FIB-SEM tomography in biology.

Three-dimensional information is much easier to understand than a set of two-dimensional images. Therefore a layman is thrilled by the pseudo-3D image taken in a scanning electron microscope (SEM) while, when seeing a transmission electron micrograph, his imagination is challenged. First approaches to gain insight in the third dimension were to make serial microtome sections of a region of interest (ROI) and then building a model of the object. Serial microtome sectioning is a tedious and skill-demanding work and therefore seldom done. In the last two decades with the increase of computer power, sophisticated display options, and the development of new instruments, an SEM with a built-in microtome as well as a focused ion beam scanning electron microscope (FIB-SEM), serial sectioning, and 3D analysis has become far easier and faster.Due to the relief like topology of the microtome trimmed block face of resin-embedded tissue, the ROI can be searched in the secondary electron mode, and at the selected spot, the ROI is prepared with the ion beam for 3D analysis. For FIB-SEM tomography, a thin slice is removed with the ion beam and the newly exposed face is imaged with the electron beam, usually by recording the backscattered electrons. The process, also called "slice and view," is repeated until the desired volume is imaged.As FIB-SEM allows 3D imaging of biological fine structure at high resolution of only small volumes, it is crucial to perform slice and view at carefully selected spots. Finding the region of interest is therefore a prerequisite for meaningful imaging. Thin layer plastification of biofilms offers direct access to the original sample surface and allows the selection of an ROI for site-specific FIB-SEM tomography just by its pronounced topographic features.

Rolf Bernander (1956-2014): pioneer of the archaeal cell cycle.

On 19 January 2014 Rolf ('Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour.

Dpp/BMP signaling in flies: From molecules to biology.

Decapentaplegic (Dpp), the fly homolog of the secreted mammalian BMP2/4 signaling molecules, is involved in almost all aspects of fly development. Dpp has critical functions at all developmental stages, from patterning of the eggshell to the determination of adult intestinal stem cell identity. Here, we focus on recent findings regarding the transcriptional regulatory logic of the pathway, on a new feedback regulator, Pentagone, and on Dpp's roles in scaling and growth of the Drosophila wing.

Molecular crime and cellular punishment: active detoxification of misfolded and aggregated proteins in the cell by the chaperone and protease networks.

Labile or mutation-sensitised proteins may spontaneously convert into aggregation-prone conformations that may be toxic and infectious. This hazardous behavior, which can be described as a form of "molecular criminality", can be actively counteracted in the cell by a network of molecular chaperone and proteases. Similar to law enforcement agents, molecular chaperones and proteases can specifically identify, apprehend, unfold and thus neutralize "criminal" protein conformers, allowing them to subsequently refold into harmless functional proteins. Irreversibly damaged polypeptides that have lost the ability to natively refold are preferentially degraded by highly controlled ATP-consuming proteases. Damaged proteins that escape proteasomal degradation can also be "incarcerated" into dense amyloids, "evicted" from the cell, or internally "exiled" to the lysosome to be hydrolysed and recycled. Thus, remarkable parallels exist between molecular and human forms of criminality, as well as in the cellular and social responses to various forms of crime. Yet, differences also exist: whereas programmed death is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom chosen by lawless societies. Significantly, there is no cellular equivalent for the role of familial care and of education in general, which is so crucial to the proper shaping of functional persons in the society. Unlike in the cell, humanism introduces a bias against radical solutions such as capital punishment, favouring crime prevention, reeducation and social reinsertion of criminals.

Hypocretin (orexin) biology and the pathophysiology of narcolepsy with cataplexy.

The discovery of hypocretins (orexins) and their causal implication in narcolepsy is the most important advance in sleep research and sleep medicine since the discovery of rapid eye movement sleep. Narcolepsy with cataplexy is caused by hypocretin deficiency owing to destruction of most of the hypocretin-producing neurons in the hypothalamus. Ablation of hypocretin or hypocretin receptors also leads to narcolepsy phenotypes in animal models. Although the exact mechanism of hypocretin deficiency is unknown, evidence from the past 20 years strongly favours an immune-mediated or autoimmune attack, targeting specifically hypocretin neurons in genetically predisposed individuals. These neurons form an extensive network of projections throughout the brain and show activity linked to motivational behaviours. The hypothesis that a targeted immune-mediated or autoimmune attack causes the specific degeneration of hypocretin neurons arose mainly through the discovery of genetic associations, first with the HLA-DQB1*06:02 allele and then with the T-cell receptor α locus. Guided by these genetic findings and now awaiting experimental testing are models of the possible immune mechanisms by which a specific and localised brain cell population could become targeted by T-cell subsets. Great hopes for the identification of new targets for therapeutic intervention in narcolepsy also reside in the development of patient-derived induced pluripotent stem cell systems.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

The role of integrins in glioma biology and anti-glioma therapies.

The tumor environment is critical for tumor maintenance and progression. Integrins are a large family of cell surface receptors mediating the interaction of tumor cells with their microenvironment and play important roles in glioma biology, including migration, invasion, angiogenesis and tumor stem cell anchorage. Here, we review preclinical and clinical data on integrin inhibition in malignant gliomas. Various pharmacological approaches to the modulation of integrin signaling have been explored including antibodies and peptide-based agents. Cilengitide, a cyclic RGD-mimetic peptide of αvβ3 and αvβ5 integrins is in advanced clinical development in glioblastoma. Cilengitide had only limited activity as a single agent in glioblastoma, but, when added to standard radiochemotherapy, appeared to prolong progression-free and overall survival in patients with newly diagnosed glioblastomas and methylation of the promoter of the O⁶ methylguanine methyltransferase (MGMT) gene. MGMT gene promoter methylation in turn predicts benefit from alkylating chemotherapy. A phase III randomized clinical trial in conjunction with standard radiochemotherapy in newly diagnosed glioblastoma patients with MGMT gene promoter methylation has recently completed accrual (EORTC 26071-22072). A companion trial explores a dose-escalated regimen of cilengitide added to radiotherapy plus temozolomide in patients without MGMT gene promoter methylation. Promising results in these trials would probably result in a broader interest in integrins as targets for glioma therapy and hopefully the development of a broader panel of anti-integrin agents.

The genome sequence of taurine cattle: a window to ruminant biology and evolution.

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.

The CD4-like molecule LAG-3, biology and therapeutic applications.

IMPORTANCE OF THE FIELD: Promising immunotherapeutic agents targeting co-stimulatory pathways are currently being tested in clinical trials. One player in this array of regulatory pathways is the LAG-3/MHC class II axis. The lymphocyte activation gene-3 (LAG-3) is a negative co-stimulatory receptor that modulates T cell homeostasis, proliferation and activation. A recombinant soluble dimeric form of LAG-3 (sLAG-3-Ig, IMP321) shows adjuvant properties and enhances immunogenicity of tumor vaccines. Recent clinical trials produced encouraging results, especially when the human dimeric soluble form of LAG-3 (hLAG-3-Ig) was used in combination with chemotherapy. AREAS COVERED IN THIS REVIEW: The biological relevance of LAG-3 in vivo. Pre-clinical data demonstrating adjuvant properties, as well as the improvement of tumor immunity by sLAG-3-Ig. Recent advances in the clinical development of the therapeutic reagent IMP321, hLAG-3-Ig, for cancer treatment. WHAT THE READER WILL GAIN: This review summarizes preclinical and clinical data on the biological functions of LAG-3. TAKE HOME MESSAGE: The LAG-3 inhibitory pathway is attracting attention, in the light of recent studies demonstrating its role in T cell unresponsiveness, and Treg function after chronic antigen stimulation. As a soluble recombinant dimer, the sLAG-3-Ig protein acts as an adjuvant for therapeutic induction of T cell responses, and may be beneficial to cancer patients when used in combination therapies.

Unification of multi-species vertebrate anatomy ontologies for comparative biology in Uberon.

BACKGROUND: Elucidating disease and developmental dysfunction requires understanding variation in phenotype. Single-species model organism anatomy ontologies (ssAOs) have been established to represent this variation. Multi-species anatomy ontologies (msAOs; vertebrate skeletal, vertebrate homologous, teleost, amphibian AOs) have been developed to represent 'natural' phenotypic variation across species. Our aim has been to integrate ssAOs and msAOs for various purposes, including establishing links between phenotypic variation and candidate genes. RESULTS: Previously, msAOs contained a mixture of unique and overlapping content. This hampered integration and coordination due to the need to maintain cross-references or inter-ontology equivalence axioms to the ssAOs, or to perform large-scale obsolescence and modular import. Here we present the unification of anatomy ontologies into Uberon, a single ontology resource that enables interoperability among disparate data and research groups. As a consequence, independent development of TAO, VSAO, AAO, and vHOG has been discontinued. CONCLUSIONS: The newly broadened Uberon ontology is a unified cross-taxon resource for metazoans (animals) that has been substantially expanded to include a broad diversity of vertebrate anatomical structures, permitting reasoning across anatomical variation in extinct and extant taxa. Uberon is a core resource that supports single- and cross-species queries for candidate genes using annotations for phenotypes from the systematics, biodiversity, medical, and model organism communities, while also providing entities for logical definitions in the Cell and Gene Ontologies. THE ONTOLOGY RELEASE FILES ASSOCIATED WITH THE ONTOLOGY MERGE DESCRIBED IN THIS MANUSCRIPT ARE AVAILABLE AT: http://purl.obolibrary.org/obo/uberon/releases/2013-02-21/ CURRENT ONTOLOGY RELEASE FILES ARE AVAILABLE ALWAYS AVAILABLE AT: http://purl.obolibrary.org/obo/uberon/releases/

Twenty years of research into Chlamydia-like organisms: a revolution in our understanding of the biology and pathogenicity of members of the phylum Chlamydiae.

Chlamydiae are obligate intracellular bacteria that share a unique but remarkably conserved biphasic developmental cycle that relies on a eukaryotic host cell for survival. Although the phylum was originally thought to only contain one family, the Chlamydiaceae, a total of nine families are now recognized. These so-called Chlamydia-like organisms (CLOs) are also referred to as 'environmental chlamydiae', as many were initially isolated from environmental sources. However, these organisms are also emerging pathogens, as many, such as Parachlamydia sp., Simkania sp. and Waddlia sp., have been associated with human disease, and others, such as Piscichlamydia sp. and Parilichlamydia sp., have been documented in association with diseases in animals. Their strict intracellular nature and the requirement for cell culture have been a confounding factor in characterizing the biology and pathogenicity of CLOs. Nevertheless, the genomes of seven CLO species have now been sequenced, providing new information on their potential ability to adapt to a wide range of hosts. As new isolation and diagnostic methods advance, we are able to further explore the richness of this phylum with further research likely to help define the true pathogenic potential of the CLOs while also providing insight into the origins of the 'traditional' chlamydiae.