Characterization of a Novel Gja8 (Cx50) Mutation in a New Cataract Rat Model.

Purpose: To describe a novel spontaneous cataract inbred strain isolated from large-scale breeding SD rats, identify the responsible gene mutation, and understand how this mutation affects lens function. Methods: Exome sequencing of 12 cataract-associated genes was performed in the affected and healthy relatives. Sequences of rat wild-type or mutant gap junction protein alpha 8 gene (Gja8) were transfected into cells. The expression level of protein was assayed by Western blot analysis. Subcellular localization of connexin 50 (Cx50) was analyzed in confocal fluorescent images. Wound-healing, 5-ethynyl-2'-deoxyuridine incorporation, and attachment assay were performed to characterize the cell migration, proliferation and adhesion. Results: The abnormality was found to be inheritable in an autosomal semi-dominant pattern through different mating patterns. We found a G to T transversion at codon 655 in Gja8, leading to a substitution of valine by phenylalanine (p.V219F). Gja8V219F/+ heterozygotes expressed nuclear cataract while Gja8V219F/V219F homozygotes manifested microphthalmia in addition to cataract. Histology revealed fiber disorders and loss of organelle-free zone in the mutant lens. Cx50V219F altered its location in HeLa cells and inhibited the proliferation, migration and adhesion abilities of HLEB3 cells. The mutation also reduced the expression of focal adhesion kinase and its phosphorylation. Conclusions: The c.655G>T mutation (p.V219F) is a novel mutation in Gja8, inducing semi-dominant nuclear cataracts in a new spontaneous cataract rat model. The p.V219F mutation altered Cx50 distribution, inhibited lens epithelial cell proliferation, migration, and adhesion, and disrupted fiber cell differentiation. As a consequence, the nuclear cataract and small lens formed.

An optimized visual loop mediated isothermal amplification assay for efficient detection of minute virus of mice with hydroxynaphthol blue dye.

Minute virus of mice (MVM) is one of the most prevalent infectious agents in laboratory mouse colonies. In this study, we optimized a loop-mediated isothermal amplification (LAMP) assay using hydroxynaphthol blue (HNB) for rapid and visual detection of MVM. The reaction, which entailed addition of HNB dye prior to amplification, was performed in one step in a single tube at 62 °C for 45 min. The limit of detection of the assay was 104 copies, which was 100-fold lower than that of conventional PCR. The assay specifically amplified MVM DNA and did not cross-react with other viruses. To validate the established LAMP system, we applied it 287 samples and detected 19 positives. In conclusion, LAMP with HNB is a sensitive, and simple assay for rapid detection of MVM infections in laboratory animals, thus offers a platform for quality monitoring.

Rapid Detection of SARS-CoV-2 Using Duplex Reverse Transcription-Multienzyme Isothermal Rapid Amplification in a Point-of-Care Testing.

In December 2019, a severe acute respiratory syndrome caused by SARS-CoV-2 spread rapidly worldwide. Portable nucleic acid tests of SARS-CoV-2 are critically important for diagnostics. In this study, we used an isothermal amplification method-Multienzyme Isothermal Rapid Amplification (MIRA)-for rapid detection of SARS-CoV-2. We designed the primers and probes in ORF1ab and N gene of SARS-CoV-2. The amplicons could be monitored by lateral flow dipsticks (LFDs). The reaction temperature, time, concentrations of primers and probes, and working volume were optimized. Four commercial swab collection buffers were used to test the amplification efficacy of our assay without RNA extraction. Our assay was able to amplify duplex targets of SARS-CoV-2 in one single reaction using one-step RT-MIRA. The assay worked well in a low volume of 10 µl at 38°C for 20 min. Using three collection buffers without guanidinium, our assay was able to amplify efficaciously without RNA extraction. The 95% limit of detection (LoD) of the RT-MIRA assay was 49.5 (95% CI, 46.8-52.7) copies/ml for ORF1ab gene and 48.8 (95% CI, 46.5-52.6) copies/ml for N gene. There is no cross-reaction with other human respiratory pathogens, such as SARS-CoV, MERS-CoV, influenza A virus, influenza B virus, human adenovirus, respiratory syncytial virus, human parainfluenza virus, and coronavirus 229E in our assay. The precision evaluation revealed that the C50-20% to C50+20% range bounds the C5-C95 interval. This assay also showed high anti-interference ability. The extraction-free RT-MIRA and qPCR detection results of 243 nucleic acid specimens from suspected patients or national references showed a 100.0% (95% confidence interval, 94.2%-100.0%) positive predictive value and a 100.0% (95% confidence interval, 92.7%-100.0%) negative predictive value. Compared with qPCR, the kappa value of the two assays was 1.00 (P < 0.0001). In conclusion, we provide a portable and visualized method for detection of SARS-CoV-2 without RNA extraction, allowing its application in SARS-CoV-2 on-site detection.

