Production and characterization of a human lysosomal recombinant iduronate-2-sulfatase produced in Pichia pastoris.

Hunter syndrome (Mucopolysaccharidosis II, MPS II) is an X-linked lysosomal storage disease produced by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Currently, MPS II patients are mainly treated with enzyme replacement therapy (ERT) using recombinant enzymes produced in mammalian cells. As an alternative, several studies have shown the production of active and therapeutic forms of lysosomal proteins in microorganisms. In this paper, we report the production and characterization of a recombinant IDS produced in the yeast Pichia pastoris (prIDS). We evaluated the effect of culture conditions and gene sequence optimization on prIDS production. The results showed that the highest production of prIDS was obtained at oxygen-limited conditions using a codon-optimized IDS cDNA. The purified enzyme showed a final activity of 12.45 nmol mg-1 H-1 and an apparent molecular mass of about 90 kDa. The highest stability was achieved at pH 6.0, and prIDS also showed high stability in human serum. Noteworthy, the enzyme was taken up by culture cells in a dose-dependent manner through mannose receptors, which allowed the delivery of the enzyme to the lysosome. In summary, these results show the potential of Pichia pastoris as a host to produce an IDS intended for a MPS II ERT.

Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia.

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

Single-domain magnetic particles with motion behavior under electromagnetic AC and DC fields are a fatal cargo in Metropolitan Mexico City pediatric and young adult early Alzheimer, Parkinson, frontotemporal lobar degeneration and amyotrophic lateral sclerosis and in ALS patients.

Metropolitan Mexico City (MMC) children and young adults exhibit overlapping Alzheimer and Parkinsons' diseases (AD, PD) and TAR DNA-binding protein 43 pathology with magnetic ultrafine particulate matter (UFPM) and industrial nanoparticles (NPs). We studied magnetophoresis, electron microscopy and energy-dispersive X-ray spectrometry in 203 brain samples from 14 children, 27 adults, and 27 ALS cases/controls. Saturation isothermal remanent magnetization (SIRM), capturing magnetically unstable FeNPs ~ 20nm, was higher in caudate, thalamus, hippocampus, putamen, and motor regions with subcortical vs. cortical higher SIRM in MMC ≤ 40y. Motion behavior was associated with magnetic exposures 25-100 mT and children exhibited IRM saturated curves at 50-300 mT associated to change in NPs position and/or orientation in situ. Targeted magnetic profiles moving under AC/AD magnetic fields could distinguish ALS vs. controls. Motor neuron magnetic NPs accumulation potentially interferes with action potentials, ion channels, nuclear pores and enhances the membrane insertion process when coated with lipopolysaccharides. TEM and EDX showed 7-20 nm NP Fe, Ti, Co, Ni, V, Hg, W, Al, Zn, Ag, Si, S, Br, Ce, La, and Pr in abnormal neural and vascular organelles. Brain accumulation of magnetic unstable particles start in childhood and cytotoxic, hyperthermia, free radical formation, and NPs motion associated to 30-50 µT (DC magnetic fields) are critical given ubiquitous electric and magnetic fields exposures could induce motion behavior and neural damage. Magnetic UFPM/NPs are a fatal brain cargo in children's brains, and a preventable AD, PD, FTLD, ALS environmental threat. Billions of people are at risk. We are clearly poisoning ourselves.

Assessing magnetic properties of biomonitors and road dust as a screening method for air pollution monitoring.

