Recruitment of Peroxin 14 to lipid droplets affects lipid storage in Drosophila.

Both peroxisomes and lipid droplets regulate cellular lipid homeostasis. Direct inter-organellar contacts as well as novel roles for proteins associated with peroxisome or lipid droplets occur when cells are induced to liberate fatty acids from lipid droplets. We have shown a non-canonical role for a subset of peroxisome-assembly [Peroxin (Pex)] proteins in this process in Drosophila. Transmembrane proteins Pex3, Pex13 and Pex14 were observed to surround newly formed lipid droplets. Trafficking of Pex14 to lipid droplets was enhanced by loss of Pex19, which directs insertion of transmembrane proteins like Pex14 into the peroxisome bilayer membrane. Accumulation of Pex14 around lipid droplets did not induce changes to peroxisome size or number, and co-recruitment of the remaining Peroxins was not needed to assemble peroxisomes observed. Increasing the relative level of Pex14 surrounding lipid droplets affected the recruitment of Hsl lipase. Fat body-specific reduction of these lipid droplet-associated Peroxins caused a unique effect on larval fat body development and affected their survival on lipid-enriched or minimal diets. This revealed a heretofore unknown function for a subset of Pex proteins in regulating lipid storage. This article has an associated First Person interview with Kazuki Ueda, joint first author of the paper.

Similar patient reported outcomes at long-term follow-up after external fixation versus internal fixation of the anterior ring component of APC injuries.

PURPOSE: Unstable pelvic ring injuries produced by external rotation of the hemipelvis and a symphyseal disruption are most often treated with internal fixation of the anterior ring, with percutaneous treatment of the posterior ring as needed. In some clinical situations, patients are treated with external fixation for their anterior injuries and the long-term functional outcomes associated with external fixation are not well understood. We ask if there is a difference in functional outcome, between treatment of these injuries with internal versus external fixation, when measured at a minimum of three years after injury. METHOD: This was a retrospective cohort study performed at a level one regional trauma center. Trauma database review identified 128 patients, with 70 subsequently excluded, with unstable anterior posterior compression (APC) pelvic ring injuries (OTA 61B2.3 & 61C1.2) treated with surgery with minimum three years of follow-up. An intervention of internal fixation versus external fixation of anterior pelvic ring was performed, and depending on the injury, supplemented with posterior iliosacral screw fixation. Main outcome was measured with the Majeed functional outcome score (0-100). RESULTS: Patients treated with external fixation reported a Majeed score of 70 (95% CI 28-100) compared to 79 (95% CI 36-100) in those with internal fixation (p-value 0.28). Subgroups of the Majeed score were not significantly different (p value > 0.05). Open fractures, severity of injury, and ISS were worse in those treated with external fixation. There was no differential loss to follow-up. Conclusion Patients with unstable pelvic ring injuries with symphyseal disruptions treated with external fixation as definitive treatment versus internal fixation may fare no different in the long term.

Nasal Chondrocyte-Derived Soluble Factors Affect Chondrogenesis of Cocultured Mesenchymal Stem Cells.

To investigate the effect of soluble factors released from human nasal chondrocytes (NCs) on cocultured human bone marrow mesenchymal stem cells (MSCs) and NC tissue-engineered constructs. Cartilage engineered from pure NCs on a three-dimensional (3D) porous collagen scaffold was cultured indirectly in a Transwell system with cartilage engineered from a direct coculture of human bone marrow-derived MSCs and NCs on a 3D porous collagen scaffold. The soluble factors were measured in the conditioned media from the different chambers of the Transwell system. Engineered cartilage from cocultures exposed to the pure NC construct exhibited reduced chondrogenic potential relative to control constructs, shown by reduced extracellular matrix deposition and increased expression of hypertrophic markers. Analysis of the soluble factors within the conditioned media showed an increase in inflammatory cytokines in the coculture chamber exposed to the pure NC construct. Principal component analysis revealed that the majority of the data variance could be explained by proinflammatory factors and hypertrophic chondrogenesis. In conclusion, our data suggest that inflammatory cytokines derived from NCs reduce the chondrogenic potential of coculture engineered cartilage through the induction of hypertrophic chondrogenesis. Impact statement The use of engineered cartilage from cocultured nasal chondrocytes (NCs) and mesenchymal stem cells for nasal cartilage reconstruction may be problematic. Our data suggest that the soluble factors from surrounding native NCs in the cartilage to be fixed can compromise the quality of the engineered cartilage if used in reconstructive surgery.

