NeuronBridge: an intuitive web application for neuronal morphology search across large data sets.

BACKGROUND: Neuroscience research in Drosophila is benefiting from large-scale connectomics efforts using electron microscopy (EM) to reveal all the neurons in a brain and their connections. To exploit this knowledge base, researchers relate a connectome's structure to neuronal function, often by studying individual neuron cell types. Vast libraries of fly driver lines expressing fluorescent reporter genes in sets of neurons have been created and imaged using confocal light microscopy (LM), enabling the targeting of neurons for experimentation. However, creating a fly line for driving gene expression within a single neuron found in an EM connectome remains a challenge, as it typically requires identifying a pair of driver lines where only the neuron of interest is expressed in both. This task and other emerging scientific workflows require finding similar neurons across large data sets imaged using different modalities. RESULTS: Here, we present NeuronBridge, a web application for easily and rapidly finding putative morphological matches between large data sets of neurons imaged using different modalities. We describe the functionality and construction of the NeuronBridge service, including its user-friendly graphical user interface (GUI), extensible data model, serverless cloud architecture, and massively parallel image search engine. CONCLUSIONS: NeuronBridge fills a critical gap in the Drosophila research workflow and is used by hundreds of neuroscience researchers around the world. We offer our software code, open APIs, and processed data sets for integration and reuse, and provide the application as a service at http://neuronbridge.janelia.org .

Rapid reconstruction of neural circuits using tissue expansion and light sheet microscopy.

Brain function is mediated by the physiological coordination of a vast, intricately connected network of molecular and cellular components. The physiological properties of neural network components can be quantified with high throughput. The ability to assess many animals per study has been critical in relating physiological properties to behavior. By contrast, the synaptic structure of neural circuits is presently quantifiable only with low throughput. This low throughput hampers efforts to understand how variations in network structure relate to variations in behavior. For neuroanatomical reconstruction, there is a methodological gulf between electron microscopic (EM) methods, which yield dense connectomes at considerable expense and low throughput, and light microscopic (LM) methods, which provide molecular and cell-type specificity at high throughput but without synaptic resolution. To bridge this gulf, we developed a high-throughput analysis pipeline and imaging protocol using tissue expansion and light sheet microscopy (ExLLSM) to rapidly reconstruct selected circuits across many animals with single-synapse resolution and molecular contrast. Using Drosophila to validate this approach, we demonstrate that it yields synaptic counts similar to those obtained by EM, enables synaptic connectivity to be compared across sex and experience, and can be used to correlate structural connectivity, functional connectivity, and behavior. This approach fills a critical methodological gap in studying variability in the structure and function of neural circuits across individuals within and between species.

Iron Deficiency Anemia Improved by Dental Implantation: A Case Report.

Treatments for improving iron deficiency anemia are generally aimed at increasing oral iron intake and/or administration. Such treatments, however, have been unsuccessful in managing nutritional disorders, including anemia, in patients with masticatory dysfunction caused by impaired occlusion. Nevertheless, few studies have assessed the potential benefits of providing optimal occlusion in such cases. Here, we report a case involving a 53-year-old woman with iron deficiency anemia, wherein we attempted to facilitate efficient mastication by establishing functional occlusion with dental implant placement. The patient was diagnosed with iron deficiency anemia and hospitalized for blood transfusion 2 years before she visited our dental clinic. At the first visit, her hemoglobin (Hb) and mean corpuscular volume values were low; sodium ferrous citrate administration and dietary guidance led to slight improvement. However, blood transfusions and iron supplementation had been ineffective over longer duration. After dental implant placement, her Hb and mean corpuscular volume values were restored and maintained for >4 years without medication. Through this report, we highlight an alternative, non-pharmacological treatment strategy for iron deficiency anemia.

Cellular-resolution gene expression profiling in the neonatal marmoset brain reveals dynamic species- and region-specific differences.

Precise spatiotemporal control of gene expression in the developing brain is critical for neural circuit formation, and comprehensive expression mapping in the developing primate brain is crucial to understand brain function in health and disease. Here, we developed an unbiased, automated, large-scale, cellular-resolution in situ hybridization (ISH)-based gene expression profiling system (GePS) and companion analysis to reveal gene expression patterns in the neonatal New World marmoset cortex, thalamus, and striatum that are distinct from those in mice. Gene-ontology analysis of marmoset-specific genes revealed associations with catalytic activity in the visual cortex and neuropsychiatric disorders in the thalamus. Cortically expressed genes with clear area boundaries were used in a three-dimensional cortical surface mapping algorithm to delineate higher-order cortical areas not evident in two-dimensional ISH data. GePS provides a powerful platform to elucidate the molecular mechanisms underlying primate neurobiology and developmental psychiatric and neurological disorders.