Particulate matter (PM) pollution is one of the world's most serious environmental challenges. Among PM components, atmospheric heavy metals (HMs) are considered one of the main pollutants responsible for causing significant negative impacts on human health, and ecological quality. This study aimed to assess environmental magnetism as a simple and rapid method that can be used to evaluate heavy metal contamination in urban areas from the relationships between magnetic properties and heavy metal concentrations. For this purpose, road dust and leaf samples of two common evergreen species (Cupressus lusitanica/Casuarina equisetifolia) were sampled simultaneously for 2 years at sites with different levels of traffic pollution. The results found significant statistical correlations between the magnetic properties and the chemical substances of the plants studied, as Fe, Cr and V showed an r ≥ 0.9 and Cr and Zn r ≥ 0.7 with χlf in C. equisetifolia. The frequency-dependent magnetic susceptibility was found to be between 0% and 14% for plants, and 0% and 2% for road dust, suggesting a rather dissimilar particle size distribution for plants, and a less important contribution from the more hazardous ultrafine superparamagnetic magnetite for both. Confirming that magnetic analyses can be used to distinguish different degrees of urban air pollution.

Assessing heavy metal pollution load index (PLI) in biomonitors and road dust from vehicular emission by magnetic properties modeling.

Vehicular traffic occupies a significant place among the sources of air pollution, due to population and urban growth that has led to an excessive increase in the vehicle fleet worldwide, and in Costa Rica as well. Vehicle emissions generate greenhouse gases (GHGs), particulate matter (PM), and heavy metals (HMs), due to combustion products from fossil-fuel engines, tire wear, and brake linings. HMs are important because they cannot be degraded or destroyed naturally; however, they can be diluted by physicochemical agents and be incorporated into trophic chains where they can be bioaccumulated causing significant negative effects on human well-being and ecological quality. This study aimed to assess the HM pollution load in biomonitors and road dust from vehicular emissions by chemical analyses and magnetic properties modeling. For this purpose, chemical and magnetic property analyses were carried out on samples of road dust and leaves of Cupressus lusitanica Mill. and Casuarina equisetifolia L., which were sampled during 2 different years in the Greater Metropolitan Area of Costa Rica known as GAM. Contamination factor (CF) and pollution load index (PLI) results showed significant metal pollution in some of the study sites. Contamination by the metals V, Cr, and Zn was most commonly present in the biomonitors, and for road dust, they were Cr, Zn, and Pb. The PLI estimates obtained with the validated support vector machine (SVM) magnetic properties models were consistent (sensitivity, specificity, and precision) with those obtained by chemical analysis, demonstrating the feasibility of this method for the identification of this index of contamination.

Comparison between machine linear regression (MLR) and support vector machine (SVM) as model generators for heavy metal assessment captured in biomonitors and road dust.

Exposure to suspended particulate matter (PM), found in the air, is one of the most acute environmental problems that affect the health of modern society. Among the different airborne pollutants, heavy metals (HMs) are particularly relevant because they are bioaccumulated, impairing the functions of living beings. This study aimed to establish a method to predict heavy metal concentrations in leaves and road dust, through their magnetic properties measurements. For this purpose, machine learning, automatic linear regression (MLR), and support vector machine (SVM) were used to establish models for the prediction of airborne heavy metals based on leaves and road dust magnetic properties. Road dust samples and leaves of two common evergreen species (Cupressus lusitanica/Casuarina equisetifolia) were sampled simultaneously during two different years in the Great Metropolitan Area (GMA) of Costa Rica. MLR and SVM algorithms were used to establish the relationship between airborne heavy metal concentrations based on single (χlf) and multiple (χlf y χdf) leaf magnetic properties and road dust. Results showed that Fe, Cu, Cr, V, and Zn concentrations were well-simulated by SVM prediction models, with adjusted R2 values ≥ 0.7 in both training and test stages. By contrast, the concentrations of Pb and Ni were not well-simulated, with adjusted R2 values < 0.7 in both training and test stages. Heavy metal predicción models using magnetic properties of leaves from Casuarina equisetifolia, as collectors, yielded better prediction results than those based on the leaves of Cupressus lusitanica and road dust, showing relatively higher adjusted R2 values and lower errors (MAE and RMSE) in both training and test stages. SVM proved to be the best prediction model with variations between single (χlf) and multiple (χlf y χdf) magnetic properties depending on the element studied.

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material.

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2% of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7% of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.

