From Monochrome to Technicolor: Simple Generic Approaches to Multicomponent Protein Nanopatterning Using Siloxanes with Photoremovable Protein-Resistant Protecting Groups.

We show that sequential protein deposition is possible by photodeprotection of films formed from a tetraethylene-glycol functionalized nitrophenylethoxycarbonyl-protected aminopropyltriethoxysilane (NPEOC-APTES). Exposure to near-UV irradiation removes the protein-resistant protecting group, and allows protein adsorption onto the resulting aminated surface. The protein resistance was tested using proteins with fluorescent labels and microspectroscopy of two-component structures formed by micro- and nanopatterning and deposition of yellow and green fluorescent proteins (YFP/GFP). Nonspecific adsorption onto regions where the protecting group remained intact was negligible. Multiple component patterns were also formed by near-field methods. Because reading and writing can be decoupled in a near-field microscope, it is possible to carry out sequential patterning steps at a single location involving different proteins. Up to four different proteins were formed into geometric patterns using near-field lithography. Interferometric lithography facilitates the organization of proteins over square cm areas. Two-component patterns consisting of 150 nm streptavidin dots formed within an orthogonal grid of bars of GFP at a period of ca. 500 nm could just be resolved by fluorescence microscopy

Isothermal by Design: Comparison with an Established Isothermal Nucleation Kinetics Analysis Method

The nucleation kinetics of the alpha form of p‐aminobenzoic acid from ethanolic and aqueous solutions is examined through a comparative examination of temperature‐jump and anti‐solvent drown‐out isothermal crystallization methodologies. Analysis of the data reveals the measured induction times, and the calculated effective interfacial tensions as a function of the supersaturation show broadly equivalent behavior for the aqueous‐ethanol mixed‐solvent drown‐out and temperature‐jump ethanol solution systems, confirming the comparability of the two methodologies. The results also demonstrate poorer agreement with the temperature‐jump pure aqueous system, highlighting the importance of the strength of solvation/desolvation as the key rate‐limiting process for the overall nucleation behavior

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Transcription of canine toll-like receptor 2, β-defensin 1 and β-defensin 103 in infected atopic skin, non-infected atopic skin, healthy skin and the CPEK cell line.

Dogs and humans with atopic dermatitis (AD) have a high prevalence of recurrent staphylococcal pyoderma. β-Defensins (BDs) and toll-like receptors (TLRs) are important in innate immunity against bacterial skin infections, and decreased BD and TLR2 expression has been associated with human AD. However, findings from recent studies of BD expression in human and canine AD have been variable and contradictory. The aim of this study was to further our understanding of the role of antimicrobial proteins in canine AD by quantifying mRNA for canine (c) BD1, cBD103 and TLR2 in healthy skin (n=17 dogs), matched samples of atopic skin with and without active infection by Staphylococcus pseudintermedius (n=13 dogs), and the canine keratinocyte cell line CPEK cultured with 5 ng/ml tumour necrosis factor alpha (TNFα) and 10 μg/ml lipopolysaccharide (LPS). mRNA for cBD1, CB103 and TLR2 were detected in all samples. TNFα significantly increased transcription of cBD1, cBD103 and TLR2 in the CPEK cells. mRNA for cBD103 was also significantly increased after stimulation with LPS. There were no significant differences in mRNA levels for cBD1, cBD103 or TLR2 in healthy, non-infected atopic or infected atopic skin. Canine AD did not appear to be associated with altered expression of cBD1, cBD103 and TLR2 in these dogs. Other studies have reported both increased and decreased expression of these antimicrobial peptides in canine AD and pyoderma, and therefore further investigation of the clinical significance of these mediators is required

PBPK Modeling as a Tool for Predicting and Understanding Intestinal Metabolism of Uridine 5′-Diphospho-glucuronosyltransferase Substrates

Uridine 5′-diphospho-glucuronosyltransferases (UGTs) are expressed in the small intestines, but prediction of first-pass extraction from the related metabolism is not well studied. This work assesses physiologically based pharmacokinetic (PBPK) modeling as a tool for predicting intestinal metabolism due to UGTs in the human gastrointestinal tract. Available data for intestinal UGT expression levels and in vitro approaches that can be used to predict intestinal metabolism of UGT substrates are reviewed. Human PBPK models for UGT substrates with varying extents of UGT-mediated intestinal metabolism (lorazepam, oxazepam, naloxone, zidovudine, cabotegravir, raltegravir, and dolutegravir) have demonstrated utility for predicting the extent of intestinal metabolism. Drug–drug interactions (DDIs) of UGT1A1 substrates dolutegravir and raltegravir with UGT1A1 inhibitor atazanavir have been simulated, and the role of intestinal metabolism in these clinical DDIs examined. Utility of an in silico tool for predicting substrate specificity for UGTs is discussed. Improved in vitro tools to study metabolism for UGT compounds, such as coculture models for low clearance compounds and better understanding of optimal conditions for in vitro studies, may provide an opportunity for improved in vitro–in vivo extrapolation (IVIVE) and prospective predictions. PBPK modeling shows promise as a useful tool for predicting intestinal metabolism for UGT substrates