Cell-based assay for ciliopathy patients to improve accurate diagnosis using ALPACA.

Skeletal ciliopathies are a group of disorders caused by dysfunction of the cilium, a small signaling organelle present on nearly every vertebrate cell. This group of disorders is marked by genetic and clinical heterogeneity, which complicates accurate diagnosis. In this study, we developed a robust, standardized immunofluorescence approach to accurately diagnose a subset of these disorders. Hereto we determined and compared the cilium phenotype of healthy individuals to patients from three different ciliopathy subgroups, using skin-derived fibroblasts. The cilium phenotype assay consists of three parameters; (1) ciliogenesis, based on the presence or absence of cilium markers, (2) cilium length, measured by the combined signal of an axonemal and a cilium membrane marker, and (3) retrograde intraflagellar transport (IFT), quantified by the area of the ciliary tip. Analysis of the cilium phenotypic data yielded comparable and reproducible results and in addition, displayed identifiable clusters for healthy individuals and two ciliopathy subgroups, i.e. ATD and CED. Our results illustrate that standardized analysis of the cilium phenotype can be used to discriminate between ciliopathy subgroups. Therefore, we believe that standardization of functional assays analyzing cilium phenotypic data can provide additional proof for conclusive diagnosis of ciliopathies, which is essential for routine diagnostic care.

Certainty-based marking in a formative assessment improves student course appreciation but not summative examination scores.

BACKGROUND: Study motivation and knowledge retention benefit from regular student self-assessments. Inclusion of certainty-based learning (CBL) in computer-assisted formative tests may further enhance this by enabling students to identify whether they are uninformed or misinformed regarding the topics tested, which may trigger future study actions including instructor consultation. METHODS: Using a cross-over study design involving two out of thirteen computer-assisted formative assessments (CAFAs) of a first-year cell biology course, we compared student-instructor interactions, student learning experiences and final exam scores between two (bio)medical science student cohorts who worked with different CBL-containing CAFAs. RESULTS: A total of 389 students participated in the study. After completion 159 (41%) filled in a questionnaire on their experience with CBL during supervised CAFAs. In the control group the median duration of student-instructor interactions was 90 s (range 60-140 s), and this increased with 20 s to 110 s (range 60-150 s) in the group working with a CBL-based CAFA. The number of interactions was similar in both groups (0.22 per student per hour, regardless of CBL inclusion). Forty percent of the students expected that CBL would positively influence their study behavior, and 23% also anticipated a positive effect on examination scores. Student examination scores, however, were not affected by CBL. Almost half of the students (43%) were in favor of CBL inclusion in future computer-assisted learning modules, whereas 33% did not see merit in including CBL in CAFAs. CONCLUSIONS: Incorporation of CBL in a single formative assessment led to a slight increase in student-instructor interaction times, but had effect neither on the number of student-instructor interactions nor on exam scores. CBL inclusion positively influenced student's appreciation of the coursework, presumably by helping students to evaluate their mastery level and identify misconceptions. A more extensive enrollment of CBL beyond an individual formative assessment, throughout a course or a curriculum, may possibly reveal positive effects on study efficacy.

Transepithelial transfer of phenanthrene, but not of benzo[a]pyrene, is inhibited by fatty acids in the proximal intestine of rainbow trout (Oncorhynchus mykiss).

