In this review, we discuss the emerging field of computational behavioral analysis-the use of modern methods from computer science and engineering to quantitatively measure animal behavior. We discuss aspects of experiment design important to both obtaining biologically relevant behavioral data and enabling the use of machine vision and learning techniques for automation. These two goals are often in conflict. Restraining or restricting the environment of the animal can simplify automatic behavior quantification, but it can also degrade the quality or alter important aspects of behavior. To enable biologists to design experiments to obtain better behavioral measurements, and computer scientists to pinpoint fruitful directions for algorithm improvement, we review known effects of artificial manipulation of the animal on behavior. We also review machine vision and learning techniques for tracking, feature extraction, automated behavior classification, and automated behavior discovery, the assumptions they make, and the types of data they work best with. Expected final online publication date for the Annual Review of Neuroscience Volume 39 is July 08, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

The brain is a network of neurons, one that generates behaviour, and knowing the former is crucial to understanding the latter. Identifying the exact network of synaptic connections, or connectome, of the fly's central nervous system is now a major objective in Drosophila neurobiology, one that has been initiated in several laboratories, especially the Janelia Research Campus of the Howard Hughes Medical Institute. Progress is most advanced in the optic neuropiles of the visual system. The effort to derive a connectome from these and other neuropile regions is proceeding by various methods of electron microscopy, especially focused-ion beam milling scanning electron microscopy, and relies upon - but is to be carefully distinguished from - published light microscopic methods that reveal the projections of genetically labelled cell types. The latter reveal those neurons that come into close proximity and are therefore candidate synaptic partners. Synaptic partnerships are not in fact reliably revealed by such candidate pairs, anatomical connections often revealing unexpected pathways. Synaptic partnerships identified from ultrastructural features provide a strong heuristic basis to interpret not only functional interactions between identified neurons, but also a powerful means to predict such interactions, and suggest functional pathways not readily predicted from existing experimental evidence. The analysis of circuit function may proceed cell by cell, by examining the behavioural outcome of either interrupting or restoring function to any one element in an anatomically defined circuit, but can be foiled by degeneracy in pathway elements. Circuit information can also be used to identify and analyse circuit motifs, and their role in higher-order network properties. These attempts in Drosophila anticipate parallel attempts in other systems, notably the inner plexiform layer of the vertebrate retina, and augment the one complete connectome already available to us, that available for 30 years in the nematode Caenorhabditis elegans.

Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles.

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.

Photolabile protecting groups (or "photocages") enable precise spatiotemporal control of chemical functionality and facilitate advanced biological experiments. Extant photocages exhibit a simple input-output relationship, however, where application of light elicits a photochemical reaction irrespective of the environment. Herein, we refine and extend the concept of photolabile groups, synthesizing the first Ca(2+) -sensitive photocage. This system functions as a chemical coincidence detector, releasing small molecules only in the presence of both light and elevated [Ca(2+) ]. Caging a fluorophore with this ion-sensitive moiety yields an "ion integrator" that permanently marks cells undergoing high Ca(2+) flux during an illumination-defined time period. Our general design concept demonstrates a new class of light-sensitive material for cellular imaging, sensing, and targeted molecular delivery.

The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent 'standard candles' for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

Primordial germ cells (PGCs) in many species associate intimately with endodermal cells, but the significance of such interactions is largely unexplored. Here, we show that Caenorhabditis elegans PGCs form lobes that are removed and digested by endodermal cells, dramatically altering PGC size and mitochondrial content. We demonstrate that endodermal cells do not scavenge lobes PGCs shed, but rather, actively remove lobes from the cell body. CED-10 (Rac)-induced actin, DYN-1 (dynamin) and LST-4 (SNX9) transiently surround lobe necks and are required within endodermal cells for lobe scission, suggesting that scission occurs through a mechanism resembling vesicle endocytosis. These findings reveal an unexpected role for endoderm in altering the contents of embryonic PGCs, and define a form of developmentally programmed cell remodelling involving intercellular cannibalism. Active roles for engulfing cells have been proposed in several neuronal remodelling events, suggesting that intercellular cannibalism may be a more widespread method used to shape cells than previously thought.

Previously, we identified that visual and olfactory associative memories of Drosophila share the mushroom body (MB) circuits (Vogt et al. 2014). Despite well-characterized odor representations in the Drosophila MB, the MB circuit for visual information is totally unknown. Here we show that a small subset of MB Kenyon cells (KCs) selectively responds to visual but not olfactory stimulation. The dendrites of these atypical KCs form a ventral accessory calyx (vAC), distinct from the main calyx that receives olfactory input. We identified two types of visual projection neurons (VPNs) directly connecting the optic lobes and the vAC. Strikingly, these VPNs are differentially required for visual memories of color and brightness. The segregation of visual and olfactory domains in the MB allows independent processing of distinct sensory memories and may be a conserved form of sensory representations among insects.

Cataloging the neuronal cell types that comprise circuitry of individual brain regions is a major goal of modern neuroscience and the BRAIN initiative. Single-cell RNA sequencing can now be used to measure the gene expression profiles of individual neurons and to categorize neurons based on their gene expression profiles. While the single-cell techniques are extremely powerful and hold great promise, they are currently still labor intensive, have a high cost per cell, and, most importantly, do not provide information on spatial distribution of cell types in specific regions of the brain. We propose a complementary approach that uses computational methods to infer the cell types and their gene expression profiles through analysis of brain-wide single-cell resolution in situ hybridization (ISH) imagery contained in the Allen Brain Atlas (ABA). We measure the spatial distribution of neurons labeled in the ISH image for each gene and model it as a spatial point process mixture, whose mixture weights are given by the cell types which express that gene. By fitting a point process mixture model jointly to the ISH images, we infer both the spatial point process distribution for each cell type and their gene expression profile. We validate our predictions of cell type-specific gene expression profiles using single cell RNA sequencing data, recently published for the mouse somatosensory cortex. Jointly with the gene expression profiles, cell features such as cell size, orientation, intensity and local density level are inferred per cell type.

Renal peritubular interstitial fibroblast-like cells are critical for adult erythropoiesis, as they are the main source of erythropoietin (EPO). Hypoxia-inducible factor 2 (HIF-2) controls EPO synthesis in the kidney and liver and is regulated by prolyl-4-hydroxylase domain (PHD) dioxygenases PHD1, PHD2, and PHD3, which function as cellular oxygen sensors. Renal interstitial cells with EPO-producing capacity are poorly characterized, and the role of the PHD/HIF-2 axis in renal EPO-producing cell (REPC) plasticity is unclear. Here we targeted the PHD/HIF-2/EPO axis in FOXD1 stroma-derived renal interstitial cells and examined the role of individual PHDs in REPC pool size regulation and renal EPO output. Renal interstitial cells with EPO-producing capacity were entirely derived from FOXD1-expressing stroma, and Phd2 inactivation alone induced renal Epo in a limited number of renal interstitial cells. EPO induction was submaximal, as hypoxia or pharmacologic PHD inhibition further increased the REPC fraction among Phd2-/- renal interstitial cells. Moreover, Phd1 and Phd3 were differentially expressed in renal interstitium, and heterozygous deficiency for Phd1 and Phd3 increased REPC numbers in Phd2-/- mice. We propose that FOXD1 lineage renal interstitial cells consist of distinct subpopulations that differ in their responsiveness to Phd2 inactivation and thus regulation of HIF-2 activity and EPO production under hypoxia or conditions of pharmacologic or genetic PHD inactivation.