Testing pterosaur ingroup relationships through broader sampling of avemetatarsalian taxa and characters and a range of phylogenetic analysis techniques.

The pterosaurs first appear in the fossil record in the middle of the Late Triassic. Their earliest representatives are known from Northern Hemisphere localities but, by the end of the Jurassic Period, this clade of flying reptiles achieved a global distribution, as well as high levels of diversity and disparity. Our understanding of early pterosaur evolution and the fundamental interrelationships within Pterosauria has improved dramatically in recent decades. However, there is still debate about how the various pterosaur subgroups relate to one another and about which taxa comprise these. Many recent phylogenetic analyses, while sampling well from among the known Triassic and Early Jurassic pterosaurs, have not included many non-pterosaurian ornithodirans or other avemetatarsalians. Given the close relationship between these groups of archosaurs, the omission of other ornithodirans and avemetatarsalians has the potential to adversely affect the results of phylogenetic analyses, in terms of character optimisation and ingroup relationships recovered. This study has addressed this issue and tests the relationships between the early diverging pterosaur taxa following the addition of avemetatarsalian taxa and anatomical characters to an existing early pterosaur dataset. This study has, for the first time, included taxa that represent the aphanosaurs, lagerpetids, silesaurids and dinosaurs, in addition to early pterosaurs. Anatomical characters used in other recent studies of archosaurs and early dinosaurs have also been incorporated. By expanding the outgroup taxa and anatomical character coverage in this pterosaur dataset, better resolution between the taxa within certain early pterosaur subclades has been achieved and stronger support for some existing clades has been found; other purported clades of early pterosaurs have not been found in this analysis-for example there is no support for a monophyletic Eopterosauria or Eudimorphodontidae. Further support has been found for a sister-taxon relationship between Peteinosaurus zambelli and Macronychoptera, a clade here named Zambellisauria (clade nov.), as well as for a monophyletic and early diverging Preondactylia. Some analyses also support the existence of a clade that falls as sister-taxon to the zambellisaurs, here named Caviramidae (clade nov.). Furthermore, some support has been found for a monophyletic Austriadraconidae at the base of Pterosauria. Somewhat surprisingly, Lagerpetidae is recovered outside of Ornithodira sensu stricto, meaning that, based upon current definitions at least, pterosaurs fall within Dinosauromorpha in this analysis. However, fundamental ornithodiran interrelationships were not the focus of this study and this particular result should be treated with caution for now. However, these results do further highlight the need for broader taxon and character sampling in phylogenetic analyses, and the effects of outgroup choice on determining ingroup relationships.

Does Coronal Plane Malalignment of the Tibial Insert in Total Ankle Arthroplasty Alter Distal Foot Bone Mechanics? A Cadaveric Gait Study.

