Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

Subtle Introgression Footprints at the End of the Speciation Continuum in a Clade of Heliconius Butterflies.

Quantifying gene flow between lineages at different stages of the speciation continuum is central to understanding speciation. Heliconius butterflies have undergone an adaptive radiation in wing color patterns driven partly by natural selection for local mimicry. Color patterns are also known to be used as assortative mating cues. Therefore, wing pattern divergence is considered to play a role in speciation. A corollary is that mimicry between closely related species may be associated with hybridization and interfere with reproductive isolation. Here, we take a multifaceted approach to explore speciation history, species boundaries, and traits involved in species differentiation between the two closely related species, Heliconius hecale and Heliconius ismenius. We focus on geographic regions where the two species mimic each other and contrast this with geographic regions where they do not mimic each other. To examine population history and patterns of gene flow, we tested and compared a four-population model accounting for linked selection. This model suggests that the two species have remained isolated for a large part of their history, yet with a small amount of gene exchange. Accordingly, signatures of genomic introgression were small except at a major wing pattern allele and chemosensing genes and stronger in the mimetic populations compared with nonmimetic populations. Behavioral assays confirm that visual confusion exists but that short-range cues determine strong sexual isolation. Tests for chemical differentiation between species identified major differences in putative pheromones which likely mediate mate choice and the maintenance of species differences.

Individual variation in reaction norms but no directional selection in reproductive plasticity of a wild passerine population.

In the plant-insect-insectivorous bird food chain, directional changes in climate can result in mismatched phenology, potentially affecting selection pressures. Phenotypic plasticity in the timing of breeding, characterized by reaction norm slopes, can help maximize fitness when faced with earlier prey emergence. In temperate passerines, the timing of tree budburst influences food availability for chicks through caterpillar phenology and the resulting food abundance patterns. Thus, the timing of tree budburst might serve as a more direct proxy for the cue to time egg-laying. The evolutionary potential of breeding plasticity relies on heritable variation, which is based upon individual variation, yet studies on individual variation in plasticity are few. Here, we tested for the laying date-budburst date and the clutch size-laying date reaction norms, and examined 1) the among-individual variance in reaction norm intercepts and slopes; and 2) the selection differentials and gradients on these intercepts and slopes. Using long-term data of oak (genus Quercus) budburst and blue tit (Cyanistes caeruleus) reproduction, we applied within-subject centering to detect reaction norms, followed by bivariate random regression to quantify among-individual variance in reaction norm properties and their covariance with fitness. Individuals significantly differed in intercepts and slopes of both laying date-budburst date and clutch size-laying date reaction norms, and directional selection was present for an earlier laying date and a larger clutch size (intercepts), but not on plasticity (slopes). We found that individuals have their own regimes for adjusting egg-laying and clutch size. This study provides further support of individual variation of phenotypic plasticity in birds.

Engineered mesenchymal stromal cell therapy during human lung ex vivo lung perfusion is compromised by acidic lung microenvironment.

Ex vivo lung perfusion (EVLP) is an excellent platform to apply novel therapeutics, such as gene and cell therapies, before lung transplantation. We investigated the concept of human donor lung engineering during EVLP by combining gene and cell therapies. Premodified cryopreserved mesenchymal stromal cells with augmented anti-inflammatory interleukin-10 production (MSCIL-10) were administered during EVLP to human lungs that had various degrees of underlying lung injury. Cryopreserved MSCIL-10 had excellent viability, and they immediately and efficiently elevated perfusate and lung tissue IL-10 levels during EVLP. However, MSCIL-10 function was compromised by the poor metabolic conditions present in the most damaged lungs. Similarly, exposing cultured MSCIL-10 to poor metabolic, and especially acidic, conditions decreased their IL-10 production. In conclusion, we found that "off-the-shelf" MSCIL-10 therapy of human lungs during EVLP is safe and feasible, and results in rapid IL-10 elevation, and that the acidic target-tissue microenvironment may compromise the efficacy of cell-based therapies.

Human Umbilical Cord Perivascular Cells and Human Bone Marrow Mesenchymal Stromal Cells Transplanted Intramuscularly Respond to a Distant Source of Inflammation.

