Crosstalk between autophagy and inflammasomes in ricin-induced inflammatory injury.

Ricin (ricin toxin, RT) has the potential to cause damage to multiple organs and systems. Currently, there are no existing antidotes, vaccinations, or effective therapies to prevent or treat RT intoxication. Apart from halting protein synthesis, RT also induces oxidative stress, inflammation and autophagy. To explore the mechanisms of RT-induced inflammatory injury and specific targets of prevention and treatment for RT poisoning, we characterized the role of cross-talk between autophagy and NLRP3 inflammasome in RT-induced damage and elucidated the underlying mechanisms. We showed that RT-induced inflammation was attributed to activation of the TLR4/MyD88/NLRP3 signaling and ROS production, evidenced by increased ASC speck formation and attenuated TXNIP/TRX-1 interaction, as well as pre-treatment with MCC950, MyD88 knockdown and NAC significantly reduced IL-1ß, IL-6 and TNF-α mRNA expression. In addition, autophagy is also enhanced in RT-triggered MLE-12 cells. RT elevated the levels of ATG5, p62 and Beclin1 protein, provoked the accumulation of LC3 puncta detected by immunofluorescence staining. Treatment with rapamycin (Rapa) reversed the RT-caused TLR4/MyD88/NLRP3 signaling activation, ASC specks formation as well as the levels of IL-1ß, IL-6 and TNF-α mRNA. In conclusion, RT promoted NLRP3 inflammasome activation and autophgay. Inflammation induced by RT was attenuated by autophagy activation, which suppressed the NLRP3 inflammasome. These findings suggest Rapa as a potential therapeutic drug for the treatment of RT-induced inflammation-related diseases.

Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone.

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir followed by sustained human-to-human infections. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 × 10(-3) substitutions per site per year (95% highest posterior density interval, 1.04 × 10(-3) to 1.41 × 10(-3) substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics.

An electrochemical aptasensor based on cocoon-like DNA nanostructure signal amplification for the detection of Escherichia coli O157:H7.

We developed an electrochemical aptasensor based on cocoon-like DNA nanostructures as signal tags for highly sensitive and selective detection of Escherichia coli O157:H7. The stable cocoon-like DNA nanostructures synthesized by the rolling circle amplification reaction were loaded with hemin as electrochemical signal tags to amplify the signals. The single-stranded DNA capture probes were modified on the surface of a Au electrode via a Au-S bond. The E. coli O157:H7 specific aptamer and capture probe formed double-stranded DNA structures on the Au electrode. The aptamer preferentially bound to E. coli O157:H7, causing the dissociation of some aptamer-capture probes and releasing some capture probes. Subsequently, the free capture probes hybridized with the DNA nanostructures through the cDNA sequence. Under optimal conditions, the change in the electrochemical signal was proportional to the logarithm of E. coli O157:H7 concentration, from 10 to 106 CFU mL-1, and the detection limit was estimated to be 10 CFU mL-1. The electrochemical aptasensor could be readily used to detect various pathogenic bacteria and to provide a new method of early diagnosis of pathogenic microorganisms.

An electrochemical biosensor based on methylene blue-loaded nanocomposites as signal-amplifying tags to detect pathogenic bacteria.

A sandwich-type electrochemical biosensor was successfully constructed for the sensitive detection of pathogenic bacteria. In this biosensor platform, methylene blue (MB) organic-inorganic nanocomposites (MB@MI) were synthesized from magainin I (MI, antimicrobial peptide specific to Escherichia coli O157:H7), Cu3(PO4)2 and MB via a one-pot method, and were explored as a novel electrochemical signal label of biosensors generating amplified electrochemical signals by differential pulse voltammetry (DPV). E. coli O157:H7 specifically sandwich bound to the aptamers on the electrode surface and MB@MI nanocomposites, and the changes in the current signal generated on the electrode surface were used for the quantitative determination of E. coli O157:H7. Under optimum conditions, the proposed biosensor showed excellent performance with a wide linear range of 102-107 CFU mL-1 and a low detection limit of 32 CFU mL-1, featuring favorable selectivity, repeatability and stability. According to the experiments conducted on real samples, the proposed approach is capable of detecting pathogenic bacteria in clinical diagnostics.

Immunoassay for foodborne pathogenic bacteria using magnetic composites Ab@Fe3O4, signal composites Ap@PtNp, and thermometer readings.

