Optical super-resolution microscopy in neurobiology

Understanding the highly plastic nature of neurons requires the dynamic visualization of their molecular and cellular organization in a native context. However, due to the limited resolution of standard light microscopy, many of the structural specializations of neurons cannot be resolved. A recent revolution in light microscopy has given rise to several super-resolution light microscopy methods yielding 2-10-fold higher resolution than conventional microscopy. We here describe the principles behind these techniques as well as their application to the analysis of the molecular architecture of the synapse. Furthermore, we discuss the potential for continued development of super-resolution microscopy as necessary for live imaging of neuronal structure and function in the brain.     

Morphological and molecular investigations of Tubulinosema ratisbonensis gen. nov., sp. nov. (Microsporidia: Tubulinosematidae fam. nov.), a parasite infecting a laboratory colony of Drosophila melanogaster (Diptera: Drosophilidae)

A new species of microsporidia from Drosophila melanogaster was investigated by light and electron microscopy and by ribosomal RNA (rRNA) sequencing. This microsporidium and the previously described Nosema kingi and Nosema acridophagus have been transferred to the new genus Tubulinosema gen. nov. with the following characters: nuclei are in diplokaryotic arrangement during the life cycle. All stages are in direct contact with the host cell cytoplasm, slightly anisofilar polar tube with the last coils being smaller in diameter arranged in one or two rows on both sides of the diplokaryon and small tubuli on the surface of late meronts. Spores are oval or slightly pyriform. Thick endospore wall, thinner over anchoring disc. This new genus and the genus Brachiola have been placed in a new family Tubulinosematidae fam. nov. Phylogenetic analysis of small subunit rRNA sequences by different methods placed Tubulinosema spp. in one clade with the genus Brachiola forming its sister clade, which is distant from the clade containing the true Nosema spp. including Nosema bombycis.     

Identification of Protor as a novel Rictor-binding component of mTOR complex-2

The mTOR (mammalian target of rapamycin) protein kinase is an important regulator of cell growth. Two complexes of mTOR have been identified: complex 1, consisting of mTOR-Raptor (regulatory associated protein of mTOR)-mLST8 (termed mTORC1), and complex 2, comprising mTOR-Rictor (rapamycininsensitive companion of mTOR)-mLST8-Sin1 (termed mTORC2). mTORC1 phosphorylates the p70 ribosomal S6K (S6 kinase) at its hydrophobic motif (Thr389), whereas mTORC2 phosphorylates PKB (protein kinase B) at its hydrophobic motif (Ser473). In the present study, we report that widely expressed isoforms of unstudied proteins termed Protor-1 (protein observed with Rictor-1) and Protor-2 interact with Rictor and are components of mTORC2. We demonstrate that immunoprecipitation of Protor-1 or Protor-2 results in the co-immunoprecipitation of other mTORC2 subunits, but not Raptor, a specific component of mTORC1. We show that detergents such as Triton X-100 or n-octylglucoside dissociate mTOR and mLST8 from a complex of Protor-1, Sin1 and Rictor. We also provide evidence that Rictor regulates the expression of Protor-1, and that Protor-1 is not required for the assembly of other mTORC2 subunits into a complex. Protor-1 is a novel Rictor-binding subunit of mTORC2, but further work is required to establish its role.     

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. Horton and Gibson contributed equally to this work.     

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.     

Mental Imagery,Impact, and Affect: A Mediation Model for Charitable Giving

One of the puzzling phenomena in philanthropy is that people can show strong compassion for identified individual victims but remain unmoved by catastrophes that affect large numbers of victims. Two prominent findings in research on charitable giving reflect this idiosyncrasy: The (1) identified victim and (2) victim number effects. The first of these suggests that identifying victims increases donations and the second refers to the finding that people’s willingness to donate often decreases as the number of victims increases. While these effects have been documented in the literature, their underlying psychological processes need further study. We propose a model in which identified victim and victim number effects operate through different cognitive and affective mechanisms. In two experiments we present empirical evidence for such a model and show that different affective motivations (donor-focused vs. victim-focused feelings) are related to the cognitive processes of impact judgments and mental imagery. Moreover, we argue that different mediation pathways exist for identifiability and victim number effects.     

Analysis of post-translational modifications in Alzheimer's disease by mass spectrometry

The roles of post-translational modifications (PTMs) in the onset and progression of disease have been extensively studied for decades. More specifically, various PTMs have been the focus of research in Alzheimer's disease (AD). The two most discussed hallmarks of the disease, senile plaques and tau tangles, are the result of PTMs of the amyloidβ protein precursor (AβPP) and the microtubule stabilizing protein: tau. While these modifications have been characterized indirectly by biochemical-based methods, the primary shortcoming to this research can be linked to a lack of a thorough molecular-based means of qualitative and quantitative analysis of many of these modifications within transgenic animal, and human samples. In this review, we discuss the important proteins and their associated PTMs linked to AD and the ways in which mass spectrometry has and will be utilized to analyze them. We also comment on novel ways in which molecular-based mass spectrometry methods should be employed going forward to resolve the interconnections of the PTMs involvement in various stages of AD pathology (preclinical, mild cognitive impairment, advanced-stage AD).     

The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.