Fluorescence immunosensor based on a specific monoclonal antibody for highly sensitive and rapid detection of gizzerosine in feed.

Gizzerosine is a biogenic amine produced in fish meal drying process and posted higher mortality due to gizzard erosion in poultry than histamine. However, it is difficult to obtain gizzerosine and achieve sensitive practical detection due to its simple structure. Herein, a monoclonal antibody (mAb) specific to gizzerosine was generated based on the new structural design and a fluorescence immunosensor for sensitive and on-site detection of gizzerosine in feed was first established. Molecular modeling of the three-dimensional (3D) structure and surface electrostatic potential of gizzerosine indicated that the carbonyl group of gizzerosine hapten might affect the important sites of antigen-antibody interactions. The proposed structure was used to obtain the sensitive and specific mAb with IC50 of 3.88 ng/mL in indirect competitive ELISA which was approximately 100-fold lower than that of direct competitive ELISA. Considering the practical application scenarios, a fluorescence immunosensor based on microporous dry method integrated with independent quality control line was established to improve detection stability. Under the optimum conditions, the proposed immunosensor showed a good linear relationship from 1.10 to 19.78 ng/mL and provided a low detection limit of 50 ng/g which was approximately 80-fold lower than the maximum recommended amount (0.4 mg/kg) of gizzerosine in feed. The recoveries of 6 kinds of feed ranged from 83.1 % to 114.3 %, which was in good consistence with that of UHPLC-MS/MS. Overall, this work provides a fast, cost-effective and reliable on-site tool for rapid screening of gizzerosine residues in feed samples.

Correlation between the facial skin microbiome and sensitive skin using the 2bRAD-M technique.

OBJECTIVE: This study aimed to expound on the correlation between facial skin microbiome and sensitive skin (SS) using a novel sequencing technique. METHODS: We applied the 2bRAD sequencing for the microbiome, which enables accurate characterization of the low-biomass microbiome at species resolution to profile facial skin microbes in SS and non-SS groups. Further, the bacterial colonies were isolated and cultured from skin surfaces to study the pro-inflammatory effect on human keratinocytes by ELISA. RESULTS: We accordingly identified 1142 genera and 4436 strains. In the SS group, the proportions of Actinomyces and Microbotryomycetes were significantly increased, whereas that of Acidimicrobiia was decreased. Kruskal-Wallis analysis revealed significant differences in 11 genera and 35 species, among which the proportions of Dermabacter, Chryseobacterium, Rhodotorula and Peptoniphilus A were increased in the SS group. Analysis of the top 10 genera revealed increased proportions of Cutibacterium, Corynebacterium and Staphylococcus. Moreover, the proportion of Dermabacter hominis was significantly increased by 18.9-fold in the SS group, whereas those of many Streptococcus strains were significantly decreased. Focus on the isolated bacterial colonies from skin surfaces, more yellow colonies were found in SS group when cultured in Tryptic Soy Broth medium for 48 h, and more interleukin-8 was detected on keratinocytes after yellow colonies stimulation, such as S.capitis, M.luteus. CONCLUSIONS: This study suggests that more SS-associated microorganisms can be identified using the 2bRAD technique even with a small sample size. Dermabacter hominis and Chryseobacterium was firstly reported with a significantly increase in SS, and the S.capitis, as well as M.luteus, but not S.aureus, may be associated with skin inflammation.

