Cytotoxic rhamnolipid micelles drive acute virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that has developed multi- or even pan-drug resistance toward most frontline and last resort antibiotics, leading to increasing frequency of infections and deaths among hospitalized patients, especially those with compromised immune systems. Further complicating treatment, P. aeruginosa produces numerous virulence factors that contribute to host tissue damage and immune evasion, promoting bacterial colonization and pathogenesis. In this study, we demonstrate the importance of rhamnolipid production in host-pathogen interactions. Secreted rhamnolipids form micelles that exhibited highly acute toxicity toward murine macrophages, rupturing the plasma membrane and causing organellar membrane damage within minutes of exposure. While rhamnolipid micelles (RMs) were particularly toxic to macrophages, they also caused membrane damage in human lung epithelial cells, red blood cells, Gram-positive bacteria, and even noncellular models like giant plasma membrane vesicles. Most importantly, rhamnolipid production strongly correlated with P. aeruginosa virulence against murine macrophages in various panels of clinical isolates. Altogether, our findings suggest that rhamnolipid micelles are highly cytotoxic virulence factors that drive acute cellular damage and immune evasion during P. aeruginosa infections.

The molecular complex of ciliary and golgin protein is crucial for skull development.

Intramembranous ossification, which consists of direct conversion of mesenchymal cells to osteoblasts, is a characteristic process in skull development. One crucial role of these osteoblasts is to secrete collagen-containing bone matrix. However, it remains unclear how the dynamics of collagen trafficking is regulated during skull development. Here, we reveal the regulatory mechanisms of ciliary and golgin proteins required for intramembranous ossification. During normal skull formation, osteoblasts residing on the osteogenic front actively secreted collagen. Mass spectrometry and proteomic analysis determined endogenous binding between ciliary protein IFT20 and golgin protein GMAP210 in these osteoblasts. As seen in Ift20 mutant mice, disruption of neural crest-specific GMAP210 in mice caused osteopenia-like phenotypes due to dysfunctional collagen trafficking. Mice lacking both IFT20 and GMAP210 displayed more severe skull defects compared with either IFT20 or GMAP210 mutants. These results demonstrate that the molecular complex of IFT20 and GMAP210 is essential for the intramembranous ossification during skull development.

Expression of Nephronectin in the Descemet's membrane of mouse corneas during development and adult homeostasis.

Nephronectin (Npnt) is an extracellular matrix (ECM) protein with pleiotropic functions during organogenesis, disease, and homeostasis. Although the ECM plays a crucial role during development and homeostasis of the adult cornea, little is known about the expression of Npnt in the mammalian cornea. Here, we investigated the expression of Npnt during early embryonic and postnatal development, and in adult mouse corneas. We combined ultrastructural and immunohistochemical analyses to study the early formation of the Descemet's membrane and how the expression of Npnt relates to key basement membrane proteins. Our section in situ hybridization and immunohistochemical analyses revealed that Npnt mRNA is expressed by the nascent corneal endothelial cells at embryonic day (E) 14.5, whereas the protein is localized in the adjacent extracellular matrix. These expression patterns were maintained in the corneal endothelium and Descemet's membrane throughout development and in adult corneas. Ultrastructural analysis revealed discontinuous electron dense regions of protein aggregates at E18.5 that was separated from the endothelial layer by an electron lucent space. At birth (postnatal day, P0), the Descemet's membrane was a single layer, which continuously thickened throughout P4, P8, P10, and P14. Npnt was localized to the Descemet's membrane by E18.5 and overlapped with Collagens IV and VIII, Laminin, and Perlecan. However, the proteins subsequently shifted and formed distinct layers in the adult cornea, whereby Npnt localized between two Collagen VIII bands and anterior to Collagen IV but overlapped with Laminin and Perlecan. Combined, our results reveal the expression of Npnt in the mouse cornea and define its spatiotemporal localization relative to key basement membrane proteins during the formation of the Descemet's membrane and in the adult cornea. Understanding the spatiotemporal expression of Npnt is important for future studies to elucidate its function in the mammalian cornea.

The Tardigrade damage suppressor protein Dsup promotes DNA damage in neurons.

