In the roots of tolerant plants, a quick response was observed with an increase in H2O2 production within 5 min after salt treatment. The expression analysis of some of the genes involved in perception, signal transduction and salt stress response confirmed their early induction in the roots of tolerant plants compared to sensitive ones. By inhibiting the synthesis of apoplastic H2O2, a reduction in the expression of these genes was detected. Our results indicate that quick H2O2 signaling in the roots is part of a coordinated response that leads to adaptation instead of senescence in salt-treated rice plants.Salinity tolerance has been extensively investigated in recent years due to its agricultural importance. Several features, such as the regulation of ionic transporters and metabolic adjustments, have been identified as salt tolerance hallmarks. Nevertheless, due to the complexity of the trait, the results achieved to date have met with limited success in improving the salt tolerance of rice plants when tested in the field, thus suggesting that a better understanding of the tolerance mechanisms is still required. In this work, differences between two varieties of rice with contrasting salt sensitivities were revealed by the imaging of photosynthetic parameters, ion content analysis and a transcriptomic approach. The transcriptomic analysis conducted on tolerant plants supported the setting up of an adaptive program consisting of sodium distribution preferentially limited to the roots and older leaves, and in the activation of regulatory mechanisms of photosynthesis in the new leaves. As a result, plants resumed grow even under prolonged saline stress. In contrast, in the sensitive variety, RNA-seq analysis revealed a misleading response, ending in senescence and cell death. The physiological response at the cellular level was investigated by measuring the intracellular profile of H Soil salinity is a major constraint for crop production worldwide, particularly on agricultural land close to the sea . Saliniz+ accumulation, affects cellular metabolism, photosynthesis and induces oxidative stress. At the whole plant level, a reduction in the growth rate and stomatal closure are the most common outcomes in response to osmotic stress. In the case of a prolonged stress, however, these mechanisms can lead to an initial biomass loss, caused by carbon starvation, followed by early senescence events leading to cell death due to ion toxicity. Recent studies demonstrate that plants undergo anticipated senescence in response to a moderate stress, in order to convey recycled nutrients to the reproductive organs and then to guarantee the survival of next generations   .2+-mediated apoplastic oxidative burst  family  may be iReactive oxygen species (ROS) generated in response to an abiotic stress, besides their well-known toxic effects, have been recently recognized as also playing a role in the complex signaling network of plant stress responses, in particular in early signaling events . In ordeSalinity tolerance is thus a complex trait in which different components are involved in counteracting all the changes induced by salt stress. This complexity is reflected in the lack of commercially available salt-resistant crops. Despite the high number of published works reporting the salt resistance of engineered plants under controlled conditions , few dat1 (Jan 2018).Further investigations into tolerance mechanisms are thus needed to identify new mechanisms of salt stress responses and to provide essential information for the production of salt-resistant crops . DevelopExploring the genetic variability with the identification of salt tolerant/sensitive couples is possible in rice. A comparison between two or more genotypes with contrasting salt responses is beneficial in the discovery of new tolerance mechanisms in rice. Many studies have compared indica and japonica varieties, and some tolerance mechanisms have been reported, thus demonstrating the usefulness of this approach .In this work, two varieties of rice belonging to the japonica group, which show a strong difference in tolerance to salinity, were selected and analyzed to reveal the mechanisms responsible for salt tolerance. The comparison was performed in the vegetative stage, which is one of the two growth phases most sensitive to salt in rice. Analysis of tolerance mechanisms during the most sensitive stages of growth is considered essential to provide new information on how to generate salt-resistant rice plants .The two varieties were initially physiologically characterized to identify the tolerance mechanisms implemented by the tolerant variety. The photosynthetic efficiency was evaluated using PAM imaging, a technique that enables the whole leaf to be examined and therefore to assess the extent of damage caused by the salt, and if necessary to see whether the damage is recoverable when the salt is removed from the culture medium. We also examined the allocation of sodium and potassium in roots and single leaves by ionomics to assess whether the tolerance was due to a recovery mechanism of ion homeostasis. We therefore performed a molecular analysis using RNA-seq to identify the metabolic and signaling pathways responsible for the physiological responses observed in the two varieties.2O2 along with the expression of genes known to be involved in the salt stress response.To reach a more complete understanding of the salt tolerance mechanism, the role of early signaling pathways induced by salt was investigated in the roots of both varieties, by analyzing the intracellular profile of the HO. sativa L. spp. japonica “temperate”) 2 were dehulled, sterilized for 1 min in 70% ethanol and rinsed five times with deionized water. Seeds were sown on water-wetted filter paper in Petri dishes and left to germinate for 48 h at 24°C in the dark. Uniformly germinated seedlings were transferred to agar-filled  seed-holders in an Araponics system 3 with a modified Hoagland solution  and Vialone Nano (VN) (solution  at appro4:CaCl2:NaNO2 = 10:2:1:1). Leaves (blade + sheath), stems and roots (thoroughly washed) were collected. For ion content analysis, samples collected at different times  from control and treated plants were dried at 40°C for 48 h and stored in plastic boxes. For RNA-seq analysis and qPCRs, samples (leaves and roots) were collected at different timepoints and frozen in liquid nitrogen.Seedlings at the V2 stage were grown with or without the saline solution . Plants were analyzed 4 h after switching on the light and were dark adapted for 30 min before collecting photosynthetic data in order to maximize the oxidation of the photosynthetic electrons transport chain. The chlorophyll fluorescence value of dark-adapted samples (F0) was measured applying a non-actinic white light source (intensity < 0.05 μmol photons m-2 s-1). The maximum chlorophyll fluorescence value (Fm) was instead measured applying a saturating light pulse (intensity = 3500 μmol photons m-2 s-1 and 800 ms duration).PSII) and was calculated according to -2 s-1 for 5 min to evaluate non-photochemical quenching (NPQ) activation kinetics. Later, the light was switched off for 3 min to evaluate NPQ relaxation. NPQ parameter was also calculated according to PSII functionality was expressed as PSII maximum quantum yield . The stomatal aperture was measured as the ratio between width and length of the stomata, as shown in Supplementary Figure 2 sections of the second leaf were cut with scissors and immediately weighed (W). Then, the sections were hydrated in water in 15 mL test tubes for 4 h in a growth chamber under light. After rapid drying, samples were weighed to obtain the turgid weight (TW). Dry weight was measured after oven-drying at 40°C for 48 h.5 cmRelative water content was determined using the following formula :Data are presented as mean ± SD of six biological replicates.n = 3) and homogenized in liquid nitrogen. 100 or 200 mg of sample  were used for total RNA extraction . 4 μg of total RNA with a RIN ≥ 8  were sent to the IGA Technology Services 4  for library preparation  and sequencing on HiSeq 2000 platform   under accession number GSE109341.After 3 days of treatment, roots and leaves were collected from 6 plants/experiments  (Preprocessed reads were mapped with TopHat  on the Oion 7.0) . Gene coion 7.0) .To expand the functional annotation of rice genes, the entire set of transcripts was annotated with the Argot web server , which aThe raw counts were used as input of a state-of-the-art differential expression analysis workflow , based oRice genes were assigned to clusters of orthologous groups from the KEGG database using blastKOALA . This inWe tested the expression pattern of 20 DEGs from the RNA profiling experiment using an OpenArray-based nanofluidic RealTime-PCR technique . A comparison of the data from the two approaches is shown in Supplementary Table 3 by applying a one-step temperature ramp (to 210°C in 10 min and maintained for 10 min). After 20 min of cooling time, the mineralized samples were transferred into polypropylene test tubes.Leaves (blade + sheath), stems and roots (washed carefully) were collected after 1, 3, and 7 days of treatment, dried at 40°C for 48 h and stored in plastic boxes. Then, dry leaf and root samples were weighed and digested by a microwave digester system  in Teflon tubes filled with 10 mL of 65% HNO-1 aliquot of an internal standard solution  was added to both samples and calibration curve to give a final concentration of 20 μg L-1.Samples were diluted 1:40 with MILLI-Q water, and the different elements concentration was measured by inductively coupled plasma mass spectrometry . A 2 mg L2 flow of 93 mL min-1 through a skimmer cone. Data are shown as mg of the ion per g of sample (dry weight).Typical polyatomic analysis interferences were removed using CRI (Collision-Reaction-Interface) with an HqPCRs were performed using the QuantStudio 12K Flex real-time PCR system, both for the OpenArray technology  and the standard protocol.