A point-of-care (POC) immunoassay was established for the sensitive and rapid detection of pathogenic Escherichia coli O157:H7, using magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4) for immunomagnetic separation, nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp) for signal amplification, and thermometer readings. Antibodies and Fe3O4 were incubated in Cu2+ phosphate buffer to synthesize the magnetic composite Ab@Fe3O4 with antibodies, to specifically capture E. coli O157:H7. Antimicrobial peptides and PtNp were incubated in Cu2+ phosphate buffer to synthesize the signal composites Ap@PtNp with antimicrobial peptides (magainin I), recognizing and labeling E. coli O157:H7. In the presence of E. coli O157:H7, magnetic microcomposites targeted bacteria and signal microcomposites to form the sandwich structure: Ab@Fe3O4-bacteria-Ap@PtNp for magnetic separation. Ap@PtNp of signal composites catalyzed H2O2 to generate thermo-signals (temperature rise), which were determined by a thermometer. This point-of-care bioassay detected E. coli O157:H7 in the linear range of 101-107 CFU mL-1 and with a detection limit of 14 CFU mL-1. One-pot process magnetic Fe3O4 organic-inorganic composites (Ab@Fe3O4, magnetic microcomposites, MMC) for immunomagnetic separation and nanozyme platinum nanoparticle (PtNp) organic-inorganic composites (Ap@PtNp, signal microcomposites, SMC) were used as signal amplification and thermometer readings for E. coli O157:H7 detection.

Ferrocene-functionalized nanocomposites as signal amplification probes for electrochemical immunoassay of Salmonella typhimurium.

An electrochemical immunosensor based on ferrocene (Fc)-functionalized nanocomposites was fabricated as an efficient electroactive signal probe to amplify electrochemical signals for Salmonella typhimurium detection. The electrochemical signal amplification probe was constructed by encapsulating ferrocene into S. typhimurium-specific antimicrobial peptides Magainin I (MI)-Cu3(PO4)2 organic-inorganic nanocomposites (Fc@MI) through a one-step process. Magnetic beads (MBs) coupled with antibody were used as capture ingredient for target magnetic separation, and Fc@MI nanoparticles were used as signal labels in the immunoassays. The sandwich of MBs-target-Fc@MI assay was performed using a screen-printed carbon electrode as transducer surface. The immunosensor platform presents a low limit of detection (LOD) of 3 CFU·mL-1 and a linear range from 10 to 107 CFU·mL-1, with good specificity and precision, and was successfully applied for S. typhimurium detection in milk. Graphical abstract One-pot process antimicrobial peptides Magainin I-Cu3(PO4)2 organic-inorganic nanocomposites (Fc@MI) were used as ideal electrochemical signal label, integrating both essential functions of biological recognition and signal amplification. Screen-printed carbon electrode (SPCE) was used as the electrochemical system for Salmonella typhimurium detection.

Immunoassay for pathogenic bacteria using platinum nanoparticles and a hand-held hydrogen detector as transducer. Application to the detection of Escherichia coli O157:H7.

An innovative approach is presented for portable and sensitive detection of pathogenic bacteria. A novel synthetic hybrid nanocomposite encapsulating platinum nanoparticles, as a highly efficient catalyst, catalyzes the hydrolysis of the ammonia-borane complex to generate hydrogen gas. The nanocomposites are used as a label for immunoassays. A portable hand-held hydrogen detector combined with nanocomposite-induced signal conversion was applied for point-of-care testing of pathogenic bacteria. A hand-held hydrogen detector was used as the transducer. Escherichia coli O157:H7 (E. coli O157: H7), as detection target, formed a sandwich structure with magnetic beads and hybrid nanocomposites. Magnetic beads were used for separation of the sandwich structure, and hybrid nanocomposites as catalysts to catalyze the generation of hydrogen from ammonia-borane. The generated hydrogen was detected by a hydrogen detector using an electrochemical method. E. coli O157:H7 has a detection limit of 10 CFU·mL-1. The immunosensor made the hand-held hydrogen detector a point-of-care meter to be used outdoors for the detection and quantification of targets beyond hydrogen. Graphical abstract Schematic presentation of one-pot synthetic peptide-Cu3(PO4)2 hybrid nanocomposites embedded PtNPs (PPNs), encapsulating many Pt particles. The PPNs acts as an ideal immunoprobe for hand-held H2 detector signal readouts, by transforming pathogenic bacteria recognition events into H2 signals.

