Sortase-Modified Cholera Toxoids Show Specific Golgi Localization

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells

Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of SARS-CoV-2 genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three available genomic nomenclature systems for SARS-CoV-2 to all sequence data from the WHO European Region available during the COVID-19 pandemic until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation. We provide a comparison of the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.Peer reviewe

Factors affecting body temperatures of toads

Factors influencing levels and rates of variation of body temperature ( T b ) in montane Bufo boreas boreas and in lowland Bufo boreas halophilus were investigated as an initial step toward understanding the role of natural thermal variation in the physiology and energetics of these ectothermic animals. Body temperatures of boreas can vary 25–30° C over 24-h periods. Such variation is primarily due to both nocturnal and diurnal activity and the physical characteristics of the montane environment. Bufo boreas halophilus are primarily nocturnal except during breeding and are voluntarily active at body temperatures ranging between 10 and 25° C. Despite variation in T b encountered in the field, boreas select a narrow range of T b in a thermal gradient, averaging 23.5 and 26.2° C for fasted individuals maintained under field conditions or acclimated to 20° C, respectively. In a thermal gradient the mean T b of fasted halophilus acclimated to 20° C is 23.9° C. Skin color of boreas varies in the field from very dark to light. The dark skins absorb approximately 4% more radiation than the light ones. Light colored boreas should absorb approximately 5% more radiation than similarly colored halophilus . Evaporative water losses increase directly with skin temperatures and vapor pressure deficit in both subspecies. Larger individuals heat and cool more slowly than smaller ones. Calculation of an enery budget for boreal toads suggests that they could sit in direct sunlight for long periods without fatally overheating, providing the skin was continually moist.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47722/1/442_2004_Article_BF00344732.pd

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases such as sortases has proved to be a popular strategy for selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially-sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using un-activated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibriums to favor product formation which greatly enhances the reaction efficiency. Subsequent optimization of the reaction allows N‑terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling

The Placental Distal Villous Hypoplasia Pattern: Interobserver Agreement and Automated Fractal Dimension as an Objective Metric

The distal villous hypoplasia (DVH) pattern is a placental correlate of fetal growth restriction. Because the pattern seems to involve less complexity than do appropriately developed placental villi, we postulated that it may be associated wi