Safety outcomes when switching between biosimilars and reference biologics: A systematic review and meta-analysis.

Biosimilars are increasingly available for the treatment of many serious disorders, however some concerns persist about switching a patient to a biosimilar whose condition is stable while on the reference biologic. Randomized controlled studies and extension studies with a switch treatment period (STP) to or from a biosimilar and its reference biologic were identified from publicly available information maintained by the U.S. Food and Drug Administration (FDA). These findings were augmented with data from peer reviewed publications containing information not captured in FDA reviews. Forty-four STPs were identified from 31 unique studies for 21 different biosimilars. Data were extracted and synthesized following PRISMA guidelines. Meta-analysis was conducted to estimate the overall risk difference across studies. A total of 5,252 patients who were switched to or from a biosimilar and its reference biologic were identified. Safety data including deaths, serious adverse events, and treatment discontinuation showed an overall risk difference (95% CI) of -0.00 (-0.00, 0.00), 0.00 (-0.01, 0.01), -0.00 (-0.01, 0.00) across STPs, respectively. Immunogenicity data showed similar incidence of anti-drug antibodies and neutralizing antibodies in patients within a STP who were switched to or from a biosimilar to its reference biologic and patients who were not switched. Immune related adverse events such as anaphylaxis, hypersensitivity reactions, and injections site reactions were similar in switched and non-switched patients. This first systematic review using statistical methods to address the risk of switching patients between reference biologics and biosimilars finds no difference in the safety profiles or immunogenicity rates in patients who were switched and those who remained on a reference biologic or a biosimilar.

Microbial Community Succession Along a Chronosequence in Constructed Salt Marsh Soils.

In this study, we examined the succession of soil microbial communities across a chronosequence of newly constructed salt marshes constructed primarily of fine-grained dredge material, using 16S rRNA amplicon sequences. Alpha diversity in the subsurface horizons was initially low and increased to reference levels within 3 years of marsh construction, while alpha diversity in the newly accumulating organic matter-rich surface soils was initially high and remained unchanged. Microbial community succession was fastest in the surface horizon (~ 24 years to reference equivalency) and became progressively slower with depth in the subsurface horizons (~ 30-67 years). Random forest linear regression analysis was used to identify important taxa driving the trajectories toward reference conditions. In the parent material, putative sulfate-reducers (Desulfobacterota), methanogens (Crenarchaeota, especially Methanosaeta), and fermenters (Chloroflexi and Clostridia) increased over time, suggesting an enrichment of these metabolisms over time, similar to natural marshes. Concurrently in the surface soils, the relative abundances of putative methane-, methyl-, and sulfide oxidizers, especially among Gammaproteobacteria, increased over time, suggesting the co-development of sulfide and methane removal metabolisms in marsh soils. Finally, we observed that the surface soil communities at one of the marshes did not follow the trajectory of the others, exhibiting a greater relative abundance of anaerobic taxa. Uniquely in this dataset, this marsh was developing signs of excessive inundation stress in terms of vegetation coverage and soil geochemistry. Therefore, we suggest that soil microbial community structure may be effective bioindicators of salt marsh inundation and are worthy of further targeted investigation.

Oocyte Development and Quality in Young and Old Mice following Exposure to Atrazine.

