DNA tension assays reveal that force-dependent integrin activation regulates neurite outgrowth in primary cortical neurons.

Biomechanical inputs are ubiquitously present in biological systems and are known to regulate various cell functions. In particular, neural cell development is sensitive to mechanical regulation, as these cells reside in one of the softest microenvironments in the body. To fully characterize and comprehend how mechanical force modulates early neuronal processes, we prepared substrates functionalized with DNA probes displaying integrin ligands, including cRGD and laminin, to quantify integrin-mediated molecular tension during neurite initiation in primary cortical neurons. Our live-cell imaging analysis reveals that integrin-mediated tension force is highly dynamic and distributed across the cell body, with the overall tension signal gradually increasing during neurite outgrowth. Notably, we detected a consistent level of mechanical force (amplitude = 4.7-12 piconewtons, pN) for cell integrin-ligand interactions. Further quantifications reveal that neurons exhibit faster cell spreading and neurite outgrowth upon interacting with ligands functionalized with 4.7 pN relative to 12 pN probes. These findings indicate that the magnitude of integrin-mediated mechanical feedback regulates neuronal activity during early neuritogenesis. Additionally, we observed that mechanical tension is correlated with calcium signaling, since inhibiting calcium influx substantially reduced mechanical tension. Thus, our findings support that the magnitude of integrin-mediated mechanical feedback regulates neuronal activity during early neuritogenesis and that mechanical force is an essential element complementing well-known biochemical regulatory mechanisms orchestrating the integrin activation machinery and controlled neurite outgrowth in cortical neurons.

Effectiveness assessment of using water environmental microHI to predict the health status of wild fish.

Aquatic wildlife health assessment is critically important for aquatic wildlife conservation. However, the health assessment of aquatic wildlife (especially aquatic wild animals) is difficult and often accompanied by invasive survey activities and delayed observability. As there is growing evidence that aquatic environmental microbiota could impact the health status of aquatic animals by influencing their symbiotic microbiota, we propose a non-invasive method to monitor the health status of wild aquatic animals using the environmental microbiota health index (microHI). However, it is unknown whether this method is effective for different ecotype groups of aquatic wild animals. To answer this question, we took a case study in the middle Yangtze River and studied the water environmental microbiota and fish gut microbiota at the fish community level, population level, and ecotype level. The results showed that the gut microHI of the healthy group was higher than that of the unhealthy group at the community and population levels, and the overall gut microHI was positively correlated with the water environmental microHI, whereas the baseline gut microHI was species-specific. Integrating these variations in four ecotype groups (filter-feeding, scraper-feeding, omnivorous, and carnivorous), only the gut microHI of the carnivorous group positively correlated with water environmental microHI. Alcaligenaceae, Enterobacteriaceae, and Achromobacter were the most abundant groups with health-negative-impacting phenotypes, had high positive correlations between gut sample group and environment sample group, and had significantly higher abundance in unhealthy groups than in healthy groups of carnivorous, filter-feeding, and scraper-feeding ecotypes. Therefore, using water environmental microHI to indicate the health status of wild fish is effective at the community level, is effective just for carnivorous fish at the ecotype level. In the middle Yangtze River, Alcaligenaceae, Enterobacteriaceae (family level), and Achromobacter (genus level) were the key water environmental microbial groups that potentially impacted wild fish health status. Of course, more data and research that test the current hypothesis and conclusion are encouraged.

Neuronal paxillin and drebrin mediate BDNF-induced force transduction and growth cone turning in a soft-tissue-like environment.

Soft tissue environments govern neuronal morphogenesis. However, the precise molecular mechanisms underlying chemotropism-directed axonal growth cone movement in extremely soft environments remain unclear. Here, we show that drebrin, a growth cone T-zone protein, modulates growth cone turning in response to brain-derived neurotrophic factor (BDNF) coated on a soft substrate. Structurally, axonal growth cones of rodent hippocampal neurons grown on 0.1 kPa hydrogels possess an expanded T zone in which drebrin is highly integrated with both F-actin and microtubules. Biochemically, we identify paxillin as interacting with drebrin in cells grown on 0.1 kPa hydrogels but not on glass coverslips. When grown on 0.1 kPa substrates, growth cones asymmetrically exposed to BDNF-bound stripes exhibit enhanced paxillin-drebrin interaction on the side facing the stripes, an activity that is PKA and AAK1 dependent but independent of Src kinase. Functionally, we show that BDNF-induced growth cone turning and force generation on soft substrates require drebrin phosphorylation and paxillin-drebrin association.