® OpenArray® Real-Time PCR Plate with Custom Gene Expression Assays (56 probes) was designed and purchased from Thermo Fisher Scientific. The following cycle was used: 10 min pre-incubation at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C  and standard qPCR protocol were used for SERF1 ; ubiquitin (NCBI: Os05g0160200) was used as reference gene (primer for TTCTACAAGGTGGACGACGC and primer rev AGATCAGAGCAAAGCGAGCA).GoTaqT method was used to analyze the gene relative expression . Thus, roots were incubated with 10 μM DHR123  for 15 min followed by 5 min rinsing in water. Roots were imaged by using a Leica B5000 fluorescence microscope  with an I3 filter. ImageJ2  was usedt-tests were applied to experiments with a sample number greater than 30. Wilcoxon–Mann–Whitney tests were applied for n < 30. For the analysis of RNA sequencing data, refer to the text above.Student’s Among the 17 Italian varieties tested , we sele8. In Italy, during prolonged drought, sea water creeps inland from the mouth of the Po river for many kilometers. The fresh water becomes brackish and the salt enters the ground by capillarity. In our experiments we simulated the seawater flooding of paddy fields, as often happens in the river deltas. Seawater contains mainly sodium, chloride, magnesium, sulfate and calcium, with Na+ and Cl- being the most predominant. The saline solution used in this work was prepared by combining different salts with the following proportion: NaCl:MgSO4:CaCl2:Na2SO4 = 10:2:1:1. Seawater consists of about 35 g/l in dissolved salts, corresponding to about 500 mM NaCl. A soil becomes salty when the salt concentration reaches about 40 mM NaCl and rice sensitivity shows up at about 60 mM NaCl . The PAM imaging technique confirmed a large decrease in PSII maximum quantum yield (ΦPSII) in all leaves of the sensitive plants after the 6th day . In contrast, in tolerant plants, only the 1st and 2nd leaves were affected by salt, whereas the 3rd and the 4th leaves , which emerged in the presence of salt, showed the same photosynthetic efficiency as the control samples . In line with this, the relative water content (RWC) of the second leaf decreased to 54.1% in the sensitive variety after 3 days of treatment , while in the tolerant plants no significant water loss was recorded.Stomatal aperture measurements after 24 h of treatment showed a reduction in the second leaf of the B plants, whereas no significant differences were observed between treated and untreated plants in VN plants . Conversely, in the sensitive variety Na+ was more uniformly partitioned between the roots and shoot, and salt accumulation was observed in all the leaves . The [K+]/[Na+] ratio was lower in the leaves of the sensitive variety than the tolerant variety , resulting in a larger ionic stress in the susceptible plants.The evaluation of KPSII were monitored for both varieties to evaluate the recovery of both the growth capacity and the photosynthetic performance .To further characterize the salt response of plants, a growth recovery experiment was conducted after 6 days of stress. After 7 days of culture in salt-free medium, the recovery capacity of the two varieties was assessed. The lengths of the 3rd and 4th leaves and the ΦFigure 4 and Table 1), whereas in the sensitive variety, the percentage of dead plants reached 76% (Table 1), and there was a negative impact on photosynthesis in the remaining plants (not shown).Our results showed that the tolerant variety recovered completely after 6 days of treatment  in B plants, and senescence in VN plants, transcriptomic analyses were conducted in roots and leaves 3 days after salt stress.A total of 542,309,740 single-end, 50 nt long reads were obtained from the sequencing experiments , which decreased to 536,477,150 after pre-processing . Most of the reads (93.1%) were successfully mapped to the rice genome and met the requirements described in the Section “Materials and Methods.” Of these, 3.48% were mapped in multiple positions and were discarded.Figure 5 and are available online at http://pathwayinspector.dmm.unipd.it/projectview/0ggWH7/ (Jan 2018).Sequencing data were analyzed as described in the Section “Materials and Methods” and exploiting metabolic pathways from the KEGG database. The results are summarized in Figure 5 and Table 2, in the sensitive variety VN, a large number of genes (DEGs) was regulated both in the leaves  and roots , almost fourfold the number of B.As shown in Table 2), gene networks (topology) were created, and finally the expression data were combined with the pathway topology to compute an enrichment score . We thus obtained a list of enriched pathways and their modulated components for each pairwise comparison. The number of enriched pathways was proportional to the number of DEGs and was therefore higher in the sensitive variety (Table 2), suggesting a wider response to salt stress in the VN variety.To better interpret the many DEGs, a pathway enrichment was performed: genes were assigned to clusters of orthologous groups from the KEGG database , for “phenylalanine, tyrosine and tryptophan biosynthesis,” the up-regulation of indole (IAA precursor) synthesis was noted , which consists of a group of 150 genes that belong mostly to the group of R genes , the pathways involved in chlorophyll metabolism, carbon fixation, oxidative stress response, autophagy, and basic metabolism  were affected by salt stress. In particular, in leaves, the genes involved in the photosystem assembly were down-regulated, whereas those implicated in nitrogen reallocation  and autophagy were up-regulated, implying that the leaves were undergoing an active chloroplast dismantling followed by reassimilation of nitrogen, typical of both natural and stress-induced leaf senescence   both in roots and leaves .In order to compare the response of the two varieties, all DEGs represented by the following GO terms were gathered: response to salt stress, osmotic stress, water stimulus, desiccation, water deprivation, and oxidative stress . In leaves, these genes were differentially expressed only in the sensitive variety. These results suggested that VN plants experienced strong oxidative stress  and cellular damage (genes for DNA helicases and heat shock proteins) in both the roots and leaves  were shared only in roots, where both varieties accumulated sodium . We thus gathered all the DEGs annotated as ion transporters/channels and carriers, in order to have a more detailed picture of the plants’ physiological conditions.As reported above, the two varieties differed in terms of sodium allocation throughout the plant , and proline and monosaccharide transporters, primarily in roots (Table 3). In addition, genes involved in Na+ and Cl- transport  were modulated only in the VN plants (Table 3), suggesting that susceptible plants were still suffering from osmotic stress, water deficit and ion toxicity after 3 days of salt treatment.After 3 days of salt stress, sensitive plants were still regulating the genes encoding mostly for aquaporins, NSCCs , ATPases, high affinity KFigure 7A) were down-regulated. Whereas in tolerant plants, only the ZEP encoding gene was down-regulated . This may correlate with PAM imaging analyses that show a different ability to activate non-photochemical quenching (NPQ). In fact the regulatory mechanism of photosynthesis reduces the photo-oxidative damage caused by the over-excitation of PSII . Using the imaging system, we observed that the high NPQ was particularly evident in areas of the leaf that maintained their PSII functionality  and Violaxanthin de-epoxidase (VDE) . In the susceptible variety, we only noted a slight increase after 1 h of treatment .The DHR123 probe was used to evaluate the intensity of HSERF1 was induced already after 5 min in roots of the B variety, with a peak around 30 min. In VN plants, however, the gene was up-regulated later and to a lesser extent .OsDREB2A (dehydration-responsive element-binding protein 2A), a gene encoding a transcription factor involved in abiotic stress response .In the signaling pathways mediated by SERF1, we studied the expression profile of response , which iresponse  during s2O2 signaling, showed a similar pattern, and were up-regulated earlier in the tolerant plants . These genes were: RbohA , whereas in VN leaves, it was up-regulated later, only 24 h after treatment .As shown in ter NHX1  was stronditions  was alsoFigures 9A,C). In leaves, no significant differences were observed between the two varieties.TPKa and TPKb, two members of the tonoplast two-pore outward potassium channel family , were upFigure 9 clearly show that B plants had early-activated mechanisms aimed at Na+ compartmentalization into vacuoles, thus improving tissue tolerance to ionic stress.The results reported in 2O2 burst in inducing an adaptive response in B plants, we performed experiments in the presence of the NADPH oxidase (NOX) inhibitor DPI. The inhibition of the plasma membrane NOX abolished the H2O2 peak usually observed after 30 min of treatment , declined by 31 and 49.1%, respectively.In order to test our hypothesis on the role of the early H2O2 peak in the tolerant variety affects the signaling pathway mediated by SERF1, which becomes more like that observed in VN roots .These results suggest that the loss of the early HToday high salinity is one of the main constraint for crop production worldwide. The concentration of salts is continuously increasing in soils that are close to coastal areas or in areas frequently subject to drought . Three q2O2, in the activation of salt tolerance mechanisms that lead to adaptation, and provide new perspectives in terms of the genetic improvement of salt tolerance.As plants are sessile organisms, they have evolved mechanisms to cope with sudden salt stress. Salinity resilience relies on different traits that were explored in the last two decades in many works  or systemically  allocates sodium differentially throughout the plant body: more in the roots than the leaves, and more in the first leaf than the second or third. Differences in sodium allocation might explain how and why the B variety is more tolerant to ionic stress than the sensitive variety. Roots are more salt tolerant than leaves : maintai+ in contrast to VN plants. This strategy is advantageous for photosynthesis: it is better to completely sacrifice a part of a leaf and keep the rest functional, rather than lose efficiency in all cells. The recovery experiments also demonstrated the capacity of B plants to limit the damage to the first leaf, which was sacrificed in order to support the photosynthetic activity of the others and to continue the photosynthetic growth of the entire plant with the emergence of new leaves.The sensitive variety VN did not show this response, and all parts of all the leaves were affected. These patterns were matched by the activation