Multiplex Surveillance Kit Using Sweetened Solid Support to Detect Arboviruses Isothermally in Mosquito Saliva from the Field.

Recently reported "displaceable probe" loop amplification (DP-LAMP) architecture has shown to amplify viral RNA from SARS-CoV-2 with little sample processing. The architecture allows signals indicating the presence of target nucleic acids to be spatially separated, and independent in sequence, from the complicated concatemer that LAMP processes create as part of their amplification process. This makes DP-LAMP an attractive molecular strategy to integrate with trap and sampling innovations to detect RNA from arboviruses carried by mosquitoes in the field. These innovations include (a) development of organically produced carbon dioxide with ethylene carbonate as a bait deployable in mosquito trap, avoiding the need for dry ice, propane tanks, or inorganic carbonates and (b) a process that induces mosquitoes to lay virus-infected saliva on a quaternary ammonium-functionalized paper (Q-paper) matrix, where (c) the matrix (i) inactivates the deposited viruses, (ii) releases their RNA, and (iii) captures viral RNA in a form that keeps it stable for days at ambient temperatures. We report this integration here, with a surprisingly simple workflow. DP-LAMP with a reverse transcriptase was found to amplify arboviral RNA directly from Q-paper, without requiring a separate elution step. This capture-amplification-detection architecture can be multiplexed, with the entire system integrated into a device that can support a campaign of surveillance, in the wild outdoors, that reports the prevalence of arboviruses from field-captured mosquitoes.

Toxicity and Biodistribution of Fragmented Polypropylene Microplastics in ICR Mice.

Currently, polypropylene (PP) is used in various products, thus leading to high daily exposure in humans. Thus, it is necessary to evaluate the toxicological effects, biodistribution, and accumulation of PP microplastics in the human body. In this study, administration of two particle sizes of PP microplastics (approximately 5 and 10-50 µm) did not lead to any significant changes in several toxicological evaluation parameters, including body weight and pathological examination, compared with the control group in ICR mice. Therefore, the approximate lethal dose and no-observed-adverse-effect level of PP microplastics in ICR mice were established as ≥2000 mg/kg. Furthermore, we manufactured cyanine 5.5 carboxylic acid (Cy5.5-COOH)-labeled fragmented PP microplastics to monitor real-time in vivo biodistribution. After oral administration of the Cy5.5-COOH-labeled microplastics to the mice, most of the PP microplastics were detected in the gastrointestinal tract and observed to be out of the body after 24 h in IVIS Spectrum CT. Therefore, this study provides a new insight into the short-term toxicity, distribution, and accumulation of PP microplastics in mammals.

Gold Standard? Method of Citric Acid Solution Swallowing Test as a Screening Test for Patients with Tracheostomy.

This study aimed to apply various ranges of citric acid levels in the mouth and T-cannula to compare the validity with instrumental aspiration measures in patients with tracheostomy. Sixty-one patients underwent the citric acid cough reflex test (CRT) and videofluoroscopic swallowing study (VFSS). Citric acid was delivered via facemask and T-cannula at concentrations of 0.4 mol/L, 0.6 mol/L, and 0.8 mol/L. Further, we recorded the coughing count and presence of ≥ 2 (C2) and ≥ 5 (C5) coughs. CRT via facemask at 0.4 mol/L C2, 0.6 mol/L C5, and 0.8 mol/L C2 and C5 were significantly associated with the presence of tracheal aspiration during VFSS. The sensitivity and specificity were optimized at 0.8 mol/L C2 for mouth inhalation and at 0.8 mol/L C5 for T-cannula inhalation. There was a significant difference in the coughing count during CRT at 0.4 mol/L and 0.8 mol/L via mouth inhalation between patients with or without tracheal aspiration, but not via T-cannula. The AUC for 0.8 mol/L facemask inhalation was 0.701. The optimal cut-off value of coughing count was thrice with 84.62% sensitivity and 50.00% specificity on the ROC curve. Afferent sensory nerve desensitization around and below the tracheostomy site could affect coughing reflex initiation and decrease the sensitivity of detecting aspiration in tracheotomized patients. The citric acid CRT via facemask can reliably detect tracheal aspiration and presence of coughing reflex compared to that via T-cannula in patients with tracheostomy.

Validity of Collagen Plugs for Ridge Preservation in a Canine Model.