Surface Viscosity-Dependent Neurite Initiation in Cortical Neurons.

Dynamic extracellular environments profoundly affect the behavior and function of cells both biochemically and mechanically. Neurite initiation is the first step for neurons to establish intricate neuronal networks. How such a process is modulated by mechanical factors is not fully understood. Particularly, it is unknown whether the molecular clutch model, which has been used to explain cell responses to matrix rigidity, also holds for neurite initiation. To study how mechanical properties modulate neurite initiation, substrates with various well-defined surface viscosities using supported lipid bilayers (SLBs) are synthesized. The results show that ligands with intermediate viscosity greatly maximize neurite initiation in primary neurons, while neurite initiation is drastically limited on substrates with higher or lower viscosity. Importantly, biochemical characterizations reveal altered focal adhesion and calpain activity are associated with distinct neurite initiation patterns. Collectively, these results indicate that neurite initiation is surface viscosity-dependent; there is an optimal range of surface viscosities to drive neurite initiation. Upon binding to ligands of varying viscosities, calpain activity is differentially triggered and leads to distinct levels of neurite outgrowth. These findings not only enhance the understanding of how extracellular environments regulate neurons, but also demonstrate the potential utility of SLBs for neural tissue engineering applications.

Modeling of a Soft-Rigid Gripper Actuated by a Linear-Extension Soft Pneumatic Actuator.

Soft robot has been one significant study in recent decades and soft gripper is one of the popular research directions of soft robot. In a static gripping system, excessive gripping force and large deformation are the main reasons for damage of the object during the gripping process. For achieving low-damage gripping to the object in static gripping system, we proposed a soft-rigid gripper actuated by a linear-extension soft pneumatic actuator in this study. The characteristic of the gripper under a no loading state was measured. When the pressure was >70 kPa, there was an approximately linear relation between the pressure and extension length of the soft actuator. To achieve gripping force and fingertip displacement control of the gripper without sensors integrated on the finger, we presented a non-contact sensing method for gripping state estimation. To analyze the gripping force and fingertip displacement, the relationship between the pressure and extension length of the soft actuator in loading state was compared with the relationship under a no-loading state. The experimental results showed that the relative error between the analytical gripping force and the measured gripping force of the gripper was ≤2.1%. The relative error between analytical fingertip displacement and theoretical fingertip displacement of the gripper was ≤7.4%. Furthermore, the low damage gripping to fragile and soft objects in static and dynamic gripping tests showed good performance of the gripper. Overall, the results indicated the potential application of the gripper in pick-and-place operations.

The American Paddlefish Genome Provides Novel Insights into Chromosomal Evolution and Bone Mineralization in Early Vertebrates.

Sturgeons and paddlefishes (Acipenseriformes) occupy the basal position of ray-finned fishes, although they have cartilaginous skeletons as in Chondrichthyes. This evolutionary status and their morphological specializations make them a research focus, but their complex genomes (polyploidy and the presence of microchromosomes) bring obstacles and challenges to molecular studies. Here, we generated the first high-quality genome assembly of the American paddlefish (Polyodon spathula) at a chromosome level. Comparative genomic analyses revealed a recent species-specific whole-genome duplication event, and extensive chromosomal changes, including head-to-head fusions of pairs of intact, large ancestral chromosomes within the paddlefish. We also provide an overview of the paddlefish SCPP (secretory calcium-binding phosphoprotein) repertoire that is responsible for tissue mineralization, demonstrating that the earliest flourishing of SCPP members occurred at least before the split between Acipenseriformes and teleosts. In summary, this genome assembly provides a genetic resource for understanding chromosomal evolution in polyploid nonteleost fishes and bone mineralization in early vertebrates.