of specific mechanisms of sodium allocation among and within leaves, revealing that B plants likely evolved mechanisms to compartmentalize NaPhyscomitrella patens, a representative of the early land colonization of plants, has NPQ been found to be enhanced in response to salt treatment  and stomata closure are the main mechanisms involved in limiting the loss of water . Our resApplying transcriptomics, we evaluated the response to stress at the molecular level after 3 days of salt exposure, when both varieties were initiating their specific programs in response to salt stress: adaptation (recovery in growth) in B plants versus senescence in VN plants. Based on the quality and quantity of differentially expressed genes (fourfold higher in VN plants), the response of the sensitive variety was broad and not specific compared to the tolerant variety. The onset of a genetically controlled cellular dismantling process (namely leaf senescence) was visible, as expected, in the VN leaves and was due to the downregulation of genes involved in carbon fixation and chloroplast assembling, and the upregulation of genes involved in nitrogen remobilization . In the This finding raises a question regarding the apparently altered pathway from the perception to the transduction of the stress signal in susceptible plants.2O2 are universal signal molecules, which transduce a stimulus after sensing. Understanding how the cell decides which pathway to activate is the next big challenge , are likely responsible for salt perception and rapid root-to-shoot signaling , was shown to be involved in ROS-dependent transcriptional regulation in rice roots and in root-to-shoot signaling , ion homeostasis  and turgor maintenance (P5CS2), thus preventing the organization of a specific response, both in the roots and leaves. This evidence supports the hypothesis that the early H2O2 burst in roots of the tolerant variety plays a role in building a rapid and specific salt stress response.Our findings suggest that the delayed H2O2 burst is generated at the plasma membrane level. The prevention of the intracellular H2O2 burst in B roots significantly affected the expression of downstream genes, e.g., SERF1, DREB2A and NHX1, resulting in a similar response to that observed in the VN variety.Experiments with the NOX inhibitor DPI demonstrated that the intracellular H2O2 signals, it is possible that the two varieties have a different membrane composition. Calcium channels and NADPH oxidases are made of subunits, and it would be interesting to explore the contribution of each subunit to the signal transduction, or to investigate whether there are subunits that are more prone to respond to the stress. In this work we show that in B plants, the expression of OSCA and NOX genes  occurred early after salt addition. The expression of these genes is not the cause but the consequence of the rapidly induced H2O2 peak in tolerant cells. However, we cannot rule out that B plants have a pre-set membrane composition that activates the adaptive mechanism well before the stress causes damage.How tolerant plants can sense and react earlier than sensitive plants is an interesting issue. As plasma membranes are involved in mediating calcium and H2O2 -dependent signals are part of the coordinated activation of downstream genes that trigger a specific salt tolerance response. In fact, tolerant plants rapidly perceived salt stress and responded by activating effective tolerance mechanisms, within the first 24 h. This response led to ionic stress tolerance in roots and adaptation in leaves, given by (i) the regulation of cytosolic [K+]/[Na+], (ii) differential sodium allocation throughout the plant, (iii) maintenance of PSII efficiency, (iv) NPQ increase, and (v) the appearance of new unaffected leaves.This report points out that early H2O2. The manipulation of early signaling events are of paramount importance for the future of plant engineering because early signals are the hub used by cells to select the right response or to cope with different stresses at the same time (Further studies are planned to identify all the players involved in the perception and transduction of the salt stress stimulus, for example by using genetically encoded probes for calcium and Hame time . ControlEF designed the experiments and wrote the manuscript. CS and EbB performed the morphological measurements. GP and TM carried out PAM imaging analyses. SR and PF performed the pathway enrichment analyses. EL and ST handled the RNA sequencing data. EnB and GS performed the ion content measurements. PS handled the openarray data. MZ reviewed the manuscript and FLS planned the experiments, wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The pathogenesis of chronic rhinosinusitis with nasal polyps is largely unknown. Previous studies have given valuable information about genetic variants associated with this disease but much is still unexplained. Our goal was to identify genetic markers and genes associated with susceptibility to chronic rhinosinusitis with nasal polyps using a family-based genome-wide association study.427 patients  with CRSwNP and 393 controls  were recruited from several Swedish hospitals. SNP association values were generated using DFAM (implemented in PLINK) and Efficient Mixed Model Association eXpedited (EMMAX). Analyses of pathway enrichment, gene expression levels and expression quantitative trait loci were then performed in turn.8. Pathway analyses using our top 1000 markers with the most significant association p-values resulted in 138 target genes. A comparison between our target genes and gene expression data from the NCBI Gene Expression Omnibus database showed significant overlap for 36 of these genes. Comparisons with data from expression quantitative trait loci showed the most skewed allelic distributions in cases with chronic rhinosinusitis with nasal polyps compared with controls for the genes HLCS, HLA-DRA, BICD2, VSIR and SLC5A1.None of the analysed SNPs reached genome wide significant association of 5.0 x 10−HLCS, HLA-DRA, BICD2, VSIR and SLC5A1 could be involved in the pathogenesis of chronic rhinosinusitis with nasal polyps. HLA-DRA has been associated with chronic rhinosinusitis with nasal polyps in previous studies and HLCS, BICD2, VSIR and SLC5A1 may be new targets for future research.Our study indicates that  Risk The aetiology of the disease is unknown. Several environmental factors have been suggested and previous studies have also shown an increased prevalence among relatives ,8 and a cystic fibrosis transmembrane conductance regulator gene (CFTR) [Genetic association can be explored using candidate gene studies or genome-wide association studies (GWAS). Candidate gene studies usually investigate a small number of single-nucleotide polymorphisms (SNPs) or other types of genetic variation, in order to find or reject associations between the genetic variants and the disease in question. These studies rely on previous knowledge and hypotheses regarding which SNPs to suspect and investigate. In comparison, a GWAS investigates hundreds of thousands of SNPs across the whole genome and is therefore not reliant on previous knowledge or hypotheses regarding the pathogenesis of the investigated disease or trait. A large number of GWASs have been performed for various complex diseases such as diabetes and asthma which has led to the finding of novel genetic pathways . There ie (CFTR) ,16 and, e (CFTR) –20, infle (CFTR) ,22, tisse (CFTR) Even though GWA studies have been successful in detecting genetic variants associated with many common diseases, the inability to explain most of the estimated heritability makes linkage analysis an alternative to detect possible rare variants. To this date, no published linkage analysis have been performed on subjects with CRSwNP. However, one such study has been performed on 8 subjects with CRS (not specified whether any of them had CRSwNP) which found a linkage signal on chromosome 7q . A combiThe aim of this study is to identify SNPs and genes associated with CRSwNP susceptibility using a family-based approach.367 patients with CRSwNP  from three Swedish ear, nose and throat clinics were recruited. These subjects were all known patients at their respective clinics, all of them fulfilled the EPOS criteria for CRSwNP [The study was carried out in accordance with the Declaration of Helsinki and was approved by the Ethics Committee at the University of Gothenburg, Sweden. Written consent was obtained from all participants.Nasal endoscopy was performed on all participants using a 2.7 mm rigid endoscope (KARL STORZ) and the participants were subsequently phenotyped as either having CRSwNP or being free from this disease. Additional data about asthma and corticosteroid medication (used to counter symptoms from either the upper or lower airways) was obtained via a structured interview. Peripheral blood samples were collected from each individual.DNA was extracted from whole blood using an in-house protocol at KBiosciences . The HiSeq Illumina platform was used for genotyping. 