Marco A. Méndez

Toxicity phenotype does not correlate with phylogeny of Cylindrospermopsis raciborskii strains

Cylindrospermopsis raciborskii is a species of freshwater, bloom-forming cyanobacterium. C. raciborskii produces toxins, including cylindrospermopsin (hepatotoxin) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed. In spite of differences in toxicity, C. raciborskii strains comprise a monophyletic group, based upon 16S rRNA gene sequence identities (greater than 99%). We performed phylogenetic analyses; 16S rRNA gene and 16S-23S rRNA gene internally transcribed spacer (ITS-1) sequence comparisons, and genomic DNA restriction fragment length polymorphism (RFLP), resolved by pulsed-field gel electrophoresis (PFGE), of strains of C. raciborskii, obtained mainly from the Australian phylogeographic cluster. Our results showed no correlation between toxic phenotype and phylogenetic association in the Australian strains. Analyses of the 16S rRNA gene and the respective ITS-1 sequences (long L, and short S) showed an independent evolution of each ribosomal operon. The genes putatively involved in the cylindrospermopsin biosynthetic pathway were present in one locus and only in the hepatotoxic strains, demonstrating a common genomic organization for these genes and the absence of mutated or inactivated biosynthetic genes in the non toxic strains. In summary, our results support the hypothesis that the genes involved in toxicity may have been transferred as an island by processes of gene lateral transfer, rather than convergent evolution.

Discriminant analysis to evaluate clustering of gene expression data

In this work we present a procedure that combines classical statistical methods to assess the confidence of gene clusters identified by hierarchical clustering of expression data. This approach was applied to a publicly released Drosophila metamorphosis data set [White et al., Science 286 (1999) 2179–2184]. We have been able to produce reliable classifications of gene groups and genes within the groups by applying unsupervised (cluster analysis), dimension reduction (principal component analysis) and supervised methods (linear discriminant analysis) in a sequential form. This procedure provides a means to select relevant information from microarray data, reducing the number of genes and clusters that require further biological analysis.

Phylogenetic relationships of Chilean leptodactylids: a molecular approach based on mitochondrial genes 12S and 16S

La mayoria de los anfibios chilenos pertenece a la subfamilia Telmatobiinae (Anura, Leptodactylidae). Varios estudios filogeneticos de Leptodactylidae y Telmatobiinae, basados principalmente en caracteres morfologicos, han sugerido implicitamente relaciones mas estrechas de algunas especies de Telmatobiinae con miembros de otras subfamilias de leptodactilidos, incluyendo el genero leptodactilino Pleurodema presente en Chile. Ademas, un numero creciente de estudios moleculares sugieren un estatus no monofiletico para Telmatobiinae, aunque ninguno de estos estudios ha investigado las relaciones filogeneticas de esta subfamilia. Secuencias parciales de los genes ribosomales mitocondriales 12S y 16S fueron comparadas para determinar las relaciones filogeneticas de los leptodactilidos chilenos y su posicion dentro de los anuros modernos (Neobatrachia). Se incluyeron 22 especies de nueve de los diez generos de telmatobinos presentes en Chile (Alsodes, Atelognathus, Batrachyla, Caudiverbera, Eupsophus, Hylorina, Insuetophrynus, Telmatobufo y Telmatobius), dos especies del genero Pleurodema y una especie de Rhinodermatidae la cual es considerada una familia derivada de los leptodactilidos por algunos autores. Se incluyeron ademas 51 especies que representan la mayoria de las familias que componen Neobatrachia. Las reconstrucciones filogeneticas se realizaron utilizando los metodos de maxima parsimonia, maxima verosimilitud e inferencia bayesiana. Las topologias obtenidas en todos los analisis indican que Telmatobiinae es un ensamblaje polifiletico, compuesto por especies que pertenecen a Hyloidea (la mayoria de los generos) y especies mas relacionadas con taxa de Australasia (el clado Caudiverbera + Telmatobufo, definido como la tribu Calyptocephalellini). Estos datos moleculares respaldan agrupaciones basadas en otro tipo de evidencia (Caudiverbera + Telmatobufo, Alsodes + Eupsophus y Batrachyla + Hylorina) y plantean nuevas hipotesis de relaciones para algunos generos de telmatobinos (Atelognathus con Batrachyla e Hylorina, Insuetophrynus + Rhinoderma). Las relaciones filogeneticas observadas en este estudio sugieren un origen multiple para los anuros del bosque templado de Chile y revelan un inesperado nivel de diversidad taxonomica y divergencia evolutiva entre los leptodactilidos chilenos

