Soluble expression and purification of Brucella cell surface protein (BCSP31) of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Adeno-associated virus serotype 2 mediated transduction and coexpression of the human apoAI and SR-BI gene in HepG2 cells

Cholesterol efflux is the first step in the reverse cholesterol transport (RCT) pathway, removing excess cholesterol from tissues, including the arterial wall, thus preventing the development of atherosclerosis. Adeno-associated virus (rAAV) has demonstrated significant promise as a DNA-delivery vector to treat serious human diseases. In this study, we constructed recombinant adeno-associated viruses coexpressing apoAI and SR-BI successfully, the double gene mRNA and protein were both strongly expressed in transduced HepG2 cells. A novel safe and efficient method of promoting the reverse cholesterol transport (RCT) may be established. These results may provide a new method for gene therapy of Arteriosclerosis.     

Diphenyl Ether Glycoside from the Stems of Ilex litseaefolia Hu et Tang

New diphenyl ether glycoside, ilexfoliaoside, was isolated from the stems of Ilex litseaefolia Hu et Tang, along with esculetin and 3,4-dimethoxybenzylacetic acid. The structure of ilexfoliaoside was determined as 6,2＇-dihydroxy-4, 4＇-di-vanilloyloxymethyl-1,1＇-diphenyl ether 2-O-β-D-glucopyranoside on the basis of chemical and spectroscopic evidence.     

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.     

Effects and molecular mechanisms of AT1-AA in retinopathy of preeclampsia

Preeclampsia not only seriously endangers maternal and fetal health during pregnancy but may incur many sequelae in postpartum women such as reduced visual acuity.Agonistic autoantibodies to the angiotensin Ⅱ type Ⅰ receptor (AT1-AA) is closely associated with preeclampsia.The aim of the present study is to determine whether AT1-AA is associated with retinal impairment during the course of preeclampsia.A preeclampsia model was established by injecting AT1-AA into pregnant rats via the tail vein.Changes in the retinal histological structure were observed.Cell apoptosis and cytokines including reactive oxygen species (ROS),as well as apoptosis-related proteins such as Bcl-2,Bax,and caspase-3 were detected.In addition,flash electroretinograms obtained at different postpartum days were analyzed.Compared with the control group,the retinal structure became edematous and the cell density was reduced significantly in preeclampsia group.The cell apoptosis rate was increased significantly.In addition,the content of ROS,the levels of Bax and caspase-3 in the retina were increased,while the content of Bcl-2 was reduced significantly.Continuous observation of the electroretinograms showed loss of retinal ganglion cells postpartum.The present study demonstrated that AT1-AA induced retinal cell apoptosis by promoting ROS release and activating caspase,suggesting that the increased postpartum susceptibility of preeclamptic women to retinopathy is related to AT1-AA-induced cell apoptosis.     

Short-term propamocarb exposure induces hepatic metabolism disorder associated with gut microbiota dysbiosis in adult male zebrafish

Propamocarb (PM) is a pesticide that is widely used to protect cucumbers and other plants from downy mildew.Recently,some studies indicated that PM exposure had potential toxic effects in animals.In this study,adult male zebrafish were exposed to 100 and 1000 μg/l PM for 7 days to assess its effects on metabolism and the gut microbiota.We observed a significant decrease in triglyceride (TG) in the livers of zebrafish that were exposed to 1000 μg/l PM for 7 days.At the same time,some genes related to glycolysis and lipid metabolism in the livers of zebrafish,including hexokinase-1 (HK1),pyruvate kinase (PK),acyi-CoA oxidase (Aco),peroxisome proliferator activated receptor alpha (Ppar-α),apolipoprotein A-Ⅳ-like (Apo),Acetyl CoA carboxylase-1 (Acc1),diacylglycerol acyltransferase (Dgat),and fatty acid synthase (Fas),were also decreased significantly after PM exposure.Based on GC-MS metabolomics analysis,a total of 48 metabolites changed significantly in the 1000 μg/l PM treatment group in comparison with the control group.These altered metabolites were mainly associated with the glycolysis,amino acid metabolism,and lipid metabolism pathways.Interestingly,we further found that the 1000 μg/l PM treatment group also showed significant elevations in Proteobacteria,Bacteroidetes,and Firmicutes at the phylum level.Sequencing of the 16S rRNA gene in the V3-V4 region also showed a significant change in the abundance and diversity of the gut microbiota in the 1000 μg/l PM treatment group.Our results indicated that exposure to PM for a short time could induce hepatic metabolic disorders and gut microbiota dysbiosis in adult male zebrafish.     