The inclusion of vegetable oils in aquafeeds introduces contaminating polycyclic aromatic hydrocarbons (PAHs) in salmonids. Since lipophilic PAHs solubilize in micelles composed of lipids, bile salts and fatty acids, dietary lipid composition can alter intestinal transepithelial PAH transfer. We studied the uptake of two PAHs, viz. benzo[a]pyrene (BaP) and phenanthrene (PHE), in rainbow trout (Oncorhynchus mykiss) intestine. We also investigated the effects of two fatty acids, viz. fish oil-derived eicosapentaenoic acid (EPA, 20:5n-3) and vegetable oil-derived oleic acid (18:1n-9) on intestinal uptake. Radiolabeled PAHs were solubilized in micelles composed of tritiated EPA and oleic acid, respectively, and administrated to intestinal segments mounted in Ussing chambers. In the absence of micelles, PHE accumulation was two times higher than BaP in the mucosal and serosal layers of proximal and distal intestine. Administration of PHE in micelles composed of oleic acid resulted in a 50% lower accumulation of PHE in the mucosal layers of the proximal intestine compared to EPA-composed micelles. Accumulation of EPA and oleic acid in the proximal intestinal mucosa correlated negatively with the transepithelial transfer of these fatty acids across the proximal intestinal epithelium. Transepithelial PHE transfer across the proximal intestine was reduced by 30% in co-exposure with EPA-composed micelles compared to 80% with oleic acid micelles. BaP was not transferred across the intestine. We conclude that the lipid composition of an aquafeed is an important determinant of PAH bioavailability. Therefore, lipid composition should be an important consideration in choosing vegetable oils as alternatives for fish oil in aquafeeds.

Design of Radioiodinated Pharmaceuticals: Structural Features Affecting Metabolic Stability towards in Vivo Deiodination.

Radioiodinated pharmaceuticals are convenient tracers for clinical and research investigations because of the relatively long half-lives of radioactive iodine isotopes (i.e., 123I, 124I, and 131I) and the ease of their chemical insertion. Their application in radionuclide imaging and therapy may, however, be hampered by poor in vivo stability of the C-I bond. After an overview of the use of iodine in biology and nuclear medicine, we present here a survey of the catabolic pathways for iodinated xenobiotics, including their biodistribution, accumulation, and biostability. We summarize successful rational improvements in the biostability and conclude with general guidelines for the design of stable radioiodinated pharmaceuticals. It appears to be necessary to consider the whole molecule, rather than the radioiodinated fragment alone. Iodine radionuclides are generally retained in vivo on sp2 carbon atoms in iodoarenes and iodovinyl moieties, but not in iodinated heterocycles or on sp3 carbon atoms. Iodoarene substituents also have an influence, with increased in vivo deiodination in the cases of iodophenols and iodoanilines, whereas methoxylation and difluorination improve biostability.

The polycyclic aromatic hydrocarbons benzo[a]pyrene and phenanthrene inhibit intestinal lipase activity in rainbow trout (Oncorhynchus mykiss).

Elevated levels of polycyclic aromatic hydrocarbons (PAHs) are detected in aquafeeds where fish oils are (partially) replaced by vegetable oils. The highly lipophilic PAHs solubilize readily in oil droplets and micelles in the intestinal lumen that can affect enzymatic lipid digestion by altering lipase activity. We therefore investigated the effect of two PAHs, benzo[a]pyrene (BaP) and phenanthrene (PHE), on bile salt-activated lipase (BAL) activity in desalted luminal extracts of the proximal intestine of rainbow trout (Oncorhynchus mykiss) using the triacylglycerides rapeseed oil and fish oil as substrates. The hydrolysis of rapeseed oil and fish oil measured at a calculated substrate concentration of 2.2mM, increased linearly up to 30min at 15°C. Substrate dependency under initial velocity conditions was described by simple Michaelis-Menten kinetics with a Km value of 1.2mM for rapeseed and fish oil. Rapeseed oil hydrolysis was inhibited by 1nM BaP and 10nM PHE. The hydrolysis of fish oil was only inhibited by 10µM BaP. The in vitro lipase activity data were corroborated by TLC/HPLC analysis of the reaction products, showing that in the presence of BaP and PHE, 46-80% less free fatty acids (FFA) were hydrolysed from rapeseed and fish oil triacylglycerides. The presence of low concentrations of BaP and PHE decreased rapeseed oil hydrolysis by BAL whereas fish oil hydrolysis was not affected. The replacement of fish oil by rapeseed oil in aquafeeds introduces PAHs that could affect lipid digestion.

Uptake of benzo[a]pyrene, but not of phenanthrene, is inhibited by fatty acids in intestinal brush border membrane vesicles of rainbow trout (Oncorhynchus mykiss).