A connectome and analysis of the adult Drosophila central brain.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.

Animal brains of all sizes, from the smallest to the largest, work in broadly similar ways. Studying the brain of any one animal in depth can thus reveal the general principles behind the workings of all brains. The fruit fly Drosophila is a popular choice for such research. With about 100,000 neurons ­ compared to some 86 billion in humans ­ the fly brain is small enough to study at the level of individual cells. But it nevertheless supports a range of complex behaviors, including navigation, courtship and learning. Thanks to decades of research, scientists now have a good understanding of which parts of the fruit fly brain support particular behaviors. But exactly how they do this is often unclear. This is because previous studies showing the connections between cells only covered small areas of the brain. This is like trying to understand a novel when all you can see is a few isolated paragraphs. To solve this problem, Scheffer, Xu, Januszewski, Lu, Takemura, Hayworth, Huang, Shinomiya et al. prepared the first complete map of the entire central region of the fruit fly brain. The central brain consists of approximately 25,000 neurons and around 20 million connections. To prepare the map ­ or connectome ­ the brain was cut into very thin 8nm slices and photographed with an electron microscope. A three-dimensional map of the neurons and connections in the brain was then reconstructed from these images using machine learning algorithms. Finally, Scheffer et al. used the new connectome to obtain further insights into the circuits that support specific fruit fly behaviors. The central brain connectome is freely available online for anyone to access. When used in combination with existing methods, the map will make it easier to understand how the fly brain works, and how and why it can fail to work correctly. Many of these findings will likely apply to larger brains, including our own. In the long run, studying the fly connectome may therefore lead to a better understanding of the human brain and its disorders. Performing a similar analysis on the brain of a small mammal, by scaling up the methods here, will be a likely next step along this path.

Conservation and divergence of related neuronal lineages in the Drosophila central brain.

Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. Drosophila central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, we mapped 18 Drosophila central brain lineages. While we found large aggregate differences between lineages, we also discovered shared patterns of morphological diversification. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.

Evaluation of alkylating pyrrole-imidazole polyamide conjugates by a novel method for high-throughput sequencer.

N-Methylpyrrole-N-methylimidazole (PI) polyamides are a class of DNA minor groove binders with DNA sequence-specificity. DNA-alkylating PI polyamide conjugates are attractive candidates as anticancer drugs acting through DNA damage and its subsequent inhibition of cell proliferation. One example is a chlorambucil-PI polyamide conjugate targeting the runt-related transcription factor (RUNX) family. RUNX1 has pro-oncogenic properties in acute myeloid leukemia, and recently the chlorambucil-PI polyamide conjugate was demonstrated to have anticancer effects. Herein, we apply another DNA-alkylating agent, seco-CBI, to target the consensus sequence of the RUNX family. Two types of CBI conjugates were prepared and their binding properties were characterized by Bind-n-Seq analysis using a high-throughput sequencer. The sequencing data were analyzed by two methods, MERMADE and our new MR (motif identification with a reference sequence), and the resultant binding motif logos were as predicted from the pairing rules proposed by Dervan et al. This is the first report to employ the MR method on alkylating PI polyamide conjugates. Moreover, cytotoxicity of conjugates 3 and 4 against a human non-small cell lung cancer, A549, were examined to show promising IC50s of 120 nm and 63 nm, respectively. These findings suggest seco-CBI-PI polyamide conjugates are candidates for oncological therapy.

The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.

Stem cell polarization is a crucial step in asymmetric cell division, which is a universal system for generating cellular diversity in multicellular organisms. Several conventional genetics studies have attempted to elucidate the mechanisms underlying cell polarization in plants, but it remains largely unknown. In plants, stomata, which are valves for gas exchange, are generated through several rounds of asymmetric divisions. In this study, we identified and characterized a chemical compound that affects stomatal stem cell polarity. High-throughput screening for bioactive molecules identified a pyridine-thiazole derivative, named bubblin, which induced stomatal clustering in Arabidopsis epidermis. Bubblin perturbed stomatal asymmetric division, resulting in the generation of two identical daughter cells. Both cells continued to express the stomatal fate determinant SPEECHLESS, and then differentiated into mispatterned stomata. Bubblin-treated cells had a defect in the polarized localization of BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), which is required for asymmetric cell fate determination. Our results suggest that bubblin induces stomatal lineage cells to divide without BASL-dependent pre-mitotic establishment of polarity. Bubblin is a potentially valuable tool for investigating cell polarity establishment in stomatal asymmetric division.