BACKGROUND: Egg development has unique features that render it vulnerable to environmental perturbation. The herbicide atrazine is an endocrine disruptor shown to have detrimental effects on reproduction across several vertebrate species. OBJECTIVES: This study was designed to determine whether exposure to low levels of atrazine impairs meiosis in female mammals, using a mouse model; in particular, the study's researchers sought to determine whether and how the fidelity of oocyte chromosome segregation may be affected and whether aging-related aneuploidy is exacerbated. METHODS: Female C57BL/6J mice were exposed to two levels of atrazine in drinking water: The higher level equaled aqueous saturation, and the lower level corresponded to detected environmental contamination. To model developmental exposure, atrazine was ingested by pregnant females at 0.5 d post coitum and continued until pups were weaned at 21 d postpartum. For adult exposure, 2-month-old females ingested atrazine for 3 months. Following exposure, various indicators of oocyte development and quality were determined, including: a) chromosome synapsis and crossing over in fetal oocytes using immunofluorescence staining of prophase-I chromosome preparations; b) sizes of follicle pools in sectioned ovaries; c) efficiencies of in vitro fertilization and early embryogenesis; d) chromosome alignment and segregation in cultured oocytes; e) chromosomal errors in metaphase-I and -II (MI and MII) preparations; and f) sister-chromatid cohesion via immunofluorescence intensity of cohesin subunit REC8 on MI-chromosome preparations, and measurement of interkinetochore distances in MII preparations. RESULTS: Mice exposed to atrazine during development showed slightly higher levels of defects in chromosome synapsis, but sizes of initial follicle pools were indistinguishable from controls. However, although more eggs were ovulated, oocyte quality was lower. At the chromosome level, frequencies of spindle misalignment and numerical and structural abnormalities were greater at both meiotic divisions. In vitro fertilization was less efficient, and there were more apoptotic cells in blastocysts derived from eggs of atrazine-exposed females. Similar levels of chromosomal defects were seen in oocytes following both developmental and adult exposure regimens, suggesting quiescent primordial follicles may be a consequential target of atrazine. An important finding was that defects were observed long after exposure was terminated. Moreover, chromosomally abnormal eggs were very frequent in older mice, implying that atrazine exposure during development exacerbates effects of maternal aging on oocyte quality. Indeed, analogous to the effects of maternal age, weaker cohesion between sister chromatids was observed in oocytes from atrazine-exposed animals. CONCLUSION: Low-level atrazine exposure caused persistent changes to the female mammalian germline in mice, with potential consequences for reproductive lifespan and congenital disease. https://doi.org/10.1289/EHP11343.

Microbial succession in a marine sediment: Inferring interspecific microbial interactions with marine cable bacteria.

Cable bacteria are long, filamentous, multicellular bacteria that grow in marine sediments and couple sulfide oxidation to oxygen reduction over centimetre-scale distances via long-distance electron transport. Cable bacteria can strongly modify biogeochemical cycling and may affect microbial community networks. Here we examine interspecific interactions with marine cable bacteria (Ca. Electrothrix) by monitoring the succession of 16S rRNA amplicons (DNA and RNA) and cell abundance across depth and time, contrasting sediments with and without cable bacteria growth. In the oxic zone, cable bacteria activity was positively associated with abundant predatory bacteria (Bdellovibrionota, Myxococcota, Bradymonadales), indicating putative predation on cathodic cells. At suboxic depths, cable bacteria activity was positively associated with sulfate-reducing and magnetotactic bacteria, consistent with cable bacteria functioning as ecosystem engineers that modify their local biogeochemical environment, benefitting certain microbes. Cable bacteria activity was negatively associated with chemoautotrophic sulfur-oxidizing Gammaproteobacteria (Thiogranum, Sedimenticola) at oxic depths, suggesting competition, and positively correlated with these taxa at suboxic depths, suggesting syntrophy and/or facilitation. These observations are consistent with chemoautotrophic sulfur oxidizers benefitting from an oxidizing potential imparted by cable bacteria at suboxic depths, possibly by using cable bacteria as acceptors for electrons or electron equivalents, but by an as yet enigmatic mechanism.

Visualizing G protein-coupled receptor homomers using photoactivatable dye localization microscopy.

G protein-coupled receptor (GPCR) di/oligomerization has revealed potential mechanisms for receptors diversification of signal selectivity, specificity, and amplitude. The use of super-resolution imaging techniques to investigate these di/oligomer molecular complexities have undoubtably provided insight to the dynamics of complexes formed at the plasma membrane. Here we describe the methodology of photoactivatable dye localization microscopy (PD-PALM) to study the spatial organization of GPCR homomers at the plasma membrane.

Quantitative methods and modeling to assess COVID-19-interrupted in vivo pharmacokinetic bioequivalence studies with two reference batches.

