Test facility management: Responsibilities and paradigms of a new era.

GLP test facility management refers to the proper management and organization of a facility that conducts studies according to GLP regulations. Compliance with GLP regulations is necessary for data generated in such facilities to be accepted by regulatory authorities. According to GLP Principles, Test facility management (TFM) is responsible for a wide range of tasks and responsibilities to ensure the smooth and efficient operation of the facility. The framework in which the TFM operates within the Test Facility is certainly much more complex than in the early days of the GLP, and moreover it is unlikely that anything will change from a scientific and technological point of view in the years to come. Several aspects have changed from a scientific and technological point of view, and we know that innovation is very rapid. From the above considerations emerges the need for a major change in the performance of the TFM's role.

Development and validation of a bioanalytical method for the quantification of CHF6550 and its metabolite (CHF6671) in rat plasma and lung homogenate using LC-MS/MS.

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for accurate determination of CHF6550 and its main metabolite in rat plasma and lung homogenate samples. All biological samples were prepared by simple protein precipitation method using deuterated internal standards. The analytes were separated on a HSS T3 analytical column with 3.2 min run time at flow rate of 0.5 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by selected-reaction monitoring of the transitions at m/z 735.3 â†’ 98.0 for CHF6550 and m/z 638.3 â†’ 319.2 and 638.3 â†’ 376.2 for CHF6671. The calibration curves for plasma samples were linear between 50 and 50000 pg/mL for both analytes. The calibration curves for lung homogenate samples were linear within 0.1-100 ng/mL for CHF6550 and 0.3-300 ng/mL for CHF6671. The method was successfully applied to a 4-week toxicity study.

Validation of an LC-MS/MS method for the quantification IOA-289 in human plasma and its application in a first-in-human clinical trial.

IOA-289 is a novel small molecule inhibitor of autotaxin developed as a first-in-class therapy of fibrotic pathologies including cancer. A method for quantitation of IOA-289 in human plasma was developed using a stable isotope labeled compound ([13C4]IOA-289) as internal standard. The analytes were extracted from human plasma by protein precipitation and the analysis was performed by liquid chromatography coupled with tandem mass spectrometric detection (LCMS/MS). The chromatographic separation was performed with a gradient elution from a BEH C18 column and under these conditions the retention time and the run time were 1 and 2 min, respectively. The assay was fully validated over the range 3-3000 ng/mL, proved to be accurate, precise and selective and was successfully applied to quantitate IOA-289 in plasma samples from subjects in a first-in-humanclinical trial.

Gene flow in phylogenomics: Sequence capture resolves species limits and biogeography of Afromontane forest endemic frogs from the Cameroon Highlands.

Puddle frogs of the Phrynobatrachus steindachneri species complex are a useful group for investigating speciation and phylogeography in Afromontane forests of the Cameroon Volcanic Line, western Central Africa. The species complex is represented by six morphologically relatively cryptic mitochondrial DNA lineages, only two of which are distinguished at the species level - southern P. jimzimkusi and Lake Oku endemic P. njiomock, leaving the remaining four lineages identified as 'P. steindachneri'. In this study, the six mtDNA lineages are subjected to genomic sequence capture analyses and morphological examination to delimit species and to study biogeography. The nuclear DNA data (387 loci; 571,936 aligned base pairs) distinguished all six mtDNA lineages, but the topological pattern and divergence depths supported only four main clades: P. jimzimkusi, P. njiomock, and only two divergent evolutionary lineages within the four 'P. steindachneri' mtDNA lineages. One of the two lineages is herein described as a new species, P. amieti sp. nov. Reticulate evolution (hybridization) was detected within the species complex with morphologically intermediate hybrid individuals placed between the parental species in phylogenomic analyses, forming a ladder-like phylogenetic pattern. The presence of hybrids is undesirable in standard phylogenetic analyses but is essential and beneficial in the network multispecies coalescent. This latter approach provided insight into the reticulate evolutionary history of these endemic frogs. Introgressions likely occurred during the Middle and Late Pleistocene climatic oscillations, due to the cyclic connections (likely dominating during cold glacials) and separations (during warm interglacials) of montane forests. The genomic phylogeographic pattern supports the separation of the southern (Mt. Manengouba to Mt. Oku) and northern mountains at the onset of the Pleistocene. Further subdivisions occurred in the Early Pleistocene, separating populations from the northernmost (Tchabal Mbabo, Gotel Mts.) and middle mountains (Mt. Mbam, Mt. Oku, Mambilla Plateau), as well as the microendemic lineage restricted to Lake Oku (Mt. Oku). This unique model system is highly threatened as all the species within the complex have exhibited severe population declines in the past decade, placing them on the brink of extinction. In addition, Mount Oku is identified to be of particular conservation importance because it harbors three species of this complex. We, therefore, urge for conservation actions in the Cameroon Highlands to preserve their diversity before it is too late.

Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries.

Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, high-content imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins ß1 and ß6 or αV and ß1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.

Evolutionary history of the Cameroon radiation of puddle frogs (Phrynobatrachidae: Phrynobatrachus), with descriptions of two critically endangered new species from the northern Cameroon Volcanic Line.

The Cameroon Volcanic Line, a mountain chain located between West and Central Africa, is a region of numerous endemic diversifications, including of puddle frogs (Phrynobatrachus). This study reviews the phylogeny and taxonomy of puddle frogs of the "Cameroon radiation," which is a clade containing mainly montane but also at least three lowland species. Molecular data revealed a novel evolutionary lineage from high altitudes in the northern part of the mountains. Puddle frogs from the new, minute-sized (SVL < 20 mm) lineage are identified using molecular, morphological and acoustic data and described as two new species, Phrynobatrachus arcanus sp. nov. (Gotel Mountains, Cameroon-Nigeria) and P. mbabo sp. nov. (Tchabal Mbabo, Cameroon). The tadpole of the first species is also described. Phylogenetic analyses placed the new lineage to the proximity of the recently described lowland small-sized taxa (P. horsti, P. ruthbeateae). Based on the inferred phylogeny, we propose five species groups within the Cameroon radiation: P. arcanus, P. chukuchuku, P. ruthbeateae, P. steindachneri, and P. werneri. The taxonomically enigmatic P. hylaios is proposed to be a member of the P. ruthbeateae species group. The basal radiation evolved during the late Miocene with subsequent diversifications occurring during the Pliocene, while closely related terminal taxa originated during the Pleistocene. We recommend that the newly described species are categorized as Critically Endangered due to their limited ranges and because recent surveys did not identify any individuals at the type localities. This further supports the need for conservation interventions in the mountains of Cameroon and Nigeria.

Liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of enmetazobactam and cefepime in human plasma.

A simple ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous analysis enmetazobactam (also known as AAI101) and cefepime in human plasma. Sample preparation was based on protein precipitation with acetonitrile. Separation was performed on Acquity BEH HILIC column (50 mm × 2.1 mm, 1.7 µm) with a mobile phase containing ammonium formate in water and acetonitrile. The analytes were analyzed with the corresponding isotopically labeled internal standards and were detected in multiple reactions monitoring (MRM) using API 5000 triple-quadrupole mass spectrometer with electrospray (ESI) source operating in positive ion mode. The calibration curves were linear over the selected ranges (r > 0.9970 for both analytes). The intra and inter-assay precision of the Quality Control samples showed CV ≤ 15% and the accuracy was within 85 and 115% in all cases for both compounds. The lower limit of quantification was 0.05 µg/mL for enmetazobactam and 0.5 µg/mL for cefepime.

Spontaneous Extracellular Matrix Accumulation in a Human in vitro Model of Renal Fibrosis Is Mediated by αV Integrins.