Tardigrades are microscopic invertebrates, which are capable of withstanding extreme environmental conditions, including high levels of radiation. A Tardigrade protein, Dsup (Damage Suppressor), protects the Tardigrade's DNA during harsh environmental stress and X-rays. When expressed in cancer cells, Dsup protects DNA from single- and double-strand breaks (DSBs) induced by radiation, increases survival of irradiated cells, and protects DNA from reactive oxygen species. These unusual properties of Dsup suggested that understanding how the protein functions may help in the design of small molecules that could protect humans during radiotherapy or space travel. Here, we investigated if Dsup is protective in cortical neurons cultured from rat embryos. We discovered that, in cortical neurons, the codon-optimized Dsup localizes to the nucleus and, surprisingly, promotes neurotoxicity, leading to neurodegeneration. Unexpectedly, we found that Dsup expression results in the formation of DNA DSBs in cultured neurons. With electron microscopy, we discovered that Dsup promotes chromatin condensation. Unlike Dsup's protective properties in cancerous cells, in neurons, Dsup promotes neurotoxicity, induces DNA damage, and rearranges chromatin. Neurons are sensitive to Dsup, and Dsup is a doubtful surrogate for DNA protection in neuronal cells.

Evolution of cisplatin resistance through coordinated metabolic reprogramming of the cellular reductive state.

BACKGROUND: Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring. METHODS: To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics. RESULTS: Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function. CONCLUSIONS: Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways.

Disruption of Trip11 in cranial neural crest cells is associated with increased ER and Golgi stress contributing to skull defects in mice.

BACKGROUND: Absence of Golgi microtubule-associated protein 210 (GMAP210), encoded by the TRIP11 gene, results in achondrogenesis. Although TRIP11 is thought to be specifically required for chondrogenesis, human fetuses with the mutation of TRIP11 also display bony skull defects where chondrocytes are usually not present. This raises an important question of how TRIP11 functions in bony skull development. RESULTS: We disrupted Trip11 in neural crest-derived cell populations, which are critical for developing skull in mice. In Trip11 mutant skulls, expression levels of ER stress markers were increased compared to controls. Morphological analysis of electron microscopy data revealed swollen ER in Trip11 mutant skulls. Unexpectedly, we also found that Golgi stress increased in Trip11 mutant skulls, suggesting that both ER and Golgi stress-induced cell death may lead to osteopenia-like phenotypes in Trip11 mutant skulls. These data suggest that Trip11 plays pivotal roles in the regulation of ER and Golgi stress, which are critical for osteogenic cell survival. CONCLUSION: We have recently reported that the molecular complex of ciliary protein and GMAP210 is required for collagen trafficking. In this paper, we further characterized the important role of Trip11 being possibly involved in the regulation of ER and Golgi stress during skull development.

Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network.

ABBREVIATIONS: atl atlastin; ALR autophagic lysosome reformation; ER endoplasmic reticulum; GFP green fluorescent protein; HSP hereditary spastic paraplegia; Lamp1 lysosomal associated membrane protein 1 PolyUB polyubiquitin; RFP red fluorescent protein; spin spinster; mTor mechanistic Target of rapamycin; VCP valosin containing protein.

50-nm Gas-Filled Protein Nanostructures to Enable the Access of Lymphatic Cells by Ultrasound Technologies.

Ultrasound imaging and ultrasound-mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need for microbubbles, which cannot transverse many biological barriers due to their large size. Here, the authors introduce 50-nm gas-filled protein nanostructures derived from genetically engineered gas vesicles(GVs) that are referred to as 50 nmGVs. These diamond-shaped nanostructures have hydrodynamic diameters smaller than commercially available 50-nm gold nanoparticles and are, to the authors' knowledge, the smallest stable, free-floating bubbles made to date. 50 nmGVs can be produced in bacteria, purified through centrifugation, and remain stable for months. Interstitially injected 50 nmGVs can extravasate into lymphatic tissues and gain access to critical immune cell populations, and electron microscopy images of lymph node tissues reveal their subcellular location in antigen-presenting cells adjacent to lymphocytes. The authors anticipate that 50 nmGVs can substantially broaden the range of cells accessible to current ultrasound technologies and may generate applications beyond biomedicine as ultrasmall stable gas-filled nanomaterials.