144 of the samples were run on Illumina Omni Express bead chips and the remaining 676 on the Illumina Core Exome array.Individuals were removed if they showed >2% missing calls , heterozygosity >3 SDs above or below mean, >100 Mendelian errors). Markers with >2% missing calls, >3 Mendelian errors or those with minor allele frequency = 0 (i.e. monomorphic loci) were excluded.2<0.2) were selected for population structure analysis. As a reference, samples from the 1000 Genomes project, Phase 3 were retrieved. Principal component analysis was then performed using GCTA, a genome-wide complex trait analysis software [E from that sample to the mass centre of the CEU reference group. Individuals with E > 5 SDE were removed (2 samples).SNPs with low linkage disequilibrium among each other (LD) , the sibling TDT and an allelic test for unrelated cases and controls in a single Cochran-Mantel-Haenszel test for each marker . The sec-8  but many2>0.25). This process was performed separately for DFAM and EMMAX association results.The top 1000 markers with the most significant association p-values were combined into intervals of SNPs in high LD . For each SNP-gene pair we have retained either LCL or skin data, corresponding to the tissue with more significant regulatory effect . We also excluded all SNPs with p-values > 0.05 or absolute effect size (regression coefficient β) of < 0.01. FDR of 0.5 was used as a cut-off for the Blood dataset, with no additional limits on effect size.To check for directed eQTL enrichment, all eQTL SNPs for each gene of interest were extracted and classified according to the direction of their regulatory effect (up-regulating or down-regulating). The frequency of the allele bearing the reported regulatory effect was then determined in our GWAS cases and controls using PLINK . The marn SNPs, 500 genes with the same number n of SNPs are retrieved; if less than 500 genes have the required number of SNPs, genes with n+1  SNPs are also retrieved, and n SNPs are randomly selected for analysis in those genes. Each gene is analysed in the same manner as the target gene. Resulting empirical distribution of p-values is used to determine the empirical significance for the gene of interest.However, Fisher’s test does not account for the effect of LD between SNPs. Therefore, the empirical significance was calculated using an iterative procedure. Genes were ordered according to the number of eQTL SNPs remaining after all filters; genes found in the differentially-expressed NP set (as described in the previous section) were removed; for each gene of interest with The workflow of the analysis is shown in Six samples were removed due to heterozygosity > 3 SDs above or below mean, three samples were removed due to > 100 Mendelian errors and one sample due to a mismatch between genotyped and indicated sex. The final dataset after quality-control consists of 782 individuals and 233 409 SNPs. Additional data about the subjects who passed quality control are presented in -8, the top ranking SNP from the DFAM analysis was rs4629180 with a p-value of 1.47x10-6, the top ranking SNP from the EMMAX analysis was rs2491026 with a p-value of 0.00014.The Manhattan plot from the DFAM analysis is provided in From the pathway enrichment analysis we extracted the top 20 gene-sets from each of the four INRICH analyses resulting in a combined list of 138 target CRSwNP genes from this study .Out of our 138 target genes from the INRICH analysis, 36 genes showed a significant difference in mRNA expression levels between nasal polyp tissue and normal tissue  Table HLCS  HLA-DRA  and BICD2  , HLCS  and BICD2 . SLC5A1 also had a skewed distribution with an empirical p-value of 0.052, these results are provided in Finally, the eQTL analysis for the Blood eQTL dataset showed significantly skewed distributions of eQTLs in cases with CRSwNP compared to controls for p 0.046) . The sam-8. However, by using pathway enrichment and post-GWAS analyses, we identified five interesting genes that could be involved in the pathogenesis of CRSwNP in this study: HLCS, HLA-DRA, BICD2, VSIR and SLC5A1. Of these, only HLA-DRA has been presented in previous studies on subjects with CRSwNP [None of the SNPs in this study reached the suggested genome-wide significance level of 5x10h CRSwNP .The present study is the first GWAS performed only on subjects with CRSwNP. Using family relationship data and non-transmitted genetic variation for control, as in the TDT, is both a strength and a potential weakness. In most chromosomal regions, we expect the related individuals to be more similar compared with completely unrelated controls. Therefore, when there are differences between related individuals these are more likely to be due to the disease than to general differences in a population (population stratification). In practice this is a strength because it would be expected to lead to less false positive results. However, due to the increased risk of CRSwNP among relatives  and the HLCS gene was the most significant gene from the eQTL analysis in the Blood dataset and the second most significant in the MuTHER dataset. HLCS is under-expressed in nasal polyp tissue with significantly increased frequency of down-regulating alleles among cases in the eQTL analysis from both the MuTHER and the Blood datasets. This gene encodes the enzyme Holocarboxylase synthetase, which is important for biotin metabolism. Holocarboxylase synthetase itself has not been implicated in CRSwNP but a study from 2013 showed that the enzyme catalyses biotinylation of heat shock protein 72 thereby inducing the expression of the gene RANTES  [RANTES is implicated in multiple studies of CRSwNP; for example a study by Chao et al. found a positive correlation between plasma RANTES protein levels and severity of disease among patients with CRSwNP[HLCS is under-expressed when RANTES protein levels are higher in polyp tissue compared with controls, the up-regulation of RANTES might be a counter reaction to initially too low levels during an early disease phase. Further studies are needed to increase the knowledge about the role of HLCS in the pathogenesis of CRSwNP.The ecreted) . RANTES th CRSwNP. RANTES th CRSwNP. AlthougHLA-DRA is over-expressed in polyp tissue and has a significantly skewed distribution of eQTLs from the Blood dataset where cases have an increased number of down-regulating alleles. HLA-DRA is one of the major histocompatibility complex, class II genes. Polymorphisms in this gene have been associated with the presence of nasal polyps in asthmatic patients [HLA class II genes have been linked to CRSwNP [patients . Additioo CRSwNP ,40. Usino CRSwNP .BICD2 is over-expressed in nasal polyps and up-regulating alleles more common in controls and down regulating slightly more common in cases. The gene product is bicaudal D homolog 2, which has been shown to induce microtubule movement [movement . It is amovement .HLA-DRA and BICD2 where cases have an increased number of down-regulating eQTLs even though the gene is over-expressed in polyp tissue. Over-production in the diseased state could possibly be the result of the body compensating for an under-production in the pre-disease state, which hypothetically could have contributed to the development of the disease.The relationship between gene-expression and eQTLs is reversed for VSIR (V-set immunoregulatory receptor) is over-expressed in polyp tissue and up-regulating alleles from the MuTHER dataset are more common in our cases with CRSwNP compared with unaffected individuals. The gene codes for the protein V-type immunoglobulin domain-containing suppressor of T-cell activation, a member of the Ig superfamily. An experimental study has suggested that it could facilitate tumour invasiveness by regulating cell surface membrane-type 1 matrix metalloproteinase [oteinase . Lines eoteinase . It has SLC5A1 as borderline significant. SLC5A1 is under-expressed in polyp tissue and also has an increased frequency of down-regulating alleles in our CRSwNP cases. The gene-product, solute carrier family 5 (sodium/glucose cotransporter) member 1 (SGLT1), is part of a family of sodium-dependent glucose transporters. Once again, this gene has not been associated with CRSwNP but one article suggests a positive substrate cross-regulation of SGLT1 and CFTR [CFTR gene is highly associated with cystic fibrosis, which often has CRSwNP as one of its clinical features [CFTR locus [Even though the empirical p-value is slightly higher than 0.05  this study also implicates and CFTR . The CFTfeatures . FurtherTR locus .In order to reach a power level necessary for genome-wide analysis we decided to include all patients with CRSwNP regardless of any other diseases which could be associated to this condition. Another reason for this is that the only largely accepted subgrouping of CRS is the division into either CRSsNP or CRSwNP. This is in all likelihood a gross simplification of the pathophysiological and genetic mechanisms behind these conditions and CRS as defined by EPOS is probably a result of a large number of different sub-diseases, each with their own genetic and/or environmental background of which little is known at the present. In this study we did not exclude participants based on potential subgroups since there is still uncertainty surrounding a division into subgroups other than CRSsNP and CRSwNP and we chose to focus on the phenotype CRSwNP itself. Similarly, we chose not to record allergy history due to the controversy surrounding allergy as a possible association to CRSwNP . HoweverHLCS, BICD2, and SLC5A1 have not been connected to asthma, VSIR was implicated with lung function decline in non-asthmatic patients in a genome-wide study published in 2012 but this association could not be confirmed by replication [HLA-DRA is associated to asthma [Since 51.5% of the participants with nasal polyps also had asthma it is possible that some of the associations could be due to an association with asthma. However, this is less likely since 17.3% of the subjects in the healthy group without polyps also had asthma. The high number of participants with both CRSwNP and asthma could have diluted the association analysis if their SNP-profiles differ significantly from subjects with CRSwNP but without asthma and potentially have made us miss markers of importance in the association analysis. This situation is likely to be countered by the gene enrichment and pathway analyses. lication . HLA-DRAo asthma .HLCS, BICD2, SLC5A1 and VSIR have not been associated with AERD in previous studies. HLA-DRA has not been linked to AERD but other HLA class II genes have [HLA-DRA to the presence of nasal polyps among asthmatic patients found two HLA-DRA polymorphisms to be potential markers for nasal polyp development in aspirin-tolerant asthma compared to the AERD subgroup [AERD is another condition linked to CRSwNP, a meta-analysis published in 2015 found a large variation in the prevalence of AERD among patients with CRSwNP among the included studies, the overall prevalence was 9.7% . One of nes have , howeversubgroup .With these caveats in mind, this study is the first of its kind. It is currently the only GWAS performed on CRSwNP and the only study that explores linkage and family-based genome-wide association with regards to this condition. Despite the issue of accurate phenotyping discussed above, this study suggests four novel genes as potential targets of interest for future research as well as once again implicate HLA-DRA.HLA-DRA as well as four additional genes; HLCS, VSIR, BICD2 and SLC5A1, which have not been previously identified as associated with chronic rhinosinusitis with nasal polyps, could be important for the development of this disease.This study suggests that S1 Table(DOCX)Click here for additional data file.