PK15 cell line stably overexpressing IL2 enhances PCV2 replication.

Porcine circovirus type 2 (PCV2) is the primary agent responsible for porcine circovirus-associated diseases (PCVADs), which is acknowledged as one of the most economically important diseases for the swine industry worldwide. Currently, the development of PCV2 vaccine against PCVADs and for other applications require large amounts of viral particles. The low propagation rate of PCV2 in vitro limits vaccine production. Previous studies showed that a cell line transfected with the porcine interleukin (IL)-2 gene gave higher PCV2 yield in vitro. However, transient transfection may become less effective and unstable after serial generations. In this work, we constructed a PK15 cell line with stable expression of porcine IL2 by lentivirus transfection. The results demonstrated that the transgenic cell line stably expressed IL2 protein significantly enhanced PCV2 replication. Thus, the transgenic PK15 cell line could be a promising cell line for vaccine production.

Effects of low molecular weight organic acids on Cu accumulation by castor bean and soil enzyme activities.

Chelating agents have been considered as an important phytoremediation strategy to enhance heavy metal extraction from contaminated soil. A pot experiment was conducted to explore the effects of low molecular weight organic acids (LMWOAs) on the phytoremediation efficiency of copper (Cu) by castor bean, and soil enzyme activities. Results indicated that the addition of all the three kinds of LMWOAs (citric, tartaric, oxalic acids) did not decrease the biomass of castor bean, despite the fact they reduced the concentration of chlorophyll-a in leaves compared to the control. The Cu concentrations in the roots and shoots significantly increased by 6-106% and 5-148%, respectively, in the LMWOAs treatments so that the total accumulation of Cu by whole plants in all the LMWOAs treatments increased by 21-189% in comparison with the control. The values of the translocation factor (TF) and bio-concentration factor (BCF) of Cu in castor bean also rose following the addition of LMWOAs, indicating that the LMWOAs enhanced the uptake and transportation of Cu. Moreover, the application of LMWOAs did not significantly change the soil pH but significantly increased the activity of soil enzymes (urease, catalase, and alkaline phosphatase). The addition of exogenous LMWOAs increased the available Cu significantly in the soil, thus promoted the phytoextraction efficiency of Cu by castor bean. These results will provide some new insights into the practical use of LMWOAs for the phytoremediation of heavy-metal-contaminated soil employing castor bean.

Effect of heat shock on murine norovirus replication in RAW264.7 cells.

Murine norovirus (MNV), is a prevalent pathogen of laboratory mice closely related to human norovirus (HuNoV), a contagious pathogen known to cause gastroenteritis worldwide; however, the mechanism of norovirus replication remains poorly understood. Both heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) play an important role in viral genome replication and viral gene expression. In this study, we first found that heat stress exerted a positive effect on the replication of MNV in the murine macrophage RAW264.7 cell line. Inhibition of Hsp70 and Hsp90 by the specific inhibitors, KNK437 and 17-AGG, respectively showed that Hsp70 and Hsp90 enhanced MNV genome replication and virion production. In addition, we found that KNK437 and 17-AGG could decrease the level of IL-1ß, IL-10, and TNF-α mRNA expression in MNV-infected cells. These data suggested that heat stress can positively regulate MNV replication, which advances our understanding of the molecular mechanism of MNV infection.

Contributions of root cell wall polysaccharides to Cu sequestration in castor (Ricinus communis L.) exposed to different Cu stresses.

Cell wall polysaccharides play a vital role in binding with toxic metals such as copper (Cu) ions. However, it is still unclear whether the major binding site of Cu in the cell wall varies with different degrees of Cu stresses. Moreover, the contribution of each cell wall polysaccharide fraction to Cu sequestration with different degrees of Cu stresses also remains to be verified. The distribution of Cu in cell wall polysaccharide fractions of castor (Ricinus communis L.) root was investigated with various Cu concentrations in the hydroponic experiment. The results showed that the hemicellulose1 (HC1) fraction fixed 44.9%-67.8% of the total cell wall Cu under Cu stress. In addition, the pectin fraction and hemicelluloses2 (HC2) fraction also contributed to the Cu binding in root cell wall, accounting for 11.0%-25.9% and 14.1%-26.6% of the total cell wall Cu under Cu treatments, respectively. When the Cu levels were ≤25 µmol/L, pectin and HC2 contributed equally to Cu storage in root cell wall. However, when the Cu level was higher than 25 µmol/L, the ability of the pectin to bind Cu was easy to reach saturation. Much more Cu ions were bound on HC1 and HC2 fractions, and the HC2 played a much more important role in Cu binding than pectin. Combining fourier transform infrared (FT-IR) and two-dimensional correlation analysis (2D-COS) techniques, the hemicellulose components were showed not only to accumulate most of Cu in cell wall, but also respond fastest to Cu stress.

