Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP

A systematic review of randomised controlled trials on the effectiveness of exercise programs on lumbo pelvic pain among postnatal women

Background: A substantial number of women tend to be affected by Lumbo Pelvic Pain (LPP) following child birth. Physical exercise is indicated as a beneficial method to relieve LPP, but individual studies appear to suggest mixed findings about its effectiveness. This systematic review aimed to synthesise evidence from randomised controlled trials on the effectiveness of exercise on LPP among postnatal women to inform policy, practice and future research. Methods: A systematic review was conducted of all randomised controlled trials published between January 1990 and July 2014, identified through a comprehensive search of following databases: PubMed, PEDro, Embase, Cinahl, Medline, SPORTDiscus, Cochrane Pregnancy and Childbirth Group’s Trials Register, and electronic libraries of authors’institutions. Randomised controlled trials were eligible for inclusion if the intervention comprised of postnatal exercise for women with LPP onset during pregnancy or within 3 months after delivery and the outcome measures included changes in LPP. Selected articles were assessed using the PEDro Scale for methodological quality and findings were synthesised narratively as meta-analysis was found to be inappropriate due to heterogeneity among included studies. Results: Four randomised controlled trials were included, involving 251 postnatal women. Three trials were rated as of ‘good’ methodological quality. All trials, except one, were at low risk of bias. The trials included physical exercise programs with varying components, differing modes of delivery, follow up times and outcome measures. Intervention in one trial, involving physical therapy with specific stabilising exercises, proved to be effective in reducing LPP intensity. An improvement in gluteal pain on the right side was reported in another trial and a significant difference in pain frequency in another. Conclusion: Our review indicates that only few randomised controlled trials have evaluated the effectiveness of exercise on LPP among postnatal women. There is also a great amount of variability across existing trials in the components of exercise programs, modes of delivery, follow up times and outcome measures. While there is some evidence to indicate the effectiveness of exercise for relieving LPP, further good quality trials are needed to ascertain the most effective elements of postnatal exercise programs suited for LPP treatment

Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent Apoptosis, but are insensitive to direct activation with exogenous fas ligand

Introduction Cholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma. Aims To determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines. Results Primary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both. Conclusions 1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes

Optimization of DNA extraction for ISSR studies in Tectona grandis L.f. - an important forest tree species

Four extraction methods and sample types were evaluated for yield, quality and suitability of genomic DNA for ISSR marker amplification in teak (Tectona grandis). Both CTAB and SDS based extraction procedures extracted better quantity and purity index of genomic DNA. Leaves of trees, bud grafts andseedlings yielded better DNA than seeds. Using identical PCR conditions, DNA extraction methods and sample types influenced amplifications of ISSR markers, with no amplifications among seed samples. DNA extraction method and sample type are very important consideration for reproducible ISSR-based molecular marker analysis in tea

Review: optical fiber sensors for civil engineering applications

Optical fiber sensor (OFS) technologies have developed rapidly over the last few decades, and various types of OFS have found practical applications in the field of civil engineering. In this paper, which is resulting from the work of the RILEM technical committee “Optical fiber sensors for civil engineering applications”, different kinds of sensing techniques, including change of light intensity, interferometry, fiber Bragg grating, adsorption measurement and distributed sensing, are briefly reviewed to introduce the basic sensing principles. Then, the applications of OFS in highway structures, building structures, geotechnical structures, pipelines as well as cables monitoring are described, with focus on sensor design, installation technique and sensor performance. It is believed that the State-of-the-Art review is helpful to engineers considering the use of OFS in their projects, and can facilitate the wider application of OFS technologies in construction industry

Antimicrobial Stewardship from Policy to Practice: Experiences from UK Antimicrobial Pharmacists

Antimicrobial stewardship in the UK has evolved dramatically in the last 15 years. Factors driving this include initial central funding for specialist pharmacists and mandatory reductions in healthcare-associated infections (particularly Clostridium difficile infection). More recently, the introduction of national stewardship guidelines, and an increased focus on stewardship as part of the UK five-year antimicrobial resistance strategy, have accelerated and embedded developments. Antimicrobial pharmacists have been instrumental in effecting changes at an organizational and national level. This article describes the evolution of the antimicrobial pharmacist role, its impact, the progress toward the actions listed in the five-year resistance strategy, and novel emerging areas in stewardship in the UK

Combined search for the quarks of a sequential fourth generation

Results are presented from a search for a fourth generation of quarks produced singly or in pairs in a data set corresponding to an integrated luminosity of 5 inverse femtobarns recorded by the CMS experiment at the LHC in 2011. A novel strategy has been developed for a combined search for quarks of the up and down type in decay channels with at least one isolated muon or electron. Limits on the mass of the fourth-generation quarks and the relevant Cabibbo-Kobayashi-Maskawa matrix elements are derived in the context of a simple extension of the standard model with a sequential fourth generation of fermions. The existence of mass-degenerate fourth-generation quarks with masses below 685 GeV is excluded at 95% confidence level for minimal off-diagonal mixing between the third- and the fourth-generation quarks. With a mass difference of 25 GeV between the quark masses, the obtained limit on the masses of the fourth-generation quarks shifts by about +/- 20 GeV. These results significantly reduce the allowed parameter space for a fourth generation of fermions.Comment: Replaced with published version. Added journal reference and DO

Outcome and factors associated with hospital mortality in patients with impaired left ventricular function undergoing coronary artery bypass grafting: where do we stand?