This review describes recent research in the development of tank systems based on complex metal hydrides for thermolysis and hydrolysis. Commercial applications using complex metal hydrides are limited, especially for thermolysis-based systems where so far only demonstration projects have been performed. Hydrolysis-based systems find their way in space, naval, military and defense applications due to their compatibility with proton exchange membrane (PEM) fuel cells. Tank design, modeling, and development for thermolysis and hydrolysis systems as well as commercial applications of hydrolysis systems are described in more detail in this review. For thermolysis, mostly sodium aluminum hydride containing tanks were developed, and only a few examples with nitrides, ammonia borane and alane. For hydrolysis, sodium borohydride was the preferred material whereas ammonia borane found less popularity. Recycling of the sodium borohydride spent fuel remains an important part for their commercial viability. Additionally, the model included two sub-models, which allowed the evaluation of non-isothermal water evaporation processes and the pressure drop of the two-phase flow through the porous catalyst. The model was one-dimensional and therefore the geometrical parameters of the reactor were not evaluated in detail and only in the flow direction. The system was initially built for vehicle applications, but it could have potential for other small portable applications [The results from Zhang or model . The modications .et al., published theoretical work on a 3-dimensional batch reactor model [4 in order to avoid problems with solubility of the hydrolysis reaction product. The data were fitted using three different kinetic models in order to evaluate the kinetics of the hydrolysis reaction. The catalyst used in the reactor was a Ru catalyst supported on Ni-foam. At low temperatures or high NaBH4 concentrations the kinetics followed a zero-order model, while at high temperatures or low NaBH4 concentrations, the reaction depended on the NaBH4 concentration and the kinetics were best described by a first order model. The experimental data were further used in a numerical 3-dimensional non-isothermal model for a pilot scale reactor. This model described the transport phenomena and also included the kinetic model for the hydrolysis reaction. Mechanical stirring in the batch reactor was shown to have an impact on the mass transfer, since NaBH4 was more easily allowed to reach the catalytic sites. Furthermore, the model showed that NaBH4 in the bottom of the reactor was used less efficiently, because the produced hydrogen decreased the stirring in the bottom. The model assumed a single-phase flow during the hydrolysis reaction, which neglected the flow of hydrogen from the catalyst after production. The gas bubbles of hydrogen were responsible for the movement of the fluid and without the incorporated artificial mixing the performance predicted by the model would be extremely low and not compatible with the experimental data obtained with the reactor. In conclusion, the 3-dimensional model was able to simulate the reactor processes and validate the kinetic model [In 2012, Sousa or model . Experimic model .In order to evaluate a two-phase flow inside a batch reactor and better describe the impact of hydrogen formation, a new two-dimensional reactor model was recently described . Data ob4 with an added catalyst. In one report, the reactor for this type of reaction consisted of brass mesh to contain the solid NaBH4 inside a cobber reactor. The steam was then passed into the reactor, where the hydrolysis reaction occurred [As mentioned earlier the hydrolysis reaction can also be carried out using steam and solid state NaBHoccurred . A two-poccurred . The modet al., was further used in a model for a PEM fuel cell power system using a NaBH4 batch reactor as hydrogen generator [4, H2O and NaBO2. This was possible because the only variable in the reactor was the hydrogen pressure. The Langmuir-Hinshelwood model was selected for describing the kinetics of the hydrogen generation rate over a range of temperatures and in time [4 hydrolysis reaction as the hydrogen source for the PEM fuel cell system. Indeed, this study had a bigger perspective, as a complete NaBH4-PEM fuel cell power system was simulated. The magnetically supported reactor described by Pozio et al., was also simulated with a simple mathematically model. This was used to evaluate the process of catalytic hydrolysis and in a later publication the complete hydrogen generator was simulated [The kinetic model described by Sousa enerator ,87. Howe in time . The resimulated ,76.4 has been given a no-go recommendation as a solid-state hydrogen storage material for automotive applications by the US DoE due to its high decomposition temperature. However, since NaBH4 releases hydrogen by hydrolysis at room temperature and the solution is both stable and non-toxic, NaBH4 has a large potential for defense and civil applications. For application purposes, hydrogen is usually catalytically generated from alkaline NaBH4 solutions. An advantage of using NaBH4 is that no external heat supply is required due to the exothermic hydrolysis reaction. A disadvantage is that the NaBO2 byproduct , needs to be collected in a separate tank in order to be recycled off board into NaBH4. For more information on the role of NaBH4 in the field of hydrogen fueled applications, the reader is referred to more in-depth articles and reviews [NaBH reviews ,89,90,91Small-unmanned aerial vehicles (UAVs) have gained much interest in the fields of defense and security in order to minimize human loses. They have been used for surveillance missions owing to the low noise and heat emissions, minimizing detection by enemies. Instead of internal combustion engines or secondary batteries, which have low thermal efficiencies and face short flight endurance (60–90 min), fuel cell systems are regarded as a more suitable alternative power source. Fuel cell systems show high thermal efficiencies, have an electrochemical reaction rather than a combustion reaction, and silent mode of operation .et al., reported 43 min of cruising flight, based on the measured capacity of the on-board hydrogen tank (192 L), which could be increased to 52 min, if the hydrogen utilization was increased to 99% [4 as their hydrogen source, are discussed as to their design, manufacture, and flight tests.As fuel cells operate on hydrogen, compressed hydrogen , liquefid to 99% . Since cd to 99% . In this4 as a hydrogen source [The US Air Force Research Laboratory in conjunction with Small Business Innovation Research partners AeroVironment, Protonex Technology Corporation  and Millennium Cell Inc. successfully demonstrated a nearly 5 h flight with Puma, an UAV, operating on a lithium ion battery and a PEM fuel cell system using NaBHn source . The flin source . The litn source .et al., also reported the use of a PEM fuel cell system operating with NaBH4 for UAVs, which consisted of a fuel cell stack, hydrogen generator and a hybrid power management system  and spent fuel (NaBO2) exchange volume within one tank hence minimizing the volumetric density . The authors claimed an estimated energy density of roughly 720 Wh·kg−1 instead of 170 Wh·kg−1 typically for commercial lithium-ion batteries, making the device competitive with existing batteries [NaBHreported . For exale phone . With a atteries . As far 4 allows a higher gravimetric hydrogen storage capacity compared to compressed hydrogen and does not have the safety and reliability constrains of compressed gases [4/liquid oxygen system the submarine could remain submerged minimally for 6.5 days with a total distance of 3500 km compared to an only 20 h submerged period for the conventional system [Submarines are valuable assets for naval applications owing to their invisibility when submerged under water ,107. Unfed gases ,109. Liqed gases . System ed gases . Further2O2 are also investigated as high-density power sources for autonomous underwater vehicles [2O2 is a thousand times denser than oxygen [4/H2O2 system [Apart for space applications, direct borohydride fuel cells (DBFCs) in combination with Hvehicles ,111,112.n oxygen . The US 2 system . Protone2 system .4 is non-reversible and recycling of the byproduct sodium metaborate (NaBO2) becomes critical. A cheap and efficient route for recycling the spent fuel  still has to be developed. For NaBH4 decomposed in water solution, its regeneration starts with dry NaBO2. Therefore, the dissolved NaBO2 has to be dried, which is an energy intense process. NaBH4 can be recovered from NaBO2 through ball milling using MgH2/Mg. Here, the small particle size and high surface area increase the yield of the reaction [NaBHreaction ,112,115.et al., developed a regenerative fuel cell, which can transform the end products back into the reactants, using an aqueous NaBH4 solution and H2O2 [4 and H2O2 (Equation (10)), which occurs at 2.23 V, is in competition with the electrolysis reaction (Equation (11)) that occurs at 1.23 V.For the direct borohydride/peroxide fuel cell, Miley and H2O2 . PreventThe authors circumvented this issue by using high over-potential materials for the electrodes and exploring the formation of perborate that hydrolyze to peroxide at 1.7 V (Equation (12)), greatly reducing the gas evolution.4 crossover, a critical issue often faced for DBFCs, can be reduced by improving the membrane electrode assembly or the liquid diffusion layer [4 hydrolysis systems for portable applications, additional research is needed to reduce the cell volume, increase the gravimetric hydrogen storage capacity and reach efficient recycling of the spent fuel.Furthermore, the inhibition of the NaBHon layer . In ordeet al., were the first to report on the design and testing of an ammonia borane hydrogen power-pack, which operated an UAV for 57 min using a 200 W PEM fuel cell. The system was operated by delivering spherical solid ammonia borane beads into a semi-batch type reactor filled with tetraethylene glycol dimethyl ether, a liquid promoter. Hydrogen purification equipment with a high filter capacity and efficient drainage system for spent fuel were also integrated for application purposes. The hydrogen generation system was the heaviest part of the hydrogen power pack taking up 64% of the total weight, whereas the fuel cell stack took up only 17.6% and the ammonia borane beads 4%. Based on the obtained results, the authors proposed an advanced reactor concept  as well as the tank design . Optimizations were then proposed on the basis of the simulation results. Several modeling tools have been built on the basis of the hierarchical methodology and resistance analysis in order to estimate performances of the system and the limiting factors. The simulations showed that a good thermal management was necessary for the absorption, whereas the pressure control was important for desorption. Still, differences exist between the simulations, which come from the choice of the kinetic model that is implemented as the governing equation for the simulations. Therefore, precautions have to be taken, since the kinetic model is built through experimental considerations. Heat transfer in the metal hydride tank can be improved using heat exchangers, multi-tubular tank geometries and heat transfer enhancers. However, this will also increase the weight of the system. Hence, optimizations are often a compromise between heat transfer and hydrogen content. The strategy based on CH/MH beds could provide a new step in tank design leading to better temperature and pressure management without decreasing the hydrogen capacity excessively.Although ample research has been performed with complex metal hydride materials on small-scale laboratory batches, considerably fewer full-scale thermolysis tanks/systems have been developed and analyzed. Improving the ratio between the mass of the complex metal hydride bed to the mass of the tank wall, by screening lighter materials for the tank wall and developing hydrogen storage materials exhibiting both higher gravimetric and volumetric storage properties, should be a goal in order to obtain lightweight storage systems. There are several design principles that must be taken into account while developing a complex metal hydride storage tank, such as packing arrangement of the material, hydrogen supply, heat transfer to the heat transfer medium, effective heat conductivity of the metal hydride bed and volume expansion. Additionally, efficient removal of the reaction heat from the metal hydride tank during refueling and how to provide heat during hydrogen delivery to the fuel cell remain an unsolved problem. Thermolysis of complex metal hydrides has yet to demonstrate the fulfillment of the DoE requirements . Especia4, it requires the recycling of NaBO2 back into NaBH4, either starting from dry NaBO2 or through the use of a regenerative fuel cell. This often means additional costs cannot be avoided. Hence, the use of NaBH4 for portable applications becomes less attractive. As the byproduct from the hydrolysis reaction can decrease the catalyst efficiency, flow reactors holds the biggest potential for reaching commercial applications as these allow to collect the byproduct in a separate tank [In hydrolysis, recycling of the spent fuel remains the single biggest challenge. For NaBHate tank ,100 The 4 and AB) have found its way into UAVs and submarine applications, the majority of these applications are currently still driven by fossil fuels. These application may benefit from the installation of filters to obtain desired the hydrogen purity, efficient drainage of spent fuel, and operation conditions . All these improvements may affect the fuel cell operation and consequently, the success of the application. Additionally, further research is needed to create a more straightforward hydrolysis system preferably with on-board recycling of the spent fuel and more robustness towards operation conditions. Indeed, the advantages such as silent mode of operation and thermal signature can be quickly overshadowed by disadvantages such as limited space in a UAVs and the weight of both the energy storage system and the auxiliary system . Therefore, any weight reduction of the hydrolysis technology that can be achieved will translate directly into an increase in flight duration.Although hydrolysis technology using complex metal hydrides (e.g., NaBHAfter 20 years of intensive research on complex metal hydrides, a widespread commercial penetration of these hydrogen storage materials in technical applications is not in sight for the next years. The research attention over the last years has been given to fundamental understanding of crystal structures, reaction kinetics and thermodynamics of these materials as well as development of materials with high hydrogen content. The development of tank systems and demonstration projects with complex metal hydrides has not always been part of the past research activities because of the challenges that come along with engineering development.Several technical barriers still exist for successful implementation of solid-state hydrogen storage systems based on thermolysis for stationary and portable applications and remain a significant challenge for transportation applications. Currently available storage tank systems typically require large volume, high weight, and have slow hydrogen charging/discharging rates and high system cost. Long-term research goals for the complex hydride materials should be to improve the gravimetric and volumetric capacities, to obtain sufficient kinetics within appropriate temperatures and pressure ranges and to lower the cost of the overall systems.It is obvious that commercial success is not only a question of high hydrogen content of hydrogen storage materials. The overall performance of the whole system, consisting of hydrogen storage material, as well as storage tank design and integration of all the components into a technical system, plays a much more important role than one particular property. Taking this into account, engineering developments for the optimization of solid-state hydrogen storage tank systems must be intensified to overcome technical barriers and to construct safe and consumer-friendly system based on renewable energy carriers.