The coronavirus disease 2019 (COVID-19) has presented unprecedented challenges to the generic drug development, including interruptions in bioequivalence (BE) studies. Per guidance published by the US Food and Drug Administration (FDA) during the COVID-19 public health emergency, any protocol changes or alternative statistical analysis plan for COVID-19-interrupted BE study should be accompanied with adequate justifications and not lead to biased equivalence determination. In this study, we used a modeling and simulation approach to assess the potential impact of study outcomes when two different batches of a Reference Standard (RS) were to be used in an in vivo pharmacokinetic BE study due to the RS expiration during the COVID-19 pandemic. Simulations were performed with hypothetical drugs under two scenarios: (1) uninterrupted study using a single batch of an RS, and (2) interrupted study using two batches of an RS. The acceptability of BE outcomes was evaluated by comparing the results obtained from interrupted studies with those from uninterrupted studies. The simulation results demonstrated that using a conventional statistical approach to evaluate BE for COVID-19-interrupted studies may be acceptable based on the pooled data from two batches. An alternative statistical method which includes a "batch" effect to the mixed effects model may be used when a significant "batch" effect was found in interrupted four-way crossover studies. However, such alternative method is not applicable for interrupted two-way crossover studies. Overall, the simulated scenarios are only for demonstration purpose, the acceptability of BE outcomes for the COVID19-interrupted studies could be case-specific.

COVID-19, Suffering and Palliative Care: A Review.

According to the WHO guideline, palliative care is an integral component of COVID-19 management. The relief of physical symptoms and the provision of psychosocial support should be practiced by all healthcare workers caring for COVID-19 patients. In this review, we aim to provide a simple outline on COVID-19, suffering in COVID-19, and the role of palliative care in COVID-19. We also introduce 3 principles of palliative care that can serve as a guide for all healthcare workers caring for COVID-19 patients, which are (1) good symptom control, (2) open and sensitive communication, and (3) caring for the whole team. The pandemic has brought immense suffering, fear and death to people everywhere. The knowledge, skills and experiences from palliative care could be used to relieve the suffering of COVID-19 patients.

Modeling Virus-Induced Inflammation in Zebrafish: A Balance Between Infection Control and Excessive Inflammation.

The inflammatory response to viral infection in humans is a dynamic process with complex cell interactions that are governed by the immune system and influenced by both host and viral factors. Due to this complexity, the relative contributions of the virus and host factors are best studied in vivo using animal models. In this review, we describe how the zebrafish (Danio rerio) has been used as a powerful model to study host-virus interactions and inflammation by combining robust forward and reverse genetic tools with in vivo imaging of transparent embryos and larvae. The innate immune system has an essential role in the initial inflammatory response to viral infection. Focused studies of the innate immune response to viral infection are possible using the zebrafish model as there is a 4-6 week timeframe during development where they have a functional innate immune system dominated by neutrophils and macrophages. During this timeframe, zebrafish lack a functional adaptive immune system, so it is possible to study the innate immune response in isolation. Sequencing of the zebrafish genome has revealed significant genetic conservation with the human genome, and multiple studies have revealed both functional conservation of genes, including those critical to host cell infection and host cell inflammatory response. In addition to studying several fish viruses, zebrafish infection models have been developed for several human viruses, including influenza A, noroviruses, chikungunya, Zika, dengue, herpes simplex virus type 1, Sindbis, and hepatitis C virus. The development of these diverse viral infection models, coupled with the inherent strengths of the zebrafish model, particularly as it relates to our understanding of macrophage and neutrophil biology, offers opportunities for far more intensive studies aimed at understanding conserved host responses to viral infection. In this context, we review aspects relating to the evolution of innate immunity, including the evolution of viral pattern recognition receptors, interferons and interferon receptors, and non-coding RNAs.

The Luteinizing Hormone Receptor Knockout Mouse as a Tool to Probe the In Vivo Actions of Gonadotropic Hormones/Receptors in Females.

Mouse models with altered gonadotropin functions have provided invaluable insight into the functions of these hormones/receptors. Here we describe the repurposing of the infertile and hypogonadal luteinizing hormone receptor (LHR) knockout mouse model (LuRKO), to address outstanding questions in reproductive physiology. Using crossbreeding strategies and physiological and histological analyses, we first addressed the physiological relevance of forced LHR homomerization in female mice using BAC expression of 2 ligand-binding and signaling deficient mutant LHR, respectively, that have previously shown to undergo functional complementation and rescue the hypogonadal phenotype of male LuRKO mice. In female LuRKO mice, coexpression of signaling and binding deficient LHR mutants failed to rescue the hypogonadal and anovulatory phenotype. This was apparently due to the low-level expression of the 2 mutant LHR and potential lack of luteinizing hormone (LH)/LHR-dependent pleiotropic signaling that has previously been shown at high receptor densities to be essential for ovulation. Next, we utilized a mouse model overexpressing human chorionic gonadotropin (hCG) with increased circulating "LH/hCG"-like bioactivity to ~40 fold higher than WT females, to determine if high circulating hCG in the LuRKO background could reveal putative LHR-independent actions. No effects were found, thus, suggesting that LH/hCG mediate their gonadal and non-gonadal effects solely via LHR. Finally, targeted expression of a constitutively active follicle stimulating hormone receptor (FSHR) progressed antral follicles to preovulatory follicles and displayed phenotypic markers of enhanced estrogenic activity but failed to induce ovulation in LuRKO mice. This study highlights the critical importance and precise control of functional LHR and FSHR for mediating ovarian functions and of the potential repurposing of existing genetically modified mouse models in answering outstanding questions in reproductive physiology.

