A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate ¹⁸O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.     

Diel and lunar variation in diving behavior of rough-toothed dolphins (Steno bredanensis) off Kauaʻi,Hawaiʻi

Observational studies describe rough-toothed dolphins (Steno bredanensis) actively foraging during the day on epipelagic species. Using data from depth-transmitting satellite tags deployed on nine individuals off Kauaʻi, we investigated diving behavior and the effects of lunar phase and solar light levels on vertical movements. Overall, tagged rough-toothed dolphins primarily used near-surface waters, spending between 83.6% and 93.7% of their time in the top 30 m of the water column. When diving, grand mean, median, and maximum dive depths were 76.9 m, 67.5 m, and 399.5 m, although individuals were in water with depths from approximately 700–1,450 m. Dive rates varied by time of day, being lowest during the day and at dawn and highest at dusk and night. Dives were deepest (M = 133.7 m, SD = 52.6 m, median = 106.5 m) and longest (M = 4.0 min, SD = 0.4 min, median = 4.0 min) at dusk, suggesting dolphins were taking advantage of prey rising to the surface in response to reduced light levels. Lunar phase indirectly affected diving, with deeper and longer dives occurring with increasing illumination. The variations in dive behavior across solar and lunar cycles indicate diving patterns shift based on the distribution of prey.     

Strategies for targeted transposon tagging of ABA biosynthetic mutants in tomato

The ABA biosynthetic pathway has been studied in detail and the steps impaired in some ABA-deficient mutants are known. However, little is known of the molecular control mechanisms regulating ABA production in planta. A direct route for improving our understanding of these mechanisms is to transposon tag and clone the wild-type counterparts of the ABA mutant alleles. On the basis of the observation that maize transposons move preferentially to linked sites in both homologous and heterologous systems and in doing so disrupt gene function, a targeted transposon mutagenesis strategy is being developed towards cloning ABA biosynthetic genes from tomato. The possibility of using marker genes to identify T-DNA insertion sites in selected parts of the genome has been examined and compared with an inverse PCR/RFLP approach to mapping T-DNAs.     

Construction of an Azospirillum brasilense Sp7 recA mutant

Summary Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant. Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A. brasilense recA gene on a 1.2 kb DNA fragment. One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A. brasilense by marker exchange. The resulting A. brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.     

Acquired Resistance to Macrolide–Lincosamide–Streptogramin Antibiotics in Lactic Acid Bacteria of Food Origin

Antibiotic resistance is a growing problem in clinical settings as well as in food industry. Lactic acid bacteria (LAB) commercially used as starter cultures and probiotic supplements are considered as reservoirs of several antibiotic resistance genes. Macrolide–lincosamide–streptogramin (MLS) antibiotics have a proven record of excellence in clinical settings. However, the intensive use of tylosin, lincomysin and virginamycin antibiotics of this group as growth promoters in animal husbandry and poultry has resulted in development of resistance in LAB of animal origin. Among the three different mechanisms of MLS resistance, the most commonly observed in LAB are the methylase and efflux mediated resistance. This review summarizes the updated information on MLS resistance genes detected and how resistance to these antibiotics poses a threat when present in food grade LAB.     

Individual variation and seasonality drive bird feeder use during winter in a Mediterranean climate

Purposeful provisioning of food to wild animals is a widespread and growing activity that has the potential to impact populations and communities. Nevertheless, studies assessing use of recreational feeders by free‐living birds during winter are surprisingly rare and largely limited to regions with continental climates characterized by freezing temperatures and snow cover. In contrast, there is little information available regarding bird use of feeders within warmer climates during winter, despite widespread recreational feeding in these areas. In this study, we quantified visitation patterns to bird feeders in a Mediterranean climate to evaluate the relationship between feeder use and several environmental variables known to influence supplemental feeder use in continental climates. We established a network of bird feeders in Corvallis, Oregon, USA, that were filled with black oil sunflower (Helianthus annuus) seeds and equipped with radio frequency identification (RFID) data loggers that recorded >315,000 visits by 70 individual Black‐capped Chickadees (Poecile atricapillus) across a 5‐month period (October 2016–March 2017). We found extensive variation in feeder use, with individuals averaging 1–406 feeder visits/day and using 1–9 of the 21 feeders that were available; individual variability was largely consistent during the course of our study. At the population level, we found that feeder use decreased from the start of our study, and this decline continued through the period when foraging was most limited by daylight, including the winter solstice. In contrast to theoretical predictions and empirical work in continental climates, we found that weather variables did not drive feeder use and that feeder visits peaked at mid‐day and gradually decreased until sunset. Our study indicates that individual‐level differences combined with seasonality to drive feeder use patterns, and we conclude that use of supplemental feeders during winter in Mediterranean climates appears to differ notably from feeder use in continental climates.     

A Dictyostelium discoideum mitochondrial fluorescent tagging vector that does not affect respiratory function

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function.