Objective: Impaired ventricular function is a known risk factor for mortality after coronary artery bypass grafting however increasingly more patients with impaired ventricular function are referred for surgery. Currently no large data is available from Pakistan regarding this aspect of coronary surgery. Our objectives were to find out the hospital mortality and mid term functional improvement in patients with impaired ventricular function undergoing coronary artery by pass grafting and identify the risk factors for mortality. Methodology: Retrospective analysis of preoperative, operative and postoperative variables of patients with impaired ventricular function who were operated for isolated first time coronary artery bypass between October 2006 to April 2009. Results: Total 190 patients with impaired ventricular function underwent isolated first time coronary artery bypass grafting during this period with a male predominance (82.6%). Mean ejection fraction of the group was 25.4±5.3%. Mean predicted mortality on logistic Euro score was 10.9±2.7%. Actual in hospital mortality of the group was 4.7% which is comparable to contemporary published results. Multivariate analysis identified use of intra aortic balloon pump, non use of internal mammary artery and preoperative NYHA functional class as factors associated with mortality. Conclusion: Coronary artery bypass grafting can be performed in patients with impaired ventricular function with acceptable hospital mortality and mid term functional improvement

Regulating Neutrophil PAD4/NOX-Dependent Cerebrovasular Thromboinflammation

Supplementary material are available online at https://www.ijbs.com/v19p0852.htm#SM0 .Copyright © 2023 The Authors. Background: Neutrophil extracellular trap (NET) production has been implicated in the pathogenesis of thromboinflammatory conditions such as Sickle Cell Disease (SCD), contributing to heightened risk for ischemic stroke. NETs are catalyzed by the enzyme Peptidyl Arginine Deiminase 4 (PAD4) and neutrophil derived reactive oxygen species (ROS), especially NADPH oxidase (NOX) which interacts with PAD4 and is therefore critical for neutrophil function. However, the role that NOX-dependent ROS and NETs play in the accelerated cerebral microvascular thrombosis associated with thromboinflammatory conditions, such as SCD, has not been fully elucidated and is the aim of this study. Methods: The in-vitro effects of targeting PAD4 and NOX were examined using physiologically relevant NET assays with neutrophils isolated from healthy volunteers (control) and SCD patients. In addition, in-vivo intravascular effects of targeting PAD4 and NOX in the cerebral microcirculation of C57BL/6 and sickle transgenic mice (STM) were assessed using a photoactivation thrombosis model (light/dye) coupled with real-time fluorescence intravital microscopy. Results: We found that targeting PAD4 and NOX in human neutrophils significantly inhibited ionomycin dependent H3cit+ neutrophils. Targeting PAD4 and NOX in-vivo resulted in prolonged blood flow cessation in cerebrovascular arterioles as well as venules. Moreover, we were able to replicate the effects of PAD4 and NOX targeting in a clinical model of accelerated thromboinflammation by increasing blood flow cessation times in cerebral microvessels in STM. These findings concurred with the clinical setting i.e. neutrophils isolated from SCD patients, which possessed an attenuation of H3cit+ neutrophil production on targeting PAD4 and NOX. Conclusions: Taken together, our compelling data suggests that PAD4 and NOX play a significant role in neutrophil driven thromboinflammation. Targeting PAD4 and NOX limits pathological H3cit+ neutrophils, which may further explain attenuation of cerebral thrombosis. Overall, this study presents a viable pre-clinical model of prevention and management of thromboinflammatory complications such as ischemic stroke.NIH/NHLBI (HL134959-01A1. JA, SV and FNEG); Royal Society Wolfson Foundation (RSWF\R3\183001. FNEG and SY)

Enzyme immobilisation on amino-functionalised multi-walled carbon nanotubes : structural and biocatalytic characterisation

BACKGROUND: The aim of this work is to investigate the structure and function of enzymes immobilised on nanomaterials. This work will allow better understanding of enzyme-nanomaterial interactions, as well as designing functional protein-nanomaterial conjugates. METHODOLOGY/PRINCIPAL FINDINGS: Multiwalled carbon nanotubes (MWNTs) were functionalised with amino groups to improve solubility and biocompatibility. The pristine and functionalised forms of MWNTs were characterised with Fourier-transform infrared spectroscopy. Thermogravimetric analysis was done to examine the degree of the functionalisation process. An immobilised biocatalyst was prepared on functionalised nanomaterial by covalent binding. Thermomyces lanuginosus lipase was used as a model enzyme. The structural change of the immobilised and free lipases were characterised with transmission electron Microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy and Circular dichroism spectroscopy. Biochemical characterisation of immobilised enzyme showed broader pH and thermal optima compared to soluble form. Reusability of the immobilised enzyme for hydrolysis of long chain esters was demonstrated up to ten cycles. CONCLUSION/SIGNIFICANCE: Lipase immobilised on MWNTs has exhibited significantly improved thermal stability. The exploration of advanced nanomaterial for enzyme immobilisation support using sophisticated techniques makes nanobiocatalyst of potential interest for biosensor applications