Enhancing Loop-Mediated Isothermal Amplification through Nonpowered Nanoelectric Preconcentration.

The global threat posed by the COVID-19 pandemic has catalyzed the development of point-of-care (POC) molecular diagnostics. While loop-mediated isothermal amplification (LAMP) stands out as a promising technique among FDA-approved methods, it is occasionally susceptible to a high risk of false positives due to nonspecific amplification of a primer dimer. In this work, we report an enhancing LAMP technique in terms of assay sensitivity and reliability through streamlined integration with a nonpowered nanoelectric preconcentration (NPP). The NPP, serving as a sample preparation tool, enriched the virus concentration in samples prior to the subsequent LAMP assay. This enrichment enabled not only to achieve more sensitive assay but also to shorten the assay time for all tested clinical samples by ∼10 min compared to the conventional LAMP. The shortened assay time suppresses the occurrence of nonspecific amplification by not providing the necessary incubation time, effectively suppressing misidentification by false positives. Utilizing this technique, we also developed a prototype of the POC NPP-LAMP kit. This kit offers a streamlined diagnostic process for nontrained individuals, from the sample enrichment, transfer of the enriched sample to LAMP assays, which facilitates on-site/on-demand diagnosis of SARS-CoV-2. This development holds the potential to contribute toward preventing not only the current outbreak but also future occurrences of pandemic viruses.

Effect of dietary concentrations of metabolizable energy and neutral detergent fiber on productive performance, egg quality, fatty liver incidence, and hepatic fatty acid metabolism in aged laying hens.

The objective of the current experiment was to investigate the effect of dietary concentrations of ME and neutral detergent fiber (NDF) on productive performance, egg quality, fatty liver incidence, and hepatic fatty acid metabolism in aged laying hens. A total of three hundred twenty 75-wk-old Hy-Line Brown laying hens were allotted to 1 of 4 dietary treatments with 8 replicates. Each replicate consisted of 10 consecutive cages with 1 hen per cage. The experiment was conducted using a completely randomized design with 2 × 2 factorial arrangement consisting of 2 levels of ME (normal [commercially recommended AMEn levels; 2,730 kcal/kg] and low [50 kcal/kg reduction in AMEn; 2,680 kcal/kg]) and 2 levels of NDF (low [9.01 and 9.61%; normal-ME and low-ME diets, respectively] and high [12.57 and 13.42%; normal-ME and low-ME diets, respectively]) in the diet. The diets and water were provided to hens on an ad libitum basis for 12 wk. Results indicated that no interactions between dietary concentrations of ME and NDF were observed for all measurements except for egg yolk color, eggshell thickness, and 2 hepatic gene expressions (i.e., carnitine palmitoyl transferase 1A and malic enzyme). For the main effects, increasing NDF concentrations in diets increased (P < 0.05) feed intake without affecting other productive performance. Hens fed normal-ME and high-NDF diets showed the darkest (P < 0.05) egg yolk color among those fed treatment diets, showing an interaction (P < 0.05). Increasing NDF concentrations in low-ME diets did not influence eggshell thickness, but those in normal-ME diets increased eggshell thickness in laying hens, showing an interaction (P < 0.05). For the main effects, increasing concentrations of dietary NDF or ME reduced (P < 0.05) hepatic fat concentrations with decreasing expressions in several genes related to fatty acid synthesis. In conclusion, increasing NDF concentrations in commercially-recommended ME diets decrease hepatic fat concentrations in aged laying hens, and therefore, may have a preventative effect on the fatty liver development in aged laying hens.

Rapid and low-cost, and disposable electrical sensor using an extended gate field-effect transistor for cardiac troponin I detection.

Field effect transistor (FET) biosensor is based on metal oxide field effect transistor that is gated by changes in the surface charges induced the reaction of biomolecules. In most cases of FET biosensor, FET biosensor is not being reused after the reaction; therefore, it is an important concept of investigate the biosensor with simplicity, cheap and reusability. However, the conventional cardiac troponin I (cTnI) sensing technique is inadequate owing to its low sensitivity and high operational time and cost. In this study, we developed a rapid and low-cost, and disposable electrical sensor using an extended gate field-effect transistor (EGFET) to detect cTnI, as a key biomarker for myocardiac infarction. We first investigated pH sensing characteristics according to the pH level, which provided a logarithmically linear sensitivity in the pH sensing buffer solution of approximately 57.9 mV/pH. Subsequently, we prepared a cTnI sample and monitored the reaction between cTnI and cTnI antibodies through the changes in the drain current and transfer curves. Our results showed that the EGFET biosensor could successfully detect the cTnI levels as well as the pH with low-cost and rapid detection.

Nanoelectrokinetic-assisted lateral flow assay for COVID-19 antibody test.