PURPOSE: The purpose of the present study was to evaluate healing outcomes after collagen plug insertion in extraction-related defects. MATERIALS AND METHODS: The third and fourth mandibular premolars in canines were extracted, and the septal bone was removed. The following treatments were performed for the defects: porcine atelo-collagen plug, bovine atelo-collagen plug, and no intervention (control). The experimental animals were killed after 6, 12, or 24 weeks (n = 4, respectively). Histologic and histomorphometric analyses were performed. RESULTS: Clinical healing was uneventful, and no difference was detected among the 3 groups. Histologically, similar healing patterns were observed in all groups. Gingival healing was complete at 6 weeks, but discontinuity in the buccal crestal bone was observed. At 12 weeks, various degrees of buccal bone depression and increase in bone marrow were observed. At 24 weeks, no further healing was observed. Histomorphometrically, the ridge width at 1, 3 and 5 mm levels below the crest and the ridge dimensions 1, 3, and 5 mm above the level were not statistically different among groups or healing periods. CONCLUSION: The healing following the use of collagen plug in the extraction socket may correspond to the natural healing after extraction.

Rapid detection of West Nile and Dengue viruses from mosquito saliva by loop-mediated isothermal amplification and displaced probes.

Arthropod-borne viruses are major causes of human and animal disease, especially in endemic low- and middle-income countries. Mosquito-borne pathogen surveillance is essential for risk assessment and vector control responses. Sentinel chicken serosurveillance (antibody testing) and mosquito pool screening (by RT-qPCR or virus isolation) are currently used to monitor arbovirus transmission, however substantial time lags of seroconversion and/or laborious mosquito identification and RNA extraction steps sacrifice their early warning value. As a consequence, timely vector control responses are compromised. Here, we report on development of a rapid arbovirus detection system whereby adding sucrose to reagents of loop-mediated isothermal amplification with displaced probes (DP-LAMP) elicits infectious mosquitoes to feed directly upon the reagent mix and expectorate viruses into the reagents during feeding. We demonstrate that RNA from pathogenic arboviruses (West Nile and Dengue viruses) transmitted in the infectious mosquito saliva was detectable rapidly (within 45 minutes) without RNA extraction. Sucrose stabilized viral RNA at field temperatures for at least 48 hours, important for transition of this system to practical use. After thermal treatment, the DP-LAMP could be reliably visualized by a simple optical image sensor to distinguish between positive and negative samples based on fluorescence intensity. Field application of this technology could fundamentally change conventional arbovirus surveillance methods by eliminating laborious RNA extraction steps, permitting arbovirus monitoring from additional sites, and substantially reducing time needed to detect circulating pathogens.

Accuracy of Real-Time Polymerase Chain Reaction in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a mild to severe respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnostic accuracy of the Centers for Disease Control and Prevention (CDC)- or World Health Organization (WHO)-recommended real-time PCR (RT-qPCR) primers in clinical practice remains unproven. We conducted a prospective study on the accuracy of RT-qPCR using an in-house-designed primer set (iNP) targeting the nucleocapsid protein as well as various recommended and commercial primers. The accuracy was assessed by culturing or seroconversion. We enrolled 12 confirmed COVID-19 patients with a total of 590 clinical samples. When a cutoff value of the cycle threshold (Ct) was set to 35, RT-qPCRs with WHO RdRp primers and CDC N1, N2, and N3 primers showed sensitivity of 42.1% to 63.2% and specificity of 90.5% to 100% in sputum, and sensitivity of 65.2% to 69.6% and specificity of 65.2% to 69.6% in nasopharyngeal samples. The sensitivity and specificity of iNP RT-qPCR in sputum and nasopharyngeal samples were 94.8%/100% and 69.6%/100%, respectively. Sputum testing had the highest sensitivity, followed by nasopharyngeal testing (P = 0.0193); self-collected saliva samples yielded better characteristics than oropharyngeal samples (P = 0.0032). Our results suggest that iNP RT-qPCR has better sensitivity and specificity than RT-PCR with WHO (P < 0.0001) or CDC (N1: P = 0.0012, N2: P = 0.0013, N3: P = 0.0012) primers. Sputum RT-qPCR analysis has the highest sensitivity, followed by nasopharyngeal, saliva, and oropharyngeal assays. Our study suggests that considerable improvement is needed for the RT-qPCR WHO and CDC primer sets for detecting SARS-CoV-2. IMPORTANCE Numerous research campaigns have addressed the vast majority of clinical and diagnostic specificity and sensitivity of various primer sets of SARS-CoV2 viral detection. Despite the impressive progress made to resolve the pandemic, there is still a need for continuous and active improvement of primers used for diagnosis in clinical practice. Our study significantly exceeds the scale of previously published research on the specificity and sensitivity of different primers comparing with different specimens and is the most comprehensive to date in terms of constant monitoring of primer sets of current usage. Henceforth, our results suggest that sputum samples sensitivity is the highest, followed by nasopharyngeal, saliva, and oropharyngeal samples. The CDC recommends the use of oropharyngeal specimens, leading to certain discrepancy between the guidelines set forth by the CDC and IDSA. We proved that the oropharyngeal samples demonstrated the lowest sensitivity for the detection of SARS-CoV-2.