Ecm29-mediated proteasomal distribution modulates excitatory GABA responses in the developing brain.

Neuronal GABAergic responses switch from excitatory to inhibitory at an early postnatal period in rodents. The timing of this switch is controlled by intracellular Cl- concentrations, but factors determining local levels of cation-chloride cotransporters remain elusive. Here, we report that local abundance of the chloride importer NKCC1 and timely emergence of GABAergic inhibition are modulated by proteasome distribution, which is mediated through interactions of proteasomes with the adaptor Ecm29 and the axon initial segment (AIS) scaffold protein ankyrin G. Mechanistically, both the Ecm29 N-terminal domain and an intact AIS structure are required for transport and tethering of proteasomes in the AIS region. In mice, Ecm29 knockout (KO) in neurons increases the density of NKCC1 protein in the AIS region, a change that positively correlates with a delay in the GABAergic response switch. Phenotypically, Ecm29 KO mice showed increased firing frequency of action potentials at early postnatal ages and were hypersusceptible to chemically induced convulsive seizures. Finally, Ecm29 KO neurons exhibited accelerated AIS developmental positioning, reflecting a perturbed AIS morphological plastic response to hyperexcitability arising from proteasome inhibition, a phenotype rescued by ectopic Ecm29 expression or NKCC1 inhibition. Together, our findings support the idea that neuronal maturation requires regulation of proteasomal distribution controlled by Ecm29.

A robust TDP-43 knock-in mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset degenerative disorder of motor neurons. The diseased spinal cord motor neurons of more than 95% of amyotrophic lateral sclerosis (ALS) patients are characterized by the mis-metabolism of the RNA/DNA-binding protein TDP-43 (ALS-TDP), in particular, the presence of cytosolic aggregates of the protein. Most available mouse models for the basic or translational studies of ALS-TDP are based on transgenic overexpression of the TDP-43 protein. Here, we report the generation and characterization of mouse lines bearing homologous knock-in of fALS-associated mutation A315T and sALS-associated mutation N390D, respectively. Remarkably, the heterozygous TDP-43 (N390D/+) mice but not those heterozygous for the TDP-43 (A315T/+) mice develop a full spectrum of ALS-TDP-like pathologies at the molecular, cellular and behavioral levels. Comparative analysis of the mutant mice and spinal cord motor neurons (MN) derived from their embryonic stem (ES) cells demonstrates that different ALS-associated TDP-43 mutations possess critical ALS-causing capabilities and pathogenic pathways, likely modified by their genetic background and the environmental factors. Mechanistically, we identify aberrant RNA splicing of spinal cord Bcl-2 pre-mRNA and consequent increase of a negative regulator of autophagy, Bcl-2, which correlate with and are caused by a progressive increase of TDP-43, one of the early events associated with ALS-TDP pathogenesis, in the spinal cord of TDP-43 (N390D/+) mice and spinal cord MN derived from their ES cells. The TDP-43 (N390D/+) knock-in mice appear to be an ideal rodent model for basic as well as translational studies of ALS- TDP.

Neurite regrowth stimulation by a red-light spot focused on the neuronal cell soma following blue light-induced retraction.

The interaction of light with biological tissues has been considered for various therapeutic applications. Light-induced neurite growth has the potential to be a clinically useful technique for neuron repair. However, most previous studies used either a large illumination area to accelerate overall neurite growth or employed a light spot to guide a growing neurite. It is not clear if optical stimulation can induce the regrowth of a retracted neurite. In the present work, we used blue light (wavelength: 473 nm) to cause neurite retraction, and we proved that using a red-light (wavelength: 650 nm) spot to illuminate the soma near the junction of the retracted neurite could induce neurite regrowth. As a comparison, we found that green light (wavelength 550 nm) had a 62% probability of inducing neurite regrowth, while red light had a 75% probability of inducing neurite regrowth at the same power level. Furthermore, the neurite regrowth length induced by red light was increased by the pre-treatment with inhibitors of myosin functions. We also observed actin propagation from the soma to the tip of the re-growing neurite following red-light stimulation of the soma. The red light-induced extension and regrowth were abrogated in the calcium-free medium. These results suggest that illumination with a red-light spot on the soma may trigger the regrowth of a neurite after the retraction caused by blue-light illumination.