OBJECTIF: Cette étude visait à expliquer la corrélation entre le microbiome de la peau du visage et la peau sensible (PS) à l'aide d'une nouvelle technique de séquençage. MÉTHODES: Nous avons appliqué le séquençage 2bRAD pour le microbiome, ce qui nous a permis de caractériser précisément le microbiome à faible biomasse à la résolution des espèces pour profiler les microbes de la peau du visage dans les groupes PS et non-PS. En outre, les colonies bactériennes ont été isolées et cultivées à partir de surfaces cutanées pour étudier l'effet pro-inflammatoire sur les kératinocytes humains par ELISA. RÉSULTATS: Nous avons donc identifié 1 142 genres et 4 436 souches. Dans le groupe PS, on a pu constater des proportions d'Actinomyces et de microbotryomycètes significativement accrues, pour de moindres proportions d'Acidimicrobiia. L'analyse de Kruskal-Wallis a révélé des différences significatives dans 11 genres et 35 espèces, parmi lesquelles des proportions de Dermabacter, Chryseobacterium, Rhodotorula et Peptoniphilus A accrues dans le groupe PS. L'analyse des 10 principaux genres a montré une augmentation des proportions de Cutibacterium, Corynebacterium et Staphylococcus. En outre, la proportion de Dermabacter hominis a été multipliée par 18,9 dans le groupe PS, soit une augmentation significative, tandis que celle de nombreuses souches de Streptococcus s'est avérée significativement plus basse. En se concentrant sur les colonies bactériennes isolées des surfaces cutanées, plus de colonies jaunes ont été trouvées dans le groupe PS lorsqu'elles étaient cultivées dans du milieu de bouillon trypticase soja pendant 48 h, et davantage d'interleukine-8 a été détectée sur les kératinocytes après la stimulation des colonies jaunes comme S. capitis, M. luteus. CONCLUSIONS: Cette étude suggère que davantage de micro-organismes associés au PS peuvent être identifiés à l'aide de la technique 2bRAD, même avec un échantillon de petite taille. Dermabacter hominis et Chryseobacterium ont été rapportés avec une augmentation significative pour les PS, et S. capitis, ainsi que M. luteus, mais pas S. aureus, pouvant être associés à une inflammation cutanée.

Dual-modular immunosensor for bongkrekic acid detection using specific monoclonal antibody.

Bongkrekic acid (BA) is a mitochondrial toxin that causes high mortality but is often mistakenly categorized as other food poisonings. The immunoassay of BA is still challenging since the specific antibody is unavailable. In this work, a monoclonal antibody specific to BA was first generated and a dual-modular immunosensor for on-site and laboratory detection was established. The antibody showed good affinity (Kd=0.33 µM) and sensitivity (IC50 =17.9 ng/mL in ELISA) with negligible cross-reactivity with common mycotoxins. In dual-modular conditions, fluorescence assay (FA) was conducted based on the inner filter effect of carbon dots (CDs) and oxidized 3,3',5,5'-tetramethylbenzidine (TMB), while the colorimetric assay (CA) was conducted using TMB2+-mediated rapid surface etching of gold nanostars (Au NSs). The proposed immunosensor showed good sensitivity and reproducibility to BA in food samples, with a limit of detection lower than 10 ng/mL and recovery ranging from 80.0% to 103.6%, which was in good consistence with that of standard LC-MS/MS. Overall, the proposed immunosensor is an ideal tool for screening BA contaminants in food with good sensitivity and high effectivity.

Modulation of TRPV1 function by Citrus reticulata (tangerine) fruit extract for the treatment of sensitive skin.

BACKGROUND: Sensitive skin (SS) is a clinical syndrome defined by the occurrence of unpleasant sensations (such as stinging, burning, pain, pruritus, and tingling) in response to stimuli that normally should not provoke them. According to growing evidence, transient receptor potential vanilloid subtype 1 (TRPV1) has elevated expression in individuals with SS and is linked with the severity of SS symptoms. However, its pathogenesis is still unknown. OBJECTIVE: Herein, Citrus reticulata (Tangerine) fruit extract (CR) was obtained and examined for its effect on SS with a focus on TRPV1 stimulation and expression. METHODS: A recombinant hTRPV1 over-expression cell line (HaCaT-TRPV1-OE cell) was constructed to screen substances and extracts from several plants. Intracellular calcium mobilization was monitored by Flexstation 3 and a fluorescence microscope using Fluo 8 AM fluorophore. Next, immunofluorescence was used to detect the TRPV1 expression under different stimulants treated for 24 h. To investigate the relief and increased tolerance of CR to lactic acid-induced skin discomfort, clinical tests were carried out on the nasolabial folds or cheek areas. RESULTS: According to the obtained results, compared to HaCaT cells, HaCaT-TRPV1-OE cells showed a higher expression of TRPV1. Neuronal hyperresponsiveness in SS triggered by capsaicin (CAP), lactic acid, phenoxyethanol or nicotinamide may be through activation of TRPV1 and increased TRPV1 expression. CAP activates TRPV1 in HaCaT-TRPV1-OE cells, and more than 100 plants or chemicals were tested for their inhibitory effects before being screened for CR. CR (1%-4%) inhibited TRPV1 activation induced by CAP or phenoxyethanol or nicotinamide. Meanwhile, CR (0.25%) suppressed TRPV1 protein expression induced by phenoxyethanol or lactic acid. In vivo results showed that CR not only instantly relieved lactic acid-induced skin discomfort under 5 min but also enhanced skin tolerance to lactic acid after 7 days of continuous use. CONCLUSIONS: Topical application of CR showed an instant and long-lasting improvement in SS by modulating the activation and expression of TRPV1. Moreover, it has been suggested that CR might act as a TRPV1 inhibitor to reduce skin irritation or sensitivity.