Molecular detection of Hsp90 inhibitor suppressing PCV2 replication in host cells.

Porcine Circovirus Type 2 (PCV2) is a pathogen that has the ability to cause devastating disease manifestations in pig populations with major economic implications. Our previous research found that Hsp90 is required for PCV2 production in PK-15 and 3D4/31 cells. The aim of this study was to evaluate the effect of Hsp90 inhibitor regulating PCV2 replication and to explore its underlying mechanism. In PK-15 and 3D4/31 cells treated with 17-AAG after viral adsorption, replication of PCV2 was attenuated as assessed by quantitating the expression of viral protein. Following NF-κB activation it was observed that 24hpi with PCV2 was significantly inhibited in the presence of 17-AAG. The expression of Hsp90 associated client proteins in PCV2-infected cells were also reduced in the presence of 17-AAG. However, treatment with MG-132 failed to rescue 17-AAG mediated reduction of PCV2 production in host cells. Thus, Hsp90 regulates PCV2 by modulating cellular signaling proteins. These results highlight the importance of cellular proteins during PCV2 infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies.

Oxalic acid activated phosphate rock and bone meal to immobilize Cu and Pb in mine soils.

The contamination of soil by copper (Cu) and lead (Pb) is a serious concern because of its high health risk via the food chain. Oxalic acid-activated phosphate rock (APR) and bone meal (BM) were applied to Cu and Pb co-contaminated soil to investigate their efficacy in the immobilization of Cu and Pb. APR and BM were applied into the contaminated soil (158.8 mg/kg total Pb and 573.2 mg/kg Cu) at four levels of dosages (0.1%, 0.5%, 2%, and 4%) and incubated for one year. The results demonstrated that the acid exchangeable Pb fraction in the soil treated with APR and BM decreased compared to the control, while there was no noticeable change in the acid-exchangeable Cu fraction in the soil treated with either APR or BM. Meanwhile, the application of BM and APR increased the fraction of residual Cu and Pb in the polluted soils. Moreover, the addition of either APR or BM at the dose of 4% decreased the concentrations of CaCl2-extractable Cu and Pb in the amended soil, and the percentages of that reduction in the APR amended soils were 56% and 91% and in BM amended soils were 67% and 64%, respectively. The immobilization of Cu and Pb by APR and BM might be induced by the increased soil pH and soluble P contents in the amended soils. In general, BM is more effective than APR on the immobilization of Cu in polluted soil, while APR had greater efficiency than BM on the immobilization of Pb when the levels of amendments were above 2%.

Effects of sulfur on toxicity and bioavailability of Cu for castor (Ricinus communis L.) in Cu-contaminated soil.

The biogeochemical cycling of sulfur (S) in soil has an important impact on the bioavailability of heavy metals and affects the utilization of soil polluted by heavy metals. In addition, S-containing compounds are involved in heavy metal detoxification. This study investigated the effects of S on the toxicity and bioavailability of copper (Cu) in castor (Ricinus communis L.) grown in Cu-contaminated mine tailings. The results showed that the application of S reduced the accumulation of Cu in castor and promoted its growth. With the addition of S, the malondialdehyde (MDA) content of castor leaves decreased significantly compared with control plants, indicating the alleviation of oxidative stress. Superoxide dismutase (SOD) and catalase (CAT) activities and glutathione (GSH) content decreased significantly with the alleviation of oxidative stress. The sequential extraction of Cu fractions showed that the application of S significantly reduced the reducible Cu fraction, and increased the oxidizable Cu fraction. It also increased the residual Cu fraction in the soil. The transformation of chemical speciation reduced the bioavailability of Cu in soil, which then reduced the accumulation of Cu in castor. Our results demonstrated that S application was effective at promoting castor growth by reducing the bioavailability and uptake of Cu in Cu-contaminated mine tailings.

The Hsp90 inhibitor 17-DMAG decreases infection of porcine circovirus type 2 in mice.