Disposable syringe-based visual immunotest for pathogenic bacteria based on the catalase mimicking activity of platinum nanoparticle-concanavalin A hybrid nanoflowers.

Disposable syringes were used in a novel point-of-care visual test for detecting pathogenic bacteria (Escherichia coli O157:H7 and Salmonella typhimurium). Hybrid nanoflowers composed of platinum nanoparticles and concanavalin A (Pt-nanoflowers) were prepared through a one-pot reaction and were found to be viable catalase mimics. They catalyze the decomposition of hydrogen peroxide (H2O2) to generate O2. When used as labels in immunoassays, they integrate both the functions of biological recognition and signal amplification. The disposable syringe pressure readout was combined with Pt-nanoflower signal conversion and successfully applied to a visual bacteria detection scheme. Both Escherichia coli O157:H7 and Salmonella typhimurium can be quantified with detection limits of as low as 15 and 7 CFU·mL-1, respectively. Graphical abstract One-pot synthetic platinum nanoparticle (PtNP)-concanavalin A hybrid nanoflowers (Pt-nanoflowers), have been used as ideal signal labels for immunoassays and integrating both essential functions of biological recognition and signal amplification. Disposable syringes were used as a readout to detect pathogenic bacteria.

Modified beacon probe assisted dual signal amplification for visual detection of microRNA.

In a recent study, we reported a novel assay for the detection of microRNA-21 based on duplex-specific nuclease (DSN)-assisted isothermal cleavage and hybridization chain reaction (HCR) dual signal amplification. The Fam modified double-stranded DNA products were generated after the HCR, another biotin modified probe was digested by DSN and released from the magnetic beads after the addition of the target miRNA. The released sequence was then combined with HCR products to form a double-tagging dsDNA, which can be recognized by the lateral flow strips. In this study, we introduced a 2-OMethyl-RNA modified beacon probe (2-OMe-MB) to make some improvements based on the previous study. Firstly, the substitution of modified probe combined on magnetic beads avoids the fussy washing steps for the separation of un-reacted probes. Furthermore, the modification of 2-OMe on the stem of the probe avoided the unnecessary cleavage by DSN, which greatly reduce the background signal. Compared to the previous work, these improvements save us a lot of steps but possess the comparable sensitivity and selectivity.

Hemin-incorporated nanoflowers as enzyme mimics for colorimetric detection of foodborne pathogenic bacteria.

Rapid, sensitive and point-of-care detection of foodborne pathogenic bacteria is essential for food safety. In this study, we found that hemin-concanavalin A hybrid nanoflowers (HCH nanoflowers), as solid mimic peroxidase, could catalyze oxidation of 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of H2O2 to a green-colored product. HCH nanoflowers, integrating the essential functions of both biological recognition and signal amplification, meet the requirements of signal labels for colorimetric immunoassay of bacteria. In view of the excellent peroxidase mimetic catalytic activity of HCH nanoflowers, a colorimetric biosensing platform was newly constructed and applied for sensitive detection of foodborne Escherichia coli O157:H7 (E. coli O157:H7). The corresponding detection limits was as low as 4.1 CFU/mL with wide linear ranges (101-106 CFU/mL).

Correction to: A pregnancy test strip for detection of pathogenic bacteria by using concanavalin A-human chorionic gonadotropin-Cu3(PO4)2 hybrid nanoflowers, magnetic separation, and smartphone readout.

The published version of this article, unfortunately, contained error. The authors are re-writing to express their sincere apology for a mistake that a mark "10-5, 10-4, 10-3, 10-2, 10-1 CFU•mL-1" in the legend of Fig. 2 was not corrected as "105, 104, 103, 102, 101 CFU•mL-1".

A pregnancy test strip for detection of pathogenic bacteria by using concanavalin A-human chorionic gonadotropin-Cu3(PO4)2 hybrid nanoflowers, magnetic separation, and smartphone readout.