BACKGROUND: Total ankle arthroplasty (TAA) is becoming a more prevalent treatment for end-stage ankle arthritis. However, the effects of malalignment on TAA remain poorly understood. QUESTIONS/PURPOSES: The purpose of this study was to quantify the mechanical effects of coronal plane malalignment of the tibial insert in TAA using cadaveric gait simulation. Specifically, we asked, is there a change in (1) ankle joint congruency, (2) kinematic joint position, (3) kinematic ROM, (4) peak plantar pressure, and (5) center of pressure with varus and valgus malalignment? METHODS: A modified TAA was implanted into seven cadaveric foot specimens. Wedges were used to simulate coronal plane malalignment of the tibial insert. The degree of malalignment (tibial insert angle [TIA] and talar component angle [TCA]) was quantified radiographically for neutral and 5°, 10°, and 15° varus and valgus wedges. Dynamic walking at 1/6 of physiological speed was simulated using a robotic gait simulator. A motion capture system was used to measure foot kinematics, and a pressure mat was used to measure plantar pressure. Joint congruency was quantified as the difference between TIA and TCA. Continuous joint position, joint ROM, peak plantar pressure, and center of pressure for varus and valgus malalignment compared with neutral alignment were estimated using linear mixed effects regression. Pairwise comparisons between malalignment conditions and neutral were considered significant if both the omnibus test for the overall association between outcome and malalignment and the individual pairwise comparison (adjusted for multiple comparisons within a given outcome) had p ≤ 0.05. RESULTS: Descriptively, the TIA and TCA were both less pronounced than the wedge angle and component incongruence was seen (R = 0.65; p < 0.001). Varus malalignment of the tibial insert shifted the tibiotalar joint into varus and internally rotated the joint. The tibiotalar joint's ROM slightly increased as the TIA shifted into varus (1.3 ± 0.7° [mean ± SD] [95% confidence interval -0.7 to 3.4]; p = 0.03), and the first metatarsophalangeal joint's ROM decreased as the TIA shifted into varus (-1.9 ± 0.9° [95% CI -5.6 to 1.7]; p = 0.007). In the sagittal plane, the naviculocuneiform joint's ROM slightly decreased as the TIA shifted into varus (-0.9 ± 0.4° [95% CI -2.1 to 0.3]; p = 0.017). Hallux pressure increased as the TIA became more valgus (59 ± 50 kPa [95% CI -88 to 207]; p = 0.006). The peak plantar pressure slightly decreased in the third and fourth metatarsals as the TIA shifted into valgus (-15 ± 17° [95% CI -65 to 37]; p = 0.03 and -8 ± 4° [95% CI -17 to 1]; p = 0.048, respectively). The fifth metatarsal's pressure slightly decreased as the TIA shifted into valgus (-18 ± 12 kPa [95% CI -51 to 15]) or varus (-7 ± 18 kPa [95% CI -58 to 45]; p = 0.002). All comparisons were made to the neutral condition. CONCLUSIONS: In this cadaver study, coronal plane malalignment in TAA altered foot kinematics and plantar pressure. In general, varus TAA malalignment led to varus shift and internal rotation of the tibiotalar joint, a slight increase in the tibiotalar ROM, and a slight decrease in the first metatarsophalangeal ROM, while a valgus TAA malalignment was manifested primarily through increased hallux pressure with a slight off-loading of the third and fourth metatarsals. CLINICAL RELEVANCE: This study may increase our understanding of the biomechanical processes that underlie the unfavorable clinical outcomes (such as, poor patient-reported outcomes or implant loosening) that have been associated with coronal plane malalignment of the tibial component in TAA.

Effect of cell seeding density on matrix-forming capacity of meniscus fibrochondrocytes and nasal chondrocytes in meniscus tissue engineering.

The presence of intact menisci is imperative for the proper function of the knee joint. Meniscus injuries are often treated by the surgical removal of the damaged tissue, which increases the likelihood of post-traumatic osteoarthritis. Tissue engineering holds great promise in producing viable engineered meniscal tissue for implantation using the patient's own cells; however, the cell source for producing the engineered tissue is unclear. Nasal chondrocytes (NC) possess many attractive features for engineering meniscus. However, in order to validate the use of NC for engineering meniscus fibrocartilage, a thorough comparison of NC and meniscus fibrochondrocytes (MFC) must be considered. Our study presents an analysis of the relative features of NC and MFC and their respective chondrogenic potential in a pellet culture model. We showed considerable differences in the cartilage tissue formed by the two different cell types. Our data showed that NC were more proliferative in culture, deposited more extracellular matrix, and showed higher expression of chondrogenic genes than MFC. Overall, our data suggest that NC produce superior cartilage tissue to MFC in a pellet culture model. In addition, NCs produce higher quality cartilage tissue at higher cell seeding densities during cell expansion.

Suppression of Hypertrophy During in vitro Chondrogenesis of Cocultures of Human Mesenchymal Stem Cells and Nasal Chondrocytes Correlates With Lack of in vivo Calcification and Vascular Invasion.

OBJECTIVE: Human nasal septal chondrocytes (NC) are a promising minimally invasive derivable chondrogenic cell source for cartilage repair. However, the quality of NC-derived cartilage is variable between donors. Coculture of NC with mesenchymal stem cells (MSCs) mitigates the variability but with undesirable markers of chondrocyte hypertrophy, such as type X collagen, and the formation of unstable calcifying cartilage at ectopic sites. In contrast, monoculture NC forms non-calcifying stable cartilage. Formation of a stable NC-MSC coculture cartilage is crucial for clinical application. The aim of this study was to explore the utility of parathyroid hormone-related peptide (PTHrP) hormone to suppress chondrocyte hypertrophy in NC-MSC cocultures and form stable non-calcifying cartilage at ectopic sites. METHODS: Human NC and bone marrow MSCs, and cocultures of NC and MSC (1:3 ratio) were aggregated in pellet form and subjected to in vitro chondrogenesis for 3 weeks in chondrogenic medium in the presence and absence of PTHrP. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks. RESULTS: Coculture of NC and MSC resulted in synergistic cartilage matrix production. PTHrP suppressed the expression of hypertrophy marker, type X collagen (COL10A1), in a dose-dependent fashion without affecting the synergism in cartilage matrix synthesis, and in vivo calcification was eradicated with PTHrP. In contrast, cocultured control (CC) pellets without PTHrP treatment expressed COL10A1, calcified, and became vascularized in vivo.

