Identification of Volatile Molecules and Bioactivity of Gruyt Craft Beer Enriched with Citrus aurantium var. dulcis L. Essential Oil.

In this work, for the first time, a gruyt beer and the same one after the addition of Citrus aurantium essential oil (AEO), were investigated to determine the composition of the volatile fraction. The applied analytical techniques, such as Head Space/Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry (HS/SPME-GC-MS) and Proton Transfer Reaction-Time of Flight-Mass Spectrometer (PTR-ToF-MS), allowed us to identify the content of volatile organic compounds (VOCs). From the comparison between the two beer samples, it showed that the one after the addition of AEO was particularly richened in limonene and a series of minor terpene compounds. AEO was also characterized by GC/MS analysis and the results showed that limonene reached 95%. Confocal microscopy was used to look at riboflavin autofluorescence in yeast cells. It was found that beer with AEO had twice as much fluorescence intensity as the control. A spectrophotometric analysis of total polyphenols, tannins, and flavonoids, and a bioactivity screening, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-Azinobis-(3-Ethylbenzthiazolin-6-Sulfonic Acid) (ABTS) radical scavenger, chelating, reducing, antiglycative ones, were also carried out. Moreover, the tolerability of the tested samples in human H69 cholangiocytes and the cytoprotection towards the tert-butyl hydroperoxide (tBOOH)-induced oxidative damage were evaluated. Under our experimental conditions, the beers were found to be able to scavenge DPPH and ABTS radicals and chelate iron ions, despite weak antiglycative and reducing properties. The tested samples did not affect the viability of H69 cholangiocytes up to the highest concentrations; moreover, no signs of cytoprotection against the damage induced by tBOOH were highlighted. Adding AEO to beer resulted in a moderate enhancement of its DPPH scavenging and chelating abilities, without improvements in the other assays. Conversely, AEO and its major compound limonene were ineffective when assessed at the concentrations added to beer. This evidence suggests that the addition of AEO may enhance the organoleptic features of the beer and slightly potentiate some of its bioactivities.

The Microalga Nannochloropsis during Transition from Quiescence to Autotrophy in Response to Nitrogen Availability.

The marine microalgae Nannochloropsis oceanica (CCMP1779) is a prolific producer of oil and is considered a viable and sustainable resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells, but the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. N deprivation-induced quiescence was accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis, leading to accumulation of triacylglycerol. Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of triacylglycerol stored in lipid droplets (LDs). Besides observing LD translocation into vacuoles, we also provide evidence for direct interaction between the LD surface protein (NoLDSP) and AUTOPHAGY-RELATED8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. This knowledge is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity.

Transcriptional analysis distinguishes breast implant-associated anaplastic large cell lymphoma from other peripheral T-cell lymphomas.

Breast implant-associated anaplastic large cell lymphoma is a new provisional entity in the revised World Health Organization classification of lymphoid malignancies, the pathogenesis and cell of origin of which are still unknown. We performed gene expression profiling of microdissected breast implant-associated anaplastic large cell lymphoma samples and compared their transcriptional profiles with those previously obtained from normal T-cells and other peripheral T-cell lymphomas and validated expression of selected markers by immunohistochemistry. Our results indicate that most breast implant-associated anaplastic large cell lymphomas exhibit an activated CD4+ memory T-cell phenotype, which is associated with CD25 and FoxP3 expression. Gene ontology analyses revealed upregulation of genes involved in cell motility programs (e.g., CCR6, MET, HGF, CXCL14) in breast implant-associated anaplastic large cell lymphomas compared to normal CD4+ T-cells and upregulation of genes involved in myeloid cell differentiation (e.g., PPARg, JAK2, SPI-1, GAB2) and viral gene transcription (e.g., RPS10, RPL17, RPS29, RPL18A) compared to other types of peripheral T-cell lymphomas. Gene set enrichment analyses also revealed shared features between the molecular profiles of breast implant-associated anaplastic large cell lymphomas and other types of anaplastic large cell lymphomas, including downregulation of T-cell receptor signaling and STAT3 activation. Our findings provide novel insights into the biology of this rare disease and further evidence that breast implant-associated anaplastic large cell lymphoma represents a distinct peripheral T-cell lymphoma entity.

Old subjects with sepsis in the emergency department: trend analysis of case fatality rate.

