Structural organization of gap junction channels

AbstractGap junctions were initially described morphologically, and identified as semi-crystalline arrays of channels linking two cells. This suggested that they may represent an amenable target for electron and X-ray crystallographic studies in much the same way that bacteriorhodopsin has. Over 30 years later, however, an atomic resolution structural solution of these unique intercellular pores is still lacking due to many challenges faced in obtaining high expression levels and purification of these structures. A variety of microscopic techniques, as well as NMR structure determination of fragments of the protein, have now provided clearer and correlated views of how these structures are assembled and function as intercellular conduits. As a complement to these structural approaches, a variety of mutagenic studies linking structure and function have now allowed molecular details to be superimposed on these lower resolution structures, so that a clearer image of pore architecture and its modes of regulation are beginning to emerge

Inner ear tissue preservation by rapid freezing: improving fixation by high-pressure freezing and hybrid methods

In the preservation of tissues in as ‘close to life’ state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues

Organizing the Curriculum for Labor Consciousness

Research on labor and its treatment in the curriculum of K-12 schools has not been a popular topic. Society’s emphasis on individualism and consumerism has fostered veneration of capitalism throughout public education, with business control of the education policy system. Critical information about the US Labor Movement has been systematically excluded from the public school curriculum, so that labor’s centrality to the flow of history and its contributions to the present status of working people are underappreciated, and neoliberalism threatens public education and teacher unionism around the world. This article describes why and how an alliance of teacher educators, teachers, and unionists are advocating for labor consciousness to be infused in to K-12 schooling. This perspective is presented in Organizing the Curriculum, an edited collection of essays, and is being implemented by the Education & Labor Collaborative, an advocacy group to promote economic, social and political empowerment through education for labor consciousness

Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and are difficult to use2. Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed3,4. MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix

Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments[superscript 1] or require light and can be difficult to use[superscript 2]. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed[superscript 3, 4]. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.National Institutes of Health (U.S.) (Grant DP1 OD003961)National Science Foundation (U.S.). Graduate Research Fellowship ProgramUnited States. Dept. of Defense (National Defense Science and Engineering Graduate (NDSEG) Fellowships

Clinical application of tumour-in-normal contamination assessment from whole genome sequencing

The unexpected contamination of normal samples with tumour cells reduces variant detection sensitivity, compromising downstream analyses in canonical tumour-normal analyses. Leveraging whole-genome sequencing data available at Genomics England, we develop a tool for normal sample contamination assessment, which we validate in silico and against minimal residual disease testing. From a systematic review of 771 patients with haematological malignancies and sarcomas, we find contamination across a range of cancer clinical indications and DNA sources, with highest prevalence in saliva samples from acute myeloid leukaemia patients, and sorted CD3+ T-cells from myeloproliferative neoplasms. Further exploration reveals 108 hotspot mutations in genes associated with haematological cancers at risk of being subtracted by standard variant calling pipelines. Our work highlights the importance of contamination assessment for accurate somatic variants detection in research and clinical settings, especially with large-scale sequencing projects being utilised to deliver accurate data from which to make clinical decisions for patient care