Serial-section atlas of the Tritonia pedal ganglion.

The pedal ganglion of the nudibranch gastropod Tritonia diomedea has been the focus of neurophysiological studies for more than 50 yr. These investigations have examined the neural basis of behaviors as diverse as swimming, crawling, reflex withdrawals, orientation to water flow, orientation to the earth's magnetic field, and learning. Despite this sustained research focus, most studies have confined themselves to the layer of neurons that are visible on the ganglion surface, leaving many neurons, which reside in deeper layers, largely unknown and thus unstudied. To facilitate work on such neurons, the present study used serial-section light microscopy to generate a detailed pictorial atlas of the pedal ganglion. One pedal ganglion was sectioned horizontally at 2-µm intervals and another vertically at 5-µm intervals. The resulting images were examined separately or combined into stacks to generate movie tours through the ganglion. These were also used to generate 3D reconstructions of individual neurons and rotating movies of digitally desheathed whole ganglia to reveal all surface neurons. A complete neuron count of the horizontally sectioned ganglion yielded 1,885 neurons. Real and virtual sections from the image stacks were used to reveal the morphology of individual neurons, as well as the major axon bundles traveling within the ganglion to and between its several nerves and connectives. Extensive supplemental data are provided, as well as a link to the Dryad Data Repository site, where the complete sets of high-resolution serial-section images can be downloaded. NEW & NOTEWORTHY Because of the large size and relatively low numbers of their neurons, gastropod mollusks are widely used for investigations of the neural basis of behavior. Most studies, however, focus on the neurons visible on the ganglion surface, leaving the majority, located out of sight below the surface, unexamined. The present light microscopy study generates the first detailed visual atlas of all neurons of the highly studied Tritonia pedal ganglion.

A Method for Bioinformatic Analysis of Transposon Insertion Sequencing (INSeq) Results for Identification of Microbial Fitness Determinants.

Transposon insertion sequencing is a process whereby microbial fitness determinants can be identified on a genome-wide scale. This process uses high-throughput next generation sequencing to screen for changes in the composition of a pool of transposon mutants after exposure to selective conditions. One commonly used process for generating transposon insertion sequencing libraries is called INSeq that works with mutant pools produced using a modified Mariner transposon. Libraries produced using the INSeq process are sequenced on the Illumina platform. In this chapter, we describe our method for processing the raw Illumina sequencing reads, aligning the reads to a reference sequence to determine read counts, and using the online transposon insertion sequencing data analysis server, ESSENTIALS, to interpret the results.

Genome-wide identification of Acinetobacter baumannii genes necessary for persistence in the lung.

UNLABELLED: Acinetobacter baumannii is a Gram-negative bacterium that causes diseases such as pneumonia, bacteremia, and soft tissue infections in hospitalized patients. Relatively little is known about how A. baumannii causes these infections. Thus, we used insertion sequencing (INSeq), a combination of transposon mutagenesis and massively parallel next-generation sequencing, to identify novel virulence factors of A. baumannii. To this end, we generated a random transposon mutant library containing 150,000 unique insertions in A. baumannii strain ATCC 17978. The INSeq analysis identified 453 genes required for growth in rich medium. The library was then used in a murine pneumonia model, and the relative levels of abundance of mutants before and after selection in the mouse were compared. When genes required for growth in rich medium were removed from the analysis, 157 genes were identified as necessary for persistence in the mouse lung. Several of these encode known virulence factors of A. baumannii, such as OmpA and ZnuB, which validated our approach. A large number of the genes identified were predicted to be involved in amino acid and nucleotide metabolism and transport. Other genes were predicted to encode an integration host factor, a transmembrane lipoprotein, and proteins involved in stress response and efflux pumps. Very few genes, when disrupted, resulted in an increase in A. baumannii numbers during host infection. The INSeq approach identified a number of novel virulence determinants of A. baumannii, which are candidate targets for therapeutic interventions. IMPORTANCE: A. baumannii has emerged as a frequent cause of serious infections in hospitals and community settings. Due to increasing antibiotic resistance, alternative approaches, such as antivirulence strategies, are desperately needed to fight A. baumannii infections. Thorough knowledge of A. baumannii pathogenicity is essential for such approaches but is currently lacking. With the increasingly widespread use of massively parallel sequencing, a class of techniques known as transposon insertion sequencing has been developed to perform comprehensive virulence screens of bacterial genomes in vivo. We have applied one of these approaches (INSeq) to uncover novel virulence factors in A. baumannii. We identified several such factors, including those predicted to encode amino acid and nucleotide metabolism proteins, an integration host factor protein, stress response factors, and efflux pumps. These results greatly expand the number of A. baumannii virulence factors and uncover potential targets for antivirulence treatments.

Novel telomere-anchored PCR approach for studying sexual stage telomeres in Aspergillus nidulans.

Telomere length varies between germline and somatic cells of the same organism, leading to the hypothesis that telomeres are lengthened during meiosis. However, little is known about the meiotic telomere length in many organisms. In the filamentous fungus Aspergillus nidulans, the telomere lengths in hyphae and asexual spores are invariant. No study using existing techniques has determined the telomere length of the sexual ascospores due to the relatively low abundance of pure meiotic cells in A. nidulans and the small quantity of DNA present. To address this, we developed a simple and sensitive PCR strategy to measure the telomere length of A. nidulans meiotic cells. This novel technique, termed "telomere-anchored PCR," measures the length of the telomere on chromosome II-L using a small fraction of the DNA required for the traditional terminal restriction fragment (TRF) Southern analysis. Using this approach, we determined that the A. nidulans ascospore telomere length is virtually identical to telomeres of other cell types from this organism, approximately 110 bp, indicating that a surprisingly strict telomere length regulation exists in the major cell types of A. nidulans. When the hyphal telomeres were measured in a telomerase reverse transcriptase (TERT) knockout strain, small decreases in length were readily detected. Thus, this technique can detect telomeres in relatively rare cell types and is particularly sensitive in measuring exceptionally short telomeres. This rapid and inexpensive telomere-anchored PCR method potentially can be utilized in other filamentous fungi and types of organisms.