A genetic screen based on in vivo RNA imaging reveals centrosome-independent mechanisms for localizing gurken transcripts in Drosophila

We have screened chromosome arm 3L for ethyl methanesulfonate-induced mutations that disrupt localization of fluorescently labeled gurken (grk) messenger (m)RNA, whose transport along microtubules establishes both major body axes of the developing Drosophila oocyte. Rapid identification of causative mutations by single-nucleotide polymorphism recombinational mapping and whole-genomic sequencing allowed us to define nine complementation groups affecting grk mRNA localization and other aspects of oogenesis, including alleles of elg1, scaf6, quemao, nudE, Tsc2/gigas, rasp, and Chd5/Wrb, and several null alleles of the armitage Piwi-pathway gene. Analysis of a newly induced kinesin light chain allele shows that kinesin motor activity is required for both efficient grk mRNA localization and oocyte centrosome integrity. We also show that initiation of the dorsoanterior localization of grk mRNA precedes centrosome localization, suggesting that microtubule self-organization contributes to breaking axial symmetry to generate a unique dorsoventral axis

Activity of Bdellovibrio Hit Locus Proteins, Bd0108 and Bd0109, Links Type IVa Pilus Extrusion/Retraction Status to Prey-Independent Growth Signalling

Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to preyindependent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation background

Age-Specific Epigenetic Drift in Late-Onset Alzheimer's Disease

Despite an enormous research effort, most cases of late-onset Alzheimer's disease (LOAD) still remain unexplained and the current biomedical science is still a long way from the ultimate goal of revealing clear risk factors that can help in the diagnosis, prevention and treatment of the disease. Current theories about the development of LOAD hinge on the premise that Alzheimer's arises mainly from heritable causes. Yet, the complex, non-Mendelian disease etiology suggests that an epigenetic component could be involved. Using MALDI-TOF mass spectrometry in post-mortem brain samples and lymphocytes, we have performed an analysis of DNA methylation across 12 potential Alzheimer's susceptibility loci. In the LOAD brain samples we identified a notably age-specific epigenetic drift, supporting a potential role of epigenetic effects in the development of the disease. Additionally, we found that some genes that participate in amyloid-β processing (PSEN1, APOE) and methylation homeostasis (MTHFR, DNMT1) show a significant interindividual epigenetic variability, which may contribute to LOAD predisposition. The APOE gene was found to be of bimodal structure, with a hypomethylated CpG-poor promoter and a fully methylated 3′-CpG-island, that contains the sequences for the ε4-haplotype, which is the only undisputed genetic risk factor for LOAD. Aberrant epigenetic control in this CpG-island may contribute to LOAD pathology. We propose that epigenetic drift is likely to be a substantial mechanism predisposing individuals to LOAD and contributing to the course of disease

A genetic screen for novel factors involved in RNA localisation in the Drosophila germline

RNA localisation is essential for the establishment of body axes in the developing Drosophila embryo. Transcripts of certain maternal genes are transported along microtubules and translated in restricted regions of the oocyte to define these axes. RNA from one such gene, gurken (grk) is first localised to the oocyte posterior, where translated Grk signals to the soma to specify the follicle cells in this region to adopt a posterior fate. Subsequently these posterior follicle cells signal back to the oocyte triggering cytoskeletal reorganisation. The nucleus and grk RNA are then transported to the dorsal-anterior region of the oocyte, where Grk signals to specify the surrounding cells to become dorsal. This two-stage grk localisation thereby determines both the anterior-posterior and dorsal-ventral axes. This thesis describes a random mutagenesis screen designed to identify novel genes on chromosome arm 3R that are involved in grk RNA localisation in the oocyte. We observed the distribution of endogenous grk RNA in vivo using a fluorescent construct. From 4943 mutagenised lines scored, I recovered 38 with reproducible grk mislocalisation and 78 lines with other defects in oogenesis. After phenotypic analyses and complementation testing, these mutants were grouped and the affected genes identified using whole genome sequencing and recombinational mapping. I focus on three groups of mutants from the screen that showed a grk mislocalisation phenotype. Mutations in karyopherin-beta3, a nuclear import factor gene, and in CG9925, encoding a TUDOR-domain containing protein, cause reduced Grk translation and axis misspecification. In two new mutant alleles of CG5508/minotaur, which encodes a protein involved in phospholipid biosynthesis, Grk translation is reduced and transposon expression levels are elevated, a typical phenotype of mutants for piRNA pathway genes. Mutants of these three genes, despite diverse predicted gene functions, share many phenotypes. I describe detailed characterisation of these novel mutants and suggest how they may all be affecting grk mRNA localisation due to disruptions in the mechanisms of piRNA biogenesis and the protection of genome integrity