Phase transition of GvpU regulates gas vesicle clustering in bacteria.

Gas vesicles (GVs) are microbial protein organelles that support cellular buoyancy. GV engineering has multiple applications, including reporter gene imaging, acoustic control and payload delivery. GVs often cluster into a honeycomb pattern to minimize occupancy of the cytosol. The underlying molecular mechanism and the influence on cellular physiology remain unknown. Using genetic, biochemical and imaging approaches, here we identify GvpU from Priestia megaterium as a protein that regulates GV clustering in vitro and upon expression in Escherichia coli. GvpU binds to the C-terminal tail of the core GV shell protein and undergoes a phase transition to form clusters in subsaturated solution. These properties of GvpU tune GV clustering and directly modulate bacterial fitness. GV variants can be designed with controllable sensitivity to GvpU-mediated clustering, enabling design of genetically tunable biosensors. Our findings elucidate the molecular mechanisms and functional roles of GV clustering, enabling its programmability for biomedical applications.

Three-dimensional printing of wood.

Natural wood has served as a foundational material for buildings, furniture, and architectural structures for millennia, typically shaped through subtractive manufacturing techniques. However, this process often generates substantial wood waste, leading to material inefficiency and increased production costs. A potential opportunity arises if complex wood structures can be created through additive processes. Here, we demonstrate an additive-free, water-based ink made of lignin and cellulose, the primary building blocks of natural wood, that can be used to three-dimensional (3D) print architecturally designed wood structures via direct ink writing. The resulting printed structures, after heat treatment, closely resemble the visual, textural, olfactory, and macro-anisotropic properties, including mechanical properties, of natural wood. Our results pave the way for 3D-printed wooden construction with a sustainable pathway to upcycle/recycle natural wood.

Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis.

Analysis of lung alveolar type 2 (AT2) progenitor stem cells has highlighted fundamental mechanisms that direct their differentiation into alveolar type 1 cells (AT1s) in lung repair and disease. However, microRNA (miRNA) mediated post-transcriptional mechanisms which govern this nexus remain understudied. We show here that the let-7 miRNA family serves a homeostatic role in governance of AT2 quiescence, specifically by preventing the uncontrolled accumulation of AT2 transitional cells and by promoting AT1 differentiation to safeguard the lung from spontaneous alveolar destruction and fibrosis. Using mice and organoid models with genetic ablation of let-7a1/let-7f1/let-7d cluster (let-7afd) in AT2 cells, we demonstrate prevents AT1 differentiation and results in aberrant accumulation of AT2 transitional cells in progressive pulmonary fibrosis. Integration of enhanced AGO2 UV-crosslinking and immunoprecipitation sequencing (AGO2-eCLIP) with RNA-sequencing from AT2 cells uncovered the induction of direct targets of let-7 in an oncogene feed-forward regulatory network including BACH1/EZH2 which drives an aberrant fibrotic cascade. Additional analyses by CUT&RUN-sequencing revealed loss of let-7afd hampers AT1 differentiation by eliciting aberrant histone EZH2 methylation which prevents the exit of AT2 transitional cells into terminal AT1s. This study identifies let-7 as a key gatekeeper of post-transcriptional and epigenetic chromatin signals to prevent AT2-driven pulmonary fibrosis.

50-nm gas-filled protein nanostructures to enable the access of lymphatic cells by ultrasound technologies.

Ultrasound imaging and ultrasound-mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need of microbubbles, which cannot transverse many biological barriers due to their large size. Here we introduce 50-nm gas-filled protein nanostructures derived from genetically engineered gas vesicles that we referred to as 50nm GVs. These diamond-shaped nanostructures have hydrodynamic diameters smaller than commercially available 50-nm gold nanoparticles and are, to our knowledge, the smallest stable, free-floating bubbles made to date. 50nm GVs can be produced in bacteria, purified through centrifugation, and remain stable for months. Interstitially injected 50nm GVs can extravasate into lymphatic tissues and gain access to critical immune cell populations, and electron microscopy images of lymph node tissues reveal their subcellular location in antigen-presenting cells adjacent to lymphocytes. We anticipate that 50nm GVs can substantially broaden the range of cells accessible to current ultrasound technologies and may generate applications beyond biomedicine as ultrasmall stable gas-filled nanomaterials.