Understanding copper homeostasis in humans and copper effects on health

Concern about human exposure to copperderives from the fact that virtually allindividuals are exposed to it in one way oranother, yet the knowledge about the effectsof chronic excess copper on human healthis clearly insufficient. Cu is an essentialmicronutrient that participates in severalprocesses crucial for life, but at the sametime, it may be toxic to cell membranes,DNA and proteins when accumulated inexcess (1, 6, 10). In man, mechanismsregulate uptake, efflux, storage andutilization of Cu preventing adverse effectsdue to excess within a rather wide range of(dietary) exposure. Cu handling appearstightly regulated, but the upper and lowerlimits of its homeostatic regulation areunclear. When exposure is high enough toinduce clinically apparent manifestations,the available markers are efficient andhelpful for diagnosis. However, thesemarkers are not sensitive enough to revealless intense changes of Cu status, whichmay be relevant in long-term human health(2, 7). Here, we review a series of studies inhumans that we have performed to explorethe capacity to handle copper loads and thelimits of copper homeostasis in humans.In a first, preliminary study, apparentlyhealthy women (20-45y) who had anestimated daily intake of 0.9 mg of copperper day (9, 11) were exposed to a three- tofourfold increase in their basal estimatedcopper exposure. Although acutegastrointestinal symptom report increased,no changes were detected in the distributioncurves of serum copper and ceruloplasminCp (3). In a second study, healthy adultswere exposed to an extra 15 to 150 µg/kg/d,provided via water consumed at home,which was ingested mixed with fluids andfood during the day (2, 3). Although in aproportion of the participants the long-termcopper consumption prior to the study wasestimated borderline low (0.9 mg Cu/day),there was no significant increase inerythrocyte superoxide dismutase (eSOD)activity after the two-month exposureperiod. This result did not support themeasurement of eSOD as a potentialindicator of marginal copper deficiency.Expressed as mean values ± SD, copperconcentrations (in serum, red blood cellsand mononuclear cells), serum/plasma Cp,and eSOD activity were within the normalrange and were not related to copper intake(ANOVA, NS). The non-Cp copper fractionwas positively correlated to serum copper (r= 0.58). Activity of liver aminotransferases,measured as indicators of liver function,remained within normal limits.In the next study, we assessed the effectof the Upper Level (UL) of dietaryrecommendations in apparently healthyadults (4, 5, 8). The UL value is a conceptdeveloped for regulatory purposes to definesafe limits of intake for the normal human. Itis the level of intake from food, water andsupplements that is unlikely to pose risks ofadverse health effects from excess over thelong term, in apparently healthy individuals,in an age- and sex-specific population group(5). Although Cp is not a good indicatorwhen infectious or inflammatory processes

PSP toxin release from the cyanobacterium Raphidiopsis brookii D9 (Nostocales) can be induced by sodium and potassium ions

