The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a ...therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Group B streptococcus (GBS) capsular polysaccharide is one of the major virulence factors underlying invasive GBS disease and a component of forthcoming vaccines. Serotype classification of GBS is ...based on the capsule polysaccharide of which ten variants are known to exist (Ia, Ib, II-IX). Current methods for GBS serotype assignment rely on latex agglutination or PCR while more recently a whole genome sequencing method was reported. In this study, three distinct algorithms for serotype assignment from genomic data were assessed using a panel of 790 clinical isolates.
The first approach utilised the entire capsular locus coupled with a mapping methodology. The second approach continues from the first and utilised a SNP-based methodology across the conserved cpsD-G region to differentiate serotypes Ia-VII and IX. Finally the third approach used the variable cpsG -K region coupled with a mapping methodology. All three approaches were assessed for typeability (percentage of isolates assigned a serotype) and concordance to the latex agglutination methodology.
Following comparisons, the third approach using the variable cpsG-K region demonstrated the best performance with 99.9% typeability and 86.7% concordance. Overall, of the 105 discordant isolates, 71 were resolved following retesting of latex agglutination and whole genome sequencing, 20 failed to assign a serotype using latex agglutination and only 14 were found to be truly discordant on re-testing. Comparison of this final approach with the previously described assembly-based approach returned identical results.
These results demonstrated that molecular capsular typing using whole genome sequencing and a mapping-based approach is a viable alternative to the traditional, latex agglutination-based serotyping method and can be implemented in a public health microbiology setting.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Purpose
Accurate modeling of the relative biological effectiveness (RBE) of particle beams requires increased systematic in vitro studies with human cell lines with care towards minimizing ...uncertainties in biologic assays as well as physical parameters. In this study, we describe a novel high‐throughput experimental setup and an optimized parameterization of the Monte Carlo (MC) simulation technique that is universally applicable for accurate determination of RBE of clinical ion beams. Clonogenic cell‐survival measurements on a human lung cancer cell line (H460) are presented using proton irradiation.
Methods
Experiments were performed at the Heidelberg Ion Therapy Center (HIT) with support from the Deutsches Krebsforschungszentrum (DKFZ) in Heidelberg, Germany using a mono‐energetic horizontal proton beam. A custom‐made variable range selector was designed for the horizontal beam line using the Geant4 MC toolkit. This unique setup enabled a high‐throughput clonogenic assay investigation of multiple, well defined dose and linear energy transfer (LETs) per irradiation for human lung cancer cells (H460) cultured in a 96‐well plate. Sensitivity studies based on application of different physics lists in conjunction with different electromagnetic constructors and production threshold values to the MC simulations were undertaken for accurate assessment of the calculated dose and the dose‐averaged LET (LETd). These studies were extended to helium and carbon ion beams.
Results
Sensitivity analysis of the MC parameterization revealed substantial dependence of the dose and LETd values on both the choice of physics list and the production threshold values. While the dose and LETd calculations using FTFP_BERT_LIV, FTFP_BERT_EMZ, FTFP_BERT_PEN and QGSP_BIC_EMY physics lists agree well with each other for all three ions, they show large differences when compared to the FTFP_BERT physics list with the default electromagnetic constructor. For carbon ions, the dose corresponding to the largest LETd value is observed to differ by as much as 78% between FTFP_BERT and FTFP_BERT_LIV. Furthermore, between the production threshold of 700 μm and 5 μm, proton dose varies by as much as 19% corresponding to the largest LETd value sampled in the current investigation. Based on the sensitivity studies, the FTFP_BERT physics list with the low energy Livermore electromagnetic constructor and a production threshold of 5 μm was employed for determining accurate dose and LETd. The optimized MC parameterization results in a different LETd dependence of the RBE curve for 10% SF of the H460 cell line irradiated with proton beam when compared with the results from a previous study using the same cell line. When the MC parameters are kept consistent between the studies, the proton RBE results agree well with each other within the experimental uncertainties.
Conclusions
A custom high‐throughput, high‐accuracy experimental design for accurate in vitro cell survival measurements was employed at a horizontal beam line. High sensitivity of the physics‐based optimization establishes the importance of accurate MC parameterization and hence the conditioning of the MC system on a case‐by‐case basis. The proton RBE results from current investigations are observed to agree with a previous measurement made under different experimental conditions. This establishes the consistency of our experimental findings across different experiments and institutions.
