While major inroads have been made in identifying the genetic causes of rare Mendelian disorders, little progress has been made in the discovery of common gene variations that predispose to complex diseases. The single gene variants that have been shown to associate reproducibly with complex diseases typically have small effect sizes or attributable risks. However, the joint actions of common gene variants within pathways may play a major role in predisposing to complex diseases (the paradigm of complex genetics). The goal of this study was to determine whether polymorphism in a candidate pathway (axon guidance) predisposed to a complex disease (Parkinson disease [PD]). We mined a whole-genome association dataset and identified single nucleotide polymorphisms (SNPs) that were within axon-guidance pathway genes. We then constructed models of axon-guidance pathway SNPs that predicted three outcomes: PD susceptibility (odds ratio = 90.8, p = 4.64 x 10(-38)), survival free of PD (hazards ratio = 19.0, p = 5.43 x 10(-48)), and PD age at onset (R(2) = 0.68, p = 1.68 x 10(-51)). By contrast, models constructed from thousands of random selections of genomic SNPs predicted the three PD outcomes poorly. Mining of a second whole-genome association dataset and mining of an expression profiling dataset also supported a role for many axon-guidance pathway genes in PD. These findings could have important implications regarding the pathogenesis of PD. This genomic pathway approach may also offer insights into other complex diseases such as Alzheimer disease, diabetes mellitus, nicotine and alcohol dependence, and several cancers.

Following injury, the mammalian spinal cord forms a glial scar and fails to regenerate. In contrast, vertebrate fish spinal cord tissue regenerates significantly to restore function. Cord transection in zebrafish (Danio rerio) initially causes paralysis and neural cell death. Subsequently, ependymal glia proliferate, bipolar glia extend across the lesion, and new neurons are born; axons from spared and nascent neurons extend along trans-lesional glial bridges to restore functional connectivity. Here we report that glucocorticoids, used in the clinical management of spinal cord injury, directly inhibit neural repair by targeting ependymal glia independently of hematogenous cells and microglia. After transecting injury, the glucocorticoid receptor in ependymal glia is regulated differentially in zebrafish (becoming inactive) vs. the rat (becoming active). Glucocorticoid blockade of neural regeneration via a direct effect on ependymal glia has important therapeutic implications for the putative benefit of corticosteroids in early management of spinal cord injury.

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

Porous titanium (P_Ti) is considered as an effective material for bone scaffold to achieve a stiffness reduction. Herein, biomimetic (bio-)scaffolds were made of sintered P_Ti, which used NaCl as the space holder and had it removed via the hydrothermal method. X-ray diffraction results showed that the subsequent sintering temperature of 1000 °C was the optimized temperature for preparing P_Ti. The compressive strength of P_Ti was measured using a compression test, which revealed an excellent load-bearing ability of above 70 MPa for that with an addition of 50 wt % NaCl (P_Ti_50). The nano-hardness of P_Ti, tested upon their solid surface, was presumably consistent with the density of pores vis-à-vis the addition of NaCl. Overall, a load-bearable P_Ti with a highly porous structure (e.g., P_Ti_50 with a porosity of 43.91% and a pore size around 340 μm) and considerable compressive strength could be obtained through the current process. Cell proliferation (MTS) and lactate dehydrogenase (LDH) assays showed that all P_Ti samples exhibited high cell affinity and low cell mortality, indicating good biocompatibility. Among them, P_Ti_50 showed relatively good in-cell morphology and viability, and is thus promising as a load-bearable bio-scaffold.

In repairing or replacing damaged bones, a dual concentric porous titanium scaffold (P-Tix-y) has emerged as a promising bio-mimic design. Herein, various P-Tix-y were made and sintered with relatively dense (x = 10, 20, or 30% porosity) and loose (y = 45, 55, or 65 porosity) structures. Firstly, NaCl was used as the pore-forming additive and followed by a hydrothermal removal method. The compressive strength of the as-formed P-Tix_y and surface morphology, nanomechanical property, and cells' affinity on the cross-sectioned surface of P-Tix_y (CP-Tix_y) were then characterized. The results demonstrate that the compressive strength of P-Ti10_45, P-Ti20_45, or P-Ti20_55 exhibits a relatively mild decline (e.g., in the range of 181 and 97 MPa, higher than the required value of 70 MPa) and suitable porosities for the intended structure. Nano-hardness on the solid surface of CP-Tix_y shows roughly consistent with that of CP-Ti (i.e., ~8.78 GPa), thus, the porous structure of CP-Tix_y remains mostly unaffected by the addition of NaCl and subsequent sintering process. Most of the surfaces of CP-Tix_y exhibit high fibroblast (L929) cell affinity with low cell mortality. Notably, in the hFOB 1.19 cell adhesion and proliferation test, CP-Ti20_55 and CP-Ti20_65 reveal high cell viability, most probably relating with the assembly of dual porosities with interconnected pores. Overall, the sample P-Ti20_55 provides a relatively load-bearable design with high cell affinity and is thus promising as a three-dimensional bio-scaffold.

Schwannomas are common peripheral nerve sheath tumors that can cause severe morbidity given their stereotypic intracranial and paraspinal locations. Similar to many solid tumors, schwannomas and other nerve sheath tumors are primarily thought to arise due to aberrant hyperactivation of the RAS growth factor signaling pathway. Here, we sought to further define the molecular pathogenesis of schwannomas.