A lateral flow assay (LFA) platform is a powerful tool for point-of-care testing (POCT), especially for self-testing. Although the LFA platform provides a simple and disposable tool for Coronavirus disease of 2019 (COVID-19) antigen (Ag) and antibody (Ab) screening tests, the lower sensitivity for low virus titers has been a bottleneck for practical applications. Herein, we report the combination of a microfluidic paper-based nanoelectrokinetic (NEK) preconcentrator and an LFA platform for enhancing the sensitivity and limit of detection (LOD). Biomarkers were electrokinetically preconcentrated onto a specific layer using the NEK preconcentrator, which was then coupled with LFA diagnostic devices for enhanced performance. Using this nanoelectrokinetic-assisted LFA (NEK-LFA) platform for self-testing, the severe acute respiratory syndrome coronavirus 2 Immunoglobulin G (SARS-CoV-2 IgG) sample was preconcentrated from serum samples. After preconcentration, the LOD of the LFA was enhanced by 32-fold, with an increase in analytical sensitivity (16.4%), which may offer a new opportunity for POCT and self-testing, especially in the COVID-19 pandemic and endemic global context.

Origami paper-based sample preconcentration using sequentially driven ion concentration polarization.

Ion concentration polarization (ICP) is one of the preconcentration techniques which can acquire a high preconcentration factor. Still, the main hurdles of ICP are its instability and low efficiency under physiological conditions with high ionic strength and abundant biomolecules. Here, we suggested a sequentially driven ICP process, which enhanced the electrokinetic force required for preconcentration, enabling enrichment of highly ionic raw samples without increasing the electric field. We acquired a 13-fold preconcentration factor (PF) in human serum using a paper-based origami structure consisting of multiple layers for three-dimensional sequential ICP (3D seq-ICP). Moreover, we demonstrated a paper-based enzyme-linked immunosorbent assay (ELISA) by 3D seq-ICP using tau protein, showing a 6-fold increase in ELISA signals.

Electrokinetic Size-Based Spatial Separation of Micro/Nanospheres Using Paper-Based 3D Origami Preconcentrator.

Sample preparation steps (e.g., preconcentration and separation) are key to enhancing sensitivity and reliability in biomedical and analytical chemistry. However, conventional methods (e.g., ultracentrifugation) cause significant loss of sample as well as their contamination. In this study, we developed a paper-based three-dimensional (3D) origami ion concentration polarization preconcentrator (POP) for highly efficient and facile sample preparation. The unique design of POP enables simultaneous preconcentration and spatial separation of target analytes rapidly and economically. The POP comprises accordion-like multifolded layers with convergent wicking areas that can separate analytes based on their sizes in different layers, which can then be easily isolated by unfolding the POP. We first demonstrated 100-fold preconcentration of albumin and its isolation on the specific layers. Then, we demonstrated the simultaneous preconcentration and spatial separation of microspheres of three different sizes (with diameters of 0.02, 0.2, and 2 µm) on the different layers.

A case of acute bilateral retrocochlear hearing loss as an initial symptom of unilateral thalamic hemorrhage.

A speech discrimination test is a test using a list of 25 phonetically balanced monosyllables. It is often overlooked but significant enough for pure tone audiometry. Many physicians have performed pure tone audiometry but without a speech discrimination test. A 73-year-old woman visited our clinic complaining of sudden bilateral hearing loss. Pure tone audiometry showed only bilateral high frequency loss. However, speech discrimination had decreased markedly. We decided to follow-up after 1 week of Ginexin-F® (ginkgo leaf extract) and Nafril® (nafronyl oxalate). She felt a gait disturbance within 2 days. Magnetic resonance imaging revealed a left thalamic hemorrhage. After a 1 month hospitalization, the hematoma subsided, and speech discrimination recovered 3 months later. Acute hearing loss due to thalamic hemorrhage that recovered has never been reported. We report the first case of retrocochlear hearing loss that occurred with a thalamic hemorrhage in a patient who recovered.

One-generation reproductive toxicity study of epichlorohydrin in Sprague-Dawley rats.

This study was conducted to evaluate the potential reproductive toxicity of epichlorohydrin in a one-generation reproduction toxicity study in compliance with OECD Test Guideline 415. Twenty-four male and female rats per group were given epichlorohydrin by gavage at 0, 3.3, 10, and 30 mg/kg/day. Males were dosed for 10 weeks prior to and during mating. Females were dosed from 2 weeks before mating to day 21 of lactation. At 30 mg/kg, an increase in the incidence of clinical signs (i.e., nasal discharge, soft feces, depression, and piloerection), gross necropsy findings (i.e., cystic pustule of the epididymidis and enlargement of the kidney) and the weights of heart, liver, and epididymidis, a decrease in male fertility, and an increased incidence of histopathological changes of the testis, epididymidis, and kidney were observed. At 10 mg/kg, decreased male fertility and increased kidney weight and incidence of histopathological changes of the epididymidis were found. There was a slight, but nonsignificant, reduction in the male fertility index at the dose of 3.3 mg/ kg. Under these experimental conditions, the lowest-observed-adverse-effect level of epichlorohydrin was 3.3 mg/kg/day for parent animals and their offspring. The absolute toxic dose for parent animals and their offspring was estimated to be 10 mg/kg/day.

