Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies.

BACKGROUND: Brucellosis is a severe zoonotic disease worldwide. Detection and identification of Brucella species are essential to prevent or treat brucellosis in humans and animals. The outer membrane protein-31 (Omp31) is a major protein of Brucellae except for B. abortus, while the Omp31 antigenic epitopes have not been extensively characterized yet. RESULTS: A total of 22 monoclonal antibodies (mAbs) were produced against Omp31 of Brucella (B.) melitensis, of which 13 recognized five linear epitopes, 7 reacted with semi-conformational epitopes and 2 reacted with conformational epitopes, respectively. The mAb isotypes were 11 (50%) IgG2a, 5 (23%) IgG1 and 6 (27%) IgM. On the basis of epitope recognition and reactivity levels, 8 mAbs including 3 IgM and 5 IgG clones were considered as highly reactive and potentially diagnostic antibodies. Among these mAbs, 7A3 (IgG1), 5B1 (IgG2a), 2C1 (IgG2a) and 5B3 (IgG2a) reacted with differently conserved linear epitopes of B. melitensis, B. ovis, B. suis and B. canis strains, while 5H3 (IgG2a) highly reacted with a conformational epitope of Omp31 when tested with several immunoassays. CONCLUSIONS: These potent monoclonal antibodies can be used for identifying Omp31 antigens or detecting B. melitensis and other Brucella species beyond B. abortus in vitro or in vivo.

Identification of abemaciclib derivatives targeting cyclin-dependent kinase 4 and 6 using molecular dynamics, binding free energy calculation, synthesis, and pharmacological evaluation.

CDK4/6 plays a crucial role in various cancers and is an effective anticancer drug target. However, the gap between clinical requirements and approved CDK4/6 drugs is unresolved. Thus, there is an urgent need to develop selective and oral CDK4/6 inhibitors, particularly for monotherapy. Here, we studied the interaction between abemaciclib and human CDK6 using molecular dynamics simulations, binding free energy calculations, and energy decomposition. V101 and H100 formed stable hydrogen bonds with the amine-pyrimidine group, and K43 interacted with the imidazole ring via an unstable hydrogen bond. Meanwhile, I19, V27, A41, and L152 interacted with abemaciclib through π-alkyl interactions. Based on the binding model, abemaciclib was divided into four regions. With one region modification, 43 compounds were designed and evaluated using molecular docking. From each region, three favorable groups were selected and combined with each other to obtain 81 compounds. Among them, C2231-A, which was obtained by removing the methylene group from C2231, showed better inhibition than C2231. Kinase profiling revealed that C2231-A showed inhibitory activity similar to that of abemaciclib; additionally, C2231-A inhibited the growth of MDA-MB-231 cells to a greater extent than did abemaciclib. Based on molecular dynamics simulation, C2231-A was identified as a promising candidate compound with considerable inhibitory effects on human breast cancer cell lines.

Detection of Brucellae in peripheral blood mononuclear cells for monitoring therapeutic efficacy of brucellosis infection.

Background: Brucellosis is one of the most severe widespread zoonoses caused by the Gram-negative bacterium Brucella species. The diagnosis and clinical assessment of human brucellosis are very important for the management of patients, while there is a lack of effective methods to detect Brucellae. Classical culture of Brucella species is time consuming and often fails. A simple and sensitive assay is needed for diagnosis of Brucella infection and monitoring of treatment in man. Methods: Blood samples and peripheral blood mononuclear cells (PBMCs) were collected from 154 patients hospitalized for brucellosis. Brucella antibodies were detected by Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Test (SAT) and enzyme-linked immunosorbent assay (ELISA). Intracellular Brucellae were detected by blood culture and immunofluorescence staining (IFS). Results: Among 154 brucellosis patients, 59.7% (92/154) were antibody reactive by RBPT, 81.8% (126/154) by SAT and 95.5% (147/154) by ELISA, respectively. Only 3.2% (5/154) of patient blood samples resulted in positive Brucella culture, while 68.8% (106/154) carried IFS detectable Brucella antigens in PBMCs. Gender (P = 0.01) but not age (P > 0.05) was a significant risk factor. The frequency of intracellular Brucella antigens was similar between patients receiving different treatment regimens (P > 0.05). However, a significant decrease of intracellular Brucellae was observed only in patients with acute brucellosis after the third course of treatment (P < 0.05), suggesting that current regimens to treat chronic brucellosis were not effective. Conclusions: IFS appears a sensitive assay for detection of Brucella antigens in PBMCs and could be used for diagnosis and therapeutic monitoring of brucellosis in clinical practice.

[Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

OBJECTIVE: To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. METHODS: Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. RESULTS: The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. CONCLUSION: The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

A simple and cost-saving phenotypic drug susceptibility testing of HIV-1.

It is essential to monitor the occurrence of drug-resistant strains and to provide guidance for clinically adapted antiviral treatment of HIV/AIDS. In this study, an individual patient's HIV-1 pol gene encoding the full length of protease and part of the reverse transcriptase was packaged into a modified lentivirus carrying dual-reporters ZsGreen and luciferase. The optimal coefficient of correlation between drug concentration and luciferase activity was optimized. A clear-cut dose-dependent relationship between lentivirus production and luciferase activity was found in the phenotypic testing system. Fold changes (FC) to a wild-type control HIV-1 strain ratios were determined reflecting the phenotypic susceptibility of treatment-exposed patient's HIV-1 strains to 12 HIV-1 inhibitors including 6 nucleoside reverse-transcriptase inhibitors (NRTIs), 4 non-nucleoside reverse transcriptase inhibitors (NNRTIs) and 2 protease inhibitors (PIs). Phenotypic susceptibility calls from 8 HIV-1 infected patients were consistent with 80-90% genotypic evaluations, while phenotypic assessments rectified 10-20% genotypic resistance calls. By a half of replacement with ZsGreen reporter, the consumption of high cost Bright-Glo Luciferase Assay is reduced, making this assay cheaper when a large number of HIV-1 infected individuals are tested. The study provides a useful tool for interpreting meaningful genotypic mutations and guiding tailored antiviral treatment of HIV/AIDS in clinical practice.