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Background The conditional reprogramming cell culture method was developed to facilitate growth of senescence-prone normal and neoplastic epithelial cells, and involves co-culture with irradiated fibroblasts and the addition of a small molecule Rho kinase (ROCK) inhibitor. The aim of this study was to determine whether this approach would facilitate the culture of compact low-grade gliomas. Methods We attempted to culture 4 pilocytic astrocytomas, 2 gangliogliomas, 2 myxopapillary ependymomas, 2 anaplastic gliomas, 2 difficult-to-classify low-grade neuroepithelial tumors, a desmoplastic infantile ganglioglioma, and an anaplastic pleomorphic xanthoastrocytoma using a modified conditional reprogramming cell culture approach. Results Conditional reprogramming resulted in robust increases in growth for a majority of these tumors, with fibroblast conditioned media and ROCK inhibition both required. Switching cultures to standard serum containing media, or serum-free neurosphere conditions, with or without ROCK inhibition, resulted in decreased proliferation and induction of senescence markers. Rho kinase inhibition and conditioned media both promoted Akt and Erk1/2 activation. Several cultures, including one derived from a NF1-associated pilocytic astrocytoma (JHH-NF1-PA1) and one from a BRAF p.V600E mutant anaplastic pleomorphic xanthoastrocytoma (JHH-PXA1), exhibited growth sufficient for preclinical testing in vitro. In addition, JHH-NF1-PA1 cells survived and migrated in larval zebrafish orthotopic xenografts, while JHH-PXA1 formed orthotopic xenografts in mice histopathologically similar to the tumor from which it was derived. Conclusions These studies highlight the potential for the conditional reprogramming cell culture method to promote the growth of glial and glioneuronal tumors in vitro, in some cases enabling the establishment of long-term culture and in vivo models.

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

Several key attributes of zebrafish make them an ideal model system for the discovery and development of regeneration promoting therapeutics; most notably their robust capacity for self-repair which extends to the central nervous system. Further, by enabling large-scale drug discovery directly in living vertebrate disease models, zebrafish circumvent critical bottlenecks which have driven drug development costs up. This review summarizes currently available zebrafish phenotypic screening platforms, HTS-ready neurodegenerative disease modeling strategies, zebrafish small molecule screens which have succeeded in identifying regeneration promoting compounds and explores how intravital imaging in zebrafish can facilitate comprehensive analysis of nanocarrier biodistribution and pharmacokinetics. Finally, we discuss the benefits and challenges attending the combination of zebrafish and nanoparticle-based drug optimization, highlighting inspiring proof-of-concept studies and looking toward implementation across the drug development community.

Müller glia (MG) function as injury-induced retinal stem cells in zebrafish but not mammals. Insights from zebrafish, however, have been used to stimulate limited regenerative responses from mammalian MG. Microglia/macrophages regulate MG stem cell activity in the chick, zebrafish and mouse. We previously showed that dexamethasone can accelerate retinal regeneration in zebrafish. Similarly, microglia ablation enhances regenerative outcomes in the mouse retina. Targeted immunomodulation may therefore enhance the regenerative potential of human MG. Nanoparticle-based immunomodulation is an emerging field with immense therapeutic potential. Here, we investigated how regeneration-enhancing dexamethasone treatments alter microglia behavior and how dendrimer-based targeting of dexamethasone to reactive microglia impact retinal regeneration kinetics. Intravital time-lapse imaging revealed specific dexamethasone-induced changes in microglia reactivity. Dendrimer-conjugated dexamethasone treatments resulted in: 1) decreased toxicity, 2) selective targeting of reactive microglia and, 3) “super-accelerated” retinal regeneration kinetics. These data support the use of dendrimer-based drug formulations for modulating microglia reactivity in degenerative disease contexts, especially as therapeutic strategies for promoting regenerative responses to neuronal cell loss.

