MS1/MMD1 homologues in the moss Physcomitrium patens are required for male and female gametogenesis.

The Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. This pollen-based male gametogenesis is a derived trait of seed plants. Male gametogenesis in the common ancestors of land plants is instead likely to have been reminiscent of that in extant bryophytes where flagellated sperms are produced by an elaborate gametophyte generation. Still, also bryophytes possess MS1/MMD1-related PHD proteins. We addressed the function of two MS1/MMD1-homologues in the bryophyte model moss Physcomitrium patens by the generation and analysis of reporter and loss-of-function lines. The two genes are together essential for both male and female fertility by providing functions in the gamete-producing inner cells of antheridia and archegonia. They are furthermore expressed in the diploid sporophyte generation suggesting a function during sporogenesis, a process proposed related by descent to pollen formation in angiosperms. We propose that the moss MS1/MMD1-related regulatory network required for completion of male and female gametogenesis, and possibly for sporogenesis, represent a heritage from ancestral land plants.

Identification of a PCSK9-LDLR disruptor peptide with in vivo function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employ an affinity-based screen of 1013in vitro-translated macrocyclic peptides to identify high-affinity PCSK9 ligands that utilize a unique, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction. Structure-based design led to molecules with enhanced function and pharmacokinetic properties (e.g., 13PCSK9i). In mice, 13PCSK9i reduces plasma cholesterol levels and increases hepatic LDLR density in a dose-dependent manner. 13PCSK9i functions by a unique, allosteric mechanism and is the smallest molecule identified to date with in vivo PCSK9-LDLR disruptor function.

The Physcomitrium patens egg cell expresses several distinct epigenetic components and utilizes homologues of BONOBO genes for cell specification.

Although land plant germ cells have received much attention, knowledge about their specification is still limited. We thus identified transcripts enriched in egg cells of the bryophyte model species Physcomitrium patens, compared the results with angiosperm egg cells, and selected important candidate genes for functional analysis. We used laser-assisted microdissection to perform a cell-type-specific transcriptome analysis on egg cells for comparison with available expression profiles of vegetative tissues and male reproductive organs. We made reporter lines and knockout mutants of the two BONOBO (PbBNB) genes and studied their role in reproduction. We observed an overlap in gene activity between bryophyte and angiosperm egg cells, but also clear differences. Strikingly, several processes that are male-germline specific in Arabidopsis are active in the P. patens egg cell. Among those were the moss PbBNB genes, which control proliferation and identity of both female and male germlines. Pathways shared between male and female germlines were most likely present in the common ancestors of land plants, besides sex-specifying factors. A set of genes may also be involved in the switches between the diploid and haploid moss generations. Nonangiosperm gene networks also contribute to the specification of the P. patens egg cell.

Autophagy Is Required for Strawberry Fruit Ripening.

Autophagy is a catabolic and recycling pathway that maintains cellular homeostasis under normal growth and stress conditions. Two major types of autophagy, microautophagy and macroautophagy, have been described in plants. During macroautophagy, cellular content is engulfed by a double-membrane vesicle called autophagosome. This vesicle fuses its outer membrane with the tonoplast and releases the content into the vacuole for degradation. During certain developmental processes, autophagy is enhanced by induction of several autophagy-related genes (ATG genes). Autophagy in crop development has been studied in relation to leaf senescence, seed and reproductive development, and vascular formation. However, its role in fruit ripening has only been partially addressed. Strawberry is an important berry crop, representative of non-climacteric fruit. We have analyzed the occurrence of autophagy in developing and ripening fruits of the cultivated strawberry. Our data show that most ATG genes are conserved in the genome of the cultivated strawberry Fragaria x ananassa and they are differentially expressed along the ripening of the fruit receptacle. ATG8-lipidation analysis proves the presence of two autophagic waves during ripening. In addition, we have confirmed the presence of autophagy at the cellular level by the identification of autophagy-related structures at different stages of the strawberry ripening. Finally, we show that blocking autophagy either biochemically or genetically dramatically affects strawberry growth and ripening. Our data support that autophagy is an active and essential process with different implications during strawberry fruit ripening.

Influence of The Segregation Phenomenon on Structural Efficiency of Lightweight Aggregate Concretes.

Lightweight aggregate concretes (LWAC) are versatile and interesting materials for projects that require greater structural efficiency. Due to the difference that exists between the densities of the materials used in these types of concrete, during transport and mainly compaction, their aggregates tend to separate from the mortar matrix, floating towards the surface, a phenomenon called segregation. Segregation in LWAC can affect its durability properties, its density, and directly affect its structural efficiency. In this work, different concrete densities (1700 kg/m3 and 1900 kg/m3) manufactured with different dosages (two different lightweight aggregates) and compaction methods (one or two layers) were analyzed to verify the impact of segregation on its structural efficiency. For this purpose, the segregation index of the LWAC was obtained by means of the image analysis technique. In addition, to obtain their structural efficiency, the density and compressive strength were obtained at different heights of the tested specimens. The results show the vibration of the samples in two layers leads to a more efficient elimination of trapped air, a reduction in the risk of segregation, and better structural efficiency.