Partial replacement of fish ingredients with vegetable ingredients has elevated levels of polycyclic aromatic hydrocarbons (PAHs) in Atlantic salmon reared on these feeds. PAH uptake in the intestinal tract is postulated to occur in association with lipid absorption and could well be affected by fatty acid composition. We therefore investigated the effects of a fish oil and vegetable oil fatty acid, eicosapentaenoic acid (EPA; 20:5n-3) and oleic acid (18:1n-9) respectively, on the uptake of benzo[a]pyrene (BaP) and phenanthrene (PHE) across the intestinal brush border membrane in the salmonid species rainbow trout (Oncorhynchus mykiss). BaP and PHE were solubilized in mixed micelles composed of either EPA or oleic acid and administrated to isolated brush border membrane vesicles (BBMV) derived from the pyloric caeca, proximal intestine and distal intestine. In the absence of free fatty acids (FFA) trans-membrane uptake of BaP and PHE was 2-7 times lower than the fraction associated to or in the membrane. In the presence of FFA, trans-membrane BaP uptake had decreased by 80 and 40% at the highest EPA and oleic acid concentration, respectively, whereas PHE uptake was virtually unaffected. In the presence of BaP, but not PHE, trans-membrane EPA uptake in BBMV had decreased. This study obtained evidence for PAH-dependent interactions with FFA uptake. We conclude that intestinal BaP uptake is reduced by luminal FFA contents whereas PHE uptake is not. A large fraction of the administrated BaP and PHE remains associated with the cellular membrane of enterocytes and may interfere with uptake of nutrients.

The teleost head kidney: Integrating thyroid and immune signalling.

The head kidney, analogous to the mammalian adrenal gland, is an organ unique for teleost fish. It comprises cytokine-producing lymphoid cells from the immune system and endocrine cells secreting cortisol, catecholamines, and thyroid hormones. The intimate organization of the immune system and endocrine system in one single organ makes bidirectional signalling between these possible. In this review we explore putative interactions between the thyroid and immune system in the head kidney. We give a short overview of the thyroid system, and consider the evidence for the presence of thyroid follicles in the head kidney as a normal, healthy trait in fishes. From mammalian studies we gather data on the effects of three important pro-inflammatory cytokines (TNFα, IL-1ß, IL-6) on the thyroid system. A general picture that emerges is that pro-inflammatory cytokines inhibit the activity of the thyroid system at different targets. Extrapolating from these studies, we suggest that the interaction of the thyroid system by paracrine actions of cytokines in the head kidney is involved in fine-tuning the availability and redistribution of energy substrates during acclimation processes such as an immune response or stress response.

Triclosan exposure results in alterations of thyroid hormone status and retarded early development and metamorphosis in Cyprinodon variegatus.

Thyroid hormones are critically involved in somatic growth, development and metamorphosis of vertebrates. The structural similarity between thyroid hormones and triclosan, an antimicrobial compound widely employed in consumer personal care products, suggests triclosan can have adverse effects on the thyroid system. The sheepshead minnow, Cyprinodon variegatus, is now used in ecotoxicological studies that have recently begun to focus on potential disruption of the thyroid axis by endocrine disrupting compounds. Here, we investigate the in vivo effects of exposure to triclosan (20, 50, and 100µgL-1) on the thyroid system and the embryonic and larval development of C. variegatus. Triclosan exposure did not affect hatching success, but delayed hatching time by 6-13h compared to control embryos. Triclosan exposure affected the ontogenetic variations of whole body thyroid hormone concentrations during the larval phase. The T3 peak around 12-15 dph, described to be indicative for the metamorphosis climax in C. variegatus, was absent in triclosan-exposed larvae. Triclosan exposure did not produce any deformity or allometric repatterning, but a delayed development of 18-32h was observed. We conclude that the triclosan-induced disruption of the thyroid system delays in vivo the start of metamorphosis in our experimental model. We observed a global developmental delay of 24-45h, equivalent to 4-7% prolongation of the developmental time in C. variegatus. The costs of delayed metamorphosis can lead to reduction of juvenile fitness and could be a determining factor in the outcome of competitive interactions.

