Physical, chemical and kinetic factors affecting prion infectivity

The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process

Calibration of the LIGO displacement actuators via laser frequency modulation

We present a frequency modulation technique for calibration of the displacement actuators of the LIGO 4-km-long interferometric gravitational-wave detectors. With the interferometer locked in a single-arm configuration, we modulate the frequency of the laser light, creating an effective length variation that we calibrate by measuring the amplitude of the frequency modulation. By simultaneously driving the voice coil actuators that control the length of the arm cavity, we calibrate the voice coil actuation coefficient with an estimated 1-sigma uncertainty of less than one percent. This technique enables a force-free, single-step actuator calibration using a displacement fiducial that is fundamentally different from those employed in other calibration methods.Comment: 10 pages, 5 figures, submitted to Classical and Quantum Gravit

A systematic investigation of production of synthetic prions from recombinant prion protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved

Exploration of Effective Spatial Performance of Accessible Neighbourhood Green (Ang), Including its Proportion and Standard Distance from User in Dhanmondi Residential Area, Dhaka

Urban physical context can be analyzed across three key matrixes (3-P) among others i.e. place, path, and people. So, pragmatic analysis of spatial effective performances connecting 3-P, require provoking frequency of people’s experience. Acknowledging this issue, the demand for accessible neighbourhood green (ANG) at an appropriate distance becomes the primary concern to enhance the quality of life and liveability in a city. However, with the continued urban growth and densification, the discrepancy between the demand and supply of open space continues to vary requiring adjustments to remain responsive. The real-life circumstance results in a shortage of parks and open areas in terms of demand and supply within accessible distance in Dhaka city. This paper intends to examine this issue through the case of the planned Dhanmondi Residential Area (DRA) in Dhaka. Despite having provision of multiple open spaces in DRA, visitor’s frequency varies due to age and gender group accessibility conditions and varying distances. Considering existing spatial norms set by Detail Area Plan (DAP), Dhaka structure plan (DSP), and numerous research works on Dhaka open spaces, the major inquiry posed here is whether these open spaces are appropriate for DRA or not. Therefore, the objective of this paper focuses on examining the quality of the physical environment of Neighbourhood public open spaces termed here as accessible neighbourhood green (ANG) in DRA to examine their adequacy concerning proportion and distance synchronized with the frequency of visits. The initial part of the paper focuses on conceptualizing the problem vis-à-vis the existing scenario.  Surveys and interviews have been conducted to assess people’s perceptions in terms of comfort, accessibility, sociability, and user frequency aligned with proximity. The result indicates that the provision of one appropriate ANG within two or three standard blocks apart contribute to enhancing the quality of life for the city dwellers and their liveability

A dynamic perfusion based blood-brain barrier model for cytotoxicity testing and drug permeation

The blood-brain barrier (BBB) serves to protect and regulate the CNS microenvironment. The development of an in-vitro mimic of the BBB requires recapitulating the correct phenotype of the in-vivo BBB, particularly for drug permeation studies. However the majority of widely used BBB models demonstrate low transendothelial electrical resistance (TEER) and poor BBB phenotype. The application of shear stress is known to enhance tight junction formation and hence improve the barrier function. We utilised a high TEER primary porcine brain microvascular endothelial cell (PBMEC) culture to assess the impact of shear stress on barrier formation using the Kirkstall QuasiVivo 600 (QV600) multi-chamber perfusion system. The application of shear stress resulted in a reorientation and enhancement of tight junction formation on both coverslip and permeable inserts, in addition to enhancing and maintaining TEER for longer, when compared to static conditions. Furthermore, the functional consequences of this was demonstrated with the reduction in flux of mitoxantrone across PBMEC monolayers. The QV600 perfusion system may service as a viable tool to enhance and maintain the high TEER PBMEC system for use in in-vitro BBB models

Precision dosing of venlafaxine during pregnancy: a pharmacokinetics modelling approach

Objectives: Venlafaxine exposure through gestation is affected by the longitudinal changes in maternal physiology. Confounding treatment is also the impact of CYP2D6 polymorphisms affecting plasma concentrations of venlafaxine. Methods: A pharmacokinetic modelling approach was employed to assess variations in maternal and foetal cord venlafaxine levels throughout gestation and to identify appropriate doses to maintain venlafaxine levels within the therapeutic range. Key findings: Throughout gestation, there was a significant decrease in simulated venlafaxine trough plasma concentrations in both extensive metaboliser (EM) and ultra-rapid metaboliser (UM) phenotypes. Approximately 70%–87% of EM and UM phenotypes exhibited trough venlafaxine plasma concentrations below the therapeutic level (<25 ng/ml), which increased to 96% at week 30. While for poor metabolizer (PM) phenotypes, the percentage was approximately 4%. Conclusion: The standard daily dose of 75 mg required adjustment for all phenotypes examined during gestation. A daily dose of 37.5–112.5 mg is appropriate for PM throughout pregnancy. For EM, a dose of 225 mg daily in the first trimester, 262.5 mg daily in the second trimester, and 375 mg daily in the third trimester is suggested to be optimal. For UM, a dose of 375 mg daily throughout gestation is suggested to be optimal

Optimization of xylanase production by filamentous fungi in solid state fermentation and scale-up to horizontal tube bioreactor

Five microorganisms, namely Aspergillus niger CECT 2700, A. niger CECT 2915, A. niger CECT 2088, Aspergillus terreus CECT 2808, and Rhizopus stolonifer CECT 2344, were grown on corncob to produce cell wall polysaccharide-degrading enzymes, mainly xylanases, by solid-state fermentation (SSF). A. niger CECT 2700 produced the highest amount of xylanases of 504±7 U/g dry corncob (dcc) after 3 days of fermentation. The optimization of the culture broth (5.0 g/L NaNO3, 1.3 g/L (NH4)2SO4, 4.5 g/L KH2PO4, and 3 g/L yeast extract) and operational conditions (5 g of bed loading, using an initial substrate to moistening medium of 1:3.6 (w/v)) allowed increasing the predicted maximal xylanase activity up to 2,452.7 U/g dcc. However, different pretreatments of materials, including destarching, autoclaving, microwave, and alkaline treatments, were detrimental. Finally, the process was successfully established in a laboratory-scale horizontal tube biore- actor, achieving the highest xylanase activity (2,926 U/g dcc) at a flow rate of 0.2 L/min. The result showed an overall 5.8-fold increase in xylanase activity after optimization of culture media, operational conditions, and scale-up.We are grateful to the Spanish Ministry of Science and Innovation for the financial support of this work (project CTQ2011-28967), which has partial financial support from the FEDER funds of the European Union; to the Leonardo da Vinci Programme for founding the stay of Felisbela Oliveira in Vigo University; to MAEC-AECID (Spanish Government) for the financial support for Perez-Bibbins, B. and to Spanish Ministry of Education, Culture and Sports for Perez-Rodriguez's FPU; and to Solla E. and Mendez J. (CACTI-University of Vigo) for their excellent technical assistance in microscopy

Purification and characterization of \u3b2-glucosidase from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of \u3b2-glucosidase from a newly isolated thermophilic fungus, Melanocarpus sp. Microbial Type Culture Collection (MTCC) 3922. The molecular weight of \u3b2-glucosidase was determined to be ~ 92 and 102 kDa with SDS PAGE and gel filtration, respectively, and pI of ~ 4.1. It was optimally active at 60\ub0C and pH 6.0, though was stable at 50\ub0C and pH 5.0 - 6.0. The presence of DTT, mercaptoethanol and metal ions such as Na+, K+, Ca2+, Mg2+ and Zn2+ positively influenced the activity of \u3b2-glucosidase but the activity was inhibited in the presence of CuSO4. \u3b2-Glucosidase recognized pNP- \u3b2-glucopyranoside (pNPG) as the preferred substrate, and showed very low affinity for pNP- \u3b2-D-cellobioside. Km and Vmax for the hydrolysis of pNPG by \u3b2-glucosidase was calculated as 3.3 mM and 43.68 \u3bcmolmin-1mg protein-1, respectively and kcat was quantified as 4 x 103 min-1. \u3b2-Glucosidase activity was enhanced appreciably in the presence of alcohols (methanol and ethanol) moreover, purified \u3b2-glucosidase showed putative transglycosylation activity that was positively catalyzed in presence of methanol as an acceptor molecule

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody lateral flow assay for antibody prevalence studies following vaccination: a diagnostic accuracy study

Background: Lateral flow immunoassays (LFIAs) are able to achieve affordable, large scale antibody testing and provide rapid results without the support of central laboratories. As part of the development of the REACT programme extensive evaluation of LFIA performance was undertaken with individuals following natural infection. Here we assess the performance of the selected LFIA to detect antibody responses in individuals who have received at least one dose of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Methods: This was a prospective diagnostic accuracy study. Sampling was carried out at renal outpatient clinic and healthcare worker testing sites at Imperial College London NHS Trust. Two cohorts of patients were recruited; the first was a cohort of 108 renal transplant patients attending clinic following two doses of SARS-CoV-2 vaccine, the second cohort comprised 40 healthcare workers attending for first SARS-CoV-2 vaccination and subsequent follow up. During the participants visit, finger-prick blood samples were analysed on LFIA device, while paired venous sampling was sent for serological assessment of antibodies to the spike protein (anti-S) antibodies. Anti-S IgG was detected using the Abbott Architect SARS-CoV-2 IgG Quant II CMIA. A total of 186 paired samples were collected. The accuracy of Fortress LFIA in detecting IgG antibodies to SARS-CoV-2 compared to anti-spike protein detection on Abbott Assay Results: The LFIA had an estimated sensitivity of 92.0% (114/124; 95% confidence interval [CI] 85.7% to 96.1%) and specificity of 93.6% (58/62; 95% CI 84.3% to 98.2%) using the Abbott assay as reference standard (using the threshold for positivity of 7.10 BAU/ml) Conclusions: Fortress LFIA performs well in the detection of antibody responses for intended purpose of population level surveillance but does not meet criteria for individual testing