Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.     

Microbial Communities in the Upper Respiratory Tract of Patients with Asthma and Chronic Obstructive Pulmonary Disease

Respiratory infections are well-known triggers of chronic respiratory diseases. Recently, culture-independent tools have indicated that lower airway microbiota may contribute to pathophysiologic processes associated with asthma and chronic obstructive pulmonary disease (COPD). However, the relationship between upper airway microbiota and chronic respiratory diseases remains unclear. This study was undertaken to define differences of microbiota in the oropharynx of asthma and COPD patients relative to those in healthy individuals. To account for the qualitative and quantitative diversity of the 16S rRNA gene in the oropharynx, the microbiomes of 18 asthma patients, 17 COPD patients, and 12 normal individuals were assessed using a high-throughput next-generation sequencing analysis. In the 259,572 total sequence reads, α and β diversity measurements and a generalized linear model revealed that the oropharynx microbiota are diverse, but no significant differences were observed between asthma and COPD patients. Pseudomonas spp. of Proteobacteria and Lactobacillus spp. of Firmicutes were highly abundant in asthma and COPD. By contrast, Streptococcus, Veillonella, Prevotella, and Neisseria of Bacteroidetes dominated in the healthy oropharynx. These findings are consistent with previous studies conducted in the lower airways and suggest that oropharyngeal airway microbiota are important for understanding the relationships between the various parts of the respiratory tract with regard to bacterial colonization and comprehensive assessment of asthma and COPD.     

Methods for Determining the Uncertainty of Population Estimates Derived from Satellite Imagery and Limited Survey Data: A Case Study of Bo City,Sierra Leone

This study demonstrates the use of bootstrap methods to estimate the total population of urban and periurban areas using satellite imagery and limited survey data. We conducted complete household surveys in 20 neighborhoods in the city of Bo, Sierra Leone, which collectively were home to 25,954 persons living in 1,979 residential structures. For five of those twenty sections, we quantized the rooftop areas of structures extracted from satellite images. We used bootstrap statistical methods to estimate the total population of the pooled sections, including the associated uncertainty intervals, as a function of sample size. Evaluations based either on rooftop area per person or on the mean number of occupants per residence both converged on the true population size. We demonstrate with this simulation that demographic surveys of a relatively small proportion of residences can provide a foundation for accurately estimating the total population in conjunction with aerial photographs.     

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.