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Silica-based scaffolds are promising in Tissue Engineering by enabling personalized scaffolds, boosting exceptional bioactivity and osteogenic characteristics. Moreover, silica materials are highly tunable, allowing for controlled drug release to enhance tissue regeneration. In this study, we developed a 3D printable silica material with controlled mesoporosity, achieved through the sol-gel reaction of tetraethyl orthosilicate (TEOS) at mild temperatures with the addition of different calcium concentrations. The resultant silica inks exhibited high printability and shape fidelity, while maintaining bioactivity and biocompatibility. Notably, the increased mesopore size enhanced the incorporation and release of large molecules, using cytochrome C as a drug model. Due to the varying surface charge of silica depending on the pH, a pH-dependent control release was obtained between pH 2.5 and 7.5, with maximum release in acidic conditions. Therefore, silica with controlled mesoporosity could be 3D printed, acting as a pH stimuli responsive platform with therapeutic potential.

Among the different layers of the cornea, the corneal endothelium, which is composed of corneal endothelial cells (CEC), plays a key role in the maintenance of cornea transparency. Based on the donor shortages and the limitations associated with transplantation, in this work we have developed collagen hydrogels with different patterned structures on the surface to provide topographies in ranges similar to the natural environment that CEC sense. This aimed at stimulating cells to maintain a typical CEC phenotype and provide alternatives to corneal transplantation. In this sense, we have elaborated curved collagen hydrogels (concave and convex) with three different topographies (50, 200 and 300 µm grooves), with the help of 3D printed mold and replicating the mold with the collagen hydrogel, onto which CEC were cultured in order to analyze its behavior. Flat hydrogels were used as controls. Cell morphology, cell circularity and gene expression of ATP1A1 and ZO-1 genes were analyzed after 3 and 6 days. Results showed an effect of the curvature and the topography compared to flat collagen hydrogels, showing higher expression of ZO-1 and ATP1A1 in curved non-patterned hydrogels at day 3. The patterned hydrogels did not have such a significant effect on gene expression compared to flat hydrogels, showing stronger effect of the curvature compared to the topography. The effect was opposite at day 6, showing higher gene expression at days 6 on the patterned hydrogels, especially for the ZO-1 gene. The gene expression results were in accordance with the cell morphology observed at the different time points, showing circularities closer to hexagon like morphology at shorter time points. Overall, this platform can serve as a system to culture cell under proper environment to further be able to transplant a CEC monolayer or to allow transplantation of thin collagen layers with cultured CEC.

The application of reliability centered maintenance onrepairable equipment requires that the reliability calculation should not to be based on failure statistics, as in traditional methods, but rather on its condition. The "load vs resistance" method presumesthe comparison of process parameters with carefully selected mechanical properties of the materials. The author proposes the calculation based on parameters monitored for diagnosis purposes. Specifically, he analyzes the erosion of steam turbine bladessubject tothe action of solid particle from the superheaters, which, under certain conditions, grow to critical thicknesses and due to stress actions caused by thermal changes, fracture and detach, acquiring such energy that then impacts the surface of the blades causing theirerosion. These phenomena are analyzed and equations are established in relation withthe mechanical properties of the blade metal, with the energy requird by oxide flakes to cause their erosion. An expression has been obtained, allowing for the application of the method, whichd has not been found in analyzed research works.

For decades, mangroves have been exposed to various pressures, resulting in the loss of large swathes around the world. For this reason, ecological restoration actions are presented as alternatives to recover mangroves that can restore their ecosystem services while helping to mitigate climate change’s effects. Mangroves are crucial, as they capture and sequester carbon in biomass and soil, highlighting their importance in environmental conservation and in the fight against climate change. In this research, the amount of carbon sequestered in a mangrove area restored eight years ago and its relationship with soil physicochemical parameters were evaluated and compared to those of a reference forest. Soil cores were collected at a depth of 30 cm from both sites, and in situ measurements of physical chemistry were made at different depths. In addition, soil salinity, bulk density, and carbon concentration were determined. The results revealed a similar amount of carbon sequestered both in the reference forest (BR) (470.17 ± 67.14 Mg C/ha) and in the restoration area (RA) (444.53 ± 86.11 Mg C/ha) (p > 0.05). A direct relationship was observed between carbon sequestration and soil depth. In the case of the RA, a direct relationship was found between carbon sequestration and soil salinity. In conclusion, the results of this study indicate that the behavior of carbon sequestration in soil is determined by the physicochemical conditions in both the BR and the RA, as well as by the presence or absence of vegetation.

