Hereditary Xerocytosis: Differential Behavior of PIEZO1 Mutations in the N-Terminal Extracellular Domain Between Red Blood Cells and HEK Cells

Hereditary Xerocytosis, a rare hemolytic anemia, is due to gain of function mutations in PIEZO1, a non-selective cation channel activated by mechanical stress. How these PIEZO1 mutations impair channel function and alter red blood cell (RBC) physiology, is not completely understood. Here, we report the characterization of mutations in the N-terminal part of the protein (V598M, F681S and the double mutation G782S/R808Q), a part of the channel that was subject of many investigations to decipher its role in channel gating. Our data show that the electrophysiological features of these PIEZO1 mutants expressed in HEK293T cells are different from previously characterized PIEZO1 mutations that are located in the pore or at the C-terminal extracellular domain of the protein. Although RBC with PIEZO1 mutations showed a dehydrated phenotype, the activity of V598M, F681S or R808Q in response to stretch was not significantly different from the WT channels. In contrast, the G782S mutant showed larger currents compared to the WT PIEZO1. Interestingly, basal activity of all the mutated channels was not significantly altered at the opposite of what was expected according to the decreased water and cation contents of resting RBC. In addition, the features of mutant PIEZO1 expressed in HEK293 cells do not always correlate with the observation in RBC where PIEZO1 mutations induced a cation leak associated with an increased conductance. Our work emphasizes the role of the membrane environment in PIEZO1 activity and the need to characterize RBC permeability to assess pathogenicity to PIEZO1 mutants associated with erythrocyte diseases.

Null Direct Object Clitics in Basque Spanish: A Study Across Four Generations

The null direct object clitic is described as one of the distinctive morphosyntactic features of Basque Spanish (e.g., compré el libro i , pero se me ha olvidado traer øi). However, no study to date has explored the variable usage of this form in cross-generational terms. The present inquiry aims to fill this void by analyzing and contrasting the results of two studies by four generations of Spanish-Basque bilinguals with the following age ranges: 85–96 (Generation 1), 55–75 (Generation 2), 35–45 (Generation 3), 18–25 (Generation 4). The education level of the participants was also taken into consideration. The first study consisted of an acceptability judgment task, in which the participants rated the acceptability of sentences with a null direct object using a 5-point Likert scale. The independent linguistic variables in this study were the semantic features [+/-definiteness] and [+/-specificity]. The acceptance rate of null direct object clitics was significantly higher among Generation 1 and 2 speakers than among those of Generation 3 and 4, with no effect of definiteness or specificity. In the second study, based on an elicitation task, older speakers (Generation 1 and 2) produced significantly more null direct object clitics than their younger counterparts (Generation 3 and 4). By contrasting the differential behavior of the participants across and within the same generation, it is shown that the generational effects observed are mainly due to the participants’ different levels of formal education.

Solitary fibrous tumor: molecular hallmarks and treatment for a rare sarcoma

Solitary fibrous tumor (SFT) is a rare soft tissue sarcoma subtype which mainly affects adults in the fifth and sixth decades of life. Originally part of a spectrum of tumors called hemangiopericytomas, classification has been refined such that SFTs now represent a distinct subtype. The identification of NAB2-STAT6 fusion in virtually all SFTs has further aided to define this rare subgroup. SFTs have a spectrum of behavior from benign to malignant, with evidence suggesting risk of metastases related to age at diagnosis, extent of necrosis, mitotic rate and tumor size. The standard treatment for localized disease is surgical excision with or without radiotherapy. Retrospective and prospective evidence suggests antiangiogenic treatment is effective for unresectable disease. Further translational work is required to understand the biology driving the differential behavior and identify more effective treatments for patients with metastatic disease.