Branchial nitrogen cycle symbionts can remove ammonia in fish gills.

Knowledge of the mechanisms by which fish excrete their metabolic nitrogenous waste and insights into nitrogen cycling in aquaculture systems is of utmost importance to improve the sustainable commercial production of fish. In fish, most nitrogenous waste is excreted via the gills as ammonia, a potentially toxic nitrogenous compound. In this study; activity assays, physiological experiments, molecular analysis and microscopy were used to show that the gills of fish harbor a unique combination of hitherto overlooked nitrogen-cycle microorganisms that can theoretically detoxify excreted ammonia by converting it into inert dinitrogen gas. By doing so, these microorganisms may benefit from the ammonia supply by the host and prevent the build-up of this compound to toxic concentrations. This novel relationship between vertebrates and microorganisms may shed new light on nitrogen handling by ammonotelic fish species.

The effect of dietary lipid composition on the intestinal uptake and tissue distribution of benzo[a]pyrene and phenanthrene in Atlantic salmon (Salmo salar).

Uptake of polycyclic aromatic hydrocarbons (PAHs) across the intestine is suggested to occur in association with dietary lipids. Partial replacement of fish ingredients by vegetable ingredients in aquafeeds has led to increased levels of PAHs in marine farmed fish. We therefore investigated, intestinal uptake, tissue distribution and PAH metabolism after a single dose of (14)C-benzo[a]pyrene (BaP) or (14)C-phenanthrene (PHE) given to Atlantic salmon (Salmo salar) acclimatized to a fish oil or vegetable oil based diet. Both BaP and PHE were absorbed along the intestine. Fish oil based feed increased BaP concentration in the pyloric caeca and that of PHE in the proximal intestine. In contrast, vegetable oil increased BaP concentrations in the distal intestine. Extraction of whole body autoradiograms removed PHE-associated radiolabeling almost completely from the intestinal mucosa, but not BaP-associated radiolabeling, indicating the presence of BaP metabolites bound to cellular macromolecules. This observation correlates with the increased cyp1a expression in the proximal intestine, distal intestine and liver in the BaP exposed group. Furthermore, BaP-induced cyp1a expression was higher in the distal intestine of salmon fed fish oil compared to the vegetable oil fed group. PHE had no significant effect on cyp1a expression in any of these tissues. We conclude that dietary lipid composition affects intestinal PAH uptake. Fish oil based feed increased intestinal PAH concentrations probably due to an enhanced solubility in micelles composed of fish oil fatty acids. Increased BaP accumulation in the distal intestine of vegetable oil fed fish seems to be associated with a reduced Cyp1a-mediated BaP metabolism.

Energy metabolism of hyperthyroid gilthead sea bream Sparus aurata L.

Thyroid hormones, in particular 3,5,3'-triiodothyronine or T3, are involved in multiple physiological processes in mammals such as protein, fat and carbohydrate metabolism. However, the metabolic actions of T3 in fish are still not fully elucidated. We therefore tested the effects of T3 on Sparus aurata energy metabolism and osmoregulatory system, a hyperthyroid-induced model that was chosen. Fish were implanted with coconut oil depots (containing 0, 2.5, 5.0 and 10.0µg T3/g body weight) and sampled at day 3 and 6 post-implantation. Plasma levels of free T3 as well as glucose, lactate and triglyceride values increased with increasing doses of T3 at days 3 and 6 post-implantation. Changes in plasma and organ metabolite levels (glucose, glycogen, triglycerides, lactate and total α amino acid) and enzyme activities related to carbohydrate, lactate, amino acid and lipid pathways were detected in organs involved in metabolism (liver) and osmoregulation (gills and kidney). Our data implicate that the liver uses amino acids as an energy source in response to the T3 treatment, increasing protein catabolism and gluconeogenic pathways. The gills, the most important extruder of ammonia, are fuelled not only by amino acids, but also by lactate. The kidney differs significantly in its substrate preference from the gills, as it obtained metabolic energy from lactate but also from lipid oxidation processes. We conclude that in S. aurata lipid catabolism and protein turnover are increased as a consequence of experimentally induced hyperthyroidism, with secondary osmoregulatory effects.