Habitat selection response of the freshwater shrimp Atyaephyra desmarestii experimentally exposed to heterogeneous copper contamination scenarios.

In contaminated aquatic ecosystems, it is expected that organisms suffer some effects caused by the contaminants. However, for mobile organisms inhabiting heterogeneously contaminated ecosystems, the continuous exposure to contaminants can be avoided by moving to less contaminated habitats. The present study evaluated the habitat selection of the freshwater shrimp Atyaephyra desmarestii experimentally exposed to different copper concentrations to verify whether the heterogeneous contamination distribution and the connectivity between habitats with different copper levels could generate a random population distribution similar to metapopulation. The experiments were performed in the HeMHAS (Heterogeneous Multi-Habitat Assay System), a non-forced multi-compartmented exposure system, in which it is possible to simulate the distribution of contaminants in a linear gradient or as patches of contamination. Copper was used to simulate a linear contamination gradient (26 to 105 µg/L Cu) and two patchy scenarios with three contamination levels [reference zone (R: 26 ±â€¯7 µg/L Cu), mixing zone (M: 61 ±â€¯2 µg/L Cu) and disturbed zone (D: 101 ±â€¯12 µg/L Cu)], with two mixing zones or one central mixing zone in a heterogeneous scenario. In the copper gradient scenario, a clear trend of shrimps (59.6 ±â€¯8.0% of the population) moving to the reference zones and an avoidance of 66.7 ±â€¯11.1% of the most contaminated zone were observed. For the patchy scenarios, a random distribution of organisms (34, 36 and 30% for R, M and D zones, respectively) was observed in the scenario with one mixing zone; on the other hand, a slight preference for the reference zones (44.9 ±â€¯4.8%) was evidenced in the scenario with two mixing zones. As shrimps are able to select less contaminated areas, it is highly important to preserve clean zones in heterogeneously contaminated environments, such as the arrangement in meta-ecosystems, as the less- or uncontaminated zones might represent less stressful areas to protect populations against continuous contamination exposure.

Influence of Crystalline Admixtures on the Short-Term Behaviour of Mortars Exposed to Sulphuric Acid.

Using durable materials is a sustainable solution for extending the lifetime of constructions. The use of crystalline admixtures makes cementitious materials more durable. They plug pores, capillary tracts and microcracks, blocking the entrance of water due to the formation of crystals that prevent the penetration of liquids. The literature has covered the performance of these admixtures on concrete, but studies on mortars are still scarce. The aim of this study is to investigate the effect of an aggressive environment (sulphuric acid solution-3 wt%) on mortars produced with different percentages of a crystalline admixture (1%, 1.5% and 2% by weight of cement content). Physical and mechanical properties were studied after immersing the mortars in a H2SO4 solution for 90 days. It was found that, after a 90-day sulphuric acid exposure, mortars with the crystalline admixture showed greater compressive strength than the control mortar, besides exhibiting lower mass loss. However, the crystalline admixture did not produce any significant effect on the capillary water absorption coefficient. In a nonaggressive environment, and in the short term, the crystalline admixture did not have a significant effect on the compressive strength, the capillary water absorption coefficient or the ultrasonic pulse velocity.

Diabetes mellitus tipo 2 en pacientes con diagnóstico de glaucoma primario de ángulo abierto durante el período 2010-2014 / Type 2 diabetes mellitus in patients diagnosed with primary open angle glaucoma during the period 2010-2014

RESUMEN Objetivo La asociación entre diabetes mellitus tipo 2 y glaucoma primario de ángulo abierto aún no está claramente establecida. Este estudio tiene como objetivo identificar la frecuencia entre ambas patologías en pacientes del Hospital Teodoro Maldonado Carbo durante el período 2010-2014 Métodos Se realizó un estudio retrospectivo, de corte transversal, de proporción, en una muestra de 283 pacientes mayores de 30 años cuyo diagnóstico fue de GPAA (Glaucoma primario de ángulo abierto) confirmado mediante estudio de tonometría, gonioscopia, campimetría y ausencia de causas secundarias de aumento de PIO. Se utilizaron criterios de la ADA/OMS para establecer el diagnóstico de DM2. Resultados La frecuencia de pacientes con diabetes en pacientes con diagnóstico de GPAA, que fueron atendidos en consultorios de Emergencia, Endocrinología y Oftalmología del Hospital Regional Teodoro Maldonado Carbo, durante el período 2010-2014, fue de 26,15% (74 de 283 pacientes), con importantes diferencias entre sexo y lugar de residencia. Conclusiones: Se demostró la existencia de una alta proporción entre DM2 y GPAA mediante razón de proporción.