A novel detection method of Cryptosporidium parvum infection in cattle based on Cryptosporidium parvum virus 1

Cryptosporidium parvum is an important zoonotic parasite that causes significant economic loss in the animal husbandry industry,especially the cattle industry.As there is no specific vaccine or drug against Cryptosporidium,a rapid and accurate method for the detection of C.parvum is of great significance.In this study,colloidal gold strips were developed based on Cryptosporidium parvum virus 1 (CSpV1) for the detection of C.parvum infection in cattle fecal samples.The colloidal gold solution was prepared by reducing trisodium citrate and the CSpV1 #5 monoclonal antibody was labeled with colloidal gold.A polyclonal antibody against the CSpV1 capsid protein and an anti-mouse IgG antibody were coated on the colloidal gold strips for use in the test and control lines,respectively.Our results showed that the detection sensitivity in fecal samples was up to a 1:64 dilution.There was no cross-reaction with Cryptosporidium andersoni or Giardia in the fecal samples.The different preservation conditions (room temperature,4℃,and 37℃) and preservation time (7,30,60,and 90 days) were analyzed.The data showed that the strips could be preserved for 90 days at 4℃ and for 60 days at room temperature or 37℃.The colloidal gold strips were used to detect the samples of 120 clinical fecal in Changchun,China.The results indicated that the rate of a positive test was 5%(6/120).This study provides a rapid and accurate method for detecting C.parvum infection in cattle and humans.     

Mitochondria-targeted antioxidant peptide SS-31 mediates neuroprotection in a rat experimental glaucoma model

To investigate the neuroprotective effects of the mitochondria-targeted antioxidant Szeto-Schiller peptide 31 (SS-31) in a rat experimental glaucoma model, SS-31 was intraperitoneally (IP) injected into Sprague-Dawley rats, followed by intracameral injection of polystyrene microspheres to induce elevated intraocular pressure (IOP). After 6 weeks, electroretinography (ERG) and flash visual-evoked potentials (F-VEPs) were recorded to assess retinal function. Hematoxylin-eosin staining was performed on retinal cross-sections to measure ganglion cell complex (GCC) thickness. Apoptotic retinal cells were assessed by TUNEL staining. Brn3a-positive retinal ganglion cells (RGCs) were counted in retinal flat mounts via immunofluorescence. The retinal total SOD, SOD2, and MDA expression levels were assessed in retinal tissue homogenates. The cyt c, Baxz and Bcl-2 protein levels in rat retinas were detected by western blot analysis. Bax and Bcl-2 expressions were also evaluated using immunohistochemistry in paraffinized sections. Our results showed that the rats that received microsphere injection developed elevated IOP. SS-31 ameliorated the reductions in the a- and b-wave amplitudes on ERG and the F-VEP amplitude in glaucomatous eyes. GCC thickness was preserved, TUNEL-positive cells were decreased in the retina, and Brn3a-positive RGCs were in creased in the SS-31-treated glaucoma group compared with those in the non-treated glaucoma group. SS-31 significantly reduced MDA levels and increased SOD2 levels after glaucoma induction. Significant suppression of cyt c release, upregulation of Bcl-2, and downregulation of Bax were observed following SS-31 administration. In summary, SS-31 exerts neuroprotective effects in this experimental glaucoma model by inhibiting mitochondrial dysfunction and therefore represents a promising therapeutic agent for glaucoma.