Working across species down on the farm: Howard S Liddell and the development of comparative psychopathology, c. 1923 to 1962

Seeking a scientific basis for understanding and treating mental illness, and inspired by the work of Ivan Pavlov, American physiologists, psychiatrists and psychologists in the 1920s turned to nonhuman animals. This paper examines how new constructs such as “experimental neurosis” emerged as tools to enable psychiatric comparison across species. From 1923 to 1962, the Cornell “Behavior Farm” was a leading interdisciplinary research center pioneering novel techniques to experimentally study nonhuman psychopathology. Led by the psychobiologist Howard Liddell, work at the Behavior Farm formed part of an ambitious program to develop new preventative and therapeutic techniques and bring psychiatry into closer relations with physiology and medicine. At the heart of Liddell’s activities were a range of nonhuman animals, including pigs, sheep, goats and dogs, each serving as a proxy for human patients. We examine how Pavlov’s conceptualization of ‘experimental neurosis’ was used by Liddell to facilitate comparison across species and communication between researchers and clinicians. Our close reading of his experimental system demonstrates how unexpected animal behaviors and emotions were transformed into experimental virtues. However, to successfully translate such behaviors from the animal laboratory into the field of human psychopathology, Liddell increasingly reached beyond, and, in effect, redefined, the Pavlovian method to make it compatible and compliant with an ethological approach to the animal laboratory. We show how the resultant Behavior Farm served as a productive “hybrid” place, containing elements of experiment and observation, laboratory and field. It was through the building of close and more naturalistic relationships with animals over extended periods of time, both normal and pathological, and within and outside of the experimental space, that Liddell could understand, manage, and make useful the myriad behavioral complexities that emerged from the life histories of experimental animals, the researchers who worked with them, and their shared relationships to the wider physical and social environments

Variability of CO concentrations in the Venus troposphere from Venus Express/VIRTIS using a Band Ratio Technique

A fast method is presented for deriving the tropospheric CO concentrations in the Venus atmosphere from near-infrared spectra using the night side 2.3 μm window. This is validated using the spectral fitting techniques of Tsang et al. [Tsang, C.C.C., Irwin, P.G.J., Taylor, F.W., Wilson, C.F., Drossart, P., Piccioni, G., de Kok, R., Lee, C., Calcutt, S.B., and the Venus Express/VIRTIS Team, 2008a. Tropospheric carbon monoxide concentrations and variability on Venus with Venus Express/VIRTIS-M observations. J. Geophys. Res. 113, doi: 10.1029/2008JE003089. E00B08] to show that monitoring CO in the deep atmosphere can be done quickly using large numbers of observations, with minimal effect from cloud and temperature variations. The new method is applied to produce some 1450 zonal mean CO profiles using data from the first eighteen months of operation from the Visible and Infrared Thermal Imaging Spectrometer infrared mapping subsystem (VIRTIS-M-IR) on Venus Express. These results show many significant long- and short-term variations from the mean equator-to-pole increasing trend previously found from earlier Earth- and space-based observations, including a possible North-South dichotomy, with interesting implications for the dynamics and chemistry of the lower atmosphere of Venus. © 2009 Elsevier Inc. All rights reserved