[Egg morphology as an indirect method to identify Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli (Diptera: Culicidae)]. / Utilidad de la morfología de los huevos como un método indirecto para identificar Anopheles benarrochi Gabaldón,Cova García & López, Anopheles oswaldoi (Peryassu) y Anopheles rangeli Gabaldón, Cova García & López, (Diptera:Culicidae) en Putumayo, Colombia.

In the Department of Putumayo in southern Colombia, malaria transmission has continued in the absence of the 4 traditional Latin American vector species--Anopheles darlingi, Anopheles nuneztovari, Anopheles albimanus or Anopheles trinkae. Human bait collections yielded Anopheles mosquitoes and a morphological variant of Anopheles benarrochi, the adult females of which can easily be misidentified as Anopheles oswaldoi. Species identification of females of Anopheles in the subgenus Nyssorhynchus is generally difficult due to overlapping morphological characters; therefore, progeny of field collected females were link-reared to assess species identity. Herein a robust method is presented to identify the species Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli from southern Colombia, using the morphology of the eggs induced from wild-caught females. Eggs of A. rangeli and A. benarrochi were differentiated on the basis of the anterior crown. In A. rangeli, this feature is positioned apically with high walls. In A. benarrochi, anterior crown is positioned more ventrally with comparatively shorter walls. No crown is present in A. oswaldoi. These differences are clear with the aid of a dissecting microscope and make accurate species determination possible even in field conditions. Egg morphology is shown to be an accurate, albeit indirect, method for the taxonomic determination for the three southern Colombian species and may also be useful in other regions of Latin America where the morphological variant of A. benarrochi is sympatric with A. oswaldoi.

Criterios campesinos para la selección de maíz (Zea mays L.) en Villaflores y Villa Corzo, Chiapas, México / Farmers' criteria for maize (Zea maysL.) selection in Villaflores and Villa Corzo, Chiapas, Mexico

RESUMEN La producción de maíz en Chiapas implica la decisión de cultivar maíces locales o híbridos. Estos últimos, tienen rendimientos más altos, pero requieren mayor cantidad de insumos, asociados con la degradación de suelos, contaminación del agua, pérdida de diversidad genética y del conocimiento sobre las variedades locales. El objetivo de esta investigación fue identificar los criterios que usan los pequeños productores, en la selección del material genético, para la siembra de maíz en los municipios de Villaflores y Villa Corzo, Chiapas. Se realizaron entrevistas semi-estructuradas a productores participantes del programa ASERCA. El maíz híbrido presentó mayor preferencia en ambos municipios, aunque algunos productores prefirieron los maíces locales o cultivaron ambos, por razones de usos y costumbres. La selección de maíces híbridos en los dos municipios se hizo principalmente con base en el rendimiento, seguido de la mayor resistencia a plagas y enfermedades. Otros criterios empleados para elegir semillas híbridas fueron: mayor resistencia al acame, mayor resistencia a la humedad, menor altura a la planta, menor pudrición de semillas y mayor tolerancia a la sequía. En maíces locales, el rendimiento fue un parámetro de importancia en ambos municipios, pero la mayor resistencia a plagas y enfermedades fue solo considerada en Villa Corzo. Y el mejor precio de venta del grano no se consideró en ninguno de los dos municipios. Otros criterios empleados para seleccionar las semillas locales fueron: mayor tolerancia a la sequía, menor costo de la semilla, mayor tamaño de la mazorca, interés por conservar dicho material, mejor sabor y permitir la rotación de variedades locales. Las seis características deseables a mejorar en el maíz local fueron: una menor altura de planta, un mayor rendimiento, una mayor resistencia a la humedad y al acame, un mayor peso del grano, un mayor tamaño de mazorca y de granos.