BACKGROUND: Tubulointerstitial fibrosis is a key feature of chronic kidney diseases leading to renal failure. It is characterised by the infiltration of fibroblasts and aberrant accumulation of extracellular matrix (ECM) proteins, which are associated with progressive loss of renal function. Integrins play a major role in fibrosis, but the mechanisms through which they do this are not fully understood. OBJECTIVE: Using a complex cell system, we test the hypothesis that integrins are pro-fibrotic via regulation of functional interactions between tubular epithelial cells and renal fibroblasts. METHOD: Contact co-culture of human primary renal proximal tubular epithelial cells and renal fibroblasts promoted the spontaneous accumulation of a mature ECM rich in interstitial collagens, which was considerably in excess of that seen in the individual mono-cultures. Both cell types persisted throughout the culture and were capable of expressing multiple ECM components. RESULTS: While ECM accumulation was inhibited by the clinically proven anti-fibrotic, nintedanib, and was partially abrogated by transforming growth factor ß neutralisation, its levels did not return to basal, indicating additional pathways were implicated in the pro-ECM response. Application of anti-integrin blocking antibodies and small molecules demonstrated a major role of the αV integrins in the ECM accumulation during fibroblast: epithelial cell interactions. CONCLUSION: Integrin-mediated pathways can facilitate the spontaneous accumulation of ECM during fibroblast: epithelial cell interactions, and this direct renal co-culture assay system could provide a translational in vitro assay for investigating novel pathways involved in the pro-ECM response and the screening of renal anti-fibrotic agents.

Development and validation of a bioanalytical method for quantification of LNA-i-miR-221, a 13-mer oligonucleotide, in rat plasma using LC-MS/MS.

LNA-i-miR-221, a 13-mer oligonucleotide, is a new miR-221 inhibitor that could be used as a novel drug for multiple myeloma. Herein, an ion-pair reversed phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of LNA-i-miR-221 in rat plasma. Plasma samples were prepared with an initial phenol/chloroform/isoamyl alcohol liquid-liquid extraction followed by a solid phase extraction. Chromatographic separation was performed with a gradient system on a HALO C18 column using hexafluoro-2-propanol/triethylamine buffer and methanol as mobile phase at a flow rate of 0.4 mL/min. Under these conditions LNA-i-miR-221 and the analogue internal standard are co-eluted at 1.2 min. The detection was carried out in multiple reaction monitoring (MRM) mode using a negative electrospray ionization (ESI) interface. The assay showed a good linearity within the calibration range 10-10000 ng/mL. The precision, accuracy, and recovery values were found to be <15% (<20% at LLOQ), 100 ±â€¯15%, and 97.6-103.7%, respectively. This method was successfully applied to measure the concentrations of LNA-i-miR-221 in plasma samples following the intravenous administration during a 4-week toxicity study in rats.

A new species of Phrynobatrachus (Anura: Phrynobatrachidae) from the Eastern Arc Mountians of Tanzania.

Phrynobatrachus discogularis sp. nov. (Anura, Phrynobatrachidae) is described from the Eastern Arc Mountains in Tanzania. It occupies upland grasslands and while it morphologically most resembles West and Central African P. gutturosus (Chabanaud, 1921), P. rungwensis (Loveridge, 1932), and P. anotis Schmidt and Inger, 1959, preliminary analysis of mitochondrial 16S rRNA indicates that the new species differs from all other species with published sequence data by a minimum distance of 4.1% and has affinities with P. rungwensis, P. uzungwensis Grandison and Howell, 1983 and P. keniensis Barbour and Loveridge, 1928-all diminutive upland eastern African taxa. The shape and colour of the male's external gular apparatus distinguish this species from all other described Phrynobatrachus species. Although geographically either sympatric or closely allopatric with P. uzungwensis, the lack of overlapping morphological characters indicates the two forms are distinct species. The new taxon is compared with other species from the region.

How many species and under what names? Using DNA barcoding and GenBank data for west Central African amphibian conservation.