Group B Streptococcus (GBS) is a common intestinal colonizer during the neonatal period, but also may cause late-onset sepsis or meningitis in up to 0.5% of otherwise healthy colonized infants after ...day 3 of life. Transmission routes and risk factors of this late-onset form of invasive GBS disease (iGBS) are not fully understood. Cases of iGBS with recurrence (n=25) and those occurring in parallel in twins/triplets (n=32) from the UK and Ireland (national surveillance study 2014/15) and from Germany and Switzerland (retrospective case collection) were analyzed to unravel shared (in affected multiples) or fixed (in recurrent disease) risk factors for GBS disease. The risk of iGBS among infants from multiple births was high (17%), if one infant had already developed GBS disease. The interval of onset of iGBS between siblings was 4.5 days and in recurrent cases 12.5 days. Disturbances of the individual microbiome, including persistence of infectious foci are suggested e.g. by high usage of perinatal antibiotics in mothers of affected multiples, and by the association of an increased risk of recurrence with a short term of antibiotics aOR 4.2 (1.3-14.2), P=0.02. Identical GBS serotypes in both recurrent infections and concurrently infected multiples might indicate a failed microbiome integration of GBS strains that are generally regarded as commensals in healthy infants. The dynamics of recurrent GBS infections or concurrent infections in multiples suggest individual patterns of exposure and fluctuations in host immunity, causing failure of natural niche occupation.
Ferrofluid lubricated squeeze film bearing design system formed by a rotating upper spherical surface and a radially rough lower flat plate considering variable magnetic field, which is oblique to ...the lower plate, have been analyzed. The variable magnetic field is important because of its advantage of generating maximum field at the required active contact zone. The problem is motivated because squeeze film behaviour is widely observed in many industrial applications like in machine tools, gears, rolling elements, hydraulic systems, engines, clutch plates, force pendulum apparatus, etc. Moreover, the squeeze film behaviour is also observed in skeletal joints of human body. On the basis of ferrohydrodynamic theory and Christensen’s stochastic theory for hydrodynamic lubrication of rough surfaces, the modified Reynolds equation is derived and expressions for squeeze film characteristics are obtained which are calculated numerically and interpreted. While deriving Reynolds equation effects of various parameters like rotation, width of the nominal minimum film thickness and squeeze velocity are also considered.
Large amounts of high quality biophysical data are needed to improve current biological effects models but such data are lacking and difficult to obtain. The present study aimed to more efficiently ...measure the spatial distribution of relative biological effectiveness (RBE) of charged particle beams using a novel high-accuracy and high-throughput experimental platform. Clonogenic survival was selected as the biological endpoint for two lung cancer cell lines, H460 and H1437, irradiated with protons, carbon, and helium ions. Ion-specific multi-step microplate holders were fabricated such that each column of a 96-well microplate is spatially situated at a different location along a particle beam path. Dose, dose-averaged linear energy transfer (LET
), and dose-mean lineal energy (y
) were calculated using an experimentally validated Geant4-based Monte Carlo system. Cells were irradiated at the Heidelberg Ion Beam Therapy Center (HIT). The experimental results showed that the clonogenic survival curves of all tested ions were y
-dependent. Both helium and carbon ions achieved maximum RBEs within specific y
ranges before biological efficacy declined, indicating an overkill effect. For protons, no overkill was observed, but RBE increased distal to the Bragg peak. Measured RBE profiles strongly depend on the physical characteristics such as y
and are ion specific.
Lipid based delivery system is gaining significant attention of researchers working on the development of novel formulations for improved therapeutic efficacy and safety of drugs. Topical drug ...delivery is needed in treatment of skin, eyes, rectum, vagina disorders and systemic disorders having skin manifestations. Lipid nanocarriers have widespread application in the topical drug delivery due to the biocompatible, biodegradable, nontoxic and nonirritating nature of the lipid. Microemulsion and nanoemulsion contain lipids in the nanosize range which can lead to penetration of drug to the deeper skin layers. Solid lipid nanoparticles and nanolipid carriers act by forming an occlusive layer on the skin leading to increased hydration and penetration of the drug. Vesicular carriers such as liposomes, niosomes, ultradeformable vesicles, cubosomes etc. are also reported to enhance the penetration of the entrapped drugs in deeper layers of skin. These carrier systems are mainly composed of lipids, surfactants, and co‐surfactants which are safe and quite acceptable by regulatory authorities. The present review article focuses on different types of lipid nanocarriers used in topical drug delivery, their advantages and limitations, mechanism of enhanced penetration, work reported in the related literature, characterization tests and their safety and toxicity concerns.
Lipid‐based nanocarriers like microemulsion and nanoemulsion, solid lipid nanoparticle (SLNs), nanostructured lipid carriers (NLCs), liposomes, niosomes, ethosomes, and transferosomes generally have been added in the different formulations like topical gel or cream. When these formulations have been applied on skin, they penetrate in the skin with different routes without systemic absorption and also provide prolonged availability at the site of action.
The document emphasizes that understanding formality in quality risk management can optimize resource usage and support risk-based decision-making by reflecting the level of importance, uncertainty, ...and complexity of the decision (1). The previous version of the guideline did not provide clear direction on the level of formality necessary for risk management processes, resulting in inconsistencies in how risk management was executed by different organizations. In QRM, low to high formality is defined by the level of structure and documentation employed in the risk management process. Changes that are well understood that have a moderate impact on the product or process may require a more structured method to document potential risks, such as FTA, risk ranking and filtering (RRF), What If tool, etc. * High formality: complex changes with minimal process knowledge that can have a significant impact on the product, process, or system may require a highly structured and use of formal tools such as process hazard analysis (PHA) or FMEA.
TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two ...cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.
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