Nano-encapsulated Escherichia coli Divisome Anchor ZipA, and in Complex with FtsZ

The E. coli membrane protein ZipA, binds to the tubulin homologue FtsZ, in the early stage of cell division. We isolated ZipA in a Styrene Maleic Acid lipid particle (SMALP) preserving its position and integrity with native E. coli membrane lipids. Direct binding of ZipA to FtsZ is demonstrated, including FtsZ fibre bundles decorated with ZipA. Using Cryo-Electron Microscopy, small-angle X-ray and neutron scattering, we determine the encapsulated-ZipA structure in isolation, and in complex with FtsZ to a resolution of 1.6 nm. Three regions can be identified from the structure which correspond to, SMALP encapsulated membrane and ZipA transmembrane helix, a separate short compact tether, and ZipA globular head which binds FtsZ. The complex extends 12 nm from the membrane in a compact structure, supported by mesoscale modelling techniques, measuring the movement and stiffness of the regions within ZipA provides molecular scale analysis and visualisation of the early divisome

An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form “SMA Lipid Particles” (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. “SMILPs” thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method

Cytokine secretion and nitric oxide production by mononuclear cells of patients with multiple sclerosis

Several experimental findings suggest a potential role of excessive nitric oxide (NO) production by macrophages, microglia and astrocytes in the pathogenesis of demyelinating lesions in MS. We assessed the production of nitrites by peripheral blood mononuclear cells (PBMCs) of 15 MS patients (10 F and 5 M) with the R-R form (EDSS: 1-3.0) and in 15 age-matched control subjects. 9 out of the 15 MS patients showed active lesions in MRI at the time of examination. 7 patients were also monitored at the onset, during and following a clinical relapse. Secretion of cytokines by PBMCs was assessed at the basal time and after 24 h of incubation with lipopolysaccharide (LPS). The production of nitrites in the supernatants of PBMCs stimulated and not stimulated with lipopolysaccharide was evaluated. The secretion of IL1 beta, IFN-gamma, TNF-alpha, IL-6 IL-10 and TGF-beta by PBMCs was detected using ELISA methods. The production of NO, both basal and stimulated, was significantly higher in the patients with active lesions than in those without active lesions (p < 0.01). No significant difference was evident between the basal and LPS-stimulated production of NO between control subjects and MS patients without active lesions. During relapses there was a significant increase in NO production by PBMCs compared to the clinical stable stage of the disease (p < 0.0001). This increase was significantly greater in the early stage of relapse than in the late stage (p < 0.04). A decline of NO levels was observed during recovery. Steroid treatment induced a significant decrease in the PBMC NO production of MS patients during exacerbations (p < 0.01). The levels of IL-1 beta, IFN-gamma and TNF-alpha are significantly higher in the supernatants of the PBMCs which produced greater amounts of NO (p < 0.02, p < 0.03, p < 0.01, respectively). On the other hand, NO levels were negatively related to IL-10 and TGF-beta production (R = -75, p < 0.0001 and R = -0.79, p < 0.0001, respectively). The increase production of NO by peripheral blood mononuclear cells demonstrated in our study to be associated with increased production of proinflammatory cytokines could therefore be considered to be a marker of mononuclear cell activation in the peripheral blood of MS patients and, indirectly, of disease activity. Its increased secretion during T cell and monocyte homing in the CNF could contribute to the damage to the blood-brain barrier and the subsequent cytokine-mediated cytotoxic effect to myelin and oligodendrocytes in the white matter of MS patients

Pd-l1/pd-1 and ctla-4 expression in equine penile squamous cell carcinomas

In horses, penile squamous cell carcinomas (epSCCs) are among the most common cutaneous neoplastic lesions. These tumors usually arise in benign lesions such as viral plaques and papillomas frequently induced by Equus caballus papillomavirus type 2 (EcPV2) infection. In the last decade, the introduction of immune checkpoint inhibitors (ICI) for the treatment of human cancers has demonstrated promising results. Among the most commonly targeted pathways, there is PD-1/PD-L1 and CTLA-4. The aim of this study is to investigate the expression of the PD-1/PD-L1 pathway and CTLA-4 in the tumor microenvironment of epSCCs to assess the feasibility of an immunotherapeutic approach. Twenty equine epithelial tumors were retrospectively selected and submitted to RT-qPCR for PD-1 and PD-L1 genes. After testing antibodies cross-reactivity by western blotting, immunohistochemistry for PD-L1 and CTLA-4 was performed. Results from RT-qPCR demonstrated that 3/20 cases expressed the PD-L1 gene, whereas the PD-1 gene was not detected. Immunohistochemical positivity for PD-L1 was found only in one case. CTLA-4-positive cells were observe in all cases but were few (Mdn = 4.8; IQR = 2.3–7.1 cells/HPF). In this study group, PD-1/PD-L1 and CTLA-4 do not appear to be highly expressed and therefore the use of ICI in epSCCs may not have promising rates of response