The thyroid gland and thyroid hormones in sheepshead minnow (Cyprinodon variegatus) during early development and metamorphosis.

The sheepshead minnow is widely used in ecotoxicological studies that only recently have begun to focus on disruption of the thyroid axis by xenobiotics and endocrine disrupting compounds. However, reference levels of the thyroid prohormone thyroxine (T4) and biologically active hormone 3,5,3'-triiodothyronine (T3) and their developmental patterns are unknown. This study set out to describe the ontogeny and morphology of the thyroid gland in sheepshead minnow, and to correlate these with whole-body concentrations of thyroid hormones during early development and metamorphosis. Eggs were collected by natural spawning in our laboratory. T4 and T3 were extracted from embryos, larvae and juveniles and an enzyme-linked immunoassay was used to measure whole-body hormone levels. Length and body mass, hatching success, gross morphology, thyroid hormone levels and histology were measured. The onset of metamorphosis at 12-day post-hatching coincided with surges in whole-body T4 and T3 concentrations. Thyroid follicles were first observed in pre-metamorphic larvae at hatching and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Follicle size and thyrocyte epithelial cell heights varied during development, indicating fluctuations in thyroid hormone synthesis activity. The increase in the whole-body T3/T4 ratio was indicative of an increase in outer ring deiodination activity. This study establishes a baseline for thyroid hormones in sheepshead minnows, which will be useful for the understanding of thyroid hormone functions and in future studies of thyroid toxicants in this species.

Knockdown of monocarboxylate transporter 8 (mct8) disturbs brain development and locomotion in zebrafish.

Allan-Herndon-Dudley syndrome (AHDS) is an inherited disorder of brain development characterized by severe psychomotor retardation. This X-linked disease is caused by mutations in the monocarboxylate transporter 8 (MCT8), an important thyroid hormone transporter in brain neurons. MCT8-knockout mice lack the 2 major neurological symptoms of AHDS, namely locomotor problems and cognitive impairment. The pathological mechanism explaining the symptoms is still obscure, and no cure for this condition is known. The development of an animal model that carries MCT8-related neurological symptoms is warranted. We have employed morpholino-based gene knockdown to create zebrafish deficient for mct8. Knockdown of mct8 results in specific symptoms in the thyroid axis and brain. The mct8-morphants showed impaired locomotor behavior and brain development. More specifically, we observed maldevelopment of the cerebellum and mid-hindbrain boundary and apoptotic clusters in the zebrafish brain. The mRNA expression of zebrafish orthologs of mammalian TSH, thyroid hormone transporters, and deiodinases was altered in mct8 morphants. In particular, deiodinase type 3 gene expression was consistently up-regulated in zebrafish mct8 morphants. The thyroid hormone metabolite tetrac, but not T3, partly ameliorated the affected phenotype and locomotion disability of morphant larvae. Our results show that mct8 knockdown in zebrafish larvae results in disturbances in the thyroid axis, brain, and locomotion behavior, which is congruent with the clinical aspect of impaired locomotion and cognition in patients with AHDS. Taken together, the zebrafish is a suitable animal model for the study of the pathophysiology of AHDS.

Voluntary timing of food intake increases weight gain and reduces basal plasma cortisol levels in common carp (Cyprinus carpio L.).

We investigated the effect of timing of food intake on growth in common carp (Cyprinus carpio L.). Juvenile carp were demand-fed for 22 days using a computerized pendulum feeder that registered meal requests. Controls were pair-fed at 10:00 h, both groups were kept at 12L:12D (lights on at 06:30 h). Demand-fed fish displayed highest food intake at 22:00 h, and the lowest at 10:00. After 22 days, demand-fed fish had grown by 20% of their initial body weight, compared to 4% of the pair-fed control. Plasma cortisol levels in demand-fed fish were remarkably low and stable, whereas in the control group levels had increased 60-fold at 10:00 h compared to 22:00 h. Hepatic mRNA expression of leptin-a1 and leptin-a2 also differed markedly between groups and time points, with leptin-a2 expression being lowest in the demand-fed group at the time point of lowest food intake. We conclude that timing of food intake is an important determinant of endocrine status, growth and welfare.