Pregnancy test strips are widely used in daily life. A commercial pregnancy test strip was modified to obtain a point-of-care device for the detection of pathogenic bacteria. Hybrid nanoflowers were prepared from concanavalin A, human chorionic gonadotropin, and Cu3(PO4)2 via a one-pot method. They were used as signaling probes in an off-the-shelf pregnancy test strip. This modified lateral flow immunoassay can detect Escherichia coli O157:H7 with a detection limit of 4 CFU·mL-1, and Salmonella typhimurium with a detection limit of 3 CFU·mL-1. Conceivably, the method has high potential as a portable and cost-effective tool for rapid determination of a wide range of analytes, especially in resource-constrained settings. Graphical abstract Hybrid nanoflower loaded human chorionic gonadotropin (hCG) and concanavalin A (hCG - nanoflowers) were synthesized via a one-pot method and used as signal labels with commercial commercial-off-the-shelf pregnancy test strips to detect pathogenic bacteria targets, thus yielding an easily smartphone readout signal.

Protective effects and plausible mechanisms of antler-velvet polypeptide against hydrogen peroxide induced injury in human umbilical vein endothelial cells.

Antler velvet polypeptide (VAP) is a prominent bioactive component of antler velvet. Whereas uncharacterized crude extracts have typically been used in pharmacological studies, in this study, the velvet polypeptide was isolated and purified by acid water extraction, ethanol precipitation, ammonium sulfate fractionation and precipitation, and chromatography, progressively. Human umbilical vein endothelial cells (HUVECs) were induced with H2O2 followed purified polypeptide treatment. Cell viability was evaluated by MTT assay. The apoptosis of cells was detected by fluorescence microscopy and flow cytometry. A cell analyzer was used to measure the mitochondrial membrane potential. The intracellular reactive oxygen species (ROS) levels were determined by flow cytometry. Oxidative stress related biochemical parameters were detected, and the expression of apoptosis-related proteins was examined by Western blot analysis. The results indicated that a 7.0 kDa polypeptide (VAP II) was isolated from antler velvet. VAP II enhanced cell viability, decreased cell apoptosis, reversed depolarization of mitochondrial membrane potential, decreased ROS levels, inhibited oxidative stress, and regulated the downstream signaling apoptotic cascade expression caused by H2O2. The protective effects of VAP II on HUVECs suggests a potential strategy for the treatment of cardiovascular disease.

Clinical and Virological Characteristics of Ebola Virus Disease Patients Treated With Favipiravir (T-705)-Sierra Leone, 2014.

BACKGROUND: During 2014-2015, an outbreak of Ebola virus disease (EVD) swept across parts of West Africa. No approved antiviral drugs are available for Ebola treatment currently. METHODS: A retrospective clinical case series was performed for EVD patients in Sierra Leone-China Friendship Hospital. Patients with confirmed EVD were sequentially enrolled and treated with either World Health Organization (WHO)-recommended supportive therapy (control group) from 10 to 30 October, or treated with WHO-recommended therapy plus favipiravir (T-705) from 1 to 10 November 2014. Survival and virological characteristics were observed for 85 patients in the control group and 39 in the T-705 treatment group. RESULTS: The overall survival rate in the T-705 treatment group was higher than that of the control group (56.4% [22/39] vs 35.3% [30/85]; P = .027). Among the 35 patients who finished all designed endpoint observations, the survival rate in the T-705 treatment group (64.8% [11/17]) was higher than that of the control group (27.8% [5/18]). Furthermore, the average survival time of the treatment group (46.9 ± 5.6 days) was longer than that of the control group (28.9 ± 4.7 days). Most symptoms of patients in the treatment group improved significantly. Additionally, 52.9% of patients who received T-705 had a >100-fold viral load reduction, compared with only 16.7% of patients in the control group. CONCLUSIONS: Treatment of EVD with T-705 was associated with prolonged survival and markedly reduced viral load, which makes a compelling case for further randomized controlled trials of T-705 for treating EVD.

Organellar proteome analyses of ricin toxin-treated HeLa cells.

Apoptosis triggered by ricin toxin (RT) has previously been associated with certain cellular organellar compartments, but the diversity in the composition of the organellar proteins remains unclear. Here, we applied a shotgun proteomics strategy to examine the differential expression of proteins in the mitochondria, nuclei, and cytoplasm of HeLa cells treated and not treated with RT. Data were combined with a global bioinformatics analysis and experimental confirmations. A total of 3107 proteins were identified. Bioinformatics predictors (Proteome Analyst, WoLF PSORT, TargetP, MitoPred, Nucleo, MultiLoc, and k-nearest neighbor) and a Bayesian model that integrated these predictors were used to predict the locations of 1349 distinct organellar proteins. Our data indicate that the Bayesian model was more efficient than the individual implementation of these predictors. Additionally, a Biomolecular Interaction Network (BIN) analysis was used to identify 149 BIN subnetworks. Our experimental confirmations indicate that certain apoptosis-related proteins (e.g. cytochrome c, enolase, lamin B, Bax, and Drp1) were found to be translocated and had variable expression levels. These results provide new insights for the systematic understanding of RT-induced apoptosis responses.

