We describe the isolation and sequencing of Middle East respiratory syndrome coronavirus (MERS-CoV) obtained from a dromedary camel and from a patient who died of laboratory-confirmed MERS-CoV infection after close contact with camels that had rhinorrhea. Nasal swabs collected from the patient and from one of his nine camels were positive for MERS-CoV RNA. In addition, MERS-CoV was isolated from the patient and the camel. The full genome sequences of the two isolates were identical. Serologic data indicated that MERS-CoV was circulating in the camels but not in the patient before the human infection occurred. These data suggest that this fatal case of human MERS-CoV infection was transmitted through close contact with an infected camel.

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here, we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 106 µm3. These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.DOI: http://dx.doi.org/10.7554/eLife.25916.001

The microenvironment of the cochlea is maintained by the barrier between the systemic circulation and the fluids inside the stria vascularis. However, the mechanisms that control the permeability of the intrastrial fluid-blood barrier remain largely unknown. The barrier comprises endothelial cells connected to each other by tight junctions and an underlying basement membrane. In a recent study, we found that the intrastrial fluid-blood barrier also includes a large number of perivascular cells with both macrophage and melanocyte characteristics. The perivascular-resident macrophage-like melanocytes (PVM/Ms) are in close contact with vessels through cytoplasmic processes. Here we demonstrate that PVM/Ms have an important role in maintaining the integrity of the intrastrial fluid-blood barrier and hearing function. Using a cell culture-based in vitro model and a genetically induced PVM/M-depleted animal model, we show that absence of PVM/Ms increases the permeability of the intrastrial fluid-blood barrier to both lowand high-molecular-weight tracers. The increased permeability is caused by decreased expression of pigment epithelial-derived factor, which regulates expression of several tight junction-associated proteins instrumental to barrier integrity. When tested for endocochlear potential and auditory brainstem response, PVM/ M-depleted animals show substantial drop in endocochlear potential with accompanying hearing loss. Our results demonstrate a critical role for PVM/Ms in regulating the permeability of the intrastrial fluid-blood barrier for establishing a normal endocochlear potential hearing threshold. mouse cochlea | paracellular permeability | tight junction | capillary T he intrastrial fluid-blood barrier separates the stria vascularis (SV) from peripheral circulation. The integrity of the barrier is critical for maintaining inner ear homeostasis, especially for sustaining the endocochlear potential (EP), an essential driving force for hearing function (1-4). Disruption of the barrier is closely associated with a number of hearing disorders, including autoimmune inner ear disease, noise-induced hearing loss, agerelated hearing loss, and several genetically linked diseases (5-10). Despite the importance of the intrastrial fluid-blood barrier, little is understood about regulation of the barrier and the mechanisms that control its permeability.In the classic view, the intrastrial fluid-blood barrier comprises basement membrane and endothelial cells (ECs) that connect to each other with tight junctions (11) to form a diffusion barrier that prevents most blood-borne substances from entering the ear (2). In a recent study, we found that the intrastrial fluid-blood barrier also includes a large number of pericytes and perivascular-resident macrophage-like melanocytes (PVM/Ms) (12, 13). The PVM/Ms are not observed in other capillary regions such as in capillary beds of the spiral ligament. The PVM/Ms are in close contact with vessels through cytoplasmic processes. The structural complexity of PVM/Ms' capillar...

Hair bundles of the inner ear have a unique structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis, and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin crosslinkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.