Primary recovery of lipase derived from Burkholderia sp. ST8 with aqueous micellar two-phase system

The partitioning and recovery of lipase derived from Burkholderia sp. ST8 strain was explored using temperature-induced aqueous micellar two-phase system (AMTPS) composed of single nonionic surfactant. Nonionic surfactant Triton X-114 and Pluronic series (triblock copolymer) were evaluated in terms of their clouding phenomenon (cloud-point temperature) and the performance of the lipase partitioning in these AMTPSs. Pluronic L81 showed the most optimum partition efficiency for the recovery of lipase to the micellar phase of the AMTPS. Based on the AMTPS which consisted of 24 (w/w) Pluronic L81 and 0.5 (w/w) potassium chloride (KCl), the selectivity of lipase partitioned to bottom phase has been enhanced to 0.035 and the lipase was purified 7.2 fold. Furthermore, the lipase from the micellar phase was consecutively extracted to a new aqueous solution, with an aim of removing the surfactant from the purified lipase. It was attained by replacing the aqueous top phase from the primary recovery of AMTPS with a new potassium thiocyanate (KSCN) solution. The lipase was then recovered in the newly formed bottom aqueous phase which culminated in the yield of 89 and partition coefficients of 0.34 and 4.50 for lipase and surfactant, respectively. AMTPS offers a convenient and efficient method for the primary recovery of lipase with low cost, large loading capacity and the potential of linear scale up. © 2011 Elsevier Ltd. All rights reserved

Discovery of ruthenium(II) metallocompound and olaparib synergy for cancer combination therapy

Synergistic drug combinations can extend the use of poly(ADP-ribose) polymerase inhibitors (PARPi) such as Olaparib to BRCA-proficient tumors and overcome acquired or de novo drug resistance. To identify new synergistic combinations for PARPi, we screened a "micro-library" comprising a mix of commercially available drugs and DNA-binding ruthenium(II) polypyridyl complexes (RPCs) for Olaparib synergy in BRCA-proficient triple-negative breast cancer cells. This identified three hits: the natural product Curcumin and two ruthenium(II)-rhenium(I) polypyridyl metallomacrocycles. All combinations identified were effective in BRCA-proficient breast cancer cells, including an Olaparib-resistant cell line, and spheroid models. Mechanistic studies indicated that synergy was achieved via DNA-damage enhancement and resultant apoptosis. Combinations showed low cytotoxicity toward non-malignant breast epithelial cells and low acute and developmental toxicity in zebrafish embryos. This work identifies RPC metallomacrocycles as a novel class of agents for cancer combination therapy and provides a proof of concept for the inclusion of metallocompounds within drug synergy screens

An experimental study of wall-plate U-bracket connection

Abstract not available

Characterization of the transient deficiency of PKC isozyme levels in immature cord blood T cells and its connection to anti-allergic cytokine profiles of the matured cells

Cord blood T cells (CBTC) from a proportion of newborns express low/deficient levels of some protein kinase C (PKC) isozymes, with low levels of PKCζ correlating with increased risk of developing allergy and associated decrease in interferon-gamma (IFN-γ) producing T cells. Interestingly, these lower levels of PKCζ were increased/normalized by supplementing women during pregnancy with n-3 polyunsaturated fatty acids. However, at present, we have little understanding of the transient nature of the deficiency in the neonate and how PKCζ relates to other PKC isozymes and whether their levels influence maturation into IFN-γ producing T cells. There is also no information on PKCζ isozyme levels in the T cell subpopulations, CD4⁺ and CD8⁺ cells. These issues were addressed in the present study using a classical culture model of neonatal T cell maturation, initiated with phytohaemagglutinin (PHA) and recombinant human interleukin-2 (rhIL-2). Of the isozymes evaluated, PKCζ, β2, δ, μ, ε, θ and λ/ι were low in CBTCs. The PKC isozyme deficiencies were also found in the CD4⁺ and CD8⁺ T cell subset levels of the PKC isozymes correlated between the two subpopulations. Examination of changes in the PKC isozymes in these deficient cells following addition of maturation signals showed a significant increase in expression within the first few hours for PKCζ, β2 and μ, and 1–2 days for PKCδ, ε, θ and λ/ι. Only CBTC PKCζ isozyme levels correlated with cytokine production, with a positive correlation with IFN-γ, interleukin (IL)-2 and tumour necrosis factor-alpha (TNF), and a negative association with IL-9 and IL-10. The findings reinforce the specificity in using CBTC PKCζ levels as a biomarker for risk of allergy development and identify a period in which this can be potentially ‘corrected’ after birth.Khalida Perveen , Alex Quach, Michael J. Stark , Susan L. Prescott, Simon C. Barry, Charles S. Hii and Antonio Ferrant