Comparative Analysis of DNA-Binding Selectivity of Hairpin and Cyclic Pyrrole-Imidazole Polyamides Based on Next-Generation Sequencing.

Many long pyrrole-imidazole polyamides (PIPs) have been synthesized in the search for higher specificity, with the aim of realizing the great potential of such compounds in biological and clinical areas. Among several types of PIPs, we designed and synthesized hairpin and cyclic PIPs targeting identical sequences. Bind-n-Seq analysis revealed that both bound to the intended sequences. However, adenines in the data analyzed by the previously reported Bind-n-Seq method appeared to be significantly higher in the motif ratio than thymines, even though the PIPs were not expected to distinguish A from T. We therefore examined the experimental protocol and analysis pipeline in detail and developed a new method based on Bind-n-Seq motif identification with a reference sequence (Bind-n-Seq-MR). High-throughput sequence analysis of the PIP-enriched DNA data by Bind-n-Seq-MR presented A and T comparably. Surface plasmon resonance assays were performed to validate the new method.

A direction-selective local-thresholding method, DSLT, in combination with a dye-based method for automated three-dimensional segmentation of cells and airspaces in developing leaves.

Quantifying the anatomical data acquired from three-dimensional (3D) images has become increasingly important in recent years. Visualization and image segmentation are essential for acquiring accurate and detailed anatomical data from images; however, plant tissues such as leaves are difficult to image by confocal or multi-photon laser scanning microscopy because their airspaces generate optical aberrations. To overcome this problem, we established a staining method based on Nile Red in silicone-oil solution. Our staining method enables color differentiation between lipid bilayer membranes and airspaces, while minimizing any damage to leaf development. By repeated applications of our staining method we performed time-lapse imaging of a leaf over 5 days. To counteract the drastic decline in signal-to-noise ratio at greater tissue depths, we also developed a local thresholding method (direction-selective local thresholding, DSLT) and an automated iterative segmentation algorithm. The segmentation algorithm uses the DSLT to extract the anatomical structures. Using the proposed methods, we accurately segmented 3D images of intact leaves to single-cell resolution, and measured the airspace volumes in intact leaves.

Microtubules contribute to tubule elongation and anchoring of endoplasmic reticulum, resulting in high network complexity in Arabidopsis.

The endoplasmic reticulum (ER) is a network of tubules and sheet-like structures in eukaryotic cells. Some ER tubules dynamically change their morphology, and others form stable structures. In plants, it has been thought that the ER tubule extension is driven by the actin-myosin machinery. Here, we show that microtubules also contribute to the ER tubule extension with an almost 20-fold slower rate than the actin filament-based ER extension. Treatment with the actin-depolymerizing drug Latrunculin B made it possible to visualize the slow extension of the ER tubules in transgenic Arabidopsis (Arabidopsis thaliana) plants expressing ER-targeted green fluorescent protein. The ER tubules elongated along microtubules in both directions of microtubules, which have a distinct polarity. This feature is similar to the kinesin- or dynein-driven ER tubule extension in animal cells. In contrast to the animal case, ER tubules elongating with the growing microtubule ends were not observed in Arabidopsis. We also found the spots where microtubules are stably colocalized with the ER subdomains during long observations of 1,040 s, suggesting that cortical microtubules contribute to provide ER anchoring points. The anchoring points acted as the branching points of the ER tubules, resulting in the formation of multiway junctions. The density of the ER tubule junction positively correlated with the microtubule density in both elongating cells and mature cells of leaf epidermis, showing the requirement of microtubules for formation of the complex ER network. Taken together, our findings show that plants use microtubules for ER anchoring and ER tubule extension, which establish fine network structures of the ER within the cell.

Identification and dynamics of Arabidopsis adaptor protein-2 complex and its involvement in floral organ development.