ABSTRACT The maize production in Chiapas implies the decision to grow local or hybrid maize. The latter present higher yields characteristics, but demand more inputs, particularly those related to soil degradation, water pollution, genetic diversity and knowledge losses over local maize varieties. The objective of this research was to identify the criteria used by small maize farmers in the election of seed for sowing in the Villaflores and Villa Corzo municipalities (Chiapas). The data were gathered through semi-structured interviews with farmers' participating in the ASERCA program and analyzed by percentages and frequencies. The hybrid maize is the most preferred variety by the small farmers in both municipalities, although some still prefer local maize (native) or bet on the cultivation of both due to use and customs reasons. The selectionof hybrid maizeseeds in both municipalities is based mainly on better yield characteristics, although in Villa Corzo, the selection of local varieties is due to their better resistance against pests and diseases. Additional criteria used to select the local seeds were: better lodging resistance, better moisture resistance, lower plant height, less seed rot and better drought tolerance. In local maize varieties, seed yield was considered an important factor in both municipalities, but a greater resistance against pests and diseases was only considered in Villa Corzo. The highest sale price was not an important consideration in neither of the two municipalities. Other criteria used to select the local seeds were better drought tolerance, seed lower cost, greater ear size, personal interest in conserving the local resource, better flavor and the possibility for the rotation of local varieties. The six wished characteristics of local maize varieties to be improved were: a lower plant height, a better yield, a better resistance to humidity and lodging, a greater grain weight, and a bigger size of ears and grains.

Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

Confirmation of Anopheles (Anopheles) calderoni Wilkerson, 1991 (Diptera: Culicidae) in Colombia and Ecuador through molecular and morphological correlation with topotypic material

The morphologically similar taxa Anopheles calderoni, Anopheles punctimacula, Anopheles malefactor and Anopheles guarao are commonly misidentified. Isofamilies collected in Valle de Cauca, Colombia, showed morphological characters most similar to An. calderoni, a species which has never previously been reported in Colombia. Although discontinuity of the postsubcostal pale spots on the costa (C) and first radial (R1) wing veins is purportedly diagnostic for An. calderoni, the degree of overlap of the distal postsubcostal spot on C and R1 were variable in Colombian specimens (0.003-0.024). In addition, in 98.2 percent of larvae, seta 1-X was located off the saddle and seta 3-C had 4-7 branches in 86.7 percent of specimens examined. Correlation of DNA sequences of the second internal transcribed spacer and mtDNA cytochrome c oxidase subunit I gene (COI) barcodes (658 bp of the COI gene) generated from Colombian progeny material and wild-caught mosquitoes from Ecuador with those from the Peruvian type series of An. calderoni confirmed new country records. DNA barcodes generated for the closely related taxa, An. malefactor and An. punctimacula are also presented for the first time. Examination of museum specimens at the University of the Valle, Colombia, revealed the presence of An. calderoni in inland localities across Colombia and at elevations up to 1113 m.

Presence of American cutaneous Leishmaniasis vectors surrounding the city of Medellín, Colombia.

The presence of Lutzomyia (Helcocyrtomyia) hartmanni, as a vector of Leishmania colombiensis and L. columbiana (Verrucarum group), recently incriminated in the transmission of leishmaniasis, and L. pia (Verrucarum group) are reported for the first time in a periurban area of Medellín city. There is thus a risk of leishmaniasis transmission in this town.

Presence of American cutaneous leishmaniasis vectors surrounding the city of Medellín, Colombia

The presence of Lutzomyia (Helcocyrtomyia) hartmanni, as a vector of Leishmania colombiensis and L. columbiana (Verrucarum group), recently incriminated in the transmission of leishmaniasis, and L. pia (Verrucarum group) are reported for the first time in a periurban area of Medellín city. There is thus a risk of leishmaniasis transmission in this town

Utilidad de la morfología de los huevos como un método indirecto para identificar Anopheles benarrochi Gabaldón, Cova García & López, Anopheles oswaldoi (Peryassu) y Anopheles rangeli Gabaldón, Cova García & López, (Diptera: Culicidae) en Putumayo, Colombia / Egg morphology as an indirect method to identify Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli (Diptera: Culicidae)