Validation of monoclonal anti-PKC isozyme antibodies for flow cytometry analyses in human T cell subsets and expression in cord blood T cells

T cells from neonates (cord blood) with a tendency to develop allergic diseases express low PKCζ levels. More extensive investigations into PKC isozyme levels in T cell subsets and changes during neonatal T cell maturation are hampered by limitations of Western blot analyses. We have undertaken to validating the specificity of commercially available antibodies marketed for flow cytometry to measure PKCα, βI, βII, δ, ε, η, θ, ζ, ι/λ and μ. Western blot analyses of human peripheral blood mononuclear cell (PBMC) lysates demonstrated that some antibodies were unsuitable for flow cytometry assays. A panel of antibodies with the desirable specificity and reliability in the flow cytometry assay were identified using both PBMC and whole blood assays. The results showed that all PKC isozymes were expressed in CD4⁺ and CD8⁺ T cells, monocytes and neutrophils. Murine lymphocytes showed similar patterns of expression. A major finding was that 35.2% and 38.5% of cord blood samples have low PKCζ (≤the 5th percentile of adult levels) in the CD4⁺ and CD8⁺ subsets, respectively, consistent with the incidence of allergy development in the population. Furthermore, these low PKCζ levels 'normalised' within 24 h after initiation of maturation of these cells in culture, providing a 'window of opportunity' for altering PKCζ levels.Khalida Perveen, Alex Quach, Andrew McPhee, Susan L. Prescott, Simon C. Barry, Charles S. Hii, Antonio Ferrant

Effect of Alirocumab on Lipoprotein(a) and Cardiovascular Risk After Acute Coronary Syndrome

BACKGROUND Lipoprotein(a) concentration is associated with cardiovascular events. Alirocumab, a proprotein convertase subtilisin/kexin type 9 inhibitor, lowers lipoprotein(a) and low-density lipoprotein cholesterol (LDL-C).OBJECTIVES A pre-specified analysis of the placebo-controlled ODYSSEY Outcomes trial in patients with recent acute coronary syndrome (ACS) determined whether alirocumab-induced changes in lipoprotein(a) and LDL-C independently predicted major adverse cardiovascular events (MACE).METHODS One to 12 months after ACS, 18,924 patients on high-intensity statin therapy were randomized to alirocumab or placebo and followed for 2.8 years (median). Lipoprotein(a) was measured at randomization and 4 and 12 months thereafter. The primary MACE outcome was coronary heart disease death, nonfatal myocardial infarction, ischemic stroke, or hospitalization for unstable angina.RESULTS Baseline lipoprotein(a) levels (median: 21.2 mg/dl; interquartile range [IQR]: 6.7 to 59.6 mg/dl) and LDL-C [corrected for cholesterol content in lipoprotein(a)] predicted MACE. Alirocumab reduced lipoprotein(a) by 5.0 mg/dl (IQR: 0 to 13.5 mg/dl), corrected LDL-C by 51.1 mg/dl (IQR: 33.7 to 67.2 mg/dl), and reduced the risk of MACE (hazard ratio [HR]: 0.85; 95% confidence interval [CI]: 0.78 to 0.93). Alirocumab-induced reductions of lipoprotein(a) and corrected LDL-C independently predicted lower risk of MACE, after adjustment for baseline concentrations of both lipoproteins and demographic and clinical characteristics. A 1-mg/dl reduction in lipoprotein(a) with alirocumab was associated with a HR of 0.994 (95% CI: 0.990 to 0.999; p = 0.0081).CONCLUSIONS Baseline lipoprotein(a) and corrected LDL-C levels and their reductions by alirocumab predicted the risk of MACE after recent ACS. Lipoprotein(a) lowering by alirocumab is an independent contributor to MACE reduction, which suggests that lipoprotein(a) should be an independent treatment target after ACS. (ODYSSEY Outcomes: Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab; NCT01663402) (C) 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.Cardiolog