Mechanistic insight into how gonadotropin hormone receptor complexes direct signaling†.

Gonadotropin hormones and their receptors play a central role in the control of male and female reproduction. In recent years, there has been growing evidence surrounding the complexity of gonadotropin hormone/receptor signaling, with it increasingly apparent that the Gαs/cAMP/PKA pathway is not the sole signaling pathway that confers their biological actions. Here we review recent literature on the different receptor-receptor, receptor-scaffold, and receptor-signaling molecule complexes formed and how these modulate and direct gonadotropin hormone-dependent intracellular signal activation. We will touch upon the more controversial issue of extragonadal expression of FSHR and the differential signal pathways activated in these tissues, and lastly, highlight the open questions surrounding the role these gonadotropin hormone receptor complexes and how this will shape future research directions.

Statistical considerations and impact of the FDA draft guidance for assessing adhesion with transdermal delivery systems and topical patches for ANDAs.

Until 2016, a ratio of means (ROM) non-inferiority (NI) test was recommended in FDA product-specific guidances (PSGs) to evaluate adhesion performance for prospective generic transdermal delivery systems (TDS). However, the ROM NI test had low power for well-adhering TDS, which were becoming increasingly prevalent. Mathematical proof and simulation revealed that the low power wasn't because the non-normality of adhesion data violated the normality assumption of parametric methods; it was because the ROM NI test was coupled with an adhesion scale where scores approached 0 as adhesion got better. In June 2016, FDA published a draft general guidance on TDS adhesion and recommended a new statistical approach, replacing the ROM NI test with a difference-of-means (DOM) NI test, using the same scale and primary endpoint (mean adhesion scores). An analysis of 40 TDS adhesion studies submitted in ANDAs after the publication of the 2016 draft guidance suggests that, consistent with simulation results, the new statistical approach markedly improves the low power, and thereby reduces the sample size required by the old approach for moderately to well-adhering TDS, while retaining comparable power for poorly adhering TDS. The new statistical approach thus enhances the potential approvability and patient access to well-adhering generic TDS.

Paired RNA Radiocarbon and Sequencing Analyses Indicate the Importance of Autotrophy in a Shallow Alluvial Aquifer.

Determining the carbon sources for active microbial populations in the subsurface is a challenging but highly informative component of subsurface microbial ecology. This work developed a method to provide ecological insights into groundwater microbial communities by characterizing community RNA through its radiocarbon and ribosomal RNA (rRNA) signatures. RNA was chosen as the biomolecule of interest because rRNA constitutes the majority of RNA in prokaryotes, represents recently active organisms, and yields detailed taxonomic information. The method was applied to a groundwater filter collected from a shallow alluvial aquifer in Colorado. RNA was extracted, radiometrically dated, and the 16S rRNA was analyzed by RNA-Seq. The RNA had a radiocarbon signature (Δ14C) of -193.4 ± 5.6‰. Comparison of the RNA radiocarbon signature to those of potential carbon pools in the aquifer indicated that at least 51% of the RNA was derived from autotrophy, in close agreement with the RNA-Seq data, which documented the prevalence of autotrophic taxa, such as Thiobacillus and Gallionellaceae. Overall, this hybrid method for RNA analysis provided cultivation-independent information on the in-situ carbon sources of active subsurface microbes and reinforced the importance of autotrophy and the preferential utilization of dissolved over sedimentary organic matter in alluvial aquifers.

Common Filing Deficiencies in Abbreviated New Drug Applications Containing Clinical Endpoint Studies.