The Assessment of Clinically Significant Differences in Treating Spinal Deformity Using the SRS Questionnaire: What Is the Threshold of Change That Is Meaningful to Patients?

BACKGROUND: The measurement of health-related quality of life is important in spinal deformity surgery. The Scoliosis Research Society questionnaire has allowed disease-specific research in this area, and determining the minimal clinically important difference (MCID) is as important as it is elusive. We seek to further refine our estimations of clinically perceived improvements by the patient. METHODS: We used an anchor-based approach for each domain of the SRS questionnaire to compare changes at 1 year after treatment. We set the MCID as the upper 95% boundary of the "no change" group bordering the "improvement" arm, where the patients may start to perceive their own change toward the better. We compared this with the mean change. RESULTS: The threshold value for the MCID was 0.54 for the pain domain, 0.31 for function, 0.62 for self-image, and 0.5 for mental health. The mean changes in our group's pain and self-image exceeded their MCID. CONCLUSIONS: Compared with our previous work, we further attempted to refine our assessment of the MCID in spinal deformity. Pain continues to show clinically significant improvement, and self-image also demonstrated mean improvement over its estimated MCID. LEVEL OF EVIDENCE: 2. CLINICAL RELEVANCE: This result in self-image is an important addition to the MCID literature, given its lack of consistency in previous work.

Peroxisome Protein Prediction in Drosophila melanogaster.

As a laboratory animal, Drosophila melanogaster has made extensive contributions to understanding many areas of fundamental biology as well as being an effective model for human disease. Until recently, there was relatively little known about fly peroxisomes. There were early studies that examined the role of peroxisome enzymes during development of organs like the eye. However, with the advent of a well-annotated, sequenced genome, several groups have collectively determined, first by sequence homology and increasingly by functional studies, Drosophila Peroxins and related peroxisome proteins. Notably, it was shown that Drosophila peroxisome biogenesis is mediated via a well-conserved PTS1 import system. Although the fly genome encodes a Pex7 homologue, a canonical PTS2 import system does not seem to be conserved in Drosophila. Given the homology between Drosophila and Saccharomyces cerevisiae or Homo sapiens peroxisome biogenesis and function, Drosophila has emerged as an effective multicellular system to model human Peroxisome Biogenesis Disorders. Finally, Drosophila peroxisome research has recently come into its own, facilitating new discoveries into the role of peroxisomes within specific tissues, such as testes or immune cells.

How has our knowledge of dinosaur diversity through geologic time changed through research history?

Assessments of dinosaur macroevolution at any given time can be biased by the historical publication record. Recent studies have analysed patterns in dinosaur diversity that are based on secular variations in the numbers of published taxa. Many of these have employed a range of approaches that account for changes in the shape of the taxonomic abundance curve, which are largely dependent on databases compiled from the primary published literature. However, how these 'corrected' diversity patterns are influenced by the history of publication remains largely unknown. Here, we investigate the influence of publication history between 1991 and 2015 on our understanding of dinosaur evolution using raw diversity estimates and shareholder quorum subsampling for the three major subgroups: Ornithischia, Sauropodomorpha, and Theropoda. We find that, while sampling generally improves through time, there remain periods and regions in dinosaur evolutionary history where diversity estimates are highly volatile (e.g. the latest Jurassic of Europe, the mid-Cretaceous of North America, and the Late Cretaceous of South America). Our results show that historical changes in database compilation can often substantially influence our interpretations of dinosaur diversity. 'Global' estimates of diversity based on the fossil record are often also based on incomplete, and distinct regional signals, each subject to their own sampling history. Changes in the record of taxon abundance distribution, either through discovery of new taxa or addition of existing taxa to improve sampling evenness, are important in improving the reliability of our interpretations of dinosaur diversity. Furthermore, the number of occurrences and newly identified dinosaurs is still rapidly increasing through time, suggesting that it is entirely possible for much of what we know about dinosaurs at the present to change within the next 20 years.