Factor analysis and environmental stratification in the assessment of grain sorghum adaptability and stability

The environmental stratification studies are crucial when releasing hybrids for different growing regions. An outstanding performance of a genotype in one environment does not qualify it for indication to all environments, due the occurrence of GxE interaction. Environmental stratification aim the breeders to form groups of environments that minimize GxE interaction. The purpose of this work was to evaluate the use of factor analysis in preliminary environmental stratification assisting at the recommendation of grain sorghum cultivars. Twenty-five hybrids were evaluated, using a randomized block design, in 12 locations during the 2015/16 season. Initially, the individual analysis of the experiments was carried out and later the joint analysis, aiming to examine the existence of G×E interaction. The means of the hybrids in the individual analyses were used to obtain the correlation matrix between pairs of environments. The factorization of this matrix was also carried out via factor analysis in order to group together the environments that most correlated with respect to the hybrids performance. Thus, differential performance between hybrids was observed through individual analyses for all the environments, with the exception of Sete Lagoas and Teresina. The joint analysis revealed the existence of a significant G×E interaction, that is, a differential behavior of the hybrids in relation to the evaluated environments. Based on the criterion of the analysis of the proportion of explained variance, it was found that six factors captured an accumulated variation of 86.29%, and the average communality observed was of 0.86. Considering the geographic and edaphoclimatic variables in the cultivation period, a pattern was not observed among the grouped places, but it is noteworthy that the grouping of places is a function of the performance of the evaluated genotypes, which can be similar even under different conditions. Given the results presented, factor analysis proved to be a tool with potential to perform environmental stratification and assist in the recommendation of grain sorghum cultivars for different regions.

Cellular Mechanisms Underlying the Low Cardiotoxicity of Istaroxime

Background Istaroxime is an inhibitor of Na + /K + ATPase with proven efficacy to increase cardiac contractility and to accelerate relaxation attributable to a relief in phospholamban‐dependent inhibition of the sarcoplasmic reticulum Ca 2+ ATPase. We have previously shown that pharmacologic Na + /K + ATPase inhibition promotes calcium/calmodulin‐dependent kinase II activation, which mediates both cardiomyocyte death and arrhythmias. Here, we aim to compare the cardiotoxic effects promoted by classic pharmacologic Na + /K + ATPase inhibition versus istaroxime. Methods and Results Ventricular cardiomyocytes were treated with ouabain or istaroxime at previously tested equi‐inotropic concentrations to compare their impact on cell viability, apoptosis, and calcium/calmodulin‐dependent kinase II activation. In contrast to ouabain, istaroxime neither promoted calcium/calmodulin‐dependent kinase II activation nor cardiomyocyte death. In addition, we explored the differential behavior promoted by ouabain and istaroxime on spontaneous diastolic Ca 2+ release. In rat cardiomyocytes, istaroxime did not significantly increase Ca 2+ spark and wave frequency but increased the proportion of aborted Ca 2+ waves. Further insight was provided by studying cardiomyocytes from mice that do not express phospholamban. In this model, the lower Ca 2+ wave incidence observed with istaroxime remains present, suggesting that istaroxime‐dependent relief on phospholamban‐dependent sarcoplasmic reticulum Ca 2+ ATPase 2A inhibition is not the unique mechanism underlying the low arrhythmogenic profile of this drug. Conclusions Our results indicate that, different from ouabain, istaroxime can reach a significant inotropic effect without leading to calcium/calmodulin‐dependent kinase II–dependent cardiomyocyte death. Additionally, we provide novel insights regarding the low arrhythmogenic impact of istaroxime on cardiac Ca 2+ handling.

Desmosterol Incorporation Into Ram Sperm Membrane Before Cryopreservation Improves in vitro and in vivo Fertility