Retinal Müller glia (MG) can act as stem-like cells to generate new neurons in both zebrafish in mice. In zebrafish, retinal regeneration is innate and robust, resulting in the replacement of lost neurons and restoration of visual function. In mice, exogenous stimulation of MG is required to reveal a dormant and, to date, limited regenerative capacity. Zebrafish studies have been key in revealing factors that promote regenerative responses in the mammalian eye. Increased understanding of how the regenerative potential of MG is regulated in zebrafish may therefore aid efforts to promote retinal repair therapeutically. Developmental signaling pathways are known to coordinate regeneration following widespread retinal cell loss. In contrast, less is known about how regeneration is regulated in the context of retinal degenerative disease, i.e., following the loss of specific retinal cell types. To address this knowledge gap, we compared transcriptomic changes between two selective cell ablation paradigms, targeted loss of rod photoreceptors or bipolar cells. Our study spanned twelve time points encompassing the entire degenerative and regenerative process of each targeted cell type. 2,531 differentially expressed genes (DEGs) were identified. Interestingly, the majority of DEGs were paradigm specific, including during MG activation phases, suggesting the nature of the injury/cell loss informs the regenerative process from initiation onward. For example, early modulation of Notch signaling was implicated in the rod but not bipolar cell ablation paradigm. Components of JAK/STAT signaling were activated in both paradigms but were more strongly induced during rod cell regeneration. We functionally validated the role of JAK/STAT signaling during rod cell regeneration using CRISPR/Cas9-based knockdown ofstat3which inhibited both MG proliferation and rod cell regeneration kinetics. Additionally, although more than a third of all DEGs are implicated as immune-system regulators, individual immune-related factors were largely paradigm specific. These data support emerging evidence that discrete “fate-biased” regenerative processes follow from selective retinal cell loss.Author SummaryBlinding diseases are linked to the loss of specific types of neurons in the retina. In humans, this eventually leads to loss of sight. In zebrafish, however, lost retinal neurons are regenerated resulting in restored vision. Our lab has developed zebrafish models that allow us to selectively induce the loss of disease-relevant retinal neurons, thereby allowing us to study how individual retinal cell types are regenerated. Here, to better understand how the regeneration of individual cell types is regulated, we compared gene expression changes occurring during the loss and regeneration of two different retinal cell types, rod photoreceptors and bipolar interneurons. We found that the majority of gene changes were specific to each cell type studied, providing strong evidence that genetic programs underlying stem cell activation in zebrafish vary depending on the cell type damaged. We also found that the immune system was strongly implicated as a regulator of regeneration in both models, but that individual genes immune-related genes tended to be more strongly associated with one of the two models.. We hope that a better understanding of how retinal cell regeneration is regulated in zebrafish will aid efforts to develop regenerative therapeutics designed to restore sight to patients who have lost their vision.

Heterologously-expressed bacterial nitroreductase (NTR) enzymes sensitize eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell ablation paradigms that have expanded studies of cell function and regeneration in vertebrate systems. However, first-generation NTRs require confoundingly toxic prodrug treatments (e.g. 10 mM MTZ) and some cell types have proven resistant. We used rational engineering and cross-species screening to develop a NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy. Toxicity tests in zebrafish showed no deleterious effects of prolonged MTZ treatments of ≤1 mM. NTR 2.0 therefore enables sustained cell loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and enable modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic resources to facilitate dissemination of NTR 2.0 to the research community.

Retinoblastoma is the most common intraocular cancer in children. While the primary tumor can often be treated by local or systemic chemotherapy, metastatic dissemination is generally resistant to therapy and remains a leading cause of pediatric cancer death in much of the world. In order to identify new therapeutic targets in aggressive tumors, we sequenced RNA transcripts in five snap frozen retinoblastomas which invaded the optic nerve and five which did not. A three-fold increase was noted in mRNA levels of ACVR1C/ALK7, a type I receptor of the TGF-β family, in invasive retinoblastomas, while downregulation of DACT2 and LEFTY2, negative modulators of the ACVR1C signaling, was observed in most invasive tumors. A two- to three-fold increase in ACVR1C mRNA was also found in invasive WERI Rb1 and Y79 cells as compared to non-invasive cells in vitro. Transcripts of ACVR1C receptor and its ligands (Nodal, Activin A/B, and GDF3) were expressed in six retinoblastoma lines, and evidence of downstream SMAD2 signaling was present in all these lines. Pharmacological inhibition of ACVR1C signaling using SB505124, or genetic downregulation of the receptor using shRNA potently suppressed invasion, growth, survival, and reduced the protein levels of the mesenchymal markers ZEB1 and Snail. The inhibitory effects on invasion, growth, and proliferation were recapitulated by knocking down SMAD2, but not SMAD3. Finally, in an orthotopic zebrafish model of retinoblastoma, a 55% decrease in tumor spread was noted (p = 0.0026) when larvae were treated with 3 µM of SB505124, as compared to DMSO. Similarly, knockdown of ACVR1C in injected tumor cells using shRNA also resulted in a 54% reduction in tumor dissemination in the zebrafish eye as compared to scrambled shRNA control (p = 0.0005). Our data support a role for the ACVR1C/SMAD2 pathway in promoting invasion and growth of retinoblastoma.