The adaptor protein-2 (AP-2) complex is a heterotetramer involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. The homologous genes of AP-2 subunits are present in the genomes of plants; however, their identities and roles in endocytic pathways are not clearly defined in plants. Here, we reveal the molecular composition of the AP-2 complex of Arabidopsis thaliana and its dynamics on the plasma membrane. We identified all of the α-, ß-, σ-, and µ-subunits of the AP-2 complex and detected a weak interaction of the AP-2 complex with clathrin heavy chain. The µ-subunit protein fused to green fluorescent protein (AP2M-GFP) was localized to the plasma membrane and to the cytoplasm. Live-cell imaging using a variable-angle epifluorescence microscope revealed that AP2M-GFP transiently forms punctate structures on the plasma membrane. Homozygous ap2m mutant plants exhibited abnormal floral structures, including reduced stamen elongation and delayed anther dehiscence, which led to a failure of pollination and a subsequent reduction of fertility. Our study provides a molecular basis for understanding AP-2-dependent endocytic pathways in plants and their roles in floral organ development and plant reproduction.

A Li rechargeable battery cathode composed of Li2MnSiO4 nanoparticles in CMK-1.

We loaded nanoparticles of lithium manganese silicate in the mesopores of CMK-1 periodic mesoporous carbon in order to obtain electrode composed of nanoparticles with a good electric conductivity. The structure was analysed by XRD, nitrogen adsorption, and TEM. Two kinds of framework CMK-1 were prepared; CMK-1 (2.0) contains more carbon than CMK-1 (1.0). XRD of these carbons provides two peaks attributed to /41/a structure. The differential curve of discharge profiles of the nanocomposite electrodes LMSx@CMK-1 (1.0) and LMSx@CMK-1 (2.0) has the peaks at 3.2-3.4 V and 4.1 V. These are assigned to the oxidation pairs of Mn2+/Mn3+ and Mn3+/Mn4+, respectively. Assuming that clogging of pores by lithium manganese silicate particles similarly causes the decrease in pore volume both for nitrogen adsorption and Li+ absorption, the net charge-discharge curves for the lithium manganese silicate particles were calculated using those for the composite electrodes and those for the framework carbons without lithium manganese silicate. Then the capacities of lithium manganese silicate nanoparticles are determined to be 240 and 230 mAh/g for LMS0.5@CMK-1 (1.0) and LMS0.5@CMK-1 (2.0), respectively. These results imply that most of the nanoparticles in CMK-1 are susceptible to the electrochemical reaction Li2MnSiO4 = MnSiO4 + 2Li+ + 2e.

[Two cases of advanced colorectal cancer which demonstrated the reversibility of oxaliplatin-mediated increase in splenic volume].

Hepatic sinusoidal injury arises occasionally after oxaliplatin-based chemotherapy. As a result, portal hypertension associated with splenomegaly occurs in some cases. We report two cases of advanced colorectal cancer which showed splenomegaly after administration of oxaliplatin-based chemotherapy. In both cases, mFOLFOX6/bevacizumab was administered as a firstline chemotherapy. Splenic volume was determined by loading the CT images onto a commercially available workstation. In case 1, initial splenic volume was 137.82mL. Two months later, it increased to 160.96mL. After six cycles of chemotherapy, we removed oxaliplatin due to peripheral neuropathy. Consequently, the splenic volume decreased to 151.58mL. Subsequent to the reintroduction of oxaliplatin, the splenic volume increased to 177.48mL. Following two cycles of mFOLFOX6/bevacizumab, oxaliplatin was removed again. In a similar way, splenic volume decreased to 158.52mL. In case 2, initial splenic volume was 105.84mL. Ten months later, it increased to 228.54mL. After administration of mFOLFOX6/bevacizumab, we continued chemotherapy with sLV5FU2/bevacizumab and irinotecan. The splenic volume decreased to 197. 06mL. In conclusion, oxaliplatin- based chemotherapy induces an increase in splenic volume, however, it may be reversible after discontinuation of oxaliplatin.

A chiral wedge molecule inhibits telomerase activity.

In addition to the Watson-Crick double helix, secondary DNA structures are thought to play important roles in a variety of biological processes. One important example is the G-quadruplex structure that is formed at the chromosome ends, which inhibits telomerase activity by blocking its access to telomeres. G-quadruplex structures represent a new class of molecular targets for DNA-interactive compounds that may be useful to target telomeres. Here, we reported the first example of enantioselective recognition of quadruplex DNA by a chiral cyclic helicene. We propose a new ligand-binding cleft between two telomeric human G-quadruplexes linked by a TTA linker. We found that the cyclic helicene M1 exhibited potent inhibitory activity against telomerase.