ABSTRACT Objective The association between diabetes mellitus type 2 (DM2) and Primary Open- Angle Glaucoma (POAG) is not clearly established yet. This study has the objective to identify the frequency of both diseases in patients of Teodoro Maldonado Carbo Hospital during the period 2010-2014. Methods A retrospective, cross-sectional frequency was conducted on a sample of 283 patients over age 30 whose diagnosis was confirmed by examination of POAG tonometry, gonioscopy, perimetry and absence of secondary causes of increased intraocular pressure. ADA/OMS criteria were used to establish the diagnosis of DM2. Results The frequency of patients with diabetes diagnosed with POAG patients who were treated at the services of Emergency, Endocrinology and Ophthalmology of the Regional Hospital Teodoro Maldonado Carbo, during the period 2010-2014, was 26.15% (74 of 283 patients) with significant differences between sex and place of residence. Conclusions The existence of a high proportion between DM2 and POAG was demonstrated by prevalence ratio.

A novel approach to assessing environmental disturbance based on habitat selection by zebra fish as a model organism.

Aquatic ecotoxicity assays used to assess ecological risk assume that organisms living in a contaminated habitat are forcedly exposed to the contamination. This assumption neglects the ability of organisms to detect and avoid contamination by moving towards less disturbed habitats, as long as connectivity exists. In fluvial systems, many environmental parameters vary spatially and thus condition organisms' habitat selection. We assessed the preference of zebra fish (Danio rerio) when exposed to water samples from two western Ecuadorian rivers with apparently distinct disturbance levels: Pescadillo River (highly disturbed) and Oro River (moderately disturbed). Using a non-forced exposure system in which water samples from each river were arranged according to their spatial sequence in the field and connected to allow individuals to move freely among samples, we assayed habitat selection by D. rerio to assess environmental disturbance in the two rivers. Fish exposed to Pescadillo River samples preferred downstream samples near the confluence zone with the Oro River. Fish exposed to Oro River samples preferred upstream waters. When exposed to samples from both rivers simultaneously, fish exhibited the same pattern of habitat selection by preferring the Oro River samples. Given that the rivers are connected, preference for the Oro River enabled us to predict a depression in fish populations in the Pescadillo River. Although these findings indicate higher disturbance levels in the Pescadillo River, none of the physical-chemical variables measured was significantly correlated with the preference pattern towards the Oro River. Non-linear spatial patterns of habitat preference suggest that other environmental parameters like urban or agricultural contaminants play an important role in the model organism's habitat selection in these rivers. The non-forced exposure system represents a habitat selection-based approach that can serve as a valuable tool to unravel the factors that dictate organisms' spatial distribution in connected ecosystems.

Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness.

Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8-PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

Autophagy is required for gamete differentiation in the moss Physcomitrella patens.

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.

Attractiveness of food and avoidance from contamination as conflicting stimuli to habitat selection by fish.

Habitat selection by fish is the outcome of a choice between different stimuli. Typically, the presence of food tends to attract organisms, while contamination triggers an avoidance response to prevent toxic effects. Given that both food and contaminants are not homogeneously distributed in the environment and that food can be available in contaminated zones, a key question has been put forward in the present study: does a higher availability of food in contaminated areas interfere in the avoidance response to contaminants regardless of the contamination level? Tilapia fry (Oreochromis sp.; 2.5-3.0 cm and 0.5-0.8 g) were exposed to two different effluent samples, diluted along a free-choice, non-forced exposure system simulating a contamination gradient. Initially, avoidance to the effluents was checked during a one hour exposure. Afterwards, food was added to the system so that the availability of food increased with the increase in the level of contamination, and the avoidance response to contamination was checked during another hour. Results clearly showed a concentration-dependent avoidance response for both effluents during the first hour (i.e., with no food). However, in presence of the food, the avoidance pattern was altered: organisms were propelled to intermittently move towards contaminated areas where food availability was higher. The incursions were taken regardless of the potential risk linked to the toxic effects. In conclusion, even when the risk of toxicity was imminent, tilapia fry were more intensively stimulated by the attractiveness of the food than by repulsion to the contamination.

EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression.

The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.

Infrahyoid flap in oropharyngeal reconstruction following carcinoma resection: A study of 6 patients and literature review.