BACKGROUND: The burden of sepsis represents a global health care problem. We aimed to assess the case fatality rate (CFR) and its predictors in subjects with sepsis admitted to a general Italian hospital from 2009 to 2016, stratified by risk score. METHODS: We performed a retrospective analysis of all sepsis-related hospitalizations after Emergency Department (ED) visit in a public Italian hospital in an 8-year period. A risk score to predict CFR was computed by logistic regression analysis of selected variables in a training set (2009-2012), and then confirmed in the whole study population. A trend analysis of CFR during the study period was performed dividing patient as high-risk (upper tertile of risk score) or low-risk. RESULTS: Two thousand four hundred ninety-two subjects were included. Over time the incidental admission rate (no. of sepsis-related admissions per 100 total admissions) increased from 4.1% (2009-2010) to 5.4% (2015-2016); P < 0.001, accompanied by a reduced CFR (from 38.0 to 18.4%; P < 0.001). A group of 10 variables (admission to intensive care unit, cardio-vascular dysfunction, HIV infection, diabetes, age ≥ 80 years, respiratory diseases, number of organ dysfunction, digestive diseases, dementia and cancer) were selected by the logistic model to predict CFR with good accuracy: AUC 0.873 [0.009]. Along the years CFR decreased from 31.8% (2009-2010) to 25.0% (2015-2016); P = 0.007. The relative proportion of subjects ≥80 years (overall, 52.9% of cases) and classified as high-risk did not change along the years. CFR decreased only in low-risk subjects (from 13.3 to 5.2%; P < 0.001), and particularly in those aged ≥80 (from 18.2 to 6.6%; P = 0.003), but not in high-risk individuals (from 69.9 to 64.2%; P = 0.713). CONCLUSION: Between 2009 and 2016 the incidence of sepsis-related hospitalization increased in a general Italian hospital, with a downward trend in CFR, only limited to low-risk patients and particularly to subjects ≥80 years.

REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment.

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and to FRIENDLY, which was previously shown to promote the normal distribution of mitochondria in Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria and chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. We conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.

The role of organizational trust and organizational support on employees' well-being.

BACKGROUND: In available scientific literature, the impact of organizational trust and perceived organizational support on employees' wellbeing has mainly been investigated without focusing on the different targets they can refer to. OBJECTIVES: The aim of the study is to test the direct role of these variables as precursors of well-being, job satisfaction and turnover intentions, by distinguishing two different levels in trust and support: the first one stemming from the employees' own team (in which employees have direct and daily interpersonal relationships, and within which they build their work experience), the second stemming from the organization itself. METHODS: In this study, a survey was administered to more than 1,000 employees of a Sicilian hospital. The relationships between the variables were studied through the use of hierarchical regression analysis. RESULTS: Trust and perceived support are both significant predictors of mental and physical health (team trust: ß=.088; organizational trust: ß=.110; team support: ß=.245; organizational support: ß=.082), job satisfaction (team trust: ß=.245; organizational trust: ß=.222; team support: ß=.209; organizational support: ß=.168) and turnover intentions (team trust: ß=- .086; organizational trust: ß=-.164; team support: ß=-.166; organizational support: ß=.064). However, the support at the team level is a more important predictor, while trust is a stronger predictor at the organizational level. CONCLUSIONS: Distinguishing between team and organizational levels of trust and support allows to better understand the relationship between trust and support and wellbeing and organizational outcomes such as job satisfaction and turnover intentions. Practical implications, as well as future research, should therefore refer to the different levels of such predictors.

Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis.

The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network.

The endoplasmic reticulum exerts control over organelle streaming during cell expansion.

Cytoplasmic streaming is crucial for cell homeostasis and expansion but the precise driving forces are largely unknown. In plants, partial loss of cytoplasmic streaming due to chemical and genetic ablation of myosins supports the existence of yet-unknown motors for organelle movement. Here we tested a role of the endoplasmic reticulum (ER) as propelling force for cytoplasmic streaming during cell expansion. Through quantitative live-cell analyses in wild-type Arabidopsis thaliana cells and mutants with compromised ER structure and streaming, we demonstrate that cytoplasmic streaming undergoes profound changes during cell expansion and that it depends on motor forces co-exerted by the ER and the cytoskeleton.

ER stress signaling requires RHD3, a functionally conserved ER-shaping GTPase.

Whether structure and function are correlated features of organelles is a fundamental question in cell biology. Here, we have assessed the ability of Arabidopsis mutants with a defective endoplasmic reticulum (ER) structure to invoke the unfolded protein response (UPR), an essential ER signaling pathway. Through molecular and genetic approaches, we show that loss of the ER-shaping GTPase Root Hair Defective 3 (RHD3) specifically disrupts the UPR by interfering with the mRNA splicing function of the master regulator IRE1. These findings establish a new role for RHD3 in the ER and support specificity of the effects of ER-shaping mutations on ER function.

Golgi traffic and integrity depend on N-myristoyl transferase-1 in Arabidopsis.

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADP-ribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes.

MAIGO5 functions in protein export from Golgi-associated endoplasmic reticulum exit sites in Arabidopsis.

Plant cells face unique challenges to efficiently export cargo from the endoplasmic reticulum (ER) to mobile Golgi stacks. Coat protein complex II (COPII) components, which include two heterodimers of Secretory23/24 (Sec23/24) and Sec13/31, facilitate selective cargo export from the ER; however, little is known about the mechanisms that regulate their recruitment to the ER membrane, especially in plants. Here, we report a protein transport mutant of Arabidopsis thaliana, named maigo5 (mag5), which abnormally accumulates precursor forms of storage proteins in seeds. mag5-1 has a deletion in the putative ortholog of the Saccharomyces cerevisiae and Homo sapiens Sec16, which encodes a critical component of ER exit sites (ERESs). mag mutants developed abnormal structures (MAG bodies) within the ER and exhibited compromised ER export. A functional MAG5/SEC16A-green fluorescent protein fusion localized at Golgi-associated cup-shaped ERESs and cycled on and off these sites at a slower rate than the COPII coat. MAG5/SEC16A interacted with SEC13 and SEC31; however, in the absence of MAG5/SEC16A, recruitment of the COPII coat to ERESs was accelerated. Our results identify a key component of ER export in plants by demonstrating that MAG5/SEC16A is required for protein export at ERESs that are associated with mobile Golgi stacks, where it regulates COPII coat turnover.

Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts.

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.

Developing and validating a high-throughput assay for salinity tissue tolerance in wheat and barley.

MAIN CONCLUSION: Leaf tissue tolerance was strongly and positively correlated with overall salt tolerance in barley, but not in wheat where the inability of sensitive varieties to exclude Na(+) is compensated by their better ability to handle Na(+) accumulated in the shoot via tissue tolerance mechanisms. A new high-throughput assay was developed to use the excised leaves to eliminate the confounding contribution of sodium exclusion mechanisms and evaluate genetic variability in salinity tissue tolerance in a large number of wheat (Triticum aestivum and Triticum turgidum ssp. durum) and barley (Hordeum vulgare) accessions. The changes in relative chlorophyll content (measured as chlorophyll content index, CCI) in excised leaves exposed to 50 mM NaCl for 48 h were found to be a reliable indicator of leaf tissue tolerance. In both wheat and barley, relative CCI correlated strongly with the overall plant salinity tolerance (evaluated in glasshouse experiments). To a large extent, this tissue tolerance was related to more efficient vacuolar Na(+) sequestration in leaf mesophyll, as revealed by fluorescent Na(+) dye imaging experiments. However, while in barley this correlation was positive, tissue tolerance in wheat correlated negatively with overall salinity tolerance. As a result, more salt-sensitive durum wheat genotypes possessed higher tissue tolerance than bread wheat plants, and this negative correlation was present within each of bread and durum wheat clusters as well. Overall, these results indicate that the lack of effective Na(+) exclusion ability in sensitive wheat varieties is compensated by their better ability to handle Na(+) accumulated in the shoot via tissue tolerance mechanisms. Implications of these findings for plant breeding for salinity tolerance are discussed.

Nodulin 22, a novel small heat-shock protein of the endoplasmic reticulum, is linked to the unfolded protein response in common bean.

The importance of plant small heat shock proteins (sHsp) in multiple cellular processes has been evidenced by their unusual abundance and diversity; however, little is known about their biological role. Here, we characterized the in vitro chaperone activity and subcellular localization of nodulin 22 of Phaseolus vulgaris (PvNod22; common bean) and explored its cellular function through a virus-induced gene silencing-based reverse genetics approach. We established that PvNod22 facilitated the refolding of a model substrate in vitro, suggesting that it acts as a molecular chaperone in the cell. Through microscopy analyses of PvNod22, we determined its localization in the endoplasmic reticulum (ER). Furthermore, we found that silencing of PvNod22 resulted in necrotic lesions in the aerial organs of P. vulgaris plants cultivated under optimal conditions and that downregulation of PvNod22 activated the ER-unfolded protein response (UPR) and cell death. We also established that PvNod22 expression in wild-type bean plants was modulated by abiotic stress but not by chemicals that trigger the UPR, indicating PvNod22 is not under UPR control. Our results suggest that the ability of PvNod22 to suppress protein aggregation contributes to the maintenance of ER homeostasis, thus preventing the induction of cell death via UPR in response to oxidative stress during plant-microbe interactions.