Aim: Oral and intravenous pharmacokinetic (PK) studies were conducted in four different minipig strains: Sinclair, Yucatan, Hanford and Göttingen after administration of metformin or R,S-verapamil (R,S-VER). Results: The results indicated that the PK of metformin was similar between all minipig strains, except for the Göttingen which had higher plasma clearance. The plasma clearance of both (+)-(R)- and (−)-(S)-VER was significantly lower in Sinclair compared with the clearance in other strains. The (+)-(R)-NOR to R,S-VER ratio was significantly higher in the Sinclair. There was a preferential conversion of R,S-VER to (+)-(R)-NOR over (−)-(S)-NOR in all minipig strains. Conclusion: This work highlights the importance of considering the impact of metabolic and dispositional differences in minipig strains when conducting PK studies.

A functional observational battery (FOB) is recommended as the first-tier neurotoxicity screening in the preclinical safety pharmacology testing guidelines. Minipigs have increasingly been used in regulatory toxicology studies; however, no current FOB protocol is available for neurotoxicity testing in these species. Hence, a minipig FOB instrument was developed. A complete crossover study with Sinclair minipigs was performed to evaluate physiologic, neurologic, and behavioral effects of amphetamine, ketamine, and diazepam. The treated minipigs were first observed in their home cage, were video-recorded for 10 minutes in an open field, and then went through a complete neurologic examination. Both ketamine and diazepam were shown to reduce the freezing and behavior shifts of treated minipigs, while increasing their exploratory behaviors. Both drugs also caused muscular and gait impairment. The effects of ketamine and diazepam were consistent with their roles as central nervous system (CNS) suppressants. Unique effects were also observed with ketamine and diazepam treatments, which may reflect their unique mechanisms of action. Consistent with its role as a CNS stimulant, amphetamine caused the treated minipigs to be hyperactive and to display increased freezing and behavior shifts and reduced exploring activities. These effects of amphetamine were opposite to those observed with ketamine and diazepam. Amphetamine also increased locomotion in the treated minipigs. The present effects of amphetamine, ketamine, and diazepam are in agreement with observations by others. In conclusion, the minipig is a suitable species for FOB evaluation of pharmaceuticals in preclinical safety pharmacology testing.

Retinoblastoma is the most common intraocular malignancy in children. We previously found that the ACVR1C/SMAD2 pathway is significantly upregulated in invasive retinoblastoma samples from patients. Here we studied the role of an ACVR1C ligand, Nodal, in regulating growth and metastatic dissemination in retinoblastoma. Inhibition of Nodal using multiple short hairpin (shRNAs) in WERI Rb1 and Y79 retinoblastoma cell cultures reduced growth by more than 90%, as determined by CCK-8 growth assay. Proliferation was also significantly inhibited, as found by Ki67 assay. These effects were paralleled by inhibition in the phosphorylation of the downstream effector SMAD2, as well as induction of apoptosis, as we observed more than three-fold increase in the percentage of cells positive for cleaved-caspase-3 or expressing cleaved-PARP1. Importantly, we found that downregulation of Nodal potently suppressed invasion in vitro, by 50 to 80%, as determined by transwell invasion assay (p = 0.02). Using an orthotopic model of retinoblastoma in zebrafish, we found 34% reduction in the ability of the cells to disseminate outside the eye, when Nodal was knocked down by shRNA (p = 0.0003). These data suggest that Nodal plays an important role in promoting growth, proliferation and invasion in retinoblastoma, and can be considered a new therapeutic target for both primary tumor growth and metastatic progression.

