New antibabesial drugs are required to fight resistant parasites, and plant-derived natural products are a robust source. Six kinds of natural product extracts derived from herbal medicines that are traditionally used for the treatment of malaria were selected to test the antibabesial effect on Babesia gibsoni in vitro and in vivo. Parasitized blood was collected from dogs infected with B. gibsoni to evaluate the inhibitory effect of verbenalin, catechin hydrate, dihydrolycorine, embelin, ursolic acid, agrimol B, and bruceine H in vitro. The expression levels of the 18S rRNA gene in all drug-treated groups were determined by relative quantification using a real-time PCR method. Significant inhibition of the in vitro growth of B. gibsoni was observed after treatment by those natural product extracts (200 nM concentration) (P < 0.05). Catechin hydrate showed the highest activity in vitro due to the lowest expression levels of the 18S rRNA gene. The IC50 value of catechin hydrate against B. gibsoni was 273 nM. In B. gibsoni infected dogs, intravenous administrations of catechin hydrate and diminazene aceturate showed significant (P < 0.05) inhibition of B. gibsoni growth at a dose of 11 mg/kg and 10 mg/kg, respectively, compared to the control group. The results of our study may suggest that catechin hydrate may be a promising alternative to treat canine babesiosis caused by B. gibsoni.
Abstract Present in all bacteria, lipoproteins are central in bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane for subsequent trafficking across the periplasm. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-Å and 3.5-Å resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE specifically recognizes its substrate by establishing multiple interactions with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.
Acinetobacter baumannii is a prevalent nosocomial pathogen that causes serious threat in health care institutions 1 .A. baumannii has demonstrated resistance to a wide array of antibiotics, including the last-resort colistin or polymyxin B 2-4 .The MlaFEDB-mediated glycerophospholipid (PL) transport was reported to play an important role in maintaining the integrity of the lipid membrane 2,3 .The MlaFEDB complex is an ATP-binding cassette transporter to actively translocate the phospholipids between the inner membrane and the periplasmic protein MlaC 4,5 .Despite the progress that has been made in understanding the function of the MlaFEDB complex by genetic and biochemical strategies in A. baumannii, questions still remain open surrounding the directionality in glycerophospholipids transport via MlaFEDB in both retrograde and anterograde transport 6,7 .The lowresolution structure of the MlaFEDB complex from A. baumannii at 8.7 Å has provided initial structural insights into this complex 8 .However, the molecular details of the transport complex assembly, the interactions with glycerophospholipids, and the transport mechanisms of the MlaFEDB in A. baumannii remain enigmatic.To chase the high-resolution structure of MlaFEDB, we overexpressed the A. baumannii mlaFEDCB operon in Escherichia coli BL21(DE3) pLyS.The MlaFEDB proteins formed a stable complex and were reconstituted into lipid nanodisc (Fig. 1a and Supplementary Fig. S1a-c).The ATPase activity of A. baumannii MlaFEDB is approximately three times that of the E. coli MlaFEDB observed in our previous study 9 (Fig. 1b, left).The ATPase activity
Neuropathic pain is one of the common surgical diseases, which leads to abnormal chronic pain and pain hypersensitivity reaction. Microglia are important glial cells in the spinal cord, which are conducive to sensitization and maintenance of chronic pain. Recently, Senkyunolide H (SNH) has been reported to play an anti-inflammatory and antioxidant role. However, the mechanisms by which SNH improves neuropathic pain symptoms remain unknown. This study aimed to evaluate and identify the role of SNH in lipopolysaccharide (LPS)-mediated neuroinflammation and oxidative stress in BV2. Western blot and immunostaining assays revealed that SNH treatment attenuated LPS-mediated activation of BV2 in a dose-dependent manner. Flow cytometry further verified that BV2 microglial cells gradually shifted from an M1 (pro-inflammatory) phenotype to an M2 (anti-inflammatory) phenotype after SNH administration. Furthermore, quantitative real-time polymerase chain reaction and ELISA assays demonstrated that SNH treatment attenuated LPS-mediated neuroinflammation and oxidative stress in BV2 microglial cells. Lastly, western blot assays suggested that SHN could inactivate the ERK and NF-κB signaling pathways. Altogether, our findings have demonstrated that SNH could reverse LPS-mediated activation of microglia, LPS-mediated neuroinflammation and oxidative stress in BV2 via regulating the ERK and NF-κB pathways.
Background: Hirsuteine is an alkaloid compound that can inhibit the proliferative activity of several cancer cell types in vitro, yet no prior reports have explored its ability to inhibit non-small cell lung cancer (NSCLC) growth. As such, herein, we sought to explore the antiproliferative activity of hirsuteine when used to treat human NSCLC A549 and NC I-H1299 cells across a range of tested concentrations (0–25 μM) and to explore the mechanisms underlying its therapeutic efficacy.Methods: The effects of hirsuteine on cell viability was examined via CCK-8 and colony formation assays, while apoptosis was assessed through Hoechst 33,258 staining and flow cytometry. Cell cycle progression was additionally evaluated via propidium iodide staining, while Western blotting and real-time quantitative polymerase chain reaction (qPCR) method were conducted to assess the levels of proteins and genes associated with apoptosis and cell cycle progression, respectively.Results: Hirsuteine markedly suppressed the proliferation of A549 and NCI-H1299 cells in a dose and time-dependent fashion and induced clear changes in cell morphology, resulting in G0-G1 phase cell cycle arrest that was related to the downregulation of Cyclin E and CDK2. Hirsuteine additionally induced robust apoptosis of A549 and NCI-H1299 cells and Bcl-2 downregulation together with the upregulation of Bax, Apaf1, cytoplasmic cytochrome C, cleaved caspase-3 and cleaved caspase-9, together driving this apoptotic cell death.Conclusion: Hirsuteine can effectively suppress the growth of human NSCLC A549 and NCI-H1299 cells and induce apoptotic death in a dose-dependent fashion in vitro , emphasizing the promising of this alkaloid compound as a potent anticancer treatment that warrants study as a treatment for human NSCLC patients.
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