The vertebrate stress response (SR) is mediated by the hypothalamic-pituitary-adrenal (HPA) axis and contributes to generating context appropriate physiological and behavioral changes. Although the HPA axis plays vital roles both in stressful and basal conditions, research has focused on the response under stress. To understand broader roles of the HPA axis in a changing environment, we characterized an adaptive behavior of larval zebrafish during ambient illumination changes. The glucocorticoid receptor (nr3c1) was necessary to maintain basal locomotor activity in light and darkness. The HPA axis was required to adapt to light more efficiently but became dispensable when longer illumination was provided. Light adaptation was more efficient in dimmer light and did not require the mineralocorticoid receptor (nr3c2). Our findings show that the HPA axis contributes to the SR at various stages, facilitating the phasic response and maintaining an adapted basal state, and that certain adaptations occur without HPA axis activity.

A method using label-free surface enhanced Raman spectroscopy (SERS) based on substrate design is provided for an early detection and differentiation of spike glycoprotein mutation sites in live SARS-CoV-2 variants. Two SERS-active substrates, Au nanocavities (Au NCs) and Au NPs on porous ZrO2 (Au NPs/pZrO2), were used to identify specific peaks of A.3, Alpha, and Delta variants at different concentrations and demonstrated the ability to provide their SERS spectra with detection limits of 0.1-1.0% (or 104-5 copies/mL). Variant identification can be achieved by cross-examining reference spectra and analyzing the substrate-analyte relationship between the suitability of the analyte upon the hotspot(s) formed at high concentrations and the effective detection distance at low concentrations. Mutation sites on the S1 chain of the spike glycoprotein for each variant may be related and distinguishable. This method does not require sample preprocessing and therefore allows for fast screening, which is of high value for more comprehensive and specific studies to distinguish upcoming variants.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of approximately 15 months. Semaphorin 3A (Sema3A), known for its axon guidance and antiangiogenic properties, has been implicated in GBM growth. We hypothesized that Sema3A directly inhibits brain tumor stem cell (BTSC) proliferation and drives invasion via Neuropilin 1 (Nrp1) and Plexin A1 (PlxnA1) receptors.

Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr(-1) s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day(-1) growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca(2+)-imaging revealed local elevation of cytoplasmic Ca(2+) concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca(2+) signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro, and complement observations made in vivo.

Glioblastoma multiforme (GBM) is the most common primary brain tumor, with a median survival of only 15 months. A subpopulation of cells, the brain tumor stem cells (BTSCs), may be responsible for the malignancy of this disease. Xenografts have proven to be a robust model of human BTSCs, but the effects of long-term passaging have yet to be determined. Here we present a study detailing changes in BTSC multipotency, invasive migration, and proliferation after serial passaging of human GBM xenografts. Immunocytochemistry and tumorsphere formation assays demonstrated the presence of BTSCs in both early generation (EG-BTSCs; less than 15 passages) and late generation (LG-BTSCs; more than 24 passages) xenografts. The EG-BTSCs upregulated expression of lineage markers for neurons and oligodendrocytes upon differentiation, indicating multipotency. In contrast, the LG-BTSCs were restricted to an astrocytic differentiation. Quantitative migration and proliferation assays showed that EG-BTSCs are more migratory and proliferative than LG-BTSCs. However, both populations respond similarly to the chemokine SDF-1 by increasing invasive migration. These differences between the EG- and LG-BTSCs were correlated with a significant decrease in nanog expression as determined by qRT-PCR. Mice implanted intracranially with EG-BTSCs showed shorter survival when compared to LG-BTSCs. Moreover, differentiation prior to implantation of EG-BTSCs, but not LG-BTSCs, led to increased survival. Thus, nanog may identify multipotent BTSCs. Furthermore, limited passaging of xenografts preserves these multipotent BTSCs, which may be an essential underlying feature of GBM lethality.

Neutrophils and other leukocytes invade the mouse uterus at term birth, which is normal for activating the uterus for labor. To better understand the regulation of this migration at term and interleukin (IL)-1β-induced preterm birth, we developed a mouse leukocyte migration assay (mLMA) and used it with rytvela, an IL-1 receptor allosteric antagonist. The mLMA uses term peripheral blood leukocytes that migrate in a Boyden chamber in response to a chemoattractant. We tested several mouse uterine tissues after homogenization and sedimentation of debris for chemoattractant activity. The most active chemoattractant homogenate came from the mouse lower uterus on gestational day (GD) 18.5. Using flow cytometry, we demonstrated that 99% of the cells that migrate are neutrophils. IL-1β administered on GD 16 stimulated neutrophil migration and invasion into the uterus and the fetal brain along with preterm birth on GD 17. Preterm birth and the increased leukocyte invasion of the maternal uterus and fetal brain were all blocked by the co-administration of rytvela. To test where the site of IL-1β action might be, we examined the potency of lower uterine chemoattractant and the activation of leukocytes following IL-1β +/- rytvela administration. IL-1β did not increase lower uterus homogenate chemoattractant activity, but it significantly (p < 0.05) increased leukocyte activation as defined by cytokine and chemokine expression. Rytvela blocked this activation of leukocytes by IL-1β. We conclude that IL-1β stimulates preterm birth in mice by increasing leukocyte activation leading to increased uterine and fetal brain leukocyte invasion.