Calidris pusilla) is a long-distance migrant that breeds in the arctic tundra of Canada and Alaska and winters on the northeast coast of South America. Its fall migration includes a 5,300-kilometer nonstop flight over the Atlantic Ocean. The avian hippocampus has been proposed to play a central role in the integration of multisensory spatial information for navigation. Hippocampal neurogenesis may contribute to hippocampal function and a variety of factors including cognitive activity, exercise, enrichment, diet and stress influence neurogenesis in the hippocampus. We quantified hippocampal neurogenesis and volume in adult migrating and wintering semipalmated sandpipers using stereological counts of doublecortin (DCX) immunolabeled immature neurons. We found that birds captured in the coastal region of Bragança, Brazil during the wintering period had more DCX positive neurons and larger volume in the hippocampus than individuals captured in the Bay of Fundy, Canada during fall migration. We also estimate the number of NeuN immunolabeled cells in migrating and wintering birds and found no significant differences between them. These findings suggest that, at this time window, neurogenesis just replaced neurons that might be lost during the transatlantic flight. Our findings also show that in active fall migrating birds, a lower level of adult hippocampal neurogenesis is associated with a smaller hippocampal formation. High levels of adult hippocampal neurogenesis and a larger hippocampal formation found in wintering birds may be late occurring effects of long distance migratory flight or the result of conditions the birds experienced while wintering.Long distance migratory birds find their way by sensing and integrating information from a large number of cues in their environment. These cues are essential to navigate over thousands of kilometers and reach the same breeding, stopover, and wintering sites every year. The semipalmated sandpiper ( The semipalmated sandpiper is a long-distance migrant that breeds in the arctic and undertakes an annual fall migration to South America. Geolocation tracking of a semipalmated sandpiper that bred on Coats Island in the Canadian arctic showed a six-day, non-stop flight from a stopover site on James Bay to the Orinoco Delta on the border of Venezuela and Guyana, followed by 11 days of further movement to northeastern Brazil where it spent the winter .The avian hippocampus plays a central role in spatial ability and spatial memory in birds as shown by single cell recording , 4, the Experiments with immediate early genes (IEGs) have showed significant changes in hippocampal activation patterns confirming the hippocampus role in navigation –30. IndeThere are many proposed functions for adult hippocampal neurogenesis. It may provide new neurons to encode new memories  or promoBecause many of these multivariate influences are present in long distance migration we hypothesized that neurogenesis in the hippocampal formation of migrating birds would be higher than that of wintering birds. Indeed, we expect that the negative influence of the extenuating exercise associated with the long distance migratory flight would be less intense than the other positive influences and this would upregulate neurogenesis. To test this hypothesis, we compared the number of new and adult neurons in the hippocampus of semipalmated sandpipers captured during fall migration in August at stopover in the Bay of Fundy, Canada, with that of individuals captured while wintering in the northeastern coastal region of Brazil near Bragança, Pará.C. pusilla  were collected in August 2012 at the Bay of Fundy, Canada (45°50'19.3" N and 64°31'5.39"W), we used C. pusilla 01, 02, 03, 04, 05 for DCX positive cell counts and volume estimation and C. pusilla 01, 11, 12, 03, 13 for NeuN cell counts. Other five wintering individuals  were captured between November and March on Canela Island , in the tropical coastal zone of northern Brazil (00°47’09.07” S and 46°43’11.29” W) and these were used only for DCX counts and volume.A total of thirteen individuals were used, eight migrating Although the number of birds are different all statistical comparisons, were made between groups of 5 (five) individuals (except for NeuN counts extracted from previous report ) assuminBirds were captured under license N° 44551–2 from the Chico Mendes Institute for conservation of Biodiversity (ICMBio) and Scientific Capture permit ST2783 from the Canadian Wildlife Service. All procedures were carried out in accordance with National Institutes of Health (USA) and Brazilian regulations for scientific procedures on animals and with approval of the Animal Users Subcommittee of the University of Western Ontario. All efforts were made to minimize the number of animals used and the stress and discomfort to animals.Immediately after capture (still on the field) and under deep isoflurane anesthesia, birds were perfused transcardially with phosphate buffered saline 0.1M followed by aldehyde fixatives . Brains were dissected, stored in phosphate buffered saline 0.1M and cut by Vibratome (Leica VT1000S) or freezing microtome  in the coronal plane. Six anatomical series of sections , cut at 60 or 80μm in the freezing microtome or vibratome respectively, were immunolabeled for NeuN or DCX or stained by Nissl. Free-floating sections were immunolabeled with anti-doublecortin antibody (Santa Cruz SC-8066) or NeuN antibody , and mounted on glass slides coated with an aqueous solution of gelatin (10%) and chromium potassium sulfate (0.5%). Sections were air-dried at room temperature, dehydrated, cleared in alcohol/xylene series and covered with 50% entellan  diluted in xylene and cover slipped.Free-floating sections were pre-treated with 0.2 M boric acid (pH 9) at 70°C for 60 min as an antigen retrieval method, washed in 1% phosphate buffer/saline/triton (PBST) and washed three times for 2 min each time in PBS. Sections were then immersed for 12 hours in 10% Normal Horse Serum Blocking Solution S-2000, Vector Laboratories (for DCX slides) and 10% Normal Goat Serum Blocking Solution