Intravenously administered mesenchymal stromal cells (MSCs) are rapidly entrapped in the lungs, where they display an anti-inflammatory phenotype. Intramuscular (IM) delivery provides an increased MSC dwell-time, which could result in a sustained modulation of an inflammatory milieu. We studied the therapeutic effects of IM delivered MSCs to treat a distant (contralateral) inflammation, and compared the efficacy of neonatal (umbilical cord) and adult bone marrow MSCs (BMMSCs). Inflammation decreased over 48 h, but neonatal cells showed an earlier response than BMMSCs. Tumor necrosis factor-induced gene-6 (TSG-6) was released at the site of MSC delivery, while neutrophil infiltration was abrogated and inflammation reduced at the contralateral site. MSCs did not distribute to the organs or to the site of inflammation. Thus, IM delivery presents a promising alternative for the treatment of inflammation, and neonatal MSCs may represent a stronger candidate than those derived from adult BM to treat inflammatory diseases.

Evaluation of biological production of lactic acid in a synthetic medium and in Aloe vera (L.) Burm. f. processing by-products / Evaluation of biological production of lactic acid in a synthetic medium and in Aloe vera (L.) Burm. f. processing by-products / Avaliação da produção biológica de ácido lático em um meio sintético e no processamento de subprodutos de Aloe vera (L.) Burm. f.

This study evaluated lactic acid production through batch fermentation in a bioreactor with Thermoanaerobacter sp. strain USBA-018 and a chemically defined culture medium and with hydrolyzed pressed extract of Aloe vera peel (AHE). The strain USBA-018 fermented various sugars, but its primary end-product was L-lactic acid. Factors which influenced L- lactic acid production were pH, addition of yeast extract (YE) and manganese chloride. Under the most favorable growing conditions for the production of lactic acid, yield (Yp/s) increased from 0.66 to 0.96 g/g with a productivity (Qp) of 0.62 g.l-1.h and a maximum lactic acid concentration of 178 mM at 26 hours of fermentation. When AHE was used, 93.3 mM, or 0.175 g.l-1.h, was obtained. These results show the potential for transformation of sugars that strain USBA-018 offers, but additional studies are needed to find out if different strategies using AHE as carbon source can produce large enough quantities of lactic acid to allow AHE to become a low-cost alternative substrate.

Este estudio evaluó la producción de ácido láctico de la cepa de Thermoanaerobacter sp. USBA-018 en un biorreactor de fermentación por lotes, utilizando como medios de cultivo una formulación químicamente definida y un extracto prensado e hidrolizado de cáscara de Aloe vera (AHE). La cepa USBA-018 fermentó varios azúcares, pero su principal producto final fue L-ácido láctico. Los factores que influyeron en la producción de L-ácido láctico fueron pH, adición de extracto de levadura (YE) y de cloruro de manganeso. Bajo las condiciones más favorables de crecimiento para la producción de ácido láctico el rendimiento (Yp/s) aumentó de 0.66 a 0.96 g/g, con una productividad (Qp) de 0.62 g.l-1.h y una máxima concentración de ácido láctico de 178 mM a las 26 horas de fermentación. Cuando se usó AHE, se obtuvieron 93.3 mM, o 0.175 g.l-1.h. Estos resultados muestran el potencial de transformación de azúcares que ofrece la cepa USBA-018, pero se requieren estudios adicionales para determinar si diferentes estrategias de uso de AHE como fuente de carbono producen cantidades suficientemente grandes de ácido láctico como para permitir que el AHE se convierta en un sustrato alternativo de bajo costo.

Este estudo avaliou a produção de ácido lático por meio de fermentação descontinua em um biorreator com Thermoanaerobacter sp. cepa USBA-018 e um meio de cultura químicamente definido e com extrato hidrolisado de casca de Aloe vera (AHE). A cepa USBA-018 fermentou vários acucares, mas seu produto final primàrio foi L-ácido lático. Os fatores que influenciaram a produção de L-ácido lático foram o pH, adição de extrato de levedura (YE) e cloreto de manganès. Sob as condicoes de crescimento mais favoráveis para a produção de ácido lático, o rendimento (Yp/S) aumentou de 0,66 a 0,96 g/g com uma produtividade (Q) de 0,62 g.L4.h e um máximo de concentração de ácido lático de 178 mM em 26 horas de fermentação. Quando o AHE foi utilizado, se obteve 93,3 mM ou 0,175 g.L-1.h. Estes resultados mostram o potencial para transformação de acucares que a cepa USBA-018 oferece, entretanto estudos adicionais sáo necessários para descobrir se diferentes estrategias utilizando o AHE como fonte de carbonos podem produzir quantidades de ácido lático grandes o suficiente para permitir que AHE se converta em um substrato alternativo de baixo custo.

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants.

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.

Symbiotic fungi alter plant chemistry that discourages leaf-cutting ants.

