene Expression in Severe Obstructive Sleep Apnea Patients.

BACKGROUND: Obstructive sleep apnea (OSA) increases the risk of Alzheimer's disease (AD), and inflammation may be involved in the early pathogenesis of AD in patients with OSA. However, the potential pathways between OSA and AD have yet to be established. In this study, we aimed to investigate differential expressions of AD-associated genes in OSA patients without evident AD or dementia. METHODS: This prospective case-control study included five patients with severe OSA and five age and sex-matched patients with non-severe OSA without evident dementia who underwent uvulopalatopharyngoplasty between 1 January 2013 and 31 December 2015. The expressions of genes associated with AD were analyzed using whole-exome sequencing. Unsupervised two-dimensional hierarchical clustering was performed on these genes. Pearson's correlation was used as the distance metric to simultaneously cluster subjects and genes. RESULTS: The expressions of CCL2, IL6, CXCL8, HLA-A, and IL1RN in the patients with severe OSA were significantly different from those in the patients with non-severe OSA and contributed to changes in the immune response, cytokine-cytokine receptor interactions, and nucleotide-binding oligomerization domain-like receptor signaling pathways. CONCLUSIONS: Inflammation may contribute to the onset of AD and physicians need to be aware of the potential occurrence of AD in patients with severe OSA.

High Amplification of the Antiviral Activity of Curcumin through Transformation into Carbon Quantum Dots.

It is demonstrated that carbon quantum dots derived from curcumin (Cur-CQDs) through one-step dry heating are effective antiviral agents against enterovirus 71 (EV71). The surface properties of Cur-CQDs, as well as their antiviral activity, are highly dependent on the heating temperature during synthesis. The one-step heating of curcumin at 180 °C preserves many of the moieties of polymeric curcumin on the surfaces of the as-synthesized Cur-CQDs, resulting in superior antiviral characteristics. It is proposed that curcumin undergoes a series of structural changes through dehydration, polymerization, and carbonization to form core-shell CQDs whose surfaces remain a pyrolytic curcumin-like polymer, boosting the antiviral activity. The results reveal that curcumin possesses insignificant inhibitory activity against EV71 infection in RD cells [half-maximal effective concentration (EC50 ) >200 µg mL-1 ] but exhibits high cytotoxicity toward RD cells (half-maximal cytotoxic concentration (CC50 ) <13 µg mL-1 ). The EC50 (0.2 µg mL-1 ) and CC50 (452.2 µg mL-1 ) of Cur-CQDs are >1000-fold lower and >34-fold higher, respectively, than those of curcumin, demonstrating their far superior antiviral capabilities and high biocompatibility. In vivo, intraperitoneal administration of Cur-CQDs significantly decreases mortality and provides protection against virus-induced hind-limb paralysis in new-born mice challenged with a lethal dose of EV71.

Nanoparticle-Based LDI-MS Immunoassay for the Multiple Diagnosis of Viral Infections.

Many serious public health emergencies around the globe are caused by viral epidemics. Thus, developing a reliable method for viral screening is in high demand. Multiplex assays for simultaneous detection and fast screening of high-risk pathogens are especially needed. This study employs metal nanoparticles to generate specific mass spectral signals for different RNA viruses, which enables simultaneous detection of whole viruses by laser desorption/ionization mass spectrometry (LDI-MS). We developed a nanoparticle-based sandwich immunosorbent assay as a sensing platform for the detection of viruses and viral nonstructural protein by LDI-MS. Cellulose acetate membrane (CAM) serves as the substrate for the fabrication of the sandwich immunosorbent assay with the advantages of clean mass spectra and high enrichment of analytes. Antibody-modified metal nanoparticles (Ab-MNPs; M = Au or Ag) act as metallic biocodes for the LDI-MS detection. The signal amplification readout for the virus is through the pulsed laser-induced formation of metal cluster ions ([M n]+; n = 1-3) from the Ab-MNPs which specifically bind on the CAM. Our sensing system is effective for the detection of intact viruses [Enterovirus 71 (EV71) and Japanese encephalitis virus (JEV)], nonstructural protein 1 (NS1) of Zika virus (ZIKV), EV71-spiked human serum samples, and the simultaneous detection of EV71 and ZIKV. Our probe efficiently detects EV71 in real clinical serum samples with >95% agreement with RT-qPCR results. This high-throughput LDI-MS viral detection system is simple, reliable, and high-throughput. We believe this platform has the potential to be employed for the routine screening of patients with viral infections.

Role of Enteroviral RNA-Dependent RNA Polymerase in Regulation of MDA5-Mediated Beta Interferon Activation.

Infection by enteroviruses can cause severe neurological complications in humans. The interactions between the enteroviral and host proteins may facilitate the virus replication and be involved in the pathogenicity of infected individuals. It has been shown that human enteroviruses possess various mechanisms to suppress host innate immune responses in infected cells. Previous studies showed that infection by enterovirus 71 (EV71) causes the degradation of MDA5, which is a critical cytoplasmic pathogen sensor in the recognition of picornaviruses for initiating transcription of type I interferons. In the present study, we demonstrated that the RNA-dependent RNA polymerase (RdRP; also denoted 3Dpol) encoded by EV71 interacts with the caspase activation and recruitment domains (CARDs) of MDA5 and plays a role in the inhibition of MDA5-mediated beta interferon (IFN-ß) promoter activation and mRNA expression. In addition, we found that the 3Dpol protein encoded by coxsackievirus B3 also interacted with MDA5 and downregulated the antiviral signaling initiated by MDA5. These findings indicate that enteroviral RdRP may function as an antagonist against the host antiviral innate immune response.IMPORTANCE Infection by enteroviruses causes severe neurological complications in humans. Human enteroviruses possess various mechanisms to suppress the host type I interferon (IFN) response in infected cells to establish viral replication. In the present study, we found that the enteroviral 3Dpol protein (or RdRP), which is a viral RNA-dependent RNA polymerase for replicating viral RNA, plays a role in the inhibition of MDA5-mediated beta interferon (IFN-ß) promoter activation. We further demonstrated that enteroviral 3Dpol protein interacts with the caspase activation and recruitment domains (CARDs) of MDA5. These findings indicate that enteroviral RdRP functions as an antagonist against the host antiviral response.

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein.

RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear region of the cell, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.

Circulating Salivary miRNA hsa-miR-221 as Clinically Validated Diagnostic Marker for Hand, Foot, and Mouth Disease in Pediatric Patients.

Enhancements in the diagnostic capabilities using host biomarkers are currently much needed where sensitivity and specificity issues plague the diagnosis of Hand, Foot and Mouth Disease (HFMD) in pediatrics clinical samples. We investigated miRNome profiles of HFMD saliva samples against healthy children and developed miRNA-based diagnosis models. Our 6-miRNA scoring model predicted HFMD with an overall accuracy of 85.11% in the training set and 92.86% in the blinded test set of Singapore cohort. Blinded evaluation of the model in Taiwan HFMD cases resulted in 77.08% accuracy with the 6-miRNA model and 68.75% with the 4-miRNA model. The strongest predictor of HFMD in all of the panels, hsa-miR-221 was found to be consistently and significantly downregulated in all of our HFMD cohorts. This is the first study to prove that HFMD infection could be diagnosed by circulating miRNAs in patient's saliva. Moreover, this study also serves as a stepping stone towards the future development of other infectious disease diagnosis workflows using novel biomarkers.

Rapid detection and quantification of Enterovirus 71 by a portable surface plasmon resonance biosensor.

This study presents the first report on a label-free detection and rapid quantification method for human enterovirus 71 (EV71) using a portable surface plasmon resonance (SPR) system. The SPR sensor instrument was configured to run on low power in a miniaturized platform to improve the device portability for a wider application both in laboratories and in the field. A color tunable organic light emitting diode in red spectrum was attached on a trapezoidal prism for the disposable light source module. The SPR signal processing using integration area under the reflectivity curve is applied for optimum signal to noise ratio (SNR) enhancement. The major capsid protein VP1 of EV71 was selected as the biomarker target in the detection study. The experimental time required for the EV71 quantification was reduced from 6 days using the conventional viral plaque assay to several minutes using the proposed method. The study results establish a detection limit of approximately 67 virus particles per milliliter (vp/ml) of EV71 in a Dulbecco's modified Eagle's medium. The VP1 detection in the portable SPR biosensor had a detection limit of approximately 4.8pg/ml in the PBS buffer. Therefore, the proposed direct EV71 viral particle quantification method can be rapidly performed in real time, with high sensitivity and less labor and without assays or fluorescence.