BACKGROUND: The objective of this report is to summarize common deficiencies identified in the filing reviews of abbreviated new drug applications (ANDAs) with clinical endpoint bioequivalence studies and skin irritation, sensitization, and adhesion (I/S/A) studies received by the US Food and Drug Administration (FDA) between 2007 and 2017, to help applicants avoid common deficiencies, minimize "refuse-to-receive" (RTR) actions, "information requests," and ANDA approval delays. METHODS: Multiple internal FDA databases were searched to evaluate and summarize common deficiencies identified in ANDA submissions containing clinical endpoint studies and skin I/S/A studies that required review by the Division of Clinical Review. A total of 275 ANDA submissions with filing reviews from January 2007 to June 2017 were analyzed in this report. RESULTS: Two hundred eighteen (79.3%) filing reviews contained one or more deficiencies. Seventy-nine (28.7%) ANDAs were issued RTR letters because of major clinical deficiencies, specifically bioequivalence and clinical deficiencies, accounting for 9% of overall identified deficiencies. Twenty-two other categories of deficiencies are summarized into 4 main categories: missing information related to the clinical studies other than data sets (38%), missing data sets (35%), formulation issues (12%), and organization/format issues (6%). CONCLUSIONS: The most common deficiency in the "missing information related to the clinical studies other than data sets" category was "missing clarification of information" (22%). We also noted that the Division of Filing Review has identified these same types of deficiencies since assuming responsibility of the filing assessment for ANDAs with clinical endpoint BE studies and skin I/S/A studies. In conclusion, to minimize "refuse-to-receive" actions, "information requests," and approval of ANDA delays for generic drug products, applicants should submit full clinical study reports, including all data sets for drug products recommending clinical studies.

Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy.

INTRODUCTION: Both genetic and infectious diseases can result in skeletal muscle degeneration, inflammation, pain, and/or weakness. Duchenne muscular dystrophy (DMD) is the most common congenital muscle disease. DMD causes progressive muscle wasting due to mutations in Dystrophin. Influenza A and B viruses are frequently associated with muscle complications, especially in children. Infections activate an immune response and immunosuppressant drugs reduce DMD symptoms. These data suggest that the immune system may contribute to muscle pathology. However, roles of the immune response in DMD and Influenza muscle complications are not well understood. Zebrafish with dmd mutations are a well-characterized model in which to study the molecular and cellular mechanisms of DMD pathology. We recently showed that zebrafish can be infected by human Influenza A virus (IAV). Thus, the zebrafish is a powerful system with which to ask questions about the etiology and mechanisms of muscle damage due to genetic and/or infectious diseases. METHODS: We infected zebrafish with IAV and assayed muscle tissue structure, sarcolemma integrity, cell-extracellular matrix (ECM) attachment, and molecular and cellular markers of inflammation in response to IAV infection alone or in the context of DMD. RESULTS: We find that IAV-infected zebrafish display mild muscle degeneration with sarcolemma damage and compromised ECM adhesion. An innate immune response is elicited in muscle in IAV-infected zebrafish: NFkB signaling is activated, pro-inflammatory cytokine expression is upregulated, and neutrophils localize to sites of muscle damage. IAV-infected dmd mutants display more severe muscle damage than would be expected from an additive effect of dmd mutation and IAV infection, suggesting that muscle damage caused by Dystrophin-deficiency and IAV infection is synergistic. DISCUSSION: These data demonstrate the importance of preventing IAV infections in individuals with genetic muscle diseases. Elucidating the mechanisms of immune-mediated muscle damage will not only apply to DMD and IAV, but also to other conditions where the immune system, inflammation, and muscle tissue are known to be affected, such as autoimmune diseases, cancer, and aging.

Evolutionary divergence of the vertebrate TNFAIP8 gene family: Applying the spotted gar orthology bridge to understand ohnolog loss in teleosts.