Diffuse intrinsic pontine glioma (DIPG) and atypical teratoid/rhabdoid tumors (AT/RT) are deadly pediatric brain tumors. There is an urgent need of better therapeutics for improving survival and quality of life for these patients. Additionally, we need improved animal models to accelerate identification of novel therapeutics and improve through-put. In line with our long-term interests of validating therapies and developing model systems for pediatric brain tumors, we tested a repurposed drug quinacrine as a potential therapy using a new and faster model system in zebrafish. Quinacrine is a safe and widely used treatment for pediatric malaria and parasitic infections. We hypothesized that quinacrine will increase tumor cell death and decrease tumorigenicity of DIPG and ATRT. We used quinacrine in six patient-derived cell lines: three AT/RT (BT37, CHLA-05, CHLA-266) and three DIPG (JHHDIPG1, SUDIPGXIII, SF7761). In all tumor cell lines, quinacrine caused a dose-dependent reduction in proliferation (BrdU) and increase in apoptosis (cleaved caspase-3 and cleaved PARP) compared to vehicle-treated cells (P Citation Format: Harpreet Kaur, Huizi Guo, Kevin Emmerich, David White, Peter Green, Alara Michelle Hector, Sepehr Akhtarkhavari, Anukriti Bhargava, Allison Martin, Smit Shah, Charles G. Eberhart, Jeffrey Mumm, Eric H. Raabe. A novel rapid zebrafish model for validation of potential therapies for fatal pediatric brain tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3449.

Diffuse intrinsic pontine glioma (DIPG) and atypical teratoid/rhabdoid tumors (AT/RT) are deadly pediatric brain tumors. Developing new targets and therapeutics are urgently needed. We have previously shown that multiple primary brain tumors and cell lines express increased amounts of the epigenetic modifier and DNA binding oncoprotein high-mobility group AT-hook 2 (HMGA2). Targeting HMGA2 using short hairpins decreased AT/RT and glioma growth in xenografted mice. We hypothesized that pharmacologic inhibition of HMGA proteins using DNA minor-groove binding drugs such as quinacrine would decrease tumor growth due to displacement of HMGA proteins from DNA. Quinacrine is a safe and widely used treatment for pediatric malaria and parasitic infections. We used quinacrine in six patient-derived cell lines: three AT/RT (BT37, CHLA-05, CHLA-266) and three DIPG (JHHDIPG1, SUDIPGXIII, SF7761). Using quinacrine fluorescence as a surrogate, we can achieve micromolar concentration of quinacrine in the mouse and zebrafish brain after oral administration without overt toxicity. In both tumor cell lines, quinacrine causes a dose-dependent reduction in proliferation (BrdU) and increase in apoptosis (cleaved caspase-3 and cleaved PARP) compared to vehicle-treated cells (P Citation Format: Harpreet Kaur, Huizi Guo, David White, Peter Green, Alara M. Hector, Sepehr Akhtarkhavari, Anukriti Bhargava, Smit Shah, Charles G. Eberhart, Jeffrey Mumm, Eric H. Raabe. Validation of potential therapies for treatment of fatal pediatric brain tumors DIPG and AT/RT using a novel rapid intracranial model in zebrafish [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A38.

Thousands of genes have been implicated in retinal regeneration, but only a few have been shown to impact the regenerative capacity of Müller glia-an adult retinal stem cell with untapped therapeutic potential. Similarly, among nearly 300 genetic loci associated with human retinal disease, the majority remain untested in animal models. To address the large-scale nature of these problems, we are applying CRISPR/Cas9-based genome modification strategies in zebrafish to target over 300 genes implicated in retinal regeneration or degeneration. Our intent is to enable large-scale reverse genetic screens by applying a multiplexed gene disruption strategy that markedly increases the efficiency of the screening process. To facilitate large-scale phenotyping, we incorporate an automated reporter quantification-based assay to identify cellular degeneration and regeneration-deficient phenotypes in transgenic fish. Multiplexed gene targeting strategies can address mismatches in scale between "big data" bioinformatics and wet lab experimental capacities, a critical shortfall limiting comprehensive functional analyses of factors implicated in ever-expanding multiomics datasets. This report details the progress we have made to date with a multiplexed CRISPR/Cas9-based gene targeting strategy and discusses how the methodologies applied can further our understanding of the genes that predispose to retinal degenerative disease and which control the regenerative capacity of retinal Müller glia cells.

Muller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration—i.e., selective cell-loss paradigms akin to degenerative disease—are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of (i) rod cell clearance, (ii) MG/progenitor cell proliferation, and (iii) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions.

At the turn of the 20th century, classical regenerative biology – the study of organismal/tissue/limb regeneration in animals such as crayfish, snails, and planaria – garnered much attention. However, scientific luminaries such as Thomas Hunt Morgan eventually turned to other fields after concluding that inquiries into regenerative mechanisms were largely intractable beyond observational intrigues. The field of regeneration has enjoyed a resurgence in research activity at the turn of the 21st century, in large part due to “the promise” of cultured stem cells regarding reparative therapeutic approaches. Additionally, genomics-based methods that allow sophisticated genetic/molecular manipulations to be carried out in nearly any species have extended organismal regenerative biology well beyond observational limits. Throughout its history, complex paradigms such as limb regeneration – involving multiple tissue/cell types, thus, potentially multiple stem cell subtypes – have predominated the regenerative biology field. Conversely, cellular regeneration – the replacement of specific cell types – has been studied from only a few perspectives (predominantly muscle and mechanosensory hair cells). Yet, many of the degenerative diseases that regenerative biology hopes to address involve the loss of individual cell types; thus, a primary emphasis of the embryonic/induced stem cell field is defining culture conditions which promote cell-specific differentiation. Here we will discuss recent methodological approaches that promote the study of cell-specific regeneration. Such paradigms can reveal how the differentiation of specific cell types and regenerative potential of discrete stem cell niches are regulated. In particular, we will focus on how the nitroreductase (NTR) system of inducible targeted cell ablation facilitates: 1) large-scale genetic and chemical screens for identifying factors that regulate regeneration and, 2) in vivo time-lapse imaging experiments aimed at investigating regenerative processes more directly. Combining powerful screening and imaging technologies with targeted ablation systems can expand our understanding of how individual stem cell niches are regulated. The former approach promotes the development of therapies aimed at enhancing regenerative potentials in humans, the latter facilitates investigation of phenomena that are otherwise difficult to resolve, such as the role of cellular transdifferentiation or the innate immune system in regenerative paradigms.

The zebrafish has emerged as an important model for whole-organism small-molecule screening. However, most zebrafish-based chemical screens have achieved only mid-throughput rates. Here we describe a versatile whole-organism drug discovery platform that can achieve true high-throughput screening (HTS) capacities. This system combines our automated reporter quantification in vivo (ARQiv) system with customized robotics, and is termed 'ARQiv-HTS'. We detail the process of establishing and implementing ARQiv-HTS: (i) assay design and optimization, (ii) calculation of sample size and hit criteria, (iii) large-scale egg production, (iv) automated compound titration, (v) dispensing of embryos into microtiter plates, and (vi) reporter quantification. We also outline what we see as best practice strategies for leveraging the power of ARQiv-HTS for zebrafish-based drug discovery, and address technical challenges of applying zebrafish to large-scale chemical screens. Finally, we provide a detailed protocol for a recently completed inaugural ARQiv-HTS effort, which involved the identification of compounds that elevate insulin reporter activity. Compounds that increased the number of insulin-producing pancreatic beta cells represent potential new therapeutics for diabetic patients. For this effort, individual screening sessions took 1 week to conclude, and sessions were performed iteratively approximately every other day to increase throughput. At the conclusion of the screen, more than a half million drug-treated larvae had been evaluated. Beyond this initial example, however, the ARQiv-HTS platform is adaptable to almost any reporter-based assay designed to evaluate the effects of chemical compounds in living small-animal models. ARQiv-HTS thus enables large-scale whole-organism drug discovery for a variety of model species and from numerous disease-oriented perspectives.

Advances in semiconductor technology have resulted in the creation of miniature medical embedded systems that can wirelessly monitor the vital signs of patients. These lightweight medical systems can aid providers in large disasters who become overwhelmed with the large number of patients, limited resources, and insufficient information. In a mass casualty incident, small embedded medical systems facilitate patient care, resource allocation, and real-time communication in the advanced health and disaster aid network (AID-N). We present the design of electronic triage tags on lightweight, embedded systems with limited memory and computational power. These electronic triage tags use noninvasive, biomedical sensors (pulse oximeter, electrocardiogram, and blood pressure cuff) to continuously monitor the vital signs of a patient and deliver pertinent information to first responders. This electronic triage system facilitates the seamless collection and dissemination of data from the incident site to key members of the distributed emergency response community. The real-time collection of data through a mesh network in a mass casualty drill was shown to approximately triple the number of times patients that were triaged compared with the traditional paper triage system.

Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. DOI: http://dx.doi.org/10.7554/eLife.08261.001, eLife digest Type 1 diabetes is caused by the body incorrectly destroying the cells in the pancreas—known as β cells—that produce insulin and so control the amount of sugar found in the bloodstream. Drugs that increase the rate at which new β cells form could therefore help to treat this disease. High-throughput screening is a technique that uses automated systems to rapidly test the effects of large numbers of drug-like compounds on living cells. Unfortunately, drugs sometimes produce different effects in animals than those they produce in isolated cells or other more simplified screening systems. Zebrafish are often used in biological studies because the larvae are transparent, making it easier to study what goes on inside them. Wang et al. have now developed a high-throughput screening system that uses genetically engineered zebrafish. The zebrafish contain ‘reporter’ genes that fluoresce when a gene is activated, and the intensity of the fluorescence can be interpreted to work out the effects of an applied drug. To search for compounds that cause β cells to grow, Wang et al. created two reporter genes: one that glows yellow when new β cells form, and one that glows red when other pancreatic cells are stimulated. An initial screen tested the effects of over 3000 drugs, most of which have been approved for use in humans. This screen identified and confirmed 24 drugs that trigger the growth of new β cells or other pancreatic cells in zebrafish larvae. Further investigation uncovered new roles for two signaling pathways that had not previously been linked to pancreatic growth. One pathway—the serotonin pathway, which is better known for transmitting signals in the brain—selectively stimulates the growth of new β cells. The work of Wang et al. therefore presents a number of possible drugs and pathways that could be targeted in the search for a new treatment for type 1 diabetes. Furthermore, this new whole-organism, high-throughput screening system could be used in the future to search for drugs that affect a range of other biological processes. DOI: http://dx.doi.org/10.7554/eLife.08261.002

Objective— To compare the fixation rigidity of recently developed external fixation systems (EFSs) to that of the traditional Kirschner-Ehmer (KE) system. Study Design— In vitro biomechanical study. Sample Population— Five different EFSs (KE, Secur-U, small SK carbon fiber, small SK titanium, large SK carbon fiber) were assembled into 7 frame geometries to stabilize Delrin plastic rods with a 1-cm gap. Methods— External skeletal fixation (ESF) constructs were tested in axial compression, torsion, medial-lateral bending, and cranial-caudal bending. Testing was conducted within the elastic range of each fixator. Mean stiffness in each mode was determined from the slope of the linear portion of the load-deformation curve. Comparison of stiffness values of each EFS within each loading mode and frame type was performed with 1-way analysis of variance (P < .05). Results— Mean stiffness values were significantly higher for the large SK EFS in all frame types compared with KE but were equal in torsional stiffness in the double-bar type 1a frame. The small SK EFS with titanium connecting bar had greater stiffness than the KE in all modes for frame types Ia, Ia-accessory bar, and II-modified. No overall difference was detected between the KE EFS and the small SK with carbon fiber rod. The stiffness of the Secur-U type Ia frame with augmentation plate was significantly greater than the KE type Ia with accessory bar. Conclusions— The newer external fixation systems evaluated in this study provided fixation rigidity equal to or greater than that of the KE system. Clinical Relevance— EFSs with increased frame rigidity should permit the use of less complex frame designs while providing fracture stability.

Background In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date. Methods Individual cell lines were stained with CM-Dil and injected into the perivitelline space of 2-day old zebrafish larvae. After 2-4 days fish were imaged using confocal microscopy and the number of metastatic cells was determined using Fiji software. Results To determine whether zebrafish can faithfully report metastatic potential in human cancer cells, we injected a series of cells with different metastatic potential into the perivitelline space of 2 day old embryos. Using cells from breast, prostate, colon and pancreas we demonstrated that the degree of cell metastasis in fish is proportional to their invasion potential in vitro. Highly metastatic cells such as MDA231, DU145, SW620 and ASPC-1 are seen in the vasculature and throughout the body of the fish after only 24–48 hours. Importantly, cells that are not invasive in vitro such as T47D, LNCaP and HT29 do not metastasize in fish. Inactivation of JAK1/2 in fibrosarcoma cells leads to loss of invasion in vitro and metastasis in vivo, and in zebrafish these cells show limited spread throughout the zebrafish body compared with the highly metastatic parental cells. Further, knockdown of WASF3 in DU145 cells which leads to loss of invasion in vitro and metastasis in vivo also results in suppression of metastasis in zebrafish. In a cancer progression model involving normal MCF10A breast epithelial cells, the degree of invasion/metastasis in vitro and in mice is mirrored in zebrafish. Using a modified version of Fiji software, it is possible to quantify individual metastatic cells in the transparent larvae to correlate with invasion potential. We also demonstrate, using lung cancers, that the zebrafish model can evaluate the metastatic ability of cancer cells isolated from primary tumors. Conclusions The zebrafish model described here offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells. Using this model it is possible to critically evaluate whether genetic manipulation of signaling pathways affects metastasis and whether primary tumors contain metastatic cells.

Alkali addition for iron precipitation, associated with sulfuric acid leaching of nickel laterites, has been considered periodically since the late 1960s, shortly after the development of the jarosite process for iron control in zinc refining.

The ARF-GEF ARNO promotes motility by activating ARF6 and a subsequent downstream activation of Rac. ARNO is shown to associate with the Rac GEF Dock180 via its coiled-coil domain. Knockdown of scaffold proteins that bind ARNO disrupts the formation of this complex and disrupts ARF-to-Rac signaling., ARFs are small GTPases that regulate vesicular trafficking, cell shape, and movement. ARFs are subject to extensive regulation by a large number of accessory proteins. The many different accessory proteins are likely specialized to regulate ARF signaling during particular processes. ARNO/cytohesin 2 is an ARF-activating protein that promotes cell migration and cell shape changes. We report here that protein–protein interactions mediated by the coiled-coil domain of ARNO are required for ARNO induced motility. ARNO lacking the coiled-coil domain does not promote migration and does not induce ARF-dependent Rac activation. We find that the coiled-coil domain promotes the assembly of a multiprotein complex containing both ARNO and the Rac-activating protein Dock180. Knockdown of either GRASP/Tamalin or IPCEF, two proteins known to bind to the coiled-coil of ARNO, prevents the association of ARNO and Dock180 and prevents ARNO-induced Rac activation. These data suggest that scaffold proteins can regulate ARF dependent processes by biasing ARF signaling toward particular outputs.

For years, emergency medical response communities have relied upon paper triage tags, clipboards of notes, and voice communications to share information during medical emergencies. This workflow, however, has proven labor intensive, time consuming, and prone to human error [1]. In collaboration with three EMS groups in the Washington, DC Metropolitan area, we have developed a next generation triage system to improve the effectiveness of emergency response. This system includes: 1) electronic triage tags, 2) wearable vital sign sensors, 3) base stations laptops to monitor and manage patients, 4) pervasive tracking software to locate patients at all stages of the disaster response process, and 5) PDAs to support documentation and communication. Our system has evolved through three iterations of rapid-development, field-studies, usability reviews, and focus-group interview. This paper summarizes engineering considerations for technologies that must operate under constraints of medical emergencies. It is our hope that the lessons reported in this paper will help technologists in developing future emergency response systems.

Real-time information communication presents a persistent challenge to the emergency response community. During a medical emergency, various first response disciplines including Emergency Medical Service (EMS), Fire, and Police, and multiple health service facilities including hospitals, auxiliary care centers and public health departments using disparate information technology systems must coordinate their efforts by sharing real-time information. This paper describes a service-oriented architecture (SOA) that uses shared data models of emergency incidents to support the exchange of data between heterogeneous systems. This architecture is employed in the Advanced Health and Disaster Aid Network (AID-N) system, a testbed investigating information technologies to improve interoperation among multiple emergency response organizations in the Washington DC Metropolitan region. This architecture allows us to enable real-time data communication between three deployed systems: 1) a pre-hospital patient care reporting software system used on all ambulances in Arlington County, Virginia (MICHAELS), 2) a syndromic surveillance system used by public health departments in the Washington area (ESSENCE), and 3) a hazardous material reference software system (WISER) developed by the National Library Medicine. Additionally, we have extended our system to communicate with three new data sources: 1) wireless automated vital sign sensors worn by patients, 2) web portals for admitting hospitals, and 3) PDAs used by first responders at emergency scenes to input data (SIRP).

We have developed a patient triage and monitoring system to facilitate effectiveness patient care during emergency medical situations. This system includes: 1) electronic triage tags, 2) location sensors, 3) vital sign sensors, and 4) robust ad-hoc mesh networking software. This paper summarizes our development of continuous, automated, real-time biomedical sensors and software for use by emergency responders during chaotic pre-hospital settings.