New frontiers in the evolution of fin development.

The locomotory appendages of vertebrates have undergone significant changes during evolution, which likely promoted a wide range of adaptive strategies. These appendages first evolved as unpaired finfolds in the dorsal midline of early chordates, more than 500 million years ago. Later on, during vertebrates' radiation, two sets of locomotory appendages emerged, developing from both sides of the latero-ventral body wall. The morphology of these paired fins in fishes at different phylogenetic positions suggests an evolutionary tendency for increasing elaboration of the endoskeleton and concomitant reduction of the distal dermoskeleton. This evolutionary process culminated with the origin of limbs in the lineages leading to tetrapods. The developmental programs responsible for the evolution of vertebrate appendages have been a major topic for evolutionary developmental biology recently. Gene expression comparisons performed in chordates explored how these mechanisms were transferred from a midline to latero-ventral position. On another front, gene function assays have begun to test classical hypotheses concerning the transition from fish fins to tetrapod limbs. In this review, we highlight these recent findings on the evolution of vertebrate fin development. First, we discuss new perspectives on the transition from midline to paired appendages focus on (i) origin and molecular regionalization of the lateral plate mesoderm and (ii) novel ectodermic competency zones for fin induction. Next, we review recent work exploring how tetrapod limbs evolved from fish fins, considering (i) molecular and structural changes in the distal ectoderm of fins and (ii) modulation of 5'HoxD transcription during fin endoskeleton development.

Hemochromatosis and pregnancy: iron stores in the Hfe-/- mouse are not reduced by multiple pregnancies.

Hereditary hemochromatosis (HH), a widespread hereditary iron metabolism disorder, is characterized by an excessive absorption of dietary iron, resulting in increased body iron stores. Some studies indicate a sex difference in disease expression, with women showing a slower disease progression and a less severe clinical profile. This is usually attributed to iron loss during menstruation and pregnancy. However, this link has not been clearly demonstrated. The Hfe-/- mouse model recapitulates key aspects of HH, including an iron overload phenotype similar to that observed in human patients. In this study, we use it to test the impact of multiple pregnancies in the iron stores. One-year-old nulliparous and pluriparous (averaging 29 weaned pups per female) C57BL/6 (B6) and Hfe-/- mice were euthanized, and blood and tissues were collected. Several serological and erythroid parameters were evaluated, as well as tissue nonheme iron content and serum ferritin. Hepcidin 1, hepcidin 2, and bone morphogenetic protein 6 (BMP6) expressions in the liver were determined by real-time PCR. No significant differences were observed for many serological and erythroid parameters although differences occurred in transferrin saturation and mean corpuscular volume in Hfe-/- mice and total iron-binding capacity in B6 mice. Hepatic iron concentration was similar for nulliparous and pluriparous mice of both genotypes, but total iron per organ (liver, spleen, heart, and pancreas) was higher overall in pluriparous females than nulliparous. Hepcidin 1 and 2 and BMP6 expressions were significantly decreased in pluriparous females, when compared with nulliparous, in both genotypes. In conclusion, multiple pregnancies do not reduce body iron stores in Hfe-/- mice.

Butyltin levels in several Portuguese coastal areas.

This work aimed to report present levels (2007-2008 sampling) of tri- (TBT), di- (DBT), and monobutyltin (MBT) in surface sediments from 11 Portuguese coastal sites and discuss the evolution of BTs contamination in the last two decades. All the samples revealed quantifiable values of TBT, DBT, and MBT with total butyltin concentrations between 1 and 565 ng/g (of Sn in dry sediment). Maximum level of TBT, 66 ng/g, was observed in Sado estuary, at Lisnave site, in the proximity of a big shipyard. MBT decreased site by site by the same order as DBT and TBT did, but its concentrations were much higher in many cases, denoting that TBT contamination was much higher in the past. A comparison with the available previous data confirmed a marked decrease of TBT contamination all over the last years, indicating that the main sources of TBT in Portuguese coastwise stopped effectively.

Increased susceptibility to Mycobacterium avium in hemochromatosis protein HFE-deficient mice.