La identificación correcta de las hembras es esencial para el éxito de cualquier estudio de epidemiología, resistencia a insecticidas o de control de vectores. En el departamento del Putumayo, en el sur de Colombia, la transmisión de malaria continúa siendo un problema, a pesar de la ausencia de los vectores principales de Latinoamérica (Anopheles darlingi Root, Anopheles nuneztovari Gabaldón, Anopheles albimanus Wideman, Anopheles trinkae Faran) en esta región. Se recolectaron. con cebo humano, hembras de Anopheles y se encontró una variante morfológica de Anopheles benarrochi, que en su estadio adulto fácilmente se confunde con Anopheles oswaldoi. La identificación de hembras de Anopheles, particularmente del subgénero Nyssorhynchus, es en general notoriamente difícil debido a la superposición de caracteres morfológicos en el estadio adulto; por tanto, las colecciones deben estar ligadas a la cría de material asociado para identificar correctamente las especies. Esto requiere tiempo y es difícil de obtener en muchas ocasiones. Se presenta un método indirecto de identificación de las especies A. benarrochi, A. oswaldoi y Anopheles rangeli del sur de Colombia usando la morfología de los huevos de hembras silvestres. Los huevos de A. rangeli y A. benarrochi se diferencian por la corona anterior, la cual es apical en A. rangeli y con paredes altas, mientras que en A. benarrochi es ventral y con paredes más cortas. Esta corona está ausente en A. oswaldoi. Estas diferencias fueron obvias incluso bajo un microscopio de luz, lo que hace posible una identificación correcta de estas especies en condiciones de campo. Se muestra cómo la observación de la morfología de los huevos puede permitir la determinación taxonómica correcta, aunque indirecta, de estas tres especies de Nyssorhynchus encontradas en el sur de Colombia, el cual puede ser útil también en otras regiones de Latinoamérica, en donde se encuentre la variante morfológica de A. benarrochi en simpatría con A. oswaldoi.Palabras clave: Nyssorhynchus, huevos, Anopheles, Colombia, microscopia electrónica.Egg morphology as an indirect method to identify Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli (Diptera: Culicidae).

Variante morfológica de adultos hembras de Anopheles / Morphological variation in Anopheles benarrochi (Diptera: Culicidae) adult females specimens from Putumayo, Colombia

Con el propósito de ampliar el conocimiento sobre las especies de Anopheles presentes en el Putumayo, sur de Colombia, y para esclarecer la identidad de los ejemplares clasificados como Anopheles (Nyssorhynchus) evansae en esta región, se recolectaron mosquitos hembra en cebo humano, se alimentaron en pequeños mamíferos y se mantuvieron vivos para la cría de isofamilias. Se realizaron observaciones de las características morfológicas de los huevos, larvas, pupas y adultos de ambos sexos, incluidas las genitalias masculinas. Se obtuvieron 247 posturas de madres identificadas preliminarmente como A. (N) evansee. A 27 de estas familias se le estudió la morfología de los estadios asociados. Todos los especímenes fueron subsecuentemente identificados como Anopheles (N:) benarrochi por la morfología de los huevos, larvas, pupas y genitalias masculinas, lo cual coincidió con las descripciones publicadas para esta especie. Sin embargo, los adultos hembra de Putumayo presentaron la proporción oscura en el tarsómero posterior 2, entre 0.17 y 0,33 de su longitud, inferior a lo informado para esta especie, lo cual se superpone con los rangos de Anopheles (N.) oswaldoi y A. (N.) evansae. Como resultado de la superposición en este carácter, es probable que hembras adultas de A. (N.) benarrochi hayan sido incorrectamente identificadas como A. (N.) evansae y A. (N.) oswaldoi en el sur de Colombia. La presencia de esta variante morfológica ha dificultado la identificación de A. (N.) benarrochi en Putumayo y, probablemente, en otras regiones de Colombia y países vecinos