Demographic profile and ocular characteristics of stage 5 retinopathy of prematurity at a referral center in Northwest China: implications for implementation.

BACKGROUND: Severe retinopathy of prematurity (ROP) with extremely unfavorable prognosis among infants can do great damage to individuals and bring tremendous social-economic burden. The purpose of this study is to describe the demographic and ocular characteristics of infants who presented with stage 5 ROP in order to identify reasons why they have become blind, and to identify contributing factors in order to focus great attention on the current ROP program and to inspire more effort in ROP screening in middle income countries. METHODS: A retrospective review of consecutive infants with stage 5 ROP from December 2010 to December 2016 in Department of Ophthalmology, Xijing Hospital. Various parameters retrieved included birthweight, gestational age, age at initial examination, postmenstrual age, screening details, check-up details and reasons for consultation. Ocular findings were recorded and also detected by ultrasonography. RESULTS: A retrospective review of 20 consecutive infants with stage 5 ROP are included. Mean birthweight was1712.3 ± 512.97 g and mean gestational age at birth was 32.1 ± 2.21 weeks. Median age at first consultancy was 9.7 month. Median postmenstrual age first consultancy was 52 weeks. All infants were never screened for ROP before they came to the referral center. Of twenty stage 5 ROP infants, 13 cases presented with bilateral stage 5 features. Of the 40 eyes of 20 infants, 33 eyes were diagnosed as stage 5. Leukocoric pupil, closed funnel configuration of retinal detachment (RD), posterior synechia, extraretinal fibrovascular proliferation and retinal folds were the most significant indicators of bad prognosis. Ten eyes appeared no fixation to light, while 30 eyes exhibited following to light or following to toys. CONCLUSIONS: Our study shows that in relatively less-developed regions of China, more needs to be done to spread awareness about the disease among pediatricians, neonatologists and ophthalmologists as well as parents of premature infants. Thus, a comprehensive control system which is a whole network of propaganda, screening, treatment and follow-up are encouraged especially in less developed areas in China as well as worldwide.

A novel class of antagonists for the FFAs receptor GPR40.

The free fatty acid receptor, GPR40, is implicated in the pathophysiology of type 2 diabetes, and is a new potential drug target for the treatment of type 2 diabetes. Its antagonist is thought to be not only a useful chemical probe for further exploring the function of GPR40 but also a lead structure for drug development. With virtual screening based on a homology model followed by a cell-based calcium mobilization assay, we found that sulfonamides are a new class of small organic antagonists for GPR40. One of the compounds, DC260126, dose-dependently inhibited GPR40-mediated Ca(2+) elevations stimulated by linoleic acid, oleic acid, palmitoleic acid and lauric acid (IC(50): 6.28+/-1.14, 5.96+/-1.12, 7.07+/-1.42, 4.58+/-1.14 microM, respectively), reduced GTP-loading and ERK1/2 phosphorylation stimulated by linoleic acid in GPR40-CHO cells, suppressed palmitic acid potentiated glucose-stimulated insulin secretion, and negatively regulated GPR40 mRNA expression induced by oleic acid in Min6 cells.