Draft Genome and Complete Hox-Cluster Characterization of the Sterlet (Acipenser ruthenus).

Background: Sturgeons (Chondrostei: Acipenseridae) are a group of "living fossil" fishes at a basal position among Actinopteri. They have raised great public interest due to their special evolutionary position, species conservation challenges, as well as their highly-prized eggs (caviar). The sterlet, Acipenser ruthenus, is a relatively small-sized member of sturgeons and has been widely distributing in both Europe and Asia. In this study, we performed whole genome sequencing, de novo assembly and gene annotation of the tarlet to construct its draft genome. Findings: We finally obtained a 1.83-Gb genome assembly (BUSCO completeness of 81.6%) from a total of 316.8-Gb raw reads generated by an Illumina Hiseq 2500 platform. The scaffold N50 and contig N50 values reached 191.06 and 18.88 kb, respectively. The sterlet genome was predicted to be comprised of 42.84% repeated sequences and to contain 22,184 protein-coding genes, of which 21,112 (95.17%) have been functionally annotated with at least one hit in public databases. A genetic phylogeny demonstrated that the sterlet is situated in the basal position among ray-finned fishes and 4dTv analysis estimated that a recent whole genome duplication occurred 21.3 million years ago. Moreover, seven Hox clusters carrying 68 Hox genes were characterized in the sterlet. Phylogeny of HoxA clusters in the sterlet and American paddlefish divided these sturgeons into two groups, confirming the independence of each lineage-specific genome duplication in Acipenseridae and Polyodontidae. Conclusions: This draft genome makes up for the lack of genomic and molecular data of the sterlet and its Hox clusters. It also provides a genetic basis for further investigation of lineage-specific genome duplication and the early evolution of ray-finned fishes.

Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.

Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties.

Ascl1 promotes tangential migration and confines migratory routes by induction of Ephb2 in the telencephalon.

During development, cortical interneurons generated from the ventral telencephalon migrate tangentially into the dorsal telencephalon. Although Achaete-scute family bHLH transcription factor 1 (Ascl1) plays important roles in the developing telencephalon, whether Ascl1 regulates tangential migration remains unclear. Here, we found that Ascl1 promoted tangential migration along the ventricular zone/subventricular zone (VZ/SVZ) and intermediate zone (IZ) of the dorsal telencephalon. Distal-less homeobox 2 (Dlx2) acted downstream of Ascl1 in promoting tangential migration along the VZ/SVZ but not IZ. We further identified Eph receptor B2 (Ephb2) as a direct target of Ascl1. Knockdown of EphB2 disrupted the separation of the VZ/SVZ and IZ migratory routes. Ephrin-A5, a ligand of EphB2, was sufficient to repel both Ascl1-expressing cells in vitro and tangentially migrating cortical interneurons in vivo. Together, our results demonstrate that Ascl1 induces expression of Dlx2 and Ephb2 to maintain distinct tangential migratory routes in the dorsal telencephalon.

Type VI adenylyl cyclase negatively regulates GluN2B-mediated LTD and spatial reversal learning.

The calcium-sensitive type VI adenylyl cyclase (AC6) is a membrane-bound adenylyl cyclase (AC) that converts ATP to cAMP under stimulation. It is a calcium-inhibited AC and integrates negative inputs from Ca(2+) and multiple other signals to regulate the intracellular cAMP level. In the present study, we demonstrate that AC6 functions upstream of CREB and negatively controls neuronal plasticity in the hippocampus. Genetic removal of AC6 leads to cyclase-independent and N-terminus of AC6 (AC6N)-dependent elevation of CREB expression, and enhances the expression of GluN2B-containing NMDA receptors in hippocampal neurons. Consequently, GluN2B-dependent calcium signaling and excitatory postsynaptic current, long-term depression, and spatial reversal learning are enhanced in the hippocampus of AC6(-/-) mice without altering the gross anatomy of the brain. Together, our results suggest that AC6 negatively regulates neuronal plasticity by modulating the levels of CREB and GluN2B in the hippocampus.