Cytotoxic rhamnolipid micelles drive acute virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that has developed multi- or even pan-drug resistance towards most frontline and last resort antibiotics, leading to increasing infections and deaths among hospitalized patients, especially those with compromised immune systems. Further complicating treatment, P. aeruginosa produces numerous virulence factors that contribute to host tissue damage and immune evasion, promoting bacterial colonization and pathogenesis. In this study, we demonstrate the importance of rhamnolipid production in host-pathogen interactions. Secreted rhamnolipids form micelles that exhibited highly acute toxicity towards murine macrophages, rupturing the plasma membrane and causing organellar membrane damage within minutes of exposure. While rhamnolipid micelles (RMs) were particularly toxic to macrophages, they also caused membrane damage in human lung epithelial cells, red blood cells, Gram-positive bacteria, and even non-cellular models like giant plasma membrane vesicles. Most importantly, rhamnolipid production strongly correlated to P. aeruginosa virulence against murine macrophages in various panels of clinical isolates. Altogether, our findings suggest that rhamnolipid micelles are highly cytotoxic virulence factors that drive acute cellular damage and immune evasion during P. aeruginosa infections.

Filamentous virus-like particles are present in coral dinoflagellates across genera and ocean basins.

Filamentous viruses are hypothesized to play a role in stony coral tissue loss disease (SCTLD) through infection of the endosymbiotic dinoflagellates (Family Symbiodiniaceae) of corals. To evaluate this hypothesis, it is critical to understand the global distribution of filamentous virus infections across the genetic diversity of Symbiodiniaceae hosts. Using transmission electron microscopy, we demonstrate that filamentous virus-like particles (VLPs) are present in over 60% of Symbiodiniaceae cells (genus Cladocopium) within Pacific corals (Acropora hyacinthus, Porites c.f. lobata); these VLPs are more prevalent in Symbiodiniaceae of in situ colonies experiencing heat stress. Symbiodiniaceae expelled from A. hyacinthus also contain filamentous VLPs, and these cells are more degraded than their in hospite counterparts. Similar to VLPs reported from SCTLD-affected Caribbean reefs, VLPs range from ~150 to 1500 nm in length and 16-37 nm in diameter and appear to constitute various stages in a replication cycle. Finally, we demonstrate that SCTLD-affected corals containing filamentous VLPs are dominated by diverse Symbiodiniaceae lineages from the genera Breviolum, Cladocopium, and Durusdinium. Although this study cannot definitively confirm or refute the role of filamentous VLPs in SCTLD, it demonstrates that filamentous VLPs are not solely observed in SCTLD-affected corals or reef regions, nor are they solely associated with corals dominated by members of a particular Symbiodiniaceae genus. We hypothesize that filamentous viruses are a widespread, common group that infects Symbiodiniaceae. Genomic characterization of these viruses and empirical tests of the impacts of filamentous virus infection on Symbiodiniaceae and coral colonies should be prioritized.

Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment.

Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in ~45% of patients. Unfortunately, TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. We previously demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual TNBC cells surviving NACT. We sought to investigate the mechanism underlying this enhanced reliance on mitochondrial metabolism. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. The functional impact of mitochondrial structure on metabolic output is highly context dependent. Several chemotherapy agents are conventionally used for neoadjuvant treatment of TNBC patients. Upon comparing mitochondrial effects of conventional chemotherapies, we found that DNA-damaging agents increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial elongation and OXPHOS. The mitochondrial effects of DNA-damaging chemotherapies were dependent on the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1). Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in an orthotopic patient-derived xenograft (PDX) model of residual TNBC. Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS, respectively, revealing longer mitochondria favor oxphos in TNBC cells. Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA-damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibit regrowth of residual tumor cells. Our data suggest that TNBC mitochondria can optimize OXPHOS through OPA1-mediated mitochondrial fusion. These findings may provide an opportunity to overcome mitochondrial adaptations of chemoresistant TNBC.