Development projects in west Central Africa are proceeding at an unprecedented rate, often with little concern for their effects on biodiversity. In an attempt to better understand potential impacts of a road development project on the anuran amphibian community, we conducted a biodiversity assessment employing multiple methodologies (visual encounter transects, auditory surveys, leaf litter plots and pitfall traps) to inventory species prior to construction of a new road within the buffer zone of Moukalaba-Doudou National Park, Gabon. Because of difficulties in morphological identification and taxonomic uncertainty of amphibian species observed in the area, we integrated a DNA barcoding analysis into the project to improve the overall quality and accuracy of the species inventory. Based on morphology alone, 48 species were recognized in the field and voucher specimens of each were collected. We used tissue samples from specimens collected at our field site, material available from amphibians collected in other parts of Gabon and the Republic of Congo to initiate a DNA barcode library for west Central African amphibians. We then compared our sequences with material in GenBank for the genera recorded at the study site to assist in identifications. The resulting COI and 16S barcode library allowed us to update the number of species documented at the study site to 28, thereby providing a more accurate assessment of diversity and distributions. We caution that because sequence data maintained in GenBank are often poorly curated by the original submitters and cannot be amended by third-parties, these data have limited utility for identification purposes. Nevertheless, the use of DNA barcoding is likely to benefit biodiversity inventories and long-term monitoring, particularly for taxa that can be difficult to identify based on morphology alone; likewise, inventory and monitoring programs can contribute invaluable data to the DNA barcode library and the taxonomy of complex groups. Our methods provide an example of how non-taxonomists and parataxonomists working in understudied parts of the world with limited geographic sampling and comparative morphological material can use DNA barcoding and publicly available sequence data (GenBank) to rapidly identify the number of species and assign tentative names to aid in urgent conservation management actions and contribute to taxonomic resolution.

Randomized Controlled Trial Comparing Collagen/Oxidized Regenerated Cellulose/Silver to Standard of Care in the Management of Venous Leg Ulcers.

OBJECTIVE: To assess healing outcomes in venous leg ulcers (VLUs) treated with a combination of collagen, oxidized regenerated cellulose, and silver in conjunction with standard of care (SOC; intervention group) compared with SOC alone (control group). Standard of care included ADAPTIC nonadhering dressing (Acelity, San Antonio, Texas) and compression. DESIGN AND SETTING: Randomized controlled trial that followed patients in 3 US facilities for 12 weeks or until complete healing. PATIENTS AND INTERVENTION: Forty-nine patients with VLUs were randomized to either the intervention group (n = 22) or the control group (n = 27). MAIN OUTCOME MEASURE: Wound healing over 12 weeks. MAIN RESULTS: Intent-to-treat analysis showed a mean percentage wound area reduction at 12 weeks of 85.6% (SD, 28.6%) for the intervention group and 72.5% (SD, 77.8%) for the control group. There was a higher healing rate in the intervention group compared with patients who received SOC only at both week 4 (23% vs 11%) and week 12 (64% vs 59%). There were no adverse events related to the study therapy. CONCLUSIONS: Although the results were not significant, there was a trend toward faster healing in the intervention group. The results of this study indicate that collagen/oxidized regenerated cellulose/silver is a suitable and safe adjunctive intervention for use with SOC to manage VLUs.

Biofilms and delayed healing - an in vitro evaluation of silver- and iodine-containing dressings and their effect on bacterial and human cells.

This study investigated whether there are differences in the ability of wound dressings to modulate certain factors known to affect wound healing. A selection of antimicrobial dressings (AQUACEL® Ag Extra™ , AQUACEL® Ag+ Extra™ , IODOFLEX™ , ACTICOAT™ 7 and PROMOGRAN PRISMA™ matrix) were tested for their effect on both bacterial bioburden and human dermal fibroblasts. Some dressings underwent further evaluation for activity against Pseudomonas aeruginosa biofilms using a colony-drip flow reactor model. The ability of in vitro biofilms to produce proteases, and the effect of PROMOGRAN PRISMA matrix on such proteases, was also investigated. All antimicrobial dressings tested reduced vegetative bacterial load; however, only PROMOGRAN PRISMA matrix was able to significantly reduce biofilm populations (P = 0·01). Additionally, PROMOGRAN PRISMA matrix was the only dressing that did not inhibit dermal fibroblast growth. All other dressings were detrimental to cell viability. In vitro biofilms of Pseudomonas aeruginosa were demonstrated as being capable of releasing bacterial proteases into their surroundings, and incubation with PROMOGRAN PRISMA matrix led to a 77% reduction in activity of such proteases (P = 0·002). The unique ability of PROMOGRAN PRISMA matrix to reduce in vitro vegetative bacteria, biofilm bacteria and bacterial proteases while still allowing dermal fibroblast proliferation may help rebalance the wound environment and reduce the occurrence of infection.