Fungal symbionts that live asymptomatically inside plant tissues (endophytes) can influence plant-insect interactions. Recent work has shown that damage by leaf-cutting ants, a major Neotropical defoliator, is reduced to almost half in plants with high densities of endophytes. We investigated changes in the phenotype of leaves that could influence ants' behavior to result in the reduction of foliar damage. We produced cucumber seedlings with high and low densities of one common endophyte species, Colletotrichum tropicale. We used the leaves in bioassays and to compare chemical and physical leaf characteristics important for ants' food selection. Ants cut about one-third more area of cucumber leaves with lower densities of endophytes and removed c. 20% more paper disks impregnated with the extracts of those leaves compared with leaves and disks from plants hosting the fungus. Colletotrichum tropicale colonization did not cause detectable changes in the composition of volatile compounds, cuticular waxes, nutrients or leaf toughness. Our study shows that endophytes changed leaf chemistry and suggests that compounds with relative low volatility released after leaf wounding are a major factor influencing foraging decisions by ants when choosing between plants with low or high endophyte loads.

Leaf endophyte load influences fungal garden development in leaf-cutting ants.

BACKGROUND: Previous work has shown that leaf-cutting ants prefer to cut leaf material with relatively low fungal endophyte content. This preference suggests that fungal endophytes exact a cost on the ants or on the development of their colonies. We hypothesized that endophytes may play a role in their host plants' defense against leaf-cutting ants. To measure the long-term cost to the ant colony of fungal endophytes in their forage material, we conducted a 20-week laboratory experiment to measure fungal garden development for colonies that foraged on leaves with low or high endophyte content. RESULTS: Colony mass and the fungal garden dry mass did not differ significantly between the low and high endophyte feeding treatments. There was, however, a marginally significant trend toward greater mass of fungal garden per ant worker in the low relative to the high endophyte treatment. This trend was driven by differences in the fungal garden mass per worker from the earliest samples, when leaf-cutting ants had been foraging on low or high endophyte leaf material for only 2 weeks. At two weeks of foraging, the mean fungal garden mass per worker was 77% greater for colonies foraging on leaves with low relative to high endophyte loads. CONCLUSIONS: Our data suggest that the cost of endophyte presence in ant forage material may be greatest to fungal colony development in its earliest stages, when there are few workers available to forage and to clean leaf material. This coincides with a period of high mortality for incipient colonies in the field. We discuss how the endophyte-leaf-cutter ant interaction may parallel constitutive defenses in plants, whereby endophytes reduce the rate of colony development when its risk of mortality is greatest.

[Urinary levels of angiotensin-(1-7) and angiotensin II in patients with severe aortic stenosis]. / Niveles urinarios de angiotensina-(1-7) y angiotensina II en pacientes con estenosis aórtica de importante repercusión hemodinámica.

OBJECTIVE: Strengthen knowledge about the pathophysiology of aortic stenosis. METHODS: Urinary levels of angiotensin-(1-7) and angiotensin II were compared between two samples: A) forty five patients with severe aortic stenosis, without systemic arterial hypertension and with normal kidney and normal left ventricular systolic function; B) control group: twenty one persons without cardiovascular disease. NULL HYPOTHESIS: there would be no difference between urinary levels. RESULTS: The average of angiotensin-(1-7) urinary concentration in severe aortic stenosis patients was 2.102 pmol/mL and 5.591 pmol/mL for the control group. The average of Ang II was 0.704 pmol/mL and 0.185 pmol/mL respectively. Using t-Student test, we determine that the difference in urinary concentration of angiotensin-(1-7) [p=0.633] and the difference of angiotensin II (p=0.631), were statistically significant. CONCLUSION: documented a statistically significant difference in urinary levels angiotensin II and angiotensin-(1-7) within the group of patients with severe aortic stenosis.

Niveles urinarios de angiotensina-(1-7) y angiotensina II en pacientes con estenosis aórtica de importante repercusión hemodinámica / Urinary levels of angiotensin-(1-7) and angiotensin II in patients with severe aortic stenosis

Objetivo:Reforzar el conocimiento sobre la fisiopatología de la estenosis aórtica. Métodos: Se compararon los niveles urinarios de angiotensina II y angiotensina-(1-7) entre dos muestras: a) 45 pacientes con estenosis aórtica de importante repercusión hemodinámica, sin hipertensión arterial sistémica y con funciones renal y sistólica ventricular izquierda normales; b) grupo de control con 21 voluntarios sin patología cardiovascular. Hipótesis nula: no habría diferencia entre los niveles urinarios. Resultados:El promedio de la concentración urinaria de angiotensina-(1-7) en pacientes con estenosis aórtica fue 2.102 pmoles/mL y de 5.591 pmoles/mL para el grupo control. La media obtenida en concentración urinaria de angiotensina II fue de 0.704 pmoles/mL en los pacientes con estenosis aórtica y 0.185 pmoles/mL en el grupo control. Utilizando la prueba t de Student determinamos que la diferencia en la concentración urinaria de angiotensina-(1-7) (p = 0.633) y la diferencia en la concentración urinaria de angiotensina II (p = 0.631), fueron estadísticamente significativas. Conclusión:Se documentó una diferencia estadísticamente significativa en los niveles urinarios de angiotensina II y angiotensina-(1-7) dentro del grupo de pacientes con estenosis aórtica de importante repercusión hemodinámica.

Objective:Strengthen knowledge about the pathophysiology of aortic stenosis. Methods: Urinary levels of angiotensin-(1-7) and angiotensin II were compared between two samples: A) forty five patients with severe aortic stenosis, without systemic arterial hypertension and with normal kidney and normal left ventricular systolic function; B) control group: twenty one persons without cardiovascular disease. Null hypothesis: there would be no difference between urinary levels. Results: The average of angiotensin-(1-7) urinary concentration in severe aortic stenosis patients was 2.102 pmol/mL and 5.591 pmol/mL for the control group. The average of Ang II was 0.704 pmol/mL and 0.185 pmol/mL respectively. Using t-Student test, we determine that the difference in urinary concentration of angiotensin-(1-7) [p = 0.633] and the difference of angiotensin II (p = 0.631), were statistically significant. Conclusion:documented a statistically significant difference in urinary levels angiotensin II and angiotensin-(1-7) within the group of patients with severe aortic stenosis.

Sexual selection drives the evolution of antiaphrodisiac pheromones in butterflies.

Competition for mates has resulted in sophisticated mechanisms of male control over female reproduction. Antiaphrodisiacs are pheromones transferred from males to females during mating that reduce attractiveness of females to subsequent courting males. Antiaphrodisiacs generally help unreceptive females reduce male harassment. However, lack of control over pheromone release by females and male control over the amount transferred provides males an opportunity to use antiaphrodisiacs to delay remating by females that have returned to a receptive state. We propose a model for the evolution of antiaphrodisiacs under the influence of intrasexual selection, and determine whether changes in this signal in 11 species of Heliconius butterflies are consistent with two predictions of the model. First, we find that as predicted, male-contributed chemical mixtures are complex and highly variable across species, with limited phylogenetic signal. Second, differences in rates of evolution in pheromone composition between two major clades of Heliconius are as expected: the clade with a greater potential for male-male competition (polyandrous) shows a faster rate of divergence than the one with typically monoandrous mating system. Taken together, our results provide evidence that for females, antiaphrodisiacs can be both honest signals of receptivity (helping reduce harassment) and chastity belts (a male-imposed reduction in remating).

Sex-specific chemical cues from immatures facilitate the evolution of mate guarding in Heliconius butterflies.

Competition for mates has substantial effects on sensory systems and often leads to the evolution of extraordinary mating behaviours in nature. The ability of males to find sexually immature females and associate with them until mating is a remarkable example. Although several aspects of such pre-copulatory mate guarding have been investigated, little is known about the mechanisms used by males to locate immature females and assess their maturity. These are not only key components of the origin and maintenance of this mating strategy, but are also necessary for inferring the level to which females cooperate and thus the incidence of sexual conflict. We investigated the cues involved in recognition of immature females in Heliconius charithonia, a butterfly that exhibits mate guarding by perching on pupae. We found that males recognized female pupae using sex-specific volatile monoterpenes produced by them towards the end of pupal development. Considering the presumed biosynthetic pathways of such compounds and the reproductive biology of Heliconius, we propose that these monoterpenes are coevolved signals and not just sex-specific cues exploited by males. Their maintenance, despite lack of female mate choice, may be explained by variation in cost that females pay with this male behaviour under heterogeneous ecological conditions.

[Penetrating atherosclerotic ulcer of the thoracic aorta: endovascular treatment, hybrid or surgery. A review]. / Aneurisma de aorta torácica por úlcera aterosclerótica penetrante: tratamiento endovascular, hibrido o cirugía. Revisión.