Induction of oxidative stress in the epididymis of rats after subchronic exposure to epichlorohydrin.

This study investigated the effects of epichlorohydrin (ECH) on spermatogenesis and antioxidant system in rats. An increase in the incidence of clinical signs, gross pathology and histopathology findings in the epididymidis, and sperm abnormalities and a decrease in the testicular spermatid counts, epididymal sperm counts, and sperm motility were observed at 30 mg/kg/day. Oxidative stress in the epididymal tissue was detected at > or =3.3 mg/kg/day. The results show that graded doses of ECH elicit depletion of antioxidant defense system and that the adverse effects on male reproductive function in ECH-treated rats may be due to the induction of oxidative stress.

Reproductive and developmental toxicity of amitraz in sprague-dawley rats.

The present study was conducted to obtain information on the effects of amitraz on reproductive and developmental parameters in rats. The test chemical was administered via the drinking water containing 0, 40, 120, and 360 ppm to male rats from 2 weeks before mating to the end of 14-day mating period and to females from 2 weeks before mating, throughout mating, gestation and up to lactational day 4. During the study period, clinical signs, body weights, food intake, organ weights, reproductive and littering findings, necropsy findings, sperm parameters, and histopathology were examined. At 360 ppm, decreases in the body weight gain, food consumption, and the number of live pups and an increase in the post-implantation loss were observed. In addition, decreases in the seminal vesicle weight and sperm motility were found in males. At 120 ppm, a decrease in the food consumption was found transiently in both males and females, but no reproductive and developmental toxicity was observed in both sexes. There were no signs of either general or reproductive and developmental toxicity in the 40 ppm group. Based on these results, it was concluded that the repeated oral administration of amitraz to rats resulted in a decrease in the food consumption at 120 ppm and decreases in the seminal vesicle weight, sperm motility, and the number of live pups and an increase in the post-implantation loss at 360 ppm in rats. Under these experimental conditions, the no-observed-adverse-effect level (NOAEL) of amitraz for general and reproduction/developmental toxicity was believed to be 120 ppm, and the no-observed-effect level (NOEL) of amitraz was believed to be 40 ppm in rats.

Dose-Response Effects of Epichlorohydrin on Male Reproductive Function in Rats.

Present study was conducted to investigate potential effects of epichlorohydrin on testicular and epididymal function in male rats. The test chemical was administered to adult male rats by gavage at dose levels of 0, 3.125, 12.5, and 50 mg/kg/day for 7 days. Testicular and epididymal function were assessed by measurement of reproductive organ weight, testicular spermatid count, epididymal sperm count, motility and morphology, and histopathology in rats. At 50 mg/kg, a decrease in the sperm motility and an increase in the incidence of sperm abnormalities were observed. Histopatho-logical examinations revealed an increase in the incidence of histopathological changes including cell debris in the ducts, vacuolization of the epithelial cells, oligospermia, and epithelial disruption in the proximal caput epididymidis. At 12.5 mg/kg, an increase in the incidence of histopathological changes of the epididymidis was found. There were no treatment-related effects at 3.125 mg/kg. These results show that 7-day repeated oral administration of epichlorohydrin to male rats results in adverse effects on sperm motility, sperm morphology, and epididymal histology at ≥ 12.5 mg/kg/day.

Effects of Exposure Period on the Developmental Toxicity of 2-Bromopropane in Sprague-Dawley Rats.

Recently we reported that 2-bromopropane (2-BP) has maternal toxicity, embryotoxicity, and teratogenicity in Sprague-Dawley rats. The aims of this study are to examine the potential effects of 2-BP administration on pregnant dams and embryo-fetal development, and to investigate the effects of metabolic activation induced by phenobarbital (PB) on developmental toxicities of 2-BP. Pregnant rats received 1000 mg/kg/day subcutaneous 2-BP injections on gestational days (GD) 6 through 10 (Group II and Group IIII) or 11 through 15 (Group IV). Pregnant rats in Group III received an intraperitoneal PB injection once daily at 80 mg/kg/day on GD 3 through 5 for induction of the liver metabolic enzyme system. Control rats received vehicle injections only on GD 6 through 15. All dams underwent caesarean sections on GD 20 and their fetuses were examined for external, visceral, and skeletal abnormalities. Significant adverse effects on pregnant dams and embryo-fetal development were observed in all the treatment groups, and the maternal and embryo-fetal effects of 2-BP observed in Group II were higher than those seen in Group IV. Conversely, maternal and embryo-fetal developmental toxicities observed in Group III were comparable to those seen in Group II. These results suggest that the potential effects of 2-BP on pregnant dams and embryo-fetal development are more likely in the first half of organogenesis (days 6~10 of pregnancy) than in the second half and that the metabolic activation induced by PB pre-treatment did not modify the developmental toxic effects of 2-BP in rats.