Enabling Solution Processable COFs through Suppression of Precipitation during Solvothermal Synthesis.

Covalent organic frameworks (COFs) are crystalline, nanoporous materials of interest for various applications, but current COF synthetic routes lead to insoluble aggregates which precludes processing for practical implementation. Here, we report a COF synthesis method that produces a stable, homogeneous suspension of crystalline COF nanoparticles that enables the preparation of COF monoliths, membranes, and films using conventional solution-processing techniques. Our approach involves the use of a polar solvent, diacid catalyst, and slow reagent mixing procedure at elevated temperatures which altogether enable access to crystalline COF nanoparticle suspension that does not aggregate or precipitate when kept at elevated temperatures. On cooling, the suspension undergoes a thermoreversible gelation transition to produce crystalline and highly porous COF materials. We further show that the modified synthesis approach is compatible with various COF chemistries, including both large- and small-pore imine COFs, hydrazone-linked COFs, and COFs with rhombic and hexagonal topologies, and in each case, we demonstrate that the final product has excellent crystallinity and porosity. The final materials contain both micro- and macropores, and the total porosity can be tuned through variation of sample annealing. Dynamic light scattering measurements reveal the presence of COF nanoparticles that grow with time at room temperature, transitioning from a homogeneous suspension to a gel. Finally, we prepare imine COF membranes and measure their rejection of polyethylene glycol (PEG) polymers and oligomers, and these measurements exhibit size-dependent rejection and adsorption of PEG solutes. This work demonstrates a versatile processing strategy to create crystalline and porous COF materials using solution-processing techniques and will greatly advance the development of COFs for various applications.

Differential responses of neurons, astrocytes, and microglia to G-quadruplex stabilization.

The G-quadruplex (G4-DNA or G4) is a secondary DNA structure formed by DNA sequences containing multiple runs of guanines. While it is now firmly established that stabilized G4s lead to enhanced genomic instability in cancer cells, whether and how G4s contribute to genomic instability in brain cells is still not clear. We previously showed that, in cultured primary neurons, small-molecule G4 stabilizers promote formation of DNA double-strand breaks (DSBs) and downregulate the Brca1 gene. Here, we determined if G4-dependent Brca1 downregulation is unique to neurons or if the effects in neurons also occur in astrocytes and microglia. We show that primary neurons, astrocytes and microglia basally exhibit different G4 landscapes. Stabilizing G4-DNA with the G4 ligand pyridostatin (PDS) differentially modifies chromatin structure in these cell types. Intriguingly, PDS promotes DNA DSBs in neurons, astrocytes and microglial cells, but fails to downregulate Brca1 in astrocytes and microglia, indicating differences in DNA damage and repair pathways between brain cell types. Taken together, our findings suggest that stabilized G4-DNA contribute to genomic instability in the brain and may represent a novel senescence pathway in brain aging.

The Prevalence of Abnormal Spinopelvic Relationships in Patients Presenting for Primary Total Hip Arthroplasty.

BACKGROUND: The prevalence of an abnormal spinopelvic relationship in patients presenting for primary total hip arthroplasty (THA) is not well known. The purpose of this study was to identify the prevalence of abnormal spinopelvic relationships in patients presenting for primary THA. METHODS: A retrospective chart review of 338 consecutive, nonselected patients undergoing primary THA from the practice of 2 fellowship-trained adult reconstruction surgeons was performed (J.E.O. and T.S.B.). Sitting and standing radiographs were measured for lumbar lordosis (LL), pelvic incidence (PI), sacral slope (SSstand), and pelvic tilt; the sacral slope was also measured on sitting radiographs (SSsit). Patients were assessed for the presence of spinopelvic imbalance, defined as PI-LL>10°, and decreased spinopelvic motion, defined as SSstand-SSsit< 10°. Descriptive statistics were reported. RESULTS: A cohort of 338 patients was identified; 110 were excluded. In total, 228 unique patients underwent measurement. One hundred one of 228 patients (44.3%) in the cohort were female. The mean age of the cohort was 60.0 ± 13 years, with the mean body mass index of 31 ± 7 mg/kg2. Spinopelvic imbalance (PI-LL > 10°) was present in 142 of 228 patients (62.3%). Decreased motion at the spinopelvic junction (SSstand-SSsit < 10°) was present in 78 of 228 patients (34.2%). Fifty (21.9%) patients had both spinopelvic imbalance and decreased spinopelvic motion. CONCLUSIONS: In a cohort of 228 patients presenting for primary THA, the prevalence of spinopelvic imbalance was 62.3%, the prevalence of decreased spinopelvic motion was 34.2%, and the prevalence of both spinopelvic imbalance and decreased spinopelvic motion was 22%. Hip surgeons are likely to encounter patients with abnormal spinopelvic relationships.