Core/shell nanoparticles for pH-sensitive delivery of doxorubicin.

Core/shell nanoparticles with lipid core were prepared and characterized as pH-sensitive delivery system of anticancer drug. The lipid core is composed of drug-loaded lecithin and the polymeric shell is composed of Pluronics (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) tri-block copolymer, F-127). Based on the preparation method in the previous report by us, the freeze-drying of drug-loaded lecithin was performed in the F-127 aqueous solution containing trehalose used as a cryoprotectant to form stabilized core/shell nanoparticles. For the application of core/shell nanoparticles as a pH-sensitive drug delivery system for anticancer drug, doxorubicin was loaded into the core/shell nanoparticles and the drug loading amount and drug release behavior in response to pH change were observed.

A potentiometric non-enzymatic glucose sensor using a molecularly imprinted layer bonded on a conducting polymer.

A non-enzymatic potentiometric glucose sensor for the determination of glucose in the micomolar level in saliva was developed based on a molecularly imprinted polymer (MIP) binding on a conducting polymer layer. A MIP containing acrylamide, and aminophenyl boronic acid, as a host molecule to glucose, was immobilized on benzoic acid-functionalized poly(terthiophene) (pTBA) by the amide bond formation onto a gold nanoparticles deposited-screen printed carbon electrode (pTBA/AuNPs/SPCE). Aromatic boronic acid was incorporated into the MIP layer to stably capture glucose and create a potentiometric signal through the changed pKa value of polymer film by the formation of boronate anion-glucose complex with generation of H+ ions by the cis-diol reaction. Reversible binding and extraction of glucose on the sensor surface was observed using a quartz crystal microbalance. Each layer of the sensor probe was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, X-ray photoelectron spectroscopy, and atomic force microscopy. The potentiometric response at the optimized conditions exhibited a wide linear dynamic range of 3.2×10-7 to 1.0×10-3M, with a detection limit of 1.9 (±0.15)×10-7M. The sensor probe revealed an excellent selectivity and sensitivity for glucose compared to other saccharides. In addition, the reliability of the proposed glucose sensor was evaluated in physiological fluid samples of saliva and finger prick blood.

Complete mitochondrial genome of the Ussuri white-toothed shrew Crocidura lasiura (Insectivora, Soricidae).

We obtained the complete mitochondrial genome of the Ussuri white-toothed shrew Crocidura lasiura (Insectivora, Soricidae) at 17 362 base pairs (bp) containing 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. Its gene order is identical to that of other vertebrates. Several repeat elements were identified in the non-coding control region (D-loop). Phylogenetic tree using mt protein-coding gene sequences showed that C. lasiura was closely related to C. attenuata. The reports of mt genome sequences of Crocidura were not enough to study phylogenetic relationships in genome levels. However, this report may help us to understand the phylogenetic relationships and evolutionary history of Crocidura.

Disposable all-solid-state pH and glucose sensors based on conductive polymer covered hierarchical AuZn oxide.

Poly(terthiophene benzoic acid) (pTBA) layered-AuZn alloy oxide (AuZnOx) deposited on the screen printed carbon electrode (pTBA/AuZnOx/SPCE) was prepared to create a disposable all-solid-state pH sensor at first. Further, FAD-glucose oxidase (GOx) was immobilized onto the pTBA/AuZnOx/SPCE to fabricate a glucose sensor. The characterizations of the sensor probe reveal that AuZnOx forms a homogeneous hierarchical structure, and that the polymerized pTBA layer on the alloy oxide surface captures GOx covalently. The benzoic acid group of pTBA coated on the probe layer synergetically improved the pH response of the alloy oxide and provide chemical binding sites to enzyme, which resulted in a Nernstian behavior (59.2 ± 0.5 mV/pH) in the pH range of 2-13. The experimental parameters affecting the glucose analysis were studied in terms of pH, temperature, humidity, and interferences. The sensor exhibited a fast response time <1s and a dynamic range between 30 and 500 mg/dL glucose with a detection limit of 17.23 ± 0.32 mg/dL. The reliabilities of the disposable pH and glucose sensors were examined for biological samples.