Leapfrogging into new territory: How Mascarene ridged frogs diversified across Africa and Madagascar to maintain their ecological niche.

The Mascarene ridged frog, Ptychadena mascareniensis, is a species complex that includes numerous lineages occurring mostly in humid savannas and open forests of mainland Africa, Madagascar, the Seychelles, and the Mascarene Islands. Sampling across this broad distribution presents an opportunity to examine the genetic differentiation within this complex and to investigate how the evolution of bioclimatic niches may have shaped current biogeographic patterns. Using model-based phylogenetic methods and molecular-clock dating, we constructed a time-calibrated molecular phylogenetic hypothesis for the group based on mitochondrial 16S rRNA and cytochrome b (cytb) genes and the nuclear RAG1 gene from 173 individuals. Haplotype networks were reconstructed and species boundaries were investigated using three species-delimitation approaches: Bayesian generalized mixed Yule-coalescent model (bGMYC), the Poisson Tree Process model (PTP) and a cluster algorithm (SpeciesIdentifier). Estimates of similarity in bioclimatic niche were calculated from species-distribution models (maxent) and multivariate statistics (Principal Component Analysis, Discriminant Function Analysis). Ancestral-area reconstructions were performed on the phylogeny using probabilistic approaches implemented in BioGeoBEARS. We detected high levels of genetic differentiation yielding ten distinct lineages or operational taxonomic units, and Central Africa was found to be a diversity hotspot for these frogs. Most speciation events took place throughout the Miocene, including "out-of-Africa" overseas dispersal events to Madagascar in the East and to São Tomé in the West. Bioclimatic niche was remarkably well conserved, with most species tolerating similar temperature and rainfall conditions common to the Central African region. The P. mascareniensis complex provides insights into how bioclimatic niche shaped the current biogeographic patterns with niche conservatism being exhibited by the Central African radiation and niche divergence shaping populations in West Africa and Madagascar. Central Africa, including the Albertine Rift region, has been an important center of diversification for this species complex.

Host preference, population growth and injuries assessment of Polyphagotarsonemus latus (banks) (ACARI: Tarsonemidae) on Capsicum annuum L. Genotypes.

Despite the continued efforts on the search for different genotypes, Capsicum annuum (L.) is quite susceptible to attack by pest arthropods, especially the broad mite Polyphagotarsonemus latus Banks. Thus, the host preference, population growth and the injuries assessment of P. latus was studied on six C. annuum genotypes used in Brazil (Atlantis, California Wonder, Impact, Palloma, Rubia and Tendence). Host preference was accessed in choice tests, pairing the several genotypes, and the population growth was observed through non-choice tests in laboratory. The injuries assessments were evaluated in the greenhouse, comparing the injury level among the six genotypes. The results indicate that California Wonder and Palloma genotypes were more preferred by P. latus, and Impact and Tendence were less preferred. P. latus presented positive population growth rates (ri) on all the genotypes, however, Palloma and California Wonder showed the highest values of population growth rate (ri = 0.344 and ri = 0.340, respectively), while Impact had the lowest value (ri = 0.281). All the evaluated C. annuum genotypes showed low tolerance to P. latus and exhibited several injuries, but there was no statistical difference between them. California Wonder had the highest average number of mites/leaf (57.15), while Impact and Tendence obtained the lowest values (36.67 and 35.12, respectively) at the end of the evaluation period. The total average of injuries notes at the end of the bioassay did not differ between the genotypes. The number of mites/leaf was growing for the injury scale to the note 3.0, but when the injury scale approached the note 4.0, there was observed a decrease in the number of mites/leaf for all the genotypes.

Defining a new diagnostic assessment parameter for wound care: Elevated protease activity, an indicator of nonhealing, for targeted protease-modulating treatment.