Comparative functional genomic studies require the proper identification of gene orthologs to properly exploit animal biomedical research models. To identify gene orthologs, comprehensive, conserved gene synteny analyses are necessary to unwind gene histories that are convoluted by two rounds of early vertebrate genome duplication, and in the case of the teleosts, a third round, the teleost genome duplication (TGD). Recently, the genome of the spotted gar, a holostean outgroup to the teleosts that did not undergo this third genome duplication, was sequenced and applied as an orthology bridge to facilitate the identification of teleost orthologs to human genes and to enhance the power of teleosts as biomedical models. In this study, we apply the spotted gar orthology bridge to help describe the gene history of the vertebrate TNFAIP8 family. Members of the TNFAIP8 gene family have been linked to regulation of immune function and homeostasis and the development of multiple cancer types. Through a conserved gene synteny analysis, we identified zebrafish orthologs to human TNFAIP8L1 and TNFAIP8L3 genes and two co-orthologs to human TNFAIP8L2, but failed to identify an ortholog to human TNFAIP8. Through the application of the orthology bridge, we determined that teleost orthologs to human TNFAIP8 genes were likely lost in a genome inversion event after their divergence from their common ancestor with spotted gar. These findings demonstrate the value of this enhanced approach to gene history analysis and support the development of teleost models to study complex questions related to an array of biomedical issues, including immunity and cancer.

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Subpallial Enhancer Transgenic Lines: a Data and Tool Resource to Study Transcriptional Regulation of GABAergic Cell Fate.

Elucidating the transcriptional circuitry controlling forebrain development requires an understanding of enhancer activity and regulation. We generated stable transgenic mouse lines that express CreERT2 and GFP from ten different enhancer elements with activity in distinct domains within the embryonic basal ganglia. We used these unique tools to generate a comprehensive regional fate map of the mouse subpallium, including sources for specific subtypes of amygdala neurons. We then focused on deciphering transcriptional mechanisms that control enhancer activity. Using machine-learning computations, in vivo chromosomal occupancy of 13 transcription factors that regulate subpallial patterning and differentiation and analysis of enhancer activity in Dlx1/2 and Lhx6 mutants, we elucidated novel molecular mechanisms that regulate region-specific enhancer activity in the developing brain. Thus, these subpallial enhancer transgenic lines are data and tool resources to study transcriptional regulation of GABAergic cell fate.

Transcriptional Networks Controlled by NKX2-1 in the Development of Forebrain GABAergic Neurons.

The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.

Advancing toxicology research using in vivo high throughput toxicology with small fish models.

Small freshwater fish models, especially zebrafish, offer advantages over traditional rodent models, including low maintenance and husbandry costs, high fecundity, genetic diversity, physiology similar to that of traditional biomedical models, and reduced animal welfare concerns. The Collaborative Workshop on Aquatic Models and 21st Century Toxicology was held at North Carolina State University on May 5-6, 2014, in Raleigh, North Carolina, USA. Participants discussed the ways in which small fish are being used as models to screen toxicants and understand mechanisms of toxicity. Workshop participants agreed that the lack of standardized protocols is an impediment to broader acceptance of these models, whereas development of standardized protocols, validation, and subsequent regulatory acceptance would facilitate greater usage. Given the advantages and increasing application of small fish models, there was widespread interest in follow-up workshops to review and discuss developments in their use. In this article, we summarize the recommendations formulated by workshop participants to enhance the utility of small fish species in toxicology studies, as well as many of the advances in the field of toxicology that resulted from using small fish species, including advances in developmental toxicology, cardiovascular toxicology, neurotoxicology, and immunotoxicology. We alsoreview many emerging issues that will benefit from using small fish species, especially zebrafish, and new technologies that will enable using these organisms to yield results unprecedented in their information content to better understand how toxicants affect development and health.

Triclosan is a mitochondrial uncoupler in live zebrafish.

Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here, we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24-hour post-fertilization (hpf) zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well. Using this method, after acute exposure to TCS, the basal oxygen consumption rate (OCR) increases, without a decrease in survival or heartbeat rate. TCS also decreases ATP-linked respiration and spare respiratory capacity and increases proton leak: all indicators of mitochondrial uncoupling. Our data indicate, that TCS is a mitochondrial uncoupler in vivo, which should be taken into consideration when assessing the toxicity and/or pharmaceutical uses of TCS. This is the first example of usage of a Seahorse Extracellular Flux Analyzer to measure bioenergetic flux of a single zebrafish embryo per well in a 96-well assay format. The method developed in this study provides a high-throughput tool to identify previously unknown mitochondrial uncouplers in a living organism. Copyright © 2016 John Wiley & Sons, Ltd.