The present study aimed to describe the techniques used and the results obtained with the infrahyoid flap for the reconstruction of medium-sized oropharyngeal defects following resection for advanced squamous cell cancer. During a period of 1 year, six patients with oropharyngeal defects were reconstructed using the infrahyoid flap. The tumor characteristics, location and size of the defect, resective and reconstructive techniques employed and the complications and outcomes of the speech and swallowing functions, as identified in the follow-up visits every 3 months, were evaluated. All flaps were performed simultaneously in association with tumoral excision and ipsilateral supraomohyoid neck dissection. The mean size of the skin paddle was 7.0×3.5 cm. The donor site was primarily sutured. The postoperative course was uneventful and all flaps were viable. One case of marginal skin paddle loss occurred without affecting the survival of the flap. Five patients received postoperative radiotherapy and one patient received concurrent postoperative chemotherapy. During the follow-up period (mean, 63 months), all patients showed excellent oral swallowing. Speech was excellent in five patients and in one patient speech was classified as good. The aesthetic results of the cervical donor site were good. Based on the present case report and the literature review, the infrahyoid flap is a simple and safe procedure for the reconstruction of the oropharynx, with a high success rate, minimal donor site morbidity and good aesthetic and functional results. The infrahyoid flap is a valid surgical option that may be considered in selected oncological patients undergoing reconstruction of medium-size oropharyngeal defects.

Predicting the effects of copper on local population decline of 2 marine organisms, cobia fish and whiteleg shrimp, based on avoidance response.

The present study focuses on avoidance response to predict population decline of the marine fish Rachycentron canadum (cobia) and larvae of the estuarine shrimp Litopenaeus vannamei (whiteleg shrimp). Avoidance of approximately 60% was recorded for the cobia fry exposed to 1.0 mg Cu/L, 1.60 mg Cu/L, and 1.80 mg Cu/L. For the shrimp larvae, avoidance was approximately 80% for all Cu concentrations. The population decline of cobia fry was conditioned by avoidance in lower concentrations. However, in higher concentrations mortality begins to play an important role. The displacement toward uncontaminated habitats might determine shrimp population decline. A Cu-contaminated environment can determine the habitat selection of both species and, therefore, their local population decline.

Autophagy and metacaspase determine the mode of cell death in plants.

Although animals eliminate apoptotic cells using macrophages, plants use cell corpses throughout development and disassemble cells in a cell-autonomous manner by vacuolar cell death. During vacuolar cell death, lytic vacuoles gradually engulf and digest the cytoplasmic content. On the other hand, acute stress triggers an alternative cell death, necrosis, which is characterized by mitochondrial dysfunction, early rupture of the plasma membrane, and disordered cell disassembly. How both types of cell death are regulated remains obscure. In this paper, we show that vacuolar death in the embryo suspensor of Norway spruce requires autophagy. In turn, activation of autophagy lies downstream of metacaspase mcII-Pa, a key protease essential for suspensor cell death. Genetic suppression of the metacaspase­autophagy pathway induced a switch from vacuolar to necrotic death, resulting in failure of suspensor differentiation and embryonic arrest. Our results establish metacaspase-dependent autophagy as a bona fide mechanism that is responsible for cell disassembly during vacuolar cell death and for inhibition of necrosis.

Detection and measurement of necrosis in plants.

Necrosis plays a fundamental role in plant physiology and pathology. When plants or plant cell cultures are subjected to abiotic stress they initiate rapid cell death with necrotic morphology. Likewise, when plants are attacked by pathogens, they develop necrotic lesions, the reaction known as hypersensitive response. Great advances in the understanding of signaling pathways that lead to necrosis during plant-pathogen interaction have been made in the last two decades using Arabidopsis thaliana as a model plant. Further understanding of these signaling pathways, as well as those regulating the execution phase of necrotic cell death per se would require a robust set of readout assays to detect and measure necrosis in various plant model systems. Here we provide description of such assays, beginning from electron microscopy, as the "gold standard" to diagnose necrosis. This is followed by two groups of biochemical and cytochemical assays used by our group to detect and quantify mitochondrial dysfunction and the loss of protoplast integrity during necrosis in Arabidopsis plants and cell suspension cultures of both Arabidopsis and Norway spruce.

Autophagy mediates caloric restriction-induced lifespan extension in Arabidopsis.

Caloric restriction (CR) extends lifespan in various heterotrophic organisms ranging from yeasts to mammals, but whether a similar phenomenon occurs in plants remains unknown. Plants are autotrophs and use their photosynthetic machinery to convert light energy into the chemical energy of glucose and other organic compounds. As the rate of photosynthesis is proportional to the level of photosynthetically active radiation, the CR in plants can be modeled by lowering light intensity. Here, we report that low light intensity extends the lifespan in Arabidopsis through the mechanisms triggering autophagy, the major catabolic process that recycles damaged and potentially harmful cellular material. Knockout of autophagy-related genes results in the short lifespan and suppression of the lifespan-extending effect of the CR. Our data demonstrate that the autophagy-dependent mechanism of CR-induced lifespan extension is conserved between autotrophs and heterotrophs.