Drosophila melanogaster as a model organism to explore olfactory function in LRRK loss-of-function mutants, which was previously demonstrated to be a useful model for PD. Surprisingly, our results showed that the LRRK mutant, compared to the wild flies, presents a dramatic increase in the amplitude of the electroantennogram responses and this is coupled with a higher number of olfactory sensilla. In spite of the above reported results, the behavioural response to olfactory stimuli in mutant flies is impaired compared to that obtained in wild type flies. Thus, behaviour modifications and morphofunctional changes in the olfaction of LRRK loss-of-function mutants might be used as an index to explore the progression of parkinsonism in this specific model, also with the aim of studying and developing new treatments. Parkinson's disease (PD) is one of the most common neurodegenerative diseases characterized by the clinical triad: tremor, akinesia, and rigidity. Several studies have suggested that PD patients show disturbances in olfaction as one of the earliest, nonspecific nonmotor symptoms of disease onset. We sought to use the fruit fly The olfactory system represents the most common and ancient sensory system within the animal kingdom, from single-celled organisms through higher animals , and oneAlthough it is now clear that PD is associated with a combination of risk factors, including environmental noxious agents and genetic predisposition , which o LRRK2 gene have been described: G2019S and G2385R [ LRRK2 G2019S mutation carriers [Furthermore, regarding genetic predisposition, only a limited percentage is due to monogenic forms of the disease . Among td G2385R . To dateAlthough the evidence about LRRK2 role in inflammatory processes and in the endolysosomal system and cytoskeleton impairment has been identified , its invSome variability has also been observed regarding nonmotor symptoms, where LRRK2 G2019S olfactory phenotype may be less pronounced than idiopathic PD and possibly G2385R mutation, and it is not clear that all G2019S mutation carriers have olfactory impairment –36.Given the above reported phenotypic variability in PD olfactory dysfunction, it seems of particular interest to have a simple model to study this nonmotor symptom, particularly in a LRRK2 mutant.The LRRK2 gene coding for unusually large protein composed of 2527 amino acids is widely expressed in the brain and other organs –40. The LRRK2 is a member of the ROCO protein family characterized by the presence of two conserved domains: a Roc (Ras in complex proteins) domain belonging to the Ras/GTPase superfamily and a COR domain . Three further conserved domains have been described: a leucine-rich repeat (LRR); a tyrosine kinase catalytic domain (MAPKKK); and finally a WD40 domain . This la Drosophila LRRK2 loss-of-function mutant for deletion of the domain WD40  sho mutant,  in anoth Drosophila LRRK2 model of PD correlated to LRRK2 mutation (LRRKWD40). This model most closely approximates human LRRK2 G2385R mutations, which occur in the human WD40 domain and are a risk factor for PD. The results showed that the LRRKWD40 mutant presents an unexpected dramatic increase in the amplitude of electrophysiological responses, but a decrease in the olfactory discrimination, with respect to wild type flies (WT).On this basis, the aim of this study was to estimate any possible olfactory dysfunction in theWD40 mutant  Drosophila melanogaster (Dm) males were used. Soon after emergence from pupae, WT or LRRKWD40 mutant males were separated from females. WT and mutant flies were reared on a standard cornmeal-yeast-agar medium in controlled environmental conditions . Flies ranging 10–15 days in age were tested according to previous experiments [For these experiments adult wild type  and LRRKeriments . in vivo as previously described [Electroantennogram (EAG) recordings were performedescribed , 48.μL truncated plastic pipette, with the head protruding at the tip. The preparation was fixed with dental wax on a microscope slide and positioned under the viewer of an Olympus BX51WI light microscope . Glass capillaries with a silver wire were filled with a conductive 0.15 M NaCl solution. The recording glass electrode was gently placed on the tip of the antennal funiculus, whereas the reference electrode was inserted in the compound eye. The EAG signal was amplified with an AC/DC probe and then acquired with an IDAC-4 interface board . A charcoal purified and humidified airflow was constantly blown over the antennae (speed 0.5 m/s) via a glass tube, placed approximately 1 cm from the antenna. The tip of a Pasteur pipette containing an odour-loaded filter paper (5 mm × 25 mm) was inserted into a small hole in the glass tube. Odour stimulation was administered by injecting a puff of purified air (0.5 s at 10 mL/s airflow) through the pipette using the stimulus delivery controller (Syntech).By taking into account the circadian cycle in olfactory sensitivity , WT and  Drosophila [Odour stimuli tested were dissolved in hexane and presented in series from minor to higher concentration . 1-Hexanol was chosen for its well-known stimulant activity inosophila –52, and osophila . As the WD40 mutant groups with the age-matched WT control group.Mean values of EAG amplitude were calculated and then analysed by comparing the results obtained in LRRK post hoc test) with a threshold level of statistical significance set at P ≤ 0.05 . EAG results are expressed as average values + SEM and represented by histograms.The significance of differences was tested by repeated-measures ANOVA or one-way ANOVA . Two 4 mL glass vials were placed symmetrically and equally spaced in a large Petri dish (the arena) and then fitted with truncated pipette tips. The vials were filled with 300 μL of water with 0.25% Triton X with or without the odorant. On the basis of the EAG response, the odour dilution chosen to trap the flies was 0.1% for both compounds. The dehydration of flies was prevented by placing a cotton ball with 3 mL of water in the arenas. Flies were starved for 9 hours prior to starting the experiments. Twelve bioassays were carried out for each experimental group of flies (15 flies per arena) and for each odour source and then replicated three times. Bioassays were performed in controlled environmental conditions and lasted 18 hours [T − C)/(T + C + NR − D), in which T is the number of flies in the treatment, C is the number in the control, NR is the number of living flies remaining in the arena, and D is the number of dead flies.Free-walking bioassays were performed following the experimental procedures used by Dekker et al. . Briefly18 hours  that com18 hours . The attt-test  with a 95% confidence level.Data obtained were expressed as average of percentages of flies reaching the treatment vial  or the control vial (water) and statistically evaluated by means of Student's WD40 mutant males was determined by counting the number of different types of sensilla present in a sample area enclosed by an electronic square frame  applied to the SEM screen in the central part of the flagellum. This area of the flagellum was chosen since it was plain displayed in all samples and the arrangement of the antennal structures allowed to count easily the number of each type of sensilla included by the frame. Moreover, in this area all kinds of sensilla were present, with the exception of one type (large basiconic sensilla) located mainly in the basal part of the flagellum [ t-test with a 95% confidence level . Higher magnification was used to study in detail the morphology of different types of sensilla.For scanning electron microscopy (SEM), 4–6-day-old flies were anesthetized with carbon dioxide, immediately immersed in hexane, shaken for 3 minutes to remove the external wax layer, then dehydrated in a graded ethanol series for ten minutes each concentration up to absolute ethanol, subsequently air-dried, and finally glued to stubs and gold coated. At least ten specimens were prepared and observed using a FEI Quanta 200 high-vacuum SEM at the Department of Tree Science, Entomology and Plant Pathology “G. Scaramuzzi,” University of Pisa. The density of antennal sensilla in both WT and LRRKlagellum . SensillWD40 mutants, the repolarization phase in LRRKWD40 being slower than WT.As shown in Figures WD40 mutants were higher than those obtained in WT specimens, exhibiting a dose response for both stimuli administered, although this tendency was more evident for 1-hexanol. In detail, LRRKWD40 flies showed a significant increase (P < 0.05) in the EAG amplitudes with respect to WT after stimulation with 1-hexanol and 1-linalool for all concentrations tested, except the lowest  in response to 1-hexanol, while the number of insects trapped in the blank did not differ in response to 1-linalool. Moreover, in the latter, no difference exists between mutant insects trapped by the odour with respect to the blank.As for the water response, the numbers of trapped flies in the blank baits were higher for LRRKThe unexpected increase in EAG response combined with the impairment of the behavioural response of mutants with respect to WT led us to investigate any possible variation in the antennal olfactory apparatus.WD40 flies in the gross structure. As shown in According to our results, no differences are found between WT and LRRKWD40 mutants.However, as shown in WD40 mutants males exceeded the WT number with highly significant difference (P ≤ 0.001). Similarly, also small basiconic and grooved sensilla were present in LRRKWD40 mutants males with a slightly higher density than in the WT males (P ≤ 0.05).In detail, as a general pattern, mutants show a higher number of all sensilla in the considered area than WT flies, although these differences are evident only at high magnification . As a ma Drosophila melanogaster any possible olfactory dysfunction correlated to a specific LRRK2 mutation (LRRKWD40) [ Drosophila model [The aim of this study was to estimate in the PD translational modelRRKWD40) , by analla model , 46.The used LRRK2 translational model most closely approximates human LRRK2 G2385R mutation, which occurs in the human WD40 domain and is a risk factor for PD, mainly in Asian populations .B9 [WD40 mutant, compared to the WT, presents an unexpected dramatic increase in the amplitude of the EAG responses and this is coupled with a higher number of olfactory sensilla.Contrary to PINK1B9 , where tB9 [WD40 mutants present an impairment in the behavioural response to odours.Nevertheless, similarly to what was observed for PINK1B9  also theWD40 shows an increase in EAG response at any concentration to both tested stimuli , at the lowest one, no differences in threshold were found between mutants and WT. Despite the great variability in the sensilla number in different strains [WD40 compared to WT, mostly in consideration of