Multiple optimality criteria support Ornithoscelida.

A recent study of early dinosaur evolution using equal-weights parsimony recovered a scheme of dinosaur interrelationships and classification that differed from historical consensus in a single, but significant, respect; Ornithischia and Saurischia were not recovered as monophyletic sister-taxa, but rather Ornithischia and Theropoda formed a novel clade named Ornithoscelida. However, these analyses only used maximum parsimony, and numerous recent simulation studies have questioned the accuracy of parsimony under equal weights. Here, we provide additional support for this alternative hypothesis using Bayesian implementation of the Mkv model, as well as through number of additional parsimony analyses, including implied weighting. Using Bayesian inference and implied weighting, we recover the same fundamental topology for Dinosauria as the original study, with a monophyletic Ornithoscelida, demonstrating that the main suite of methods used in morphological phylogenetics recover this novel hypothesis. This result was further scrutinized through the systematic exclusion of different character sets. Novel characters from the original study (those not taken or adapted from previous phylogenetic studies) were found to be more important for resolving the relationships within Dinosauromorpha than the relationships within Dinosauria. Reanalysis of a modified version of the character matrix that supports the Ornithischia-Saurischia dichotomy under maximum parsimony also supports this hypothesis under implied weighting, but not under the Mkv model, with both Theropoda and Sauropodomorpha becoming paraphyletic with respect to Ornithischia.

Investigations to extend viability of a rainbow trout primary gill cell culture.

The primary culture of fish gill cells can provide functional, cell diverse, model in vitro platforms able to tolerate an aqueous exposure analogous to in vivo tissues. The utility of such models could be extended to a variety of longer term exposure scenarios if a method could be established to extend culture viability when exposed to water for longer periods. Here we report findings of a series of experiments to establish increased longevity, as monitored by culture transepithelial electrical resistance (TEER) and concurrent histological developments. Experimental cultures improved TEER during apical freshwater exposure for a mean of twelve days, compared to previous viabilities of up to 3 days. Cultures with larger surface areas and the use of trout serum rather than foetal bovine serum (FBS) contributed to the improvement, while perfusion of the intact gill prior to cell harvest resulted in a significantly faster preparation. Detailed scanning electron microscopy analysis of cultures revealed diverse surface structures that changed with culture age. Cultures grown on membranes with an increased porosity, collagen coating or 3D structure were of no benefit compared to standard membranes. Increased culture longevity, achieved in this study and reported for the first time, is a significant breakthrough and opens up a variety of future experimentation that has previously not been possible. The extended viability facilitates exploration of in vitro chronic or pulse-exposure test paradigms, longer term physiological and environmental monitoring studies and the potential for interactive co-culture with other organoid micro-tissues.

A dinosaur missing-link? Chilesaurus and the early evolution of ornithischian dinosaurs.

The enigmatic dinosaur taxon Chilesaurus diegosuarezi was originally described as a tetanuran theropod, but this species possesses a highly unusual combination of features that could provide evidence of alternative phylogenetic positions within the clade. In order to test the relationships of Chilesaurus, we added it to a new dataset of early dinosaurs and other dinosauromorphs. Our analyses recover Chilesaurus in a novel position, as the earliest diverging member of Ornithischia, rather than a tetanuran theropod. The basal position of Chilesaurus within the clade and its suite of anatomical characters suggest that it might represent a 'transitional' taxon, bridging the morphological gap between Theropoda and Ornithischia, thereby offering potential insights into the earliest stages of ornithischian evolution, which were previously obscure. For example, our results suggest that pubic retroversion occurred prior to some of the craniodental and postcranial modifications that previously diagnosed the clade (e.g. the presence of a predentary bone and ossified tendons).

A Teachable Moment After Orthopaedic Fracture in the Smoking Patient: A Randomized, Controlled Trial.