It is widely accepted that elevated protease activity (EPA) in chronic wounds impedes healing. However, little progress has occurred in quantifying the level of protease activity that is detrimental for healing. The aim of this study was to determine the relationship between inflammatory protease activity and wound healing status, and to establish the level of EPA above which human neutrophil-derived elastase (HNE) and matrix metalloproteases (MMP) activities correlate with nonhealing wounds. Chronic wound swab samples (n = 290) were collected from four wound centers across the USA to measure HNE and MMP activity. Healing status was determined according to percentage reduction in wound area over the previous 2-4 weeks; this was available for 211 wounds. Association between protease activity and nonhealing wounds was determined by receiver operating characteristic analysis (ROC), a statistical technique used for visualizing and analyzing the performance of diagnostic tests. ROC analysis showed that area under the curve (AUC) for HNE were 0.69 for all wounds and 0.78 for wounds with the most reliable wound trajectory information, respectively. For MMP, the corresponding AUC values were 0.70 and 0.82. Analysis suggested that chronic wounds having values of HNE >5 and/or MMP ≥13, should be considered wound healing impaired. EPA is indicative of nonhealing wounds. Use of a diagnostic test to detect EPA in clinical practice could enable clinicians to identify wounds that are nonhealing, thus enabling targeted treatment with protease modulating therapies.

Sublethal Effects of Essential Oils From Eucalyptus staigeriana (Myrtales: Myrtaceae), Ocimum gratissimum (Lamiales: Laminaceae), and Foeniculum vulgare (Apiales: Apiaceae) on the Biology of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Spodoptera frugiperda (Smith 1797) (Lepidoptera: Noctuidae) is a major pest of maize, Zea mays L. Its control is often achieved through repeated applications per season of insecticides, which may lead to adverse effects on the ecosystem. Thus, the study of alternative methods with less environmental impact has expanded to include the use of essential oils. These oils are products of the secondary metabolism in plants, and their insecticidal activity has been widely demonstrated in populations of many pest insects. This study evaluated the insecticidal activities of essential oils from Eucalyptus staigeriana, Ocimum gratissimum, and Foeniculum vulgare on Spodoptera frugiperda. Gas chromatography­mass spectrometry profiles and contact toxicity of these oils as well as their sublethal effects on larvae and reproductive parameters in adults were evaluated. All three oils had sublethal effects on S. frugiperda; however, the oil of O. gratissimum showed the best results at all doses tested. These essential oils may have promise for control of S. frugiperda.

Biomarker responses to estrogen and androgen exposure in the brook stickleback (Culaea inconstans): A new bioindicator species for endocrine disrupting compounds.

Small-bodied freshwater fish are commonly used in regulatory testing for endocrine disrupting compounds (EDCs) but most lack a sensitive and quantifiable androgen-specific biomarker. Brook stickleback (Culaea inconstans) are a North American freshwater fish whose males produce an androgen-regulated glycoprotein in the kidney called spiggin. Although spiggin induction in females has been used as an androgen-specific biomarker of exposure in other stickleback species it has not been characterized in brook stickleback. Therefore, our objective was to develop a bioassay using brook stickleback to measure estrogenic and androgenic responses and establish the sensitivity of traditional and novel biomarkers of exposure. We first developed and optimized a qPCR assay to measure spiggin and vitellogenin transcript levels in kidney and liver tissue, respectively. Basal levels were differentially expressed in mature wild-caught male and female brook stickleback. To determine their sensitivity to EDCs, fish were exposed to nominal concentrations of 1, 10 and 100ng/L of 17α-methyltestosterone (MT) or 17α-ethinylestradiol (EE2) for 21days (sampled at 7 and 21days) under semi-static renewal conditions. MT and EE2 exposure induced spiggin and vitellogenin transcripts in female kidneys and male livers, respectively. Exposure to EE2 also increased hepatosomatic index in both sexes and decreased gonadosomatic index in females. Histopathological alterations were observed in the kidney of EE2-exposed fish and an increase in kidney epithelium cell height occurred in MT-exposed females. Given the sensitivity of these endpoints, the brook stickleback is a promising new freshwater fish model for EDC evaluation and a potential bioindicator for EDCs in North American freshwater environments.

hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature.

To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.