The aortic aneurysm is part of the acute aortic syndromes (AAS). Aortic aneurysms have a weakened tunica media. Acute aneurysm expansion may herald rupture with high morbility and mortality. Five percent of AAS are diagnosed as pentetrating atherosclerotic ulcer which is an ulceration of an atherosclerotic lesion of the aorta that penetrates the internal elastic lamina and allows hematoma formation within the tunica media of the aortic wall. Endovascular treatment is an alternative to surgery and has provided an adequate rate of successful repair. There is another type of treatment which combines surgery and endovascular repair (the hybrid open-endovascular repair) which provides adequate results. The afford mention case is about a patient with a penetrating atherosclerotic ulcer. The hematoma that results from this ulcer extends and self-contains the aneurysm, with a high risk of rupture. We will also describe the aneurysm treatment options.

Aneurisma de aorta torácica por úlcera aterosclerótica penetrante: tratamiento endovascular, hibrido o cirugía: revisión / Penetrating atherosclerotic ulcer of the thoracic aorta: endovascular treatment, hybrid or surgery: a review

The aortic aneurysm is part of the acute aortic syndromes (AAS). Aortic aneurysms have a weakened tunica media. Acute aneurysm expansion may herald rupture with high morbility and mortality. Five percent of AAS are diagnosed as pentetrating atherosclerotic ulcer which is an ulceration of an atherosclerotic lesion of the aorta that penetrates the internal elastic lamina and allows hematoma formation within the tunica media of the aortic wall. Endovascular treatment is an alternative to surgery and has provided an adequate rate of successful repair. There is another type of treatment which combines surgery and endovascular repair (the hybrid open-endovascular repair) which provides adequate results. The afford mention case is about a patient with a penetrating atherosclerotic ulcer. The hematoma that results from this ulcer extends and self-contains the aneurysm, with a high risk of rupture. We will also describe the aneurysm treatment options.

[Prevention and treatment of hypertension and coronary heart disease]. / Prevención y tratamiento de la hipertensión arterial sistémica en el paciente con enfermedad arterial coronaria.

The association between arterial systemic hypertension arterial coronary disease has been demonstrated by cumulated evidence of several epidemiological studies. Hypertension is an important independent risk factor for the development of coronary artery disease, vascular cerebral disease and nephropathy. Important advances exist in the knowledge of neurohumoral and hemodynamic factors that come together in the pathophysiology of the hypertension and in the development of coronary disease that allow to establish better strategies not only of treatment, but also of prevention, with the purpose of diminishing the cardiovascular mortality. The spectrum of the coronary artery disease secondary to atherosclerosis is wide and the strategies of treatment of hypertension must be adapted to each particular case. The treatment of both conditions needs of specific limits of agreement to the conditions of the patient and the form of presentation of each one of these disease.

[Extreme elevation of the blood pressure (hypertensive crisis): recommendations for its clinical-therapeutic approach]. / Elevación extrema de la presión arterial (crisis hipertensiva): recomendaciones para su abordaje clínico-terapéutico.

From beginnings of last century the hypertensive emergency was defined as the association of acutely elevation from the arterial pressure and the appearance of damage to end organ. At present is recognized the effects of the hypertensive emergency, the aspects of its patophysiology in which are included phenomenon of vasomotricity and the participation of different substances with vasoactives properties. The clinical presentation includes not only the manifestations of the increase of the arterial pressure, the end organ damage too; for this reason the hypertensive emergency needs the immediate reduction of the arterial tension to prevent the damage to specific organs. The treatment in every case will have to be individualized, with a wide knowledge of the characteristics of every medicament to obtain the best results. The diagnosis and treatment of the hypertensive emergencies needs often of the attention of its complications if they have appeared and later, of a treatment of support for the arterial hypertension.

[Left posterolateral extensive myocardial infarct. An electroanatomical comparison]. / Infarto posterolateral izquierdo extenso. Cotejo electro-anatómico.

A complete ECG thoracic circle allows exploring some heart structures not explored by the conventional electrocardiogram. It provides a direct indication on the location of the damaged myocardium. In fact, posterolateral infarctions can be limited to the inferior third of the left ventricle or can cover the entire free left ventricular wall from the base up to the heart apex and can be univentricular or biventricular. On the other side, the unipolar thoracic leads and the high abdominal leads MD, ME, MI show the evolution of the signs of injury, characteristic of the acute stage of infarction, toward necrosis. We present the example of a 61-year-old man, whose ECG shows signs of subepicardial or transmural injury and of necrosis in the low precordial leads V5 and V6, as well as in the high left posterior leads V8 and V9. This fact suggests the presence of an acute extensive myocardial infarction extending from the base to the heart apex. Moreover, the moderate elevation of the RS-T segment from to V9R to V7R indicates the presence of subepicardial injury in the high posterior regions of the right ventricular wall. These electrocardiographic data were confirmed by the radioactive isotope study and, definitively, by the anatomical findings.