OBJECTIVES: To assess whether education during hospitalization after an acute fracture changes patient attitudes toward smoking-related complications and to assess whether this change persists into the first outpatient follow-up visit. DESIGN: Prospective, randomized, controlled trial. SETTING: Level 1 trauma center. PATIENTS: Inpatients with fractures who identified as smokers: 40 assessed for inclusion and randomized, 30 completed inpatient assessments, and 20 completed outpatient follow-up. INTERVENTION: An educational intervention by the researcher to teach the patient about the harms of smoking regarding fracture healing. MAIN OUTCOME MEASURES: A novel questionnaire to assess the intervention via Likert scale responses, evaluating perceived risk, affective response, and self-role. RESULTS: Education resulted in an increase in perceived risk and affective response within the cohort and an increase in perceived risk when compared with control subjects. No significant differences persisted into outpatient follow-up. CONCLUSIONS: This trial demonstrated that a teachable moment can have an early effect on certain attitudes toward smoking after an acute fracture. These changes did not persist at the first follow-up visit. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Traumatic Cervical Unilateral and Bilateral Facet Dislocations Treated With Anterior Cervical Discectomy and Fusion Has a Low Failure Rate.

STUDY DESIGN: Retrospective radiographic and chart review. OBJECTIVE: To define the rate and associated risk factors of treatment failure of anterior cervical fusion for treatment of cervical facet dislocations. METHODS: Between 2004 and 2014, a retrospective review at a single level 1 trauma center identified 38 patients with unilateral or bilateral dislocated facet(s) treated with anterior cervical discectomy and fusion (ACDF). Two patients were eliminated due to less than 30-day follow-up. Demographic data, initial neurological exams, surgical data, radiographic findings, and follow-up records were reviewed. RESULTS: Of the 36 patients with facet dislocations treated with ACDF using a fixed locking plate, 16 were unilateral and 20 were bilateral. The mean age was 35 years (range 13-58). Mean follow-up was 323 days (range 30-1998). There were 3 treatment failures (8%). Three of 7 (43%) endplate fractures failed (P < .01), and 1/28 (4%) facet fractures failed (P = .13). The mean time to failure was 4 weeks (1-7 weeks). One treatment failure had a facet fracture, and all 3 failures had an associated endplate fracture. CONCLUSION: Treatment failure occurred in 3 out of 36 (8%) patients with facet fracture dislocations treated with anterior cervical discectomy, fusion, and plating. Rates of failure are lower than has been previously reported. Endplate fractures of the inferior level in jumped facets appears to be a major risk factor of biomechanical failure. However, a facet fracture may not be a risk factor for failure. In the absence of an endplate fracture, ACDF is a reasonable treatment option in patients with single-level cervical facet dislocation.

A new hypothesis of dinosaur relationships and early dinosaur evolution.

For 130 years, dinosaurs have been divided into two distinct clades-Ornithischia and Saurischia. Here we present a hypothesis for the phylogenetic relationships of the major dinosaurian groups that challenges the current consensus concerning early dinosaur evolution and highlights problematic aspects of current cladistic definitions. Our study has found a sister-group relationship between Ornithischia and Theropoda (united in the new clade Ornithoscelida), with Sauropodomorpha and Herrerasauridae (as the redefined Saurischia) forming its monophyletic outgroup. This new tree topology requires redefinition and rediagnosis of Dinosauria and the subsidiary dinosaurian clades. In addition, it forces re-evaluations of early dinosaur cladogenesis and character evolution, suggests that hypercarnivory was acquired independently in herrerasaurids and theropods, and offers an explanation for many of the anatomical features previously regarded as notable convergences between theropods and early ornithischians.

Pharmaceutical Metabolism in Fish: Using a 3-D Hepatic In Vitro Model to Assess Clearance.

At high internal doses, pharmaceuticals have the potential for inducing biological/pharmacological effects in fish. One particular concern for the environment is their potential to bioaccumulate and reach pharmacological levels; the study of these implications for environmental risk assessment has therefore gained increasing attention. To avoid unnecessary testing on animals, in vitro methods for assessment of xenobiotic metabolism could aid in the ecotoxicological evaluation. Here we report the use of a 3-D in vitro liver organoid culture system (spheroids) derived from rainbow trout to measure the metabolism of seven pharmaceuticals using a substrate depletion assay. Of the pharmaceuticals tested, propranolol, diclofenac and phenylbutazone were metabolised by trout liver spheroids; atenolol, metoprolol, diazepam and carbamazepine were not. Substrate depletion kinetics data was used to estimate intrinsic hepatic clearance by this spheroid model, which was similar for diclofenac and approximately 5 fold higher for propranolol when compared to trout liver microsomal fraction (S9) data. These results suggest that liver spheroids could be used as a relevant and metabolically competent in vitro model with which to measure the biotransformation of pharmaceuticals in fish; and propranolol acts as a reproducible positive control.

A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins.

Peroxisomes are membrane-bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence-based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope-tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well-developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease.