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.

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.

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.

Elevated expression of circulating miR876-5p is a specific response to severe EV71 infections.

Human enterovirus 71 (EV71) is a major causative agent of hand, foot, and, mouth disease, accounting for more than 65% of recent outbreaks. Following enteroviral infection, the host responses are crucial indicators for the development of a diagnosis regarding the clinical severity of EV71 infections. In this study, we implemented NanoString nCounter technology to characterize the responses of serum microRNA (miRNA) profiles to various EV71 infection diseases. Upon EV71 infection, 44 miRNAs were observed in patients with EV71 infections, with at least a 2-fold elevation and 133 miRNAs with a 2-fold reduction compared with the same miRNAs in healthy controls. Further detailed work with miR876-5p, a 9.5-fold change of upregulated miR-876-5p expression was observed in cases with severe EV71 symptoms, revealed that in vitro and in vivo knockdown of miR876-5p reduced viral RNA in cultured cells, and attenuated the severity of symptoms in EV71-infected mice. Altogether, we demonstrated that the elevated expression of circulating miR876-5p is a specific response to severe EV71 infections.

Proteome demonstration of alpha-1-acid glycoprotein and alpha-1-antichymotrypsin candidate biomarkers for diagnosis of enterovirus 71 infection.

BACKGROUND: Human enterovirus 71 (EV71) is the major causative agents of hand-foot-and-mouth disease and frequently associated with severe complications such as encephalitis and death. Understanding the host response following enteroviral infection may facilitate the development of biomarkers for EV71 infections. METHODS: We implemented two-dimensional gel electrophoresis technology on proteins prepared from serum obtained from 4 mild and 4 severe cases of EV71 infections and 4 healthy control children, to investigate the differentially expressed proteins. The differential expressed proteins were further identified with liquid chromatography-mass spectrometry/mass spectrometry analysis and western blotting validation. RESULTS: A total of 27 differentially expressed proteins were picked and identified with liquid chromatography-mass spectrometry/mass spectrometry. Of the 27 identified proteins, 6 proteins were up-regulated in the mild-infected and severe EV71-infected patients in comparison to the healthy control group. Two proteins, alpha-1-acid-glycoprotein (AGP1) and alpha-antichymotrypsin (AACT), were not detected in the EV71-infected patients, but appeared in the control patient. Western blotting analysis demonstrated that AGP1 and AACT proteins were negatively associated with the clinical severity of EV71 infection. Similarly, both of the proteins were not detected in the secretion medium from the EV71-infected neuroblastoma cells, but detected in the mock-infected cells, suggesting that differentially expressed AGP1/AACT protein levels are in response to EV71 infections. CONCLUSIONS: Two candidate proteins AGP1 and AACT, whose expression levels were reduced under the EV71 infection pathological condition, provide useful source of information for potential diagnostic biomarkers of EV71 infection in children.