Paralytic shellfish poisoning (PSP) toxins are a group of naturally occurring neurotoxic alkaloids produced among several genera of primarily freshwater cyanobacteria and marine dinoflagellates. Although saxitoxin (STX) and analogs are all potent Na(+) channel blockers in vertebrate cells, the functional role of these compounds for the toxigenic microorganisms is unknown. Based upon the known importance of monovalent cations (such as sodium) in the maintenance of cellular homeostasis and ion channel function, we examined the effect of high extracellular concentrations of these ions on growth, cellular integrity, toxin production and release to the external medium in the filamentous freshwater cyanobacterium, Raphidiopsis brookii D9; a gonyautoxins (GTX2/3) and STX producing toxigenic strain. We observed a toxin export in response to high (17 mM) NaCl and KCl concentrations in the growth medium that was not primarily related to osmotic stress effects, compared to the osmolyte mannitol. Addition of exogenous PSP toxins with the same compositional profile as the one produced by R. brookii D9 was able to partially mitigate this effect of high Na⁺ (17 mM). The PSP toxin biosynthetic gene cluster (sxt) in D9 has two genes (sxtF and sxtM) that encode for a MATE (multidrug and toxic compound extrusion) transporter. This protein family, represented by NorM in the bacterium Vibrio parahaemolyticus, confers resistance to multiple cationic toxic agents through Na⁺/drug antiporters. Conserved domains for Na⁺ and drug recognition have been described in NorM. For the D9 sxt cluster, the Na⁺ recognition domain is conserved in both SxtF and SxtM, but the drug recognition domain differs between them. These results suggest that PSP toxins are exported directly in response to the presence of monovalent cations (Na⁺, K⁺) at least at elevated concentrations. Thus, the presence of both genes in the sxt cluster from strain D9 can be explained as a selective recognition mechanism by the SxtF/M transporters for GTX2/3 and STX. We propose that these toxins in cyanobacteria could act extracellularly as a protective mechanism to ensure homeostasis against extreme salt variation in the environment.

Micronutrient deficiencies in patients with typical and atypical celiac disease.

Objective: The extent of the digestive/absorptive involvement in atypical presentation of celiac disease (CD) is not always clear. The aim of the study was to assess nutritional status of iron (Fe), copper (Cu), and zinc (Zn) in patients with typical CD (TCD) and atypical CD (ACD). Patients and Methods: A cross-sectional study was done in patients with TCD, ACD, and healthy controls (HC). Hemoglobin, serum ferritin, free erythrocyte protoporphyrin, Fe, Cu, ceruloplasmin, Zn, anti-endomysial antibodies, and anti-tissue transglutaminase antibodies were measured. Data were analyzed by Kruskal-Wallis, principal component analysis, and linear discriminant analysis. Results: One hundred nine individuals were studied (54 TCD, 19 ACD, 36 HC); mean age ± standard deviation was 23 ± 15.8 (range 1.6–75.4) years. Median and range of hemoglobin were 12.8 g/dL (8.1–17.6) in TCD, 12.4 g/dL (10.5–14.5) in ACD, and 13.6 g/dL (11.1–16.7) in HC (P < 0.0001); serum ferritin was 17.7 &mgr;g/L (2.9–157), 10.8 &mgr;g/L (2.7–39.8), and 28.7 &mgr;g/L (4.5–127.2), respectively (P < 0.01). Cu was 105 &mgr;g/dL (60–185), 97.5 &mgr;g/dL (40–130), and 125 &mgr;g/dL (80–205), respectively (P < 0.05). Ceruloplasmin was 21.6 mg/dL (14.2–73.2), 22.6 mg/dL (0.9–34.3), and 32.1 mg/dL (5.8–72.6), respectively (P < 0.01). There were no differences in Fe, free erythrocyte protoporphyrin, and Zn. Principal component analysis showed that 58% of observed variability was explained by Fe and Cu indicators. Linear discriminant analysis revealed differences between CD and HC (P < 0.0001), with high values of correct classification for TCD (73%) and HC (72%), but not ACD (16%), which were mostly classified as TCD (79%). Conclusions: Deficiency of micronutrients was found both in typical as well as in atypical cases.