Stage-Dependent Axon Transport of Proteasomes Contributes to Axon Development.

Axon extension at the growing tip requires elevated local protein supply, with a capability sustainable over long axons in varying environments. The exact mechanisms, however, remain elusive. Here we report that axon-promoting factors elicited a retrograde transport-dependent removal of proteasomes from nascent axon terminals, thereby increasing protein stability at axon tips. Such an effect occurred through phosphorylation of a dynein-interacting proteasome adaptor protein Ecm29. During the transition from immature neurites to nascent axons in cultured hippocampal neurons, live-cell imaging revealed a significant increase of the retrograde axonal transport of fluorescently labeled 20S proteasomes. This retrograde proteasome transport depended on neuron stage and increased with axon lengths. Blockade of retrograde transport caused accumulation of proteasomes, reduction of axon growth, and attenuation of growth-associated Par6 at the axon tip of newly polarized neurons. Our results delineate a regulatory mechanism that controls proteasome abundance via preferential transport required for axon development in newborn neurons.

Relay of cyclin-dependent kinases in the regulation of axonal growth.

One of the most perplexing problems in neuronal morphogenesis is how local polarity signals echo genetic instructions to establish structural and functional asymmetry of neuronal compartments, i.e., axons, dendrites, and synapses. However studying these phenomena is complicated because both genes and the local environment influence the phenotype of developing neurons. Cell cycle-associated nuclear transcription regulators involved in axon extension, for example Cdk12 and Cdk13, thus provide ideal models for connecting spatially separated events at specific developmental time points.

Second messenger signaling for neuronal polarization: cell mechanics-dependent pattern formation.

Neuronal polarization is a critical step in the neuronal morphogenesis. Despite the identification of several evolutionarily conserved factors for neural polarization, the exact mechanisms by which cells initiate and maintain polarity remain to be characterized. Here, we review the recent progress on the roles of second messengers, specifically the cyclic nucleotides and membrane-associated phospholipids, in the initiation, propagation, and integration of polarization signals, and propose an inhibitor-free model for neural polarization. The characteristic features of neuron polarization include the formation of single axon and multiple dendrites. These features involve chemical and mechanical mechanisms such as reaction-diffusion and tug-of-war, by which second messengers can act in concert to initiate and stabilize the cellular asymmetry. Nevertheless, biochemical factors eliciting the long-range inhibition remain ambiguous. Thus, we provide a simple, inhibitor-free model that can incorporate known cytochemical and cytomechanical factors, and produce features of neuronal polarization in environments provided with minimized extracellular regulators.

Molecular phylogeny and conservation priorities of the subfamily Acheilognathinae (Teleostei: Cyprinidae).

It is increasingly accepted that conservation work should consider the evolutionary history of target species. Fishes in the subfamily Acheilognathinae, family Cyprinidae, are, with the exception of three species exclusively distributed in Europe, restricted to Asia and show a distinct spawning behavior in laying their eggs in gill chambers of freshwater mussels. At present, many of the 70 species recognized in this group are facing with serious population decline in China and Japan, and their phylogenetic relationships are not well resolved. In the present study, based on mtDNA cyt b and 12S rRNA gene sequences, we reconstructed a more detailed species-level phylogenetic tree of this group, and assessed species conservation priorities based on their evolutionary distinctiveness. Molecular phylogenetic analyses showed that the Acheilognathinae contains two major clades: Acheilognathus clade and Tanakia-Rhodeus clade. Based on this phylogenetic result, conservation priority analyses were conducted using ED (evolutionary distinctiveness)/HED (heightened evolutionary distinctiveness), and EDGE (evolutionary distinctiveness and global endangeredness)/HEDGE (heightened evolutionary distinctiveness and global endangeredness) methods. The results suggested that T. himantegus, T. lanceolata, A. gracilis, A. imberbis, T. tanago, and A. longipinnis should be ranked as the top-priority species for conservation. According to our results, we also discussed the current conservation efforts of the bitterling fishes and gave suggestions for future work.