Mycobacterium avium is an opportunistic infectious agent in immunocompromised patients, living inside macrophage phagosomes. As for other mycobacterial species, iron availability is a critical factor for M. avium survival and multiplication. Indeed, an association between host secondary iron overload and increased susceptibility to these mycobacteria is generally acknowledged. However, studies on the impact of primary iron overload on M. avium infection have not been performed. In this work, we used animal models of primary iron overload that mimic the human disease hereditary hemochromatosis. This pathology is characterized by increased serum transferrin saturation with iron deposition in parenchymal cells, mainly in the liver, and is most often associated with mutations in the gene encoding the molecule HFE. In this paper, we demonstrate that mice of two genetically determined primary iron overload phenotypes, Hfe(-/-) and beta2m(-/-), show an increased susceptibility to experimental infection with M. avium and that during infection these animals accumulate iron inside granuloma macrophages. beta2m(-/-) mice were found to be more susceptible than Hfe(-/-) mice, but depleting Hfe(-/-) mice of CD8(+) cells had no effect on resistance to infection. Overall, our results suggest that serum iron, rather than total liver iron, levels have a considerable impact on susceptibility to M. avium infection.

HoxA Genes and the Fin-to-Limb Transition in Vertebrates.

HoxA genes encode for important DNA-binding transcription factors that act during limb development, regulating primarily gene expression and, consequently, morphogenesis and skeletal differentiation. Within these genes, HoxA11 and HoxA13 were proposed to have played an essential role in the enigmatic evolutionary transition from fish fins to tetrapod limbs. Indeed, comparative gene expression analyses led to the suggestion that changes in their regulation might have been essential for the diversification of vertebrates' appendages. In this review, we highlight three potential modifications in the regulation and function of these genes that may have boosted appendage evolution: (1) the expansion of polyalanine repeats in the HoxA11 and HoxA13 proteins; (2) the origin of +a novel long-non-coding RNA with a possible inhibitory function on HoxA11; and (3) the acquisition of cis-regulatory elements modulating 5' HoxA transcription. We discuss the relevance of these mechanisms for appendage diversification reviewing the current state of the art and performing additional comparative analyses to characterize, in a phylogenetic framework, HoxA11 and HoxA13 expression, alanine composition within the encoded proteins, long-non-coding RNAs and cis-regulatory elements.

Iron overload favors the elimination of Leishmania infantum from mouse tissues through interaction with reactive oxygen and nitrogen species.

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host's iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host's oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Organochlorine pesticides levels in Portuguese coastal areas.

Data on levels of persistent organochlorine pesticides (OCPs) that exhibit potentially harmful effects to the environment all over the Portuguese coastal sediments were scarce. Therefore, in 2007-2008 12 OCPs were surveyed in surface sediments from 11 coastal sites (estuarine or lagoonal) covering north, centre and south areas of Portugal. Analyzes were performed using a previously validated method, involving pre-extraction of OCPs from sediments assisted by microwave and headspace solid-phase microextraction followed by gas chromatography with electron capture detection for quantification and mass spectrometry for OCPs identification. Measurable levels (0.08-26ng g(-1)) of alpha-hexachlorocyclohexane (alpha -HCH), gamma-hexachlorocyclohexane (lindane), aldrin, dieldrin, endrin, 4,4'-DDT, 4,4'-DDD, 4,4'-DDE, heptachlor, heptachlor epoxide, endosulfan I and methoxychlor were observed in all cases. In few cases, the found levels denote the probability of occurrence of adverse biological effects.

An expeditious method for the determination of organochlorine pesticides residues in estuarine sediments using microwave assisted pre-extraction and automated headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry.

Determination of organochlorine pesticides (OCPs) in sediments implicates extraction of these compounds from the matrix, which is difficult owing to strong interaction among OCPs and different constituents of the sediments, particularly organic content. The method here described is a combination of microwave assisted extraction (MAE), headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS), acting in selected-ion storage mode, or GC-electron capture detector (ECD, for routine analysis). Methanol was used as extracting solvent and aliquots of the MAE extracts (after inclusion of a step for sulfur elimination when required) were used to prepare aqueous solutions for HS-SPME. A complete automation of the SPME procedure increases the sample throughput, including standard addition for calibration purpose. The procedure has the advantage of exclude additional clean-up steps and pre-concentration before SPME. Application to reference sediments of different characteristics revealed absence of significant interferences from the matrix for alpha-lindane, gamma-lindane, aldrin, dieldrin, endrin, 4,4'-DDT, 4,4'-DDD, 4,4'-DDE, heptachlor, heptachlor epoxide and good sensitivity. Detection limits ranged from 0.005 to 0.11 ng of OCP per gram of dried sediment using GC-MS and from 0.01 to 0.26 ngg(-1) using GC-ECD. The linear response ranges embraced 5-6 orders of magnitude (up to 1000 ngg(-1)) in GC-MS, being narrower for GC-ECD. The method was successfully applied to sandy and muddy sediments from Portuguese rivers estuaries, enabling quantification of seven OCPs. The method resulted effective, relatively simple and fast, being suitable for routine monitoring of residues of OCPs from sediments of different grain size and organic matter content, which influence concentration, mobility and availability of contaminants.