Historia natural cuantitativa de una relación parásito-hospedero: el sistema Tristerix-cactáceas en Chile semiárido

Presentamos informacion cuantitativa de la historia natural de la relacion parasito-hospedero constituida por el muerdago holoparasito Tristerix aphyllus (Loranthaceae) y sus hospederos cactaceas. Mas especificamente, indagamos en los determinantes historicos y biogeograficos de la interaccion y cuantificamos la autoecologia de la biologia floral, polinizacion, dispersion y parasitismo en este sistema. El impacto del parasitismo sobre la evolucion de sistemas defensivos en las cactaceas hospederas es considerado tanto a nivel intraespecifico como interespecifico, tomando en cuenta el potencial para seleccion mediada por parasitos y la estructura geografica de la interaccion. Finalmente, sugerimos futuras avenidas de investigacion en este sistema que incluyen los topicos de: (i) evolucion de la virulencia, (ii) estructuracion de la interaccion en mosaico geografico y, (iii) pruebas historicas de adaptacion. Estos aspectos permitiran adquirir un mayor conocimiento de la sutileza ecologica y de la evolucion de esta especial interaccion en los sistemas naturales de Chile semiarido

Sex and Ceruloplasmin Modulate the Response to Copper Exposure in Healthy Individuals

Previous studies indicated that sex might influence the response to copper exposure. Ceruloplasmin (Cp) is an indicator of Cu status, but it is not clear whether and how it reflects changes of Cu status among healthy individuals. In this study, 82 apparently healthy women and men were chosen from 800 individuals because their Cp values belonged to the higher and lower 10% of the group Cp distribution curve. Before and after receiving a supplement of 10 mg Cu/day (upper limit of daily intake) for 2 months, we performed blood and urinary biochemical measurement of potential Cu markers. We used principal component analysis and linear discriminant analysis to identify blood and/or urinary Cu indicators that showed a differential response to copper. Results showed that Cp values in serum represent a reliable indicator to differentiate subgroups within the normal population in their response to Cu exposure. The response depends on Cp values and on sex, such that women with higher and men with lower Cp values exhibit the greatest response.

Phylogenetic and phylogeographic analysis of the genus Orestias (Teleostei: Cyprinodontidae) in the southern Chilean Altiplano: the relevance of ancient and recent divergence processes in speciation

This study presents phylogenetic molecular data of the Chilean species of Orestias to propose an allopatric divergence hypothesis and phylogeographic evidence that suggests the relevance of abiotic factors in promoting population divergence in this complex. The results reveal that diversification is still ongoing, e.g. in the Ascotán salt pan, where populations of Orestias ascotanensis restricted to individual freshwater springs exhibit strong genetic differentiation, reflecting putative independent evolutionary units. Diversification of Orestias in the southern Altiplano may be linked to historical vicariant events and contemporary variation in water level; these processes may have affected the populations from the Plio-Pleistocene until the present.

Cop-like operon: Structure and organization in species of the Lactobacillale order

Copper is an essential and toxic trace metal for bacteria and, therefore, must be tightly regulated in the cell. Enterococcus hirae is a broadly studied model for copper homeostasis. The intracellular copper levels in E. hirae are regulated by the cop operon, which is formed by four genes: copA and copB that encode ATPases for influx and efflux of copper, respectively; copZ that encodes a copper chaperone; and copY, a copper responsive repressor. Since the complete genome sequence for E. hirae is not available, it is possible that other genes may encode proteins involved in copper homeostasis. Here, we identified a cop-like operon in nine species of Lactobacillale order with a known genome sequence. All of them always encoded a CopY-like repressor and a copper ATPase. The alignment of the cop-like operon promoter region revealed two CopY binding sites, one of which was conserved in all strains, and the second was only present in species of Streptococcus genus and L. johnsonii. Additional proteins associated to copper metabolism, CutC and Cupredoxin, also were detected. This study allowed for the description of the structure and organization of the cop operon and discussion of a phylogenetic hypothesis based on the differences observed in this operons organization and its regulation in Lactobacillale order.