Pregnancy rates in ewes are markedly low after cervical insemination with frozen-thawed sperm. Sensitivity of ram sperm to freeze-thawing is related to the lipid composition of the membrane, particularly to its low sterol content. Recently, we proved that sterol content of ram sperm can be increased by treatment with methyl-β-cyclodextrin-sterol complexes and we provided mechanistic based evidence on the differential behavior of cholesterol and desmosterol in the ram sperm membrane. In the present study, we evaluated the role of increasing cholesterol and desmosterol content of ram sperm before cryopreservation, on the extent and distribution of sterols, cryocapacitation status, acrosome integrity, DNA damage associated with apoptosis and fertility competence in vitro and in vivo of post-thawed sperm. After freeze-thawing, similar levels of sterol content were evidenced in control sperm cells and in those pre-incubated with either cholesterol or desmosterol. Still, moderately higher levels of sterols were registered in treated sperm compared to the control, indicating no physiological excess of sterols after thawing or sterol losses that exceed the control. Live cell imaging of fluorescent cholesterol evidenced the presence of sperm sub-populations differentially affected by freeze-thawing. Similar unimodal frequency profiles were observed between sterol-enriched groups, while the control exhibited a sub-population of sperm compatible with low sterol content. Tyrosine phosphorylation was significantly lower when ram sperm incorporated cholesterol compared to the control. No difference in this capacitation parameter was found between the latter and desmosterol-enriched sperm. The percentage of sperm with damaged acrosomes post-thawing, assessed by a fluorescent lectin, was reduced in sperm that incorporated sterols before freezing, irrespective of the sterol class. These results suggest that sterols exert a stabilizing effect on the acrosome. No differences were found in levels of apoptotic DNA fragmentation among experimental groups. As to fertility trials, desmosterol-enriched sperm gave rise to higher rates of in vitro activated oocytes by heterologous fertilization and to significantly lower pregnancy loss in vivo. Our research provides new insights on sterol incorporation into ram sperm prior to cryopreservation, in particular on the additional benefit of incorporating desmosterol as a strategy to improve fertility outcome.

Human cultured IMR-32 neuronal-like and U87 glial-like cells have different patterns of toxicity under fluoride exposure

Background Fluoride (F) is a naturally exists in nature but several studies have indicated it as an environmental toxicant to all leaving beings. Human F exposure has increased over the years since this ion has been used by industry on foods, beverages, toothpastes and on water supply. Although F is safe at optimal concentrations in water supply, human exposure to high levels could trigger neurofunctional deficits. Materials and methods In this study, human glial-like (U87) and neuronal-like (IMR-32) cells lineages were used to access F toxicity and CNS cell sensibility on both cell facing the same protocol. Cells were exposed to F over 3, 5 and 10 days on two different F concentrations. Fluoride exposed cells were evaluated by standard toxicity assays to cell viability, apoptosis, necrosis and general cell metabolism. Oxidative stress parameters were evaluated by ATP and ROS levels, lipid peroxidation, GSH/GSSG ratio and comet assay. Results No changes were observed in IMR-32 at any given time while after 10 days of exposure to 0.22μg/mL, U87 glial-like cells showed signs of toxicity such as decreased cell viability by necrosis while general cell metabolism was increased. Oxidative stress parameters were next evaluated only on U87 glial-like cells after 10 days of exposure. F induced a decrease on ATP levels while no changes were observed on reactive oxygen species and lipid peroxidation. GSH/GSSG ratio was decreased followed by DNA damage both on 0.22μg/mL F. Conclusions Our results suggest an important differential behavior of the distinct types of cells exposed to the different fluoride concentrations, pointing that the U87 glial-like cells as more susceptible to damage triggered by this ion.

Energy Cane x Sugarcane Microregion Interaction in the State of Pernambuco: Sugarcane for Production of Bioenergy and Renewable Fuels

Assessing the differential behavior of a group of genotypes in various environments is fundamentally important in any breeding program. As sugarcane is the most important crop in the state of Pernambuco, it is of great relevance to study its performance in different cultivation sites to assist in the recommendation of new cultivars that increase the productivity of the cane fields. In view of the new demand from the sugar-energy sector for cultivars with high energy potential, this work aimed to select and recommend new genotypes with high fiber and sucrose percentage in the sugarcane microregions of the state of Pernambuco. The methodologies used to classify genotypes for adaptability and stability were as follows: simple linear regression, the modified centroid method, additive main effects, multiplicative interaction analysis, and linear mixed models. Genotypes with higher productivity and specific adaptability to the tested microregions were identified. The methodologies applied were efficient and complementary in recommending genotypes with favorable prospects for increasing sugar productivity, cogeneration of electric energy and the production of renewable fuels. Genotypes 6, 7, 9, 14, 16, and 18 stand out in terms of the productivity of sugar and fiber, with high potential to be released as commercial cultivars.