Food deprivation induces chronic stress and affects thyroid hormone metabolism in Senegalese sole (Solea senegalensis) post-larvae.

In vertebrates, stress and thyroid systems interact closely, most likely because of the involvement of both systems in energy metabolism. However, studies on these interactions, especially during larval development, are scarce. Recently, cDNAs coding for corticotropin-releasing hormone (CRH) and CRH-binding protein (CRH-BP), two key players in the regulation of the neuroendocrine stress response, were characterized for the flatfish Senegalese sole (Solea senegalensis). To investigate the involvement of stress and thyroid systems in this species, the effects of food deprivation during early development of S. senegalensis were assessed. Growth was arrested in food-deprived post-larvae, which was also reflected by decreased carbon and nitrogen contents, indicating increased catabolism. Food deprivation induces chronic stress, as illustrated by enhanced whole-body cortisol levels, as well as an up regulation of crh and a decrease of crh-bp expression levels. Furthermore, whole-body total T3 concentrations of food-deprived post-larvae were reduced, although tshß subunit expression levels remained unaffected. Our results show that food deprivation is a chronic stressor that induces energy-releasing catabolic processes that compensate for the reduced energy intake, and inhibits anabolic processes via the peripheral thyroid system.

Environmental factors affecting thyroid function of wild sea bass (Dicentrarchuslabrax) from European coasts.

Thyroid functional status of wild fish in relation with the contamination of their environment deserves further investigation. We here applied a multi-level approach of thyroid function assessment in 87 wild sea bass collected near several estuaries: namely the Scheldt, the Seine, the Loire, the Charente and the Gironde. Thyroxine (T(4)) and triiodothyronine (T(3)) concentrations in muscle were analyzed by radioimmunoassay. The activity of hepatic enzymes involved in extrathyroidal pathways of thyroid hormone metabolism, viz. deiodination, glucuronidation and sulfatation were analyzed. Last, follicle diameter and epithelial cell heights were measured. We observed changes that are predicted to lead to an increased conversion of T(4)-T(3) and lowered thyroid hormone excretion. The changes in the metabolic pathways of thyroid hormones can be interpreted as a pathway to maintain thyroid hormone homeostasis. From all compounds tested, the higher chlorinated PCBs seemed to be the most implicated in this perturbation.

Kinetics and thiol requirements of iodothyronine 5'-deiodination are tissue-specific in common carp (Cyprinus carpio L.).

Iodothyronine deiodinases determine the biological activity of thyroid hormones. Despite the homology of the catalytic sites of mammalian and teleostean deiodinases, in-vitro requirements for the putative thiol co-substrate dithiothreitol (DTT) vary considerably between vertebrate species. To further our insights in the interactions between the deiodinase protein and its substrates: thyroid hormone and DTT, we measured enzymatic iodothyronine 5'-deiodination, Dio1 and Dio2 mRNA expression, and Dio1 affinity probe binding in liver and kidney preparations from a freshwater teleost, the common carp (Cyprinus carpio L.). Deiodination rates, using reverse T3 (rT3, 3,3',5'-triiodothyronine) as the substrate, were analysed as a function of the iodothyronine and DTT concentrations. In kidney rT3 5'-deiodinase activity measured at rT3 concentrations up to 10 µM and in the absence of DTT does not saturate appreciably. In the presence of 1mM DTT, renal rT3 deiodination rates are 20-fold lower. In contrast, rT3 5'-deiodination in liver is potently stimulated by 1mM DTT. The marked biochemical differences between 5'-deiodination in liver and kidney are not associated with the expression of either Dio1 or Dio2 mRNA since both organs express both deiodinase types. In liver and kidney, DTT stimulates the incorporation of N-bromoacetylated affinity labels in proteins with estimated molecular masses of 57 and 55, and 31 and 28 kDa, respectively. Although primary structures are highly homologous, the biochemistry of carp deiodinases differs markedly from their mammalian counterparts.