Proteomic study of differential protein expression in mouse lung tissues after aerosolized ricin poisoning.

Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning.

The current landscape of the antimicrobial peptide melittin and its therapeutic potential.

Melittin, a main component of bee venom, is a cationic amphiphilic peptide with a linear α-helix structure. It has been reported that melittin can exert pharmacological effects, such as antitumor, antiviral and anti-inflammatory effects in vitro and in vivo. In particular, melittin may be beneficial for the treatment of diseases for which no specific clinical therapeutic agents exist. Melittin can effectively enhance the therapeutic properties of some first-line drugs. Elucidating the mechanism underlying melittin-mediated biological function can provide valuable insights for the application of melittin in disease intervention. However, in melittin, the positively charged amino acids enables it to directly punching holes in cell membranes. The hemolysis in red cells and the cytotoxicity triggered by melittin limit its applications. Melittin-based nanomodification, immuno-conjugation, structural regulation and gene technology strategies have been demonstrated to enhance the specificity, reduce the cytotoxicity and limit the off-target cytolysis of melittin, which suggests the potential of melittin to be used clinically. This article summarizes research progress on antiviral, antitumor and anti-inflammatory properties of melittin, and discusses the strategies of melittin-modification for its future potential clinical applications in preventing drug resistance, enhancing the selectivity to target cells and alleviating cytotoxic effects to normal cells.

Expression of Toll-like receptors in the cerebellum during pathogenesis of prion disease.

Prion diseases, such as scrapie, entail the accumulation of disease-specific prion protein (PrPSc) within the brain. Toll-like receptors (TLRs) are crucial components of the pattern recognition system. They recognize pathogen-associated molecular patterns (PAMPs) and play a central role in orchestrating host innate immune responses. The expression levels of Toll-like receptors (TLRs) in the central nervous system (CNS) were not well-defined. To establish a model of prion diseases in BALB/C mice, the 22L strain was employed. The features of the 22L strain were analyzed, and the cerebellum exhibited severe pathological changes. TLR1-13 levels in the cerebellum were measured using quantitative polymerase chain reaction (qPCR) at time points of 60, 90, 120, and the final end point (145 days post-infection). During the pathogenesis, the expression levels of Toll-like receptors (TLRs) 1, 2, 7, 8, and 9 increased in a time-dependent manner. This trend mirrored the expression patterns of PrPSc (the pathological isoform of the prion protein) and glial fibrillary acidic protein. Notably, at the end point, TLR1-13 levels were significantly elevated. Protein level of TLR7 and TLR9 showed increasing at the end point of the 22L-infected mice. A deeper understanding of the increased Toll-like receptors (TLRs) in prion diseases could shed light on their role in initiating immune responses at various stages during pathogenesis. This insight is particularly relevant when considering TLRs as potential therapeutic targets for prion diseases.

Caspase-3/Gasdermin E-mediated pyroptosis contributes to Ricin toxin-induced inflammation.

Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may aggravate the harm to the body. Pyroptosis, a type of gasdermin-mediated cell death, is a contributor to the exacerbation of inflammation. Accumulating evidence indicate that pyroptosis plays a significant role in the pathogen infection and tissue injury, suggesting a potential correlation between pyroptosis and RT-induced inflammation. Here, we aim to demonstrate this correlation and explore its molecular mechanisms. Results showed that RT triggers mouse alveolar macrophage MH-S cells pyroptosis by activating caspase-3 and cleaving Gasgermin E (GSDME). In contrast, inhibition of caspase-3 with Z-DEVD-FMK (inhibitor of caspase-3) or knockdown of GSDME attenuates this process, suggesting the essential role of caspase-3/GSDME-mediated pyroptosis in contributing to RT-induced inflammation. Collectively, our study enhances our understanding of a novel mechanism of ricin cytotoxicity, which may emerge as a potential target in immunotherapy to control the RT-induced inflammation.