Temperature-induced gel formation of core/shell nanoparticles for the regeneration of ischemic heart.

Vascular endothelial growth factor (VEGF)-loaded core/shell nanoparticles were prepared and their gelation behavior in response to temperature was characterized for the regeneration of ischemic heart. The core is composed of lecithin containing VEGF and the shell is composed of Pluronic F-127 (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer). When Capryol 90 (propylene glycol monocaprylate) was added to an aqueous solution of the core/shell nanoparticles, a temperature-induced gel composed of core/shell nanoparticles was observed to form at body temperature. This phenomenon was utilized for the stable localization of core/shell nanoparticles at the ischemic area. For an in vivo assessment, VEGF-loaded core/shell nanoparticles with and without inducement of the gel formation were applied to a subacute myocardial infarction model in rats and functional analysis of the heart was monitored by means of a PV catheter four weeks later. The results showed that the VEGF-loaded core/shell nanoparticles and their gel improved the heart functions, particularly with regard to the ejection fraction and cardiac output.

An amperometric chloramphenicol immunosensor based on cadmium sulfide nanoparticles modified-dendrimer bonded conducting polymer.

An amperometric chloramphenicol (CAP) immunosensor was fabricated by covalently immobilizing anti-chloramphenicol acetyl transferase (anti-CAT) antibody on cadmium sulfide nanoparticles (CdS) modified-dendrimer that was bonded to the conducting polymer (poly 5, 2': 5', 2''-terthiophene-3'-carboxyl acid (poly-TTCA)) layer. The AuNPs, dendrimers, and CdS nanoparticles were deposited onto the polymer layer in order to enhance the sensitivity of the sensor probes. The particle sizes were determined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The immobilization of dendrimers, CdS, and anti-CAT were confirmed using energy disruptive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and quartz crystal microbalance (QCM) techniques. The detection of CAP was based on the competitive immuno-interaction between the free- and labeled-CAP for active sites of the anti-CAT. Hydrazine was used as the label for CAP, and it electrochemically catalyzed the reduction of H(2)O(2) at -0.35 V vs. Ag/AgCl. Under optimized conditions, the proposed immunosensor exhibited a linear range of CAP detection between 50 pg/mL and 950 pg/mL, and the detection limit was 45 pg/mL. The immunosensor was examined in real meat samples for the analysis of CAP.

Heparin-immobilized pluronic/PVA composite microparticles for the sustained delivery of ionic drug.

Heparin-immobilized Pluronic (F-68)/Polyvinylalcohol (PVA) composite microparticles were designed and characterized for the sustained drug delivery of ionic drug. Venlafaxine, antidepressant medication, was used as a model drug. For the efficient loading of ionic drug, heparin was immobilized into F-68/PVA composite microparticles. Differential scanning calorimetry (DSC) was used to understand the intra/intermolecular interactions in the heparin-immobilized F-68/PVA composite gels containing model drug. For the application as a sustained drug delivery system, the loading amount and release pattern of loaded drug were measured using high performance liquid chromatography (HPLC).

Formation of core/shell nanoparticles with a lipid core and their application as a drug delivery system.

A novel preparation method for core/shell nanoparticles with a drug-loaded lipid core was designed and characterized. The lipid core is composed of lecithin and a drug, and the polymeric shell is composed of Pluronics (poly(ethylene oxide)-poly (propylene oxide)-poly(ethylene oxide) triblock copolymer, F-127). For the formation of stabilized core/shell nanoparticles, freeze-drying was performed in the presence of trehalose used as a cryoprotectant. Cryogenic transmittance electron microscopy (cryo-TEM), differential scanning calorimetry (DSC), and a particle size analyzer were used to observe the formation of the stabilized core/shell nanoparticles. For the application of the core/shell nanoparticles as a drug carrier, paclitaxel, a potent anticancer drug, was loaded into the core/shell nanoparticles, and the drug loading amount and the drug release pattern were observed.