the relevant number of trichoid sensilla which can cause an increased summed antennal sensitivity. Further, we cannot exclude a role of the perireceptor environment in the chemoreceptor response. In this respect, it is possible that in mutants more odorant binding proteins could be present and/or interact with the odorants more tightly, but the presence of a diminished concentration of the odour degrading enzymes is also possible [olf) that was first identified in the olfactory epithelium [ Drosophila antennae.In detail, even if LRRK strains , we cannpossible –58. Accopossible  found inithelium , 61 homoMore recently, Yun and Park , analysiWD40 mutants trapped is lower than WT regardless of the stimulus tested. As for the functional significance of the two stimulants, 1-hexanol was reported to involve both the appetitive and the aversive response in flies, even if with great variability in its effects [Looking at the behavioural response to the odours, 1-hexanol attracts a number of insects statistically higher than 1-linalool, in both WT and mutant flies, but the number of LRRK effects , 63, and effects , 53, 64. effects . We cann effects  but thatAs for the response to water, in our opinion, results showing that LRRK2 mutants are trapped by water in the 1-hexanol experiments and especially the absence of difference between water and the odour in the case of 1-linalool point to an impairment in the discrimination capability of LRRK2 mutants. In fact, even if mutants present a higher electrophysiological response to both stimuli with respect to WT, the percentage of mutants which goes to the odours under a double-choice situation is about half that of WT flies. These results can be attributable to an impairment we found in the mitochondrial morphology in the antennal lobes , where o Drosophila WT males showed the presence of four types of sensilla as previously reported [WD40 mutant did not display noticeable gross morphological differences with respect to the WT, but a general increase in the number of sensilla in the central part of the flagellum was highlighted. As far as we know, no data are available to correlate the number of sensilla with LRRK function; we recall that LRRK2 is known to be involved in cytoskeleton assembly [ lozenge Drosophila mutants showed changes at different extent in morphological features of antennal sensilla, including a reduction or an increase in the number and/or lack of some sensilla [Our observations on antennal flagellum ofreported  and mappreported ; furtherassembly , but howsensilla .WD40.Our functional approach to merging olfactory sensory electrophysiology with behavioural response was used to go in depth in the analysis of odour detection with the aim of considering olfaction as a diagnostic marker for PD in the LRRKWD40 mutants present an impairment in the behavioural response to odours. In this respect in a pioneeristic work on human odour stimuli detection in correlating electrophysiological activity from human olfactory bulb and the subjective response to stimuli, Hughes et al. [Our results show that, despite the dose-response relationship and the unexpected high EAG amplitude which point to a “normal” olfactory function, the LRRKs et al.  stated “ Drosophila PD models such as LRRKWD40 and PINK1B9 [ Drosophila is a powerful model also for the LRRK2 loss-of-function variant.In conclusion, our results suggest that olfactory behavioural impairment is a common feature for two PINK1B9  despite


Patients with human epidermal growth factor receptor 2 (HER2) -positive metastatic tumors treated in the public health system in Brazil do not have access to trastuzumab. This study aimed to estimate the impact of the lack of access to anti-HER2 therapies on the mortality of these patients.On the basis of published data, the number of patients with HER2-positive advanced breast cancer in 2016 who should receive anti-HER2 targeted therapy was estimated. Three different treatment groups were considered for this hypothetical cohort: chemotherapy alone, chemotherapy plus trastuzumab, and chemotherapy plus trastuzumab and pertuzumab. The number of patients alive after 2 years of follow-up was estimated on the basis of the efficacy results of the pivotal trials considering these interventions.It was calculated that 2,008 women will be diagnosed with advanced HER2-positive breast cancer in Brazil in 2016. It was estimated that only 808 women would be alive in 2018 if they receive only chemotherapy . On the other hand, the bar rises to 1,408 women alive in 2018 if they receive chemotherapy plus trastuzumab and 1,576 women alive in 2018 if they receive the gold standard of chemotherapy plus trastuzumab and pertuzumab.Trastuzumab is included in the WHO’s list of essential medications, but the Brazilian public health system does not yet provide this treatment to its population with advanced disease. The introduction of trastuzumab and pertuzumab would have a positive effect, preventing premature deaths in women with metastatic HER2-positive breast cancer in Brazil. Cancer is the second leading cause of death in the country after cardiovascular disease.2 The Brazilian National Cancer Institute (INCA) estimates there will be 57,960 new cases of breast cancer in 2016,3 and approximately 15,000 Brazilian women die as a result of breast cancer each year.4 Access to health care delivery is associated with cancer outcomes.5Breast cancer is the most common cancer in women worldwide, and 70% of breast cancer deaths occur in women from low- and middle-income countries.6 It is now clear that the advent and routine use of targeted anti-HER2 therapies have dramatically improved disease control and survival in patients with HER2-positive breast cancer. The list of essential medicines of the WHO includes trastuzumab both for early-stage as well as for metastatic HER2-positive breast cancer.7 The world cancer nongovernmental community, represented at World Health Assembly by the Union for International Cancer Control board members, is playing a key role in decreasing inequity in health care services. For several years, national advocacy patient coalitions, through collaborative initiatives, have been trying to influence public policies for the access to innovative therapies for patients with advanced breast cancer, with no success.8Roughly 15% to 20% of human breast cancers are classified as human epidermal growth factor receptor 2 (HER2) positive, a subgroup of tumors with a more aggressive clinical course and worse prognosis. The discovery of HER2 led to the development and approval of the first HER2-targeted therapy, trastuzumab.Within Brazil’s public health system, which covers approximately three-fourths of the country’s population, access to HER2-targeted therapy is restricted. The current internationally accepted standard of care for first-line therapy in metastatic HER2-positive breast cancer is trastuzumab plus pertuzumab plus chemotherapy. Up to 2 years ago, the standard had been trastuzumab plus chemotherapy for more than a decade. In Brazil, trastuzumab has been available for the adjuvant treatment of patients with early-stage disease in the public health system only since 2013, but it is yet to be offered for the treatment of women with advanced disease. Patients with private insurance (including one-fourth of the population) do have access to all approved anti-HER2 agents available, which include trastuzumab, pertuzumab, trastuzumab emtansine (T-DM1), and lapatinib. Lack of access to effective therapy is a common issue in low- and middle-income compared with high-income countries. The consequences of this discrepancy can be estimated.This study aimed to estimate the impact of the lack of access to anti-HER2 therapies on the mortality of patients with advanced HER2-positive breast cancer treated in the public health system in Brazil.3 were used to define the incidence of breast cancer in Brazil. According to official information from the Instituto Brasileiro de Geografia e Estatística,9 73.7% of the population is covered under the public health system, and 27.3% has some form of private insurance.The statistics published by INCA10 we assumed that approximately 20% of patients have HER2-positive disease and that 94% present with stages I to III, whereas 6% are diagnosed with stage IV disease. Assuming a constant incidence within the last 5 years, we calculated the number of women diagnosed with metastatic breast cancer in 2016 as the sum of the de novo diagnoses in 2016 plus the estimated recurrences in patients diagnosed in the last 5 years. Recurrence rates for patients diagnosed with stages I to III HER2-positive breast cancers treated with chemotherapy plus trastuzumab were calculated based on published data from the HERA trial.12 Although in the public health system, trastuzumab has only been used for early-stage disease since 2013, we assumed a conservative recurrence rate as if all patients had received trastuzumab-based (neo) adjuvant therapy.On the basis of the findings of the epidemiologic study AMAZONE,We considered three potential treatment groups for this analysis. Patients could be treated with chemotherapy alone, chemotherapy plus trastuzumab, or chemotherapy plus trastuzumab and pertuzumab.6 in 2001. After the publication of this trial, the use of chemotherapy alone was no longer included as part of any randomized clinical trial in this setting because it was considered unethical in view of the superiority of the trastuzumab-containing arm. Survival data for the other two groups, chemotherapy plus trastuzumab versus chemotherapy plus trastuzumab and pertuzumab, were based on the results of the CLEOPATRA trial.13Survival data for the chemotherapy alone arm were based on the results of the randomized clinical trial published by Slamon et alINCA estimates there will be 57,960 new cases of breast cancer in 2016, of which 73.7%  will be covered by the public health system. According to the AMAZONE trial, roughly 20% of these cases are HER2-positive , of which 94%  present with stages I to III disease and 6% (513) with stage IV.12 These data are summarized in We estimated that 2,008 women would be diagnosed with HER2-positive metastatic breast cancer in 2016. This number was obtained by adding the patients presenting with stage IV disease in 2016 (513 women) to those developing a recurrence of the disease after receiving (neo) adjuvant treatment  in the previous 5 years. The number of recurrences within the last 5 years was estimated on the basis of the results of the HERA trial.13 and the trial published by Slamon et al6 in 2001, median overall survival for the treatment groups was estimated as follows: 20.3 months for chemotherapy alone; 40.8 months for chemotherapy plus trastuzumab; and 56.5 months for chemotherapy plus trastuzumab and pertuzumab.On the basis of the results of the CLEOPATRA trialConsidering 2,008 women with metastatic HER2-positive breast cancer in 2016 and on the basis of the median overall survival results, mortality rates of 50, 25, and 18 deaths per month were estimated for the treatment arms chemotherapy alone, chemotherapy plus trastuzumab, and chemotherapy plus trastuzumab and pertuzumab, respectively.Applying these monthly mortality rates to the next 24 months, we estimated that of the 2,008 women with metastatic disease in 2016, 808 women would be alive after 2 years of follow-up if treated with chemotherapy alone. If these same women were treated with chemotherapy plus trastuzumab , the number of patients alive after 2 years would increase to 1,408. This represents an absolute gain of 600 more women alive after 2 years as the result of treatment with trastuzumab. Furthermore, treatment with chemotherapy plus trastuzumab and pertuzumab would result in a total of 1,576 women alive after 2 years, an absolute gain of 768 patients. 