Mast cell activation triggered by SARS-CoV-2 causes inflammation in brain microvascular endothelial cells and microglia.

SARS-CoV-2-induced excessive inflammation in brain leads to damage of blood-brain barrier, hypoxic-ischemic injury, and neuron degeneration. The production of inflammatory cytokines by brain microvascular endothelial cells and microglia is reported to be critically associated with the brain pathology of COVID-19 patients. However, the cellular mechanisms for SARS-CoV-2-inducing activation of brain cells and the subsequent neuroinflammation remain to be fully delineated. Our research, along with others', has recently demonstrated that SARS-CoV-2-induced accumulation and activation of mast cells (MCs) in mouse lung could further induce inflammatory cytokines and consequent lung damages. Intracerebral MCs activation and their cross talk with other brain cells could induce neuroinflammation that play important roles in neurodegenerative diseases including virus-induced neuro-pathophysiology. In this study, we investigated the role of MC activation in SARS-CoV-2-induced neuroinflammation. We found that (1) SARS-CoV-2 infection triggered MC accumulation in the cerebrovascular region of mice; (2) spike/RBD (receptor-binding domain) protein-triggered MC activation induced inflammatory factors in human brain microvascular endothelial cells and microglia; (3) MC activation and degranulation destroyed the tight junction proteins in brain microvascular endothelial cells and induced the activation and proliferation of microglia. These findings reveal a cellular mechanism of SARS-CoV-2-induced neuroinflammation.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China.

BACKGROUND: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. RESULTS: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. CONCLUSIONS: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

Toxoplasma gondii infection induces cell apoptosis via multiple pathways revealed by transcriptome analysis.

Toxoplasma gondii is a worldwide parasite that can infect almost all kinds of mammals and cause fatal toxoplasmosis in immunocompromised patients. Apoptosis is one of the principal strategies of host cells to clear pathogens and maintain organismal homeostasis, but the mechanism of cell apoptosis induced by T. gondii remains obscure. To explore the apoptosis influenced by T. gondii, Vero cells infected or uninfected with the parasite were subjected to apoptosis detection and subsequent dual RNA sequencing (RNA-seq). Using high-throughput Illumina sequencing and bioinformatics analysis, we found that pro-apoptosis genes such as DNA damage-inducible transcript 3 (DDIT3), growth arrest and DNA damage-inducible α (GADD45A), caspase-3 (CASP3), and high-temperature requirement protease A2 (HtrA2) were upregulated, and anti-apoptosis genes such as poly(adenosine diphosphate (ADP)-ribose) polymerase family member 3 (PARP3), B-cell lymphoma 2 (Bcl-2), and baculoviral inhibitor of apoptosis protein (IAP) repeat containing 5 (BIRC5) were downregulated. Besides, tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1), TRAF2, TNF receptor superfamily member 10b (TNFRSF10b), disabled homolog 2 (DAB2)|-interacting protein (DAB2IP), and inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) were enriched in the upstream of TNF, TNF-related apoptosis-inducing ligand (TRAIL), and endoplasmic reticulum (ER) stress pathways, and TRAIL-receptor 2 (TRAIL-R2) was regarded as an important membrane receptor influenced by T. gondii that had not been previously considered. In conclusion, the T. gondii RH strain could promote and mediate apoptosis through multiple pathways mentioned above in Vero cells. Our findings improve the understanding of the T. gondii infection process through providing new insights into the related cellular apoptosis mechanisms.

Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips.

The coronavirus disease 2019 (COVID-19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS-CoV-2, constructing the pET-28a (+)-N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS-CoV-2-N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high-titer and high-specificity monoclonal antibodies, and the test strips exclusively react with the SARS-CoV-2-N protein and no cross-reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS-CoV-2 infection through serological antigen.

A novel loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) device for rapid detection of Toxoplasma gondii in the blood of stray cats and dogs.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.

TITLE: Un nouveau dispositif de bandelette à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) pour la détection rapide de Toxoplasma gondii dans le sang des chats et chiens errants. ABSTRACT: Toxoplasma gondii est un parasite protozoaire intracellulaire obligatoire qui provoque la toxoplasmose et menace la santé humaine et les animaux à sang chaud dans le monde entier. Des méthodes de diagnostic simples et applicables sont nécessaires de toute urgence pour guider le développement d'approches efficaces pour la prévention de la toxoplasmose. La plupart des outils de diagnostic moléculaire pour l'infection par T. gondii nécessitent des compétences techniques élevées, un équipement sophistiqué et un environnement de laboratoire contrôlé. Dans cette étude, nous avons développé un test par bandelettes à flux latéral d'amplification isotherme médiée par les boucles (LAMP-LFD) qui cible spécifiquement les 529 pb qui détectent une infection par T. gondii. Ce nouvel appareil portable est universel, rapide, convivial et garantit une sensibilité expérimentale ainsi qu'un faible risque de contamination par aérosol. Notre test LAMP-LFD a une limite de détection de 1 fg d'ADN de T. gondii et ne montre aucune réaction croisée avec d'autres pathogènes parasites, y compris Cryptosporidium parvum, Leishmania donovani et Plasmodium vivax. Nous avons validé le test en détectant T. gondii dans l'ADN extrait d'échantillons de sang prélevés sur 318 chats et chiens errants prélevés dans les villes de Deqing, Wenzhou, Yiwu, Lishui et Zhoushan dans la province du Zhejiang, dans l'est de la Chine. Le dispositif LAMP-LFD a détecté la prévalence de l'ADN de T. gondii chez respectivement 4,76 et 4,69% des chats et chiens errants. En conclusion, le test LAMP-LFD développé est efficace, minimise la contamination par les aérosols et convient donc à la détection de T. gondii dans les établissements médicaux simples et sur le terrain.

Hydrologic performance of bioretention in an expressway service area.

Bioretention can be an effective measure for stormwater treatment. However, there is a lack of systematic analysis of the impact of bioretention design parameters on hydrologic performance. Herein, SWMM and RECARGA models were applied to generate the typical annual rainfall runoff and simulate the water balance of the bioretention system in an expressway service area. The purpose of the investigation was to identify key design parameters for the bioretention system and delineate the priorities in developing the design. Results showed that the average groundwater recharge ratios for bioretention basins with and without an underdrain were 58.29% and 92.27%, respectively, the average overflow ratios were 4.13% and 4.19%, the average evapotranspiration ratios were 4.48% and 4.47%, and the average outflow ratio for bioretention with an underdrain was 33.94%. The ratio of the bioretention area to drainage area, and the saturated infiltration rates of planting soil and native soil were the main factors influencing water balance, while the underdrain diameter and gravel layer depth exerted little effect. Based on the impact analysis, multivariate nonlinear regression models of runoff reduction rate for two types of bioretention basin were established, which both exhibited high determination coefficients and acceptable Nash-Sutcliffe coefficients.