Subfunctionalization of POMC paralogues in Senegalese sole (Solea senegalensis).

The precursor protein proopiomelanocortin (POMC) gives rise to a variety of biologically active peptides through cell-specific posttranslational processing. Two transcripts of pomc were found in the flatfish Solea senegalensis (ssePOMC-A and ssePOMC-B), that most likely represent subfunctionalized paralogues: ssePOMC-A lacks the N-terminal cleavage site for ß-MSH, whereas ssePOMC-B cannot yield ACTH and completely lacks the opioid consensus sequence in the ß-END region. An analysis of nucleotide substitution rates shows that the POMC-derived peptides possess well-conserved regions under purifying selection, except the ß-END derived from POMC-B, which has undergone positive selection. The calculated K(s) values for ssePOMC-A versus ssePOMC-B and zebrafish POMCαversus zebrafish POMCß are 0.40 and 0.72, respectively, indicating that the zebrafish POMC paralogues started to evolve almost twice as early in evolution, and that the Solea POMC paralogues arose independently from the whole genome duplication event that gave rise to the zebrafish paralogues. This makes ssePOMC-B the first identified POMCα orthologue that lacks the opioid consensus. Furthermore, pomc-a expression is down-regulated in chronic stressed S. senegalensis juveniles, whereas pomc-b expression levels remain unaffected, indicating different physiological roles for both POMC paralogues. The distribution of functional POMC-derived peptide hormones over two pomc genes in S. senegalensis suggests subfunctionalization of the paralogues, a relevant notion when studying POMC function in endocrine responses.

Anammox bacteria in different compartments of recirculating aquaculture systems.

Strict environmental restrictions force the aquaculture industry to guarantee optimal water quality for fish production in a sustainable manner. The implementation of anammox (anaerobic ammonium oxidation) in biofilters would result in the conversion of both ammonium and nitrite (both toxic to aquatic animals) into harmless dinitrogen gas. Both marine and freshwater aquaculture systems contain populations of anammox bacteria. These bacteria are also present in the faeces of freshwater and marine fish. Interestingly, a new planctomycete species appears to be present in these recirculation systems too. Further exploitation of anammox bacteria in different compartments of aquaculture systems can lead to a more environmentally friendly aquaculture practice.

Identification and functional characterization of zebrafish solute carrier Slc16a2 (Mct8) as a thyroid hormone membrane transporter.

Most components of the thyroid system in bony fish have been described and characterized, with the notable exception of thyroid hormone membrane transporters. We have cloned, sequenced, and expressed the zebrafish solute carrier Slc16a2 (also named monocarboxylate transporter Mct8) cDNA and established its role as a thyroid hormone transport protein. The cloned cDNA shares 56-57% homology with its mammalian orthologs. The 526-amino-acid sequence contains 12 predicted transmembrane domains. An intracellular N-terminal PEST domain, thought to be involved in proteolytic processing of the protein, is present in the zebrafish sequence. Measured at initial rate and at the body/rearing temperature of zebrafish (26 C), T(3) uptake by zebrafish Slc16a2 is a saturable process with a calculated Michaelis-Menten constant of 0.8 µM T(3). The rate of T(3) uptake is temperature dependent and Na(+) independent. Interestingly, at 26 C, zebrafish Slc16a2 does not transport T(4). This implies that at a normal body temperature in zebrafish, Slc16a2 protein is predominantly involved in T(3) uptake. When measured at 37 C, zebrafish Slc16a2 transports T(4) in a Na(+)-independent manner. In adult zebrafish, the Slc16a2 gene is highly expressed in brain, gills, pancreas, liver, pituitary, heart, kidney, and gut. Beginning from the midblastula stage, Slc16a2 is also expressed during zebrafish early development, the highest expression levels occurring 48 h after fertilization. This is the first direct evidence for thyroid hormone membrane transporters in fish. We suggest that Slc16a2 plays a key role in the local availability of T(3) in adult tissues as well as during the completion of morphogenesis of primary organ systems.