Although several factors affecting porcine circovirus type 2 (PCV2) infection have been reported, their precise roles are far from clear. The aim of this study was to determine whether 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90, could significantly affect PCV2 infection and immune responses in BALB/c mice. Intraperitoneal injection of 17-DMAG significantly reduced viral loads in the blood and tissues of mice infected with PCV2, compared with control groups. The 17-DMAG treatment decreased serum interleukin (IL)-10 and tumor necrosis factor(TNF)-α levels, but it did not have a significant effect on the IL-1ß level. These data demonstrate that 17-DMAG is highly effective in suppressing PCV2 replication in BALB/c mice, indicating that it has potential value as an antiviral drug against PCV2 infection.

Differentially expressed microRNAs in the corpus cavernosum from a murine model with type 2 diabetes mellitus-associated erectile dysfunction.

To better understand the molecular aetiology of type 2 diabetes mellitus-associated erectile dysfunction (T2DMED) and to provide candidates for further study of its diagnosis and treatment, this study was designed to investigate differentially expressed microRNAs (miRNAs) in the corpus cavernosum (CC) of mice with T2DMED using GeneChip array techniques (Affymetrix miRNA 4.0 Array) and to predict target genes and signalling pathways regulated by these miRNAs based on bioinformatic analysis using TargetScan, the DAIAN web platform and DAVID. In the initial screening, 21 miRNAs appeared distinctly expressed in the T2DMED group (fold change ≥3, p ≤ 0.01). Among them, the differential expression of miR-18a, miR-206, miR-122, and miR-133 were confirmed by qRT-PCR (p < 0.05 and FDR <5 %). According to bioinformatic analysis, the four miRNAs were speculated to play potential roles in the mechanisms of T2DMED via regulating 28 different genes and several pathways, including apoptosis, fibrosis, eNOS/cGMP/PKG, and vascular smooth muscle contraction processes, which mainly focused on influencing the functions of the endothelium and smooth muscle in the CC. IGF-1, as one of the target genes, was verified to decrease in the CCs of T2DMED animals via ELISA and was confirmed as the target of miR-18a or miR-206 via luciferase assay. Finally, these four miRNAs deserve further confirmation as biomarkers of T2DMED in larger studies. Additionally, miR-18a and/or miR-206 may provide new preventive/therapeutic targets for ED management by targeting IGF-1.

Role of intestinal flora imbalance in pathogenesis of pouchitis.

OBJECTIVE: To discuss the role of intestinal flora imbalance in the pathogenesis of pouchitis. METHODS: The pouchitis rat model was established and the faeces sample and the mucous membrane sample were collected regularly, in which the bacterial nucleic acids were extracted for quantitative analysis of the intestinal flora in the samples through using the real-time quantitative PCR technique and high energy sequencing technology. RESULTS: The disorder phenomenon of the intestinal flora appeared at the 7th day of the experiment, and the pouchitis was presented at the 21st day of the experiment. At the 31st day of the experiment, compared to control group and non-pouchitis group, the quantity of Bifidobacterium and the Lactobacillus of the pouchitis model rats in the mucous membrane sample and the faeces sample were significantly decreased (P < 0.05), and the Bacteroidetes, Faecalibacterium prausnitzii and XIV Clostridium leptum subgroup in the mucous membrane of pouchitis were significantly decreased (P < 0.05). The IV Clostridium coccoides group was the main flora in the mucous membrane of pouchitis, the bacterial diversity of non-pouchitis group and control group was significantly higher than that of the pouchitis group (P < 0.05). CONCLUSIONS: The intestinal flora imbalance is one of the factors that cause the incidence of the pouchitis; this study provides a clue of the pathogenesis and treatment direction of the intestinal inflammatory disease.

Heat shock protein 90 is essential for replication of porcine circovirus type 2 in PK-15 cells.

Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated disease (PCVAD). However, the mechanism of PCV2 replication has not been understood completely. Heat shock protein 90 (Hsp90) plays an important role in viral genome replication, viral genes expression, and viral particle packaging. In this study, we firstly found that inhibition of Hsp90 by pretreatment of host cells with 17-AAG, a specific inhibitor of Hsp90, or blocking Hsp90α/Hsp90ß with siRNA, resulted in significantly reduced viral replication in PK-15 cells. But inhibition of Hsp90 by 17-AAG did not affect PCV2 entry into the host cells. Meanwhile, over-expression of Hsp90α/Hsp90ß enhanced PCV2 genome replication and virion production. In addition, Hsp90ß was enriched in the nuclear zone in the cells infected with PCV2. But it did not interact with the viral Cap/Rep proteins. It suggested that Hsp90 is required for PCV2 production in PK-15 cells culture. It should be helpful for further evaluating the mechanism of replication and pathogenesis of PCV2 and developing novel antiviral therapies.