On the advantages and disadvantages of choice: future research directions in choice overload and its moderators.

Researchers investigating the psychological effects of choice have provided extensive empirical evidence that having choice comes with many advantages, including better performance, more motivation, and greater life satisfaction and disadvantages, such as avoidance of decisions and regret. When the decision task difficulty exceeds the natural cognitive resources of human mind, the possibility to choose becomes more a source of unhappiness and dissatisfaction than an opportunity for a greater well-being, a phenomenon referred to as choice overload. More recently, internal and external moderators that impact when choice overload occurs have been identified. This paper reviews seminal research on the advantages and disadvantages of choice and provides a systematic qualitative review of the research examining moderators of choice overload, laying out multiple critical paths forward for needed research in this area. We organize this literature review using two categories of moderators: the choice environment or context of the decision as well as the decision-maker characteristics.

ER-associated VAP27-1 and VAP27-3 proteins functionally link the lipid-binding ORP2A at the ER-chloroplast contact sites.

The plant endoplasmic reticulum (ER) contacts heterotypic membranes at membrane contact sites (MCSs) through largely undefined mechanisms. For instance, despite the well-established and essential role of the plant ER-chloroplast interactions for lipid biosynthesis, and the reported existence of physical contacts between these organelles, almost nothing is known about the ER-chloroplast MCS identity. Here we show that the Arabidopsis ER membrane-associated VAP27 proteins and the lipid-binding protein ORP2A define a functional complex at the ER-chloroplast MCSs. Specifically, through in vivo and in vitro association assays, we found that VAP27 proteins interact with the outer envelope membrane (OEM) of chloroplasts, where they bind to ORP2A. Through lipidomic analyses, we established that VAP27 proteins and ORP2A directly interact with the chloroplast OEM monogalactosyldiacylglycerol (MGDG), and we demonstrated that the loss of the VAP27-ORP2A complex is accompanied by subtle changes in the acyl composition of MGDG and PG. We also found that ORP2A interacts with phytosterols and established that the loss of the VAP27-ORP2A complex alters sterol levels in chloroplasts. We propose that, by interacting directly with OEM lipids, the VAP27-ORP2A complex defines plant-unique MCSs that bridge ER and chloroplasts and are involved in chloroplast lipid homeostasis.

In Arabidopsis, the spatial and dynamic organization of the endoplasmic reticulum and Golgi apparatus is influenced by the integrity of the C-terminal domain of RHD3, a non-essential GTPase.

The mechanisms underlying the organization and dynamics of plant endomembranes are largely unknown. Arabidopsis RHD3, a distant member of the dynamin superfamily, has recently been implicated in plant ER morphology and Golgi movement through analyses of dominant-negative mutants of the putative GTPase domain in a heterologous system. Whether RHD3 is indispensable for ER architecture and what role regions other than the putative GTPase domain play in RHD3 function are unanswered questions. Here we characterized an EMS mutant, gom8, with disrupted Golgi movement and positioning and compromised ER shape and dynamics. gom8 mapped to a missense mutation in the RHD3 hairpin loop domain, causing accumulation of the mutant protein into large structures, a markedly different distribution compared with wild-type RHD3 over the ER network. Despite the GOM8 distribution, tubules fused in the peripheral ER of the gom8 mutant. These data imply that integrity of the hairpin region is important for the subcellular distribution of RHD3, and that reduced availability of RHD3 over the ER can cause ER morphology defects, but does not prevent peripheral fusion between tubules. This was confirmed by evidence that gom8 was phenocopied in an RHD3 null background. Furthermore, we established that the region encompassing the RHD3 hairpin domain and the C-terminal cytosolic domain is necessary for RHD3 function. We conclude that RHD3 is important in ER morphology, but is dispensable for peripheral ER tubulation in an endogenous context, and that its activity relies on the C-terminal region in addition to the GTPase domain.