14 as a driver of disease biology in up to one-quarter of breast cancers represents a paradigm shift in the way this malignancy is understood. It is now widely recognized that breast cancer comprises heterogeneous subtypes that require different treatment approaches.15 The successful development of trastuzumab, the first drug to target HER2-positive cancer, validates the concept that we can improve outcomes by treating an underlying molecular driver. In the past decade, three other HER2-directed therapies—lapatinib, pertuzumab, and T-DM1—have earned regulatory approval based on consistent and reproducible data indicating benefits both in the metastatic and early-disease settings.16Major efforts and achievements in medical research in the last decades have led to a better understanding of the complex molecular heterogeneity of cancer in general and breast cancer in particular. Arguably, the concept of heterogeneity is one of the important contributions to our approach to the disease. The discovery of the HER2 alteration18 The advent of anti-HER therapies has significantly overridden this poor prognosis, so that patients with HER2-positive metastatic breast cancer treated with modern HER2-targeted therapies now experience outcomes comparable, if not superior, to their counterparts with HER2-negative disease.19Several studies have identified HER2-positive breast cancers as having a more aggressive biology and being associated with inferior outcomes, including shorter time to disease progression and shorter overall survival.6 More recently, patients treated with chemotherapy and trastuzumab in the CLEOPATRA trial had a median overall survival of > 40 months. In the same trial, patients who received double anti-HER blockade with pertuzumab and trastuzumab in combination with taxane chemotherapy demonstrated an overall survival of > 56 months.13 These clinically significant improvements in outcomes have been associated with acceptable toxicity profiles, as demonstrated by patient-reported outcomes from randomized clinical trial data. In particular, long-term administration of anti-HER2 monoclonal antibodies has been found to be much more tolerable than protracted chemotherapy. Routine follow-up of cardiac function, albeit the most serious potential adverse event, leads to early detection of cardiac dysfunction, allowing for appropriate and safe management of most affected patients. Recognizing these significant benefits, the WHO included trastuzumab in its list of essential oncology medicines.Pivotal trials illustrate the evolution of the treatment of advanced HER2-positive breast cancer in terms of overall survival benefits. In the late 1990s, patients who were randomly assigned to chemotherapy had a median overall survival of 20.3 months.20 Among other factors, financial status and access to health care delivery are associated with cancer outcomes. Striking income disparities are identified in Brazil. It is understandable that wealth distribution in the country affects the distribution of disease, access to health care, and consequently the overall health and disease-specific survival rates of the population. Several factors that explain some of these disparities in outcome have been previously analyzed. These include delays in diagnosis as a result of low awareness of cancer and implementation of mammography screening, and treatment-related issues such as quality of surgery as well as limited access to radiotherapy and modern systemic therapies.1Five-year breast cancer survival rates vary from approximately 80% in high-income countries to 60% in middle-income countries and 40% in low-income countries.21The Brazilian health system has two major components: the public system, Sistema Único de Saúde (SUS), with funding provided by the Ministry of Health, and the supplemental or private system, comprising voluntary and noncompulsory health plans and insurances. Approximately 73% of the population has access to health care only through SUS.22 Once registration is granted through this process, most drugs (with few exceptions) become available to patients in the private health system. For patients in the public system, however, an additional approval step where cost-effectiveness analysis is performed by another commission, Comissão Nacional de Incorporação de Tecnologias (CONITEC), is necessary. Most modern high-cost medications evaluated by CONITEC have not been approved for use by patients in the public system on account of allegedly insufficient scientific evidence of benefit, lack of cost effectiveness, or incomplete documentation on the request file.For a new drug to be approved in Brazil, it must follow a strict regulatory process coordinated by the Agencia Nacional de Vigilância Sanitária (ANVISA).23Anti-HER2 medications are a clear example of such misguided decisions. Patients in the private system have had access to trastuzumab (since 1999), lapatinib (since 2007), pertuzumab (since 2013), and T-DM1 (since 2014). In the public system, patients with early-stage HER2-positive breast cancer have had access to trastuzumab for only the last 3 years; those with metastatic disease still do not receive any form of targeted anti-HER2 therapy, even though reproducible and consistent benefits have been clearly demonstrated for almost two decades. Estimation of the number of deaths as a consequence of the late introduction to adjuvant trastuzumab has previously been presented.22 delays in treatment, and insufficient radiotherapy. In 2006, women with HER2-positive breast cancer were 10 times more likely to receive trastuzumab if they were privately insured. At the time, only 6% of patients with HER2-positive breast cancer received trastuzumab in the public system versus 56% in the private sector.24 The more advanced stage at diagnosis, coupled with insufficient access to modern systemic therapies, dooms patients to a worse survival outcome when compared with those with private health coverage.This enormous gap in the quality of breast cancer treatment between public and private settings is yet another cause of the significant disparities on top of late diagnosis,25 The legal basis for these litigations is the Brazilian constitution, which guarantees universal medical care to all Brazilian citizens.26 A second partial solution has been found by some state governments, who supplement the list of cancer therapies with a few high-cost modern therapies (including trastuzumab). These local solutions end up being only temporary and partial.One alternative patients have sought while facing these restrictions to access is to file specific lawsuits against the government to receive trastuzumab, a common situation in almost all Latin American countries.27 The lack of access to potentially beneficial treatment has consequences that can be estimated.Although trastuzumab received ANVISA’s approval for treatment of metastatic HER2-positive disease in 1999, it still has not become available for patients in the public system. Its approval for the treatment of early disease was granted in 2007. Patients with private insurance have had access to treatment since that date, but those with public coverage have only had access to trastuzumab in the adjuvant setting since the end of 2012.13 led to a change in the gold standard treatment of HER2-positive advanced breast cancer: dual blockade with trastuzumab and pertuzumab. According to our analysis, if dual HER2 blockade plus chemotherapy were used, the difference in the number of women alive after 2 years would be even higher: 768 patients.Our results show that approximately 2,008 women will be diagnosed with metastatic HER2-positive breast cancer in 2016. On the basis of our estimates, if treated with chemotherapy without any HER2-targeted agents, only 808 of these women will be alive in 2018. On the other hand, if trastuzumab is incorporated, 1,408 of them are expected to be alive. The availability of trastuzumab in the public health system for patients with metastatic disease would result in an additional 600 women alive at the end of 2 years. Making this situation even more dramatic, the publication of the CLEOPATRA trialWe acknowledge that delivering on the promise of precision medicine to all patients will require a massive effort, not only from doctors and researchers, but also from pharmaceutical companies and governmental agencies, among others. At the same time, it is imperative to recognize that the consequences of depriving a significant proportion of patients from low- and middle-income countries of effective therapy can be estimated. This exercise should be an important component to be taken into account when discussing what will or what will not be offered as treatment.This analysis has significant limitations. Specific data from low- and middle-income countries are sparse and under-reported, meaning we had to rely mostly on estimates from INCA. Additionally, it is important to state that we are extrapolating survival differences directly from randomized clinical trials, and it is possible that these differences are less pronounced in general clinical practice. This assumption might introduce a bias favoring the difference between treatments in real life. On the other hand, because trastuzumab was introduced in the adjuvant treatment only in 2013, our estimates are conservative by assuming a lower relapse rate than that of patients who do not receive trastuzumab in this setting. In conclusion, 15 years after the worldwide approval of trastuzumab for metastatic breast cancer and 1 year after its inclusion in the WHO essential medicines list, the Brazilian public health system does not yet provide this treatment to its population. The introduction of trastuzumab and pertuzumab would significantly increase the life span of women with metastatic HER2-positive breast cancer in Brazil and prevent 768 premature deaths in the next 2 years.