Tissue defects can lead to serious health problems and often require grafts or transplants to repair damaged soft tissues. However, these procedures can be complex and may not always be feasible due to a lack of available tissue. Hydrogels have shown potential as a replacement for tissue grafts due to their ability to support cell survival and encapsulate biomolecules such as growth factors. In particular, guanosine-based hydrogels have been explored as a potential solution, but they often exhibit limited stability which hampers their use in the biofabrication of complex grafts. To address this issue, we explored the use of borate ester chemistry and more complex boric acid derivatives to improve the stability and properties of guanosine-based hydrogels. We hypothesized that the aromatic rings in these derivatives would enhance the stability and printability of the hydrogels through added π-π stack interactions. After optimization, 13 compositions containing either 2-naphthylboronic acid or boric acid were selected. Morphology studies shows a well-defined nanofibrilar structure with good printable properties (thixotropic behaviour, print fidelity and printability). Moreover, the pH of all tested hydrogels was within the range suitable for cell viability (7.4–8.3). Nevertheless, only the boric acid-based formulations were stable for at least 7 days. Thus, our results clearly demonstrated that the presence of additional aromatic rings did actually impair the hydrogel properties. We speculate that this is due to steric hindrance caused by adjacent groups, which disrupt the correct orientation of the aromatic groups required for effective π-π stack interactions of the guanosine building block. Despite this drawback, the developed guanosine-boric acid hydrogel exhibited good thixotropic properties and was able to support cell survival, proliferation, and migration. For instance, SaOS-2 cells planted on these printed structures readily migrated into the hydrogel and showed nearly 100% cell viability after 7 days. In conclusion, our findings highlight the potential of guanosine-boric acid hydrogels as tissue engineering scaffolds that can be readily enhanced with living cells and bioactive molecules. Thus, our work represents a significant advancement towards the development of functionalized guanosine-based hydrogels.

Bacterial infection of implanted scaffolds may have fatal consequences and, in combination with the emergence of multidrug bacterial resistance, the development of advanced antibacterial biomaterials and constructs is of great interest. Since decades ago, metals and their ions had been used to minimize bacterial infection risk and, more recently, metal-based nanomaterials, with improved antimicrobial properties, have been advocated as a novel and tunable alternative. A comprehensive review is provided on how metal ions and ion nanoparticles have the potential to decrease or eliminate unwanted bacteria. Antibacterial mechanisms such as oxidative stress induction, ion release and disruption of biomolecules are currently well accepted. However, the exact antimicrobial mechanisms of the discussed metal compounds remain poorly understood. The combination of different metal ions and surface decorations of nanoparticles will lead to synergistic effects and improved microbial killing, and allow to mitigate potential side effects to the host. Starting with a general overview of antibacterial mechanisms, we subsequently focus on specific metal ions such as silver, zinc, copper, iron and gold, and outline their distinct modes of action. Finally, we discuss the use of these metal ions and nanoparticles in tissue engineering to prevent implant failure.

Abstract Biomaterials and scaffolds for Tissue Engineering are widely used for an effective healing and regeneration. However, the implantation of these scaffolds causes an innate immune response in which the macrophage polarization from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype is crucial to avoid chronic inflammation. Recent studies have showed that the use of bioactive ions such as cobalt (Co2+), copper (Cu2+) and magnesium (Mg2+) could improve tissue regeneration, although there is limited evidence on their effect on the macrophage response. Therefore, we investigated the immunomodulatory potential of Co2+, Cu2+ and Mg2+ in macrophage polarization. Our results indicate that Mg2+ and concentrations of Cu2+ lower than 10 μM promoted the expression of M2 related genes. However, higher concentrations of Cu2+ and Co2+ (100 μM) stimulated pro-inflammatory marker expression, indicating a concentration dependent effect of these ions. Furthermore, Mg2+ were able to decrease M1 marker expression in presence of a mild pro-inflammatory stimulus, showing that Mg2+ can be used to modulate the inflammatory response, even though their application can be limited in a strong pro-inflammatory environment.

The productivity of mangrove ecosystems is associated with litterfall production, which continuously contributes large quantities of organic matter, in the form of detritus, to the food web via adjacent ecosystems. However, the degree of deterioration of mangrove ecosystems worldwide has been increasing due to anthropogenic activities, leading to the loss of vegetation cover and changes in hydrological patterns, the chemical conditions of interstitial water and soil, and the litterfall degradation rate and, thus, the integration of organic matter into the ecosystem. In this study, we investigate the relationship between leaf degradation and interstitial water chemistry in two mangrove forests located in Oaxaca, Mexico, that are characterized by differences in environmental conditions, species, and anthropogenic activity. Forty-two 10 cm × 20 cm nylon mesh bags were installed in the Rhizophora mangle forest along two flood-associated lines (21 per line), and twenty-one bags were installed centrally in the Avicennia germinans forest because of the flood conditions in this area. Three bags per line were collected each month. This material was then dried and calcined for determination of the decomposition rate (k). The in situ redox potential and interstitial water salinity of mangrove forests were measured using a HACH HQ40d multiparametric probe and A&O refractometer, and the sulfate concentration was determined by ion chromatography. The results show that daily average decomposition rates were higher in the Salina lagoon (k = 0.01 g·day−1) than in the Chacahua lagoon (k = 0.004 g·day−1). The degradation model was Y = 66.054e−0.010t, R2 = 0.89, p ≤ 0.05, for the Salina lagoon and Y = 67.75e−0.004t, R2 = 0.76, p ≤ 0.05, for the Chacahua lagoon. Leaf decomposition rates differed between the Salina and Chacahua lagoons (F1,206 = 4.8, p < 0.03). In the Salina lagoon, dominated by A. germinans, an inverse relationship was established between the percentage of degraded biomass with respect to salinity concentration (R2 = 0.82, p < 0.013) and redox potential (R2 = 0.89, p < 0. 015), and for the Chacahua lagoon, dominated by R. mangle, the percentage of degraded litter biomass was found to be inversely correlated with redox potential (R2 = 0.94, p < 0.005) and sulfate concentration (R2 = 0.88, p < 0.017). Based on the results obtained in this study, we conclude that variations in the chemical conditions of interstitial water and hydrological patterns can affect the process of mangrove leaf degradation based on species and the integration of organic matter in the soil and in adjacent ecosystems. These findings are potentially useful for mangrove management because they advance understanding of the dynamics of organic matter in mangroves and the importance of maintaining the health of these ecosystems, which is necessary for the maintenance of coastal fishing production.

Los autores comentan el concepto de Raunkiaer del fitoclima de los caméfitos. También se dirigió la atención al problema de los límites biogeográficos, especialmente en lo que se refiere a la parte sur de América del Sur y la Antártida en general. Además, se agrega un bosquejo geográfico y climatológico de la Antártida y los territorios circundantes. Se llega a la conclusión de que el concepto de Raunkiaer no puede ser aceptado en su limitación austral, habiendo utilizado aquí elementos correspondientes a las comunidades vegetales del cordón insular, las cuales pertenecen, según los autores, a los subantárticos. Tras manifestar sus opiniones sobre el concepto de Raunkiaer del fitoclima de los caméfitos antárticos, muestran mediante un mapa las provincias naturales y fijan los nuevos límites del fitoclima de las Malvinas, Staten-Island, Fireland, Picton, Navarino, etc. que debe incluirse en la provincia climatológica frío-templado y, por lo tanto, no puede llamarse subantártico.

El autor, teniendo en cuenta los conceptos publicados por el Dr. W. Knoche referidos a la "temperatura activa" y "precipitación efectiva", que calcula a partir de la fórmula de Thornthwaite, modificada por el ingeniero JM Fraga, propone una variación para el gráfico o diagrama hidrotérmico de Raunkiaer, sustituyendo las curvas de las temperaturas medias mensuales (T) y las precipitaciones medias mensuales (P) observadas, por las de la "temperatura activa" media mensual (Ta) y la "precipitación efectiva" media mensual (Pe). El trabajo incluye también un mapa, donde se ubican las estaciones meteorológicas, cuyos promedios se han utilizado para estimar las curvas de los 44 gráficos agregados a esta publicación, y que se han ordenado en provincias botánicas según las consignadas por Castellanos y Pérez-Moreau en "Los tipos de vegetación de la República Argentina".

En el presente trabajo, el autor primero da cuenta de los diferentes significados que muchos viajeros y científicos le dan a la palabra pampa. Como es una palabra de origen peruano utilizada por los indios khechuwa, cuyas tribus que vivían allí nunca llegaron a los llanos de Buenos Aires, es evidente que fue traída por los españoles que también llamaron indios pampas a un grupo de tribus que habitaban el llano bonaerense, de las cuales ninguna se llamaba así. La palabra pampa se refiere a llanuras montañosas, que fisiográficamente no se pueden comparar con las llanuras de Buenos Aires; estas tienen una pequeña altura sobre el nivel del mar, y por ello sólo se puede aplicar a la región al oeste de la llanura bonaerense.

El autor da una reseña de la Lilaeopsis mencionada para Argentina. Este género cuenta en nuestro país con 8 especies, 2 variedades y 1 forma, las cuales se ilustran en estas publicaciones. Algunas especies y variedades ya han sido descritas por el autor en la Review Physis, números 40 y 42; por otro lado, en este artículo, se elaboran nov. comb. L. andina A. W. Hill. form. subulata (Wedd.), L. attenuata (Hook. et Arn.) y L. minor (A. W. Hill). Refiriéndose a Lilaeopsis attenuata Fernald (conf. Contrib. Gray Herb. Harvard Univ., New Ser., no 72, p. 94) el autor no lo cuenta porque se ha probado que bajo esta designación, el Dr. Fernald ha determinado varios ejemplos de Lilaeopsis andina A. W. Hill, existente en el Herbario Grey. Además, el Dr. Hill da como sinónimo de esa combinación con algunas de sus especies (véase Journ. of Linn. Soc. Bot., t. 47. p. 540 y 543). El artículo contiene una clave para la determinación de las especies argentinas.

Mangrove forests have proven to be resilient to most environmental change, surviving catastrophic climate events over time. Our study aimed to evaluate the chemical variability of pore water and its effect on phenological production in three mangrove species (Rhizophora mangle, Avicennia germinans, and Laguncularia racemosa) along the coast of the state of Campeche during a year of severe drought (2009) and a year of average precipitation (2010). Pore water salinity and redox potential were measured monthly in a mangrove forest in 2009 and 2010. Litterfall production and reproductive phenology was measured monthly. We determined the relationships among litterfall production, reproductive phenology, pore water chemistry and precipitation of three species between years. Precipitation, pore water salinity and redox potential significantly differed among years, seasons and sites, and also showed significant interaction between years and seasons (p < 0.05). Significant variation was observed in litterfall production, propagules, flowers, and leaf litter among sites (p < 0.05). A significant change was observed in propagules and flowers among years, and in total litterfall and leaf litter between seasons and species (p < 0.05). Under severe drought, salinity had the strongest effect on total litterfall and propagules in R. mangle, while A. germinans, had the strongest effect on propagule/flower precipitation. Both A. germinans and L. racemosa showed higher resilience than R. mangle at all sites under severe drought conditions. These findings can support activity allocation for mangrove conservation and restoration by providing the tolerance thresholds of the three species that dominate in the regional area of Campeche state. Likewise, this research provides knowledge to the Intergovernmental Experts Group on climate change about drought intensity and its magnitude of impact on mangrove productivity, reproduction and integrity.

Cardiovascular disease (CVD) is the leading cause of death among persons aged 65 and older in the United States and many other developed countries. Tissue engineered vascular systems (TEVS) can serve as grafts for CVD treatment and be used as in vitro model systems to examine the role of various genetic factors during the CVD progressions. Current focus in the field is to fabricate TEVS that more closely resembles the mechanical properties and extracellular matrix environment of native vessels, which depends heavily on the advance in biofabrication techniques and discovery of novel biomaterials. In this review, we outline the mechanical and biological design requirements of TEVS and explore the history and recent advances in biofabrication methods and biomaterials for tissue engineered blood vessels and microvascular systems with special focus on in vitro applications. In vitro applications of TEVS for disease modeling are discussed.

Protein-protein interactions (PPIs) mediate a large number of important regulatory pathways. Their modulation represents an important strategy for discovering novel therapeutic agents. However, the features of PPI binding surfaces make the use of structure-based drug discovery methods very challenging. Among the diverse approaches used in the literature to tackle the problem, linear peptides have demonstrated to be a suitable methodology to discover PPI disruptors. Unfortunately, the poor pharmacokinetic properties of linear peptides prevent their direct use as drugs. However, they can be used as models to design enzyme resistant analogs including, cyclic peptides, peptide surrogates or peptidomimetics. Small molecules have a narrower set of targets they can bind to, but the screening technology based on virtual docking is robust and well tested, adding to the computational tools used to disrupt PPI. We review computational approaches used to understand and modulate PPI and highlight applications in a few case studies involved in physiological processes such as cell growth, apoptosis and intercellular communication.

Bone is the most studied tissue in the field of tissue regeneration. Even though it has intrinsic capability to regenerate upon injury, several pathologies and injuries could hamper the highly orchestrated bone formation and resorption process. Bone tissue engineering seeks to mimic the extracellular matrix of the tissue and the different biochemical pathways that lead to successful regeneration. For many years, the use of extrinsic factors (i.e., growth factors and drugs) to modulate these biological processes have been the preferred choice in the field. Even though it has been successful in some instances, this approach presents several drawbacks, such as safety-concerns, short release profile and half-time life of the compounds. On the other hand, the use of inorganic ions has attracted significant attention due to their therapeutic effects, stability and lower biological risks. Biomaterials play a key role in such strategies where they serve as a substrate for the incorporation and release of the ions. In this review, the methodologies used to incorporate ions in biomaterials is presented, highlighting the osteogenic properties of such ions and the roles of biomaterials in controlling their release.

Failure of corneal endothelium cell monolayer is the main cause leading to corneal transplantation. Autologous cell-based therapies are required to reconstruct in vitro the cell monolayer. Several strategies have been proposed using embryonic stem cells and induced pluripotent stem cells, although their use has ethical issues as well as limited clinical applications. For this purpose, we propose the use of dental pulp stem cells isolated from the third molars to form the corneal endothelium cell monolayer. We hypothesize that using dental pulp stem cells that share an embryological origin with corneal endothelial cells, as they both arise from the neural crest, may allow a direct differentiation process avoiding the use of reprogramming techniques, such as induced pluripotent stem cells. In this work, we report a two-step differentiation protocol, where dental pulp stem cells are derived into neural crest stem-like cells and, then, into corneal endothelial-like cells. Initially, for the first-step we used an adhesion culture and compared two initial cell sources: a direct formation from dental pulp stem cells with the differentiation from induced pluripotent stem cells. Results showed significantly higher levels of early stage marker AP2 for the dental pulp stem cells compared to induced pluripotent stem cells. In order to provide a better environment for neural crest stem cells generation, we performed a suspension method, which induced the formation of neurospheres. Results showed that neurosphere formation obtained the peak of neural crest stem cell markers expression after 4 days, showing overexpression of AP2, Nestin, and p75 markers, confirming the formation of neural crest stem-like cells. Furthermore, pluripotent markers Oct4, Nanog, and Sox2 were as well-upregulated in suspension culture. Neurospheres were then directly cultured in corneal endothelial conditioned medium for the second differentiation into corneal endothelial-like cells. Results showed the conversion of dental pulp stem cells into polygonal-like cells expressing higher levels of ZO-1, ATP1A1, COL4A2, and COL8A2 markers, providing a proof of the conversion into corneal endothelial-like cells. Therefore, our findings demonstrate that patient-derived dental pulp stem cells may represent an autologous cell source for corneal endothelial therapies that avoids actual transplantation limitations as well as reprogramming techniques.

The use of copper (Cu2+) and cobalt (Co2+) has been described to stimulate blood vessel formation, a key process for the success of tissue regeneration. However, understanding how different concentrations of these ions affect cellular response is important to design scaffolds for their delivery to better fine tune the angiogenic response. On the one hand, gene expression analysis and the assessment of tubular formation structures with human umbilical vein endothelial cells (HUVEC) revealed that high concentrations (10μM) of Cu2+ in early times and lower concentrations (0.1 and 1μM) at later times (day 7) enhanced angiogenic response. On the other hand, higher concentrations (25μM) of Co2+ during all time course increased the angiogenic gene expression and 0.5, 5 and 25μM enhanced the ability to form tubular structures. To further explore synergistic effects combining both ions, the non-toxic concentrations were used simultaneously, although results showed an increased cell toxicity and no improvement of angiogenic response. These results provide useful information for the design of Cu2+ or Co2+ delivery scaffolds in order to release the appropriate concentration during time course for blood vessel stimulation.

Macrophages have been deemed crucial for correct tissue regeneration, which is a complex process with multiple overlapping phases, including inflammation. Previous studies have suggested that divalent ions are promising cues that can induce an anti-inflammatory response, since they are stable cues that can be released from biomaterials. However, their immunomodulatory potential is limited in a pro-inflammatory environment. Therefore, we investigated whether copper and magnesium ions combined with low concentrations of the anti-inflammatory drug, dexamethasone (dex), could have a synergistic effect in macrophage, with or without pro-inflammatory stimulus, in terms of morphology, metabolic activity and gene expression. Our results showed that the combination of copper and dex strongly decreased the expression of pro-inflammatory markers, while the combination with magnesium upregulated the expression of IL-10. Moreover, in the presence of a pro-inflammatory stimulus, the combination of copper and dex induced a strong TNF-α response, suggesting an impairment of the anti-inflammatory actions of dex. The combination of magnesium and dex in the presence of a pro-inflammatory stimulus did not promote any improvement in comparison to dex alone. The results obtained in this study could be relevant for tissue engineering applications and in the design of platforms with a dual release of divalent ions and small molecules.

The seeds germination, stem and root elongation of water lettuces (Lactuca sativa L) were used to evaluate the phytotoxicity of water and soil of Champotón River, Campeche, Mexico. water and soil sample were collected from two sampling sites. Lettuce seeds were exposed to three different dilutions of water and aqueous extract of soil. Significant differences on germination, root and stem elongation of lettuce were detected. Water showed more phytotoxic effect than aqueous extract of soil and inhibitory concentration 50 (IC50) for radicle exposed to river water was 52% while stem elongation was 69%. Due to paucity the previously available phytotoxicological studies onto Champotón River, it is now essential to allocate time and resources to consider development of suitable chronic phytotoxicity tests. Keywords: Bioassay, Lactuca sativa, Champotón river, Phytotoxicity, Soil–water solution, Environmental toxicology

Mangroves are considered one of the most productive ecosystems worldwide, providing multiple environmental goods and services; however, in recent years, there have been modifications and deterioration in the structure and function of these ecosystems, caused by various natural events and anthropic activities, such as the construction of roads, wastewater discharge, unsustainable livestock, and agricultural practices, as well as the impact of chemicals, such as heavy metals, oil spills, and the use of herbicides. In this research, phytotoxic effects on seedlings of Rhizophora mangle were evaluated at an exposure of five dilutions w/v (5%, 10%, 25%, 50%, and 100%) of the commercial presentation of 2,4-dichlorophenoxyacetic acid (2,4-D). Propagules grown in a greenhouse under local tidal regimes were used, so the growth of stem diameter, height, biomass production in root, leaves, and stems, as well as the concentration of chlorophyll a of the exposed seedlings were measured. The comparison of these parameters in seedlings with only seawater presented significant differences (p ≤ 0.05) and inhibitory effects on growth (diameter), the stem concentration of chlorophyll a, and the production of biomass of leaves, stems, and roots. The inhibitory effect of exposure to 2,4-D on chlorophyll production and root biomass is highlighted, with an average decrease of 45% relative to the control. The sensitivity of the Rhizophora mangle seedlings to the applied concentrations of herbicide evidence the inhibitory effects on the morphological variables of biomass production and chlorophyll a production in mangrove leaves.

El autor describe una nueva especie de Umbelliferae (Spermolepis castellanosii), encontrada en Río Negro, Neuquén y Mendoza (Argentina). *RÉSUMÉ: ?Une nouvelle espèce des Ombellifères?. L?auteur décrit une nouvelle espèce des Ombellifères (Spermolepis castellanosii), trouvée dans le Río Negro, Neuquén et Mendoza (Argentine).

Revisión de las Hydrocotyle (Apiales) argentinas. * Presentado a la Facultad de Ciencias Exactas, Físicas y Naturales de la Universidad de Buenos Aires, para optar al grado de Doctor en Ciencias Naturales.