Immunohistochemical and ultrastructural analysis of the maturing larval zebrafish enteric nervous system reveals the formation of a neuropil pattern.

The gastrointestinal tract is constructed with an intrinsic series of interconnected ganglia that span its entire length, called the enteric nervous system (ENS). The ENS exerts critical local reflex control over many essential gut functions; including peristalsis, water balance, hormone secretions and intestinal barrier homeostasis. ENS ganglia exist as a collection of neurons and glia that are arranged in a series of plexuses throughout the gut: the myenteric plexus and submucosal plexus. While it is known that enteric ganglia are derived from a stem cell population called the neural crest, mechanisms that dictate final neuropil plexus organization remain obscure. Recently, the vertebrate animal, zebrafish, has emerged as a useful model to understand ENS development, however knowledge of its developing myenteric plexus architecture was unknown. Here, we examine myenteric plexus of the maturing zebrafish larval fish histologically over time and find that it consists of a series of tight axon layers and long glial cell processes that wrap the circumference of the gut tube to completely encapsulate it, along all levels of the gut. By late larval stages, complexity of the myenteric plexus increases such that a layer of axons is juxtaposed to concentric layers of glial cells. Ultrastructurally, glial cells contain glial filaments and make intimate contacts with one another in long, thread-like projections. Conserved indicators of vesicular axon profiles are readily abundant throughout the larval plexus neuropil. Together, these data extend our understanding of myenteric plexus architecture in maturing zebrafish, thereby enabling functional studies of its formation in the future.

Spatial and temporal patterns of surface water quality and ichthyotoxicity in urban and rural river basins in Texas.

The Double Mountain Fork Brazos River (Texas, USA) consists of North (NF) and South Forks (SF). The NF receives urban runoff and twice-reclaimed wastewater effluent, whereas the SF flows through primarily rural areas. The objective of this study was to determine and compare associations between standard water quality variables and ichthyotoxicity at a landscape scale that included urban (NF) and rural (SF) sites. Five NF and three SF sites were sampled quarterly from March 2008 to March 2009 for specific conductance, salinity, hardness, pH, temperature, and turbidity; and a zebrafish (Danio rerio) embryo bioassay was used to determine ichthyotoxicity. Metal and nutrient concentrations at all sites were also measured in addition to standard water quality variables in spring 2009. Principal component analyses identified hardness, specific conductance, and salinity as the water variables that best differentiate the urban NF (higher levels) from rural SF habitat. Nutrient levels were also higher in the NF, but no landscape scale patterns in metal concentrations were observed. Ichthyotoxicity was generally higher in NF water especially in winter, and multiple regression analyses suggested a positive association between water hardness and ichthyotoxicity. To test for the potential influence of the toxic golden alga (Prymnesium parvum) on overall ichthyotoxicity, a cofactor known to enhance golden alga toxin activity was used in the bioassays. Golden alga ichthyotoxicity was detected in the NF but not the SF, suggesting golden alga may have contributed to overall ichthyotoxicity in the urban but not in the rural system. In conclusion, the physicochemistry of the urban-influenced NF water was conducive to the expression of ichthyotoxicity and also point to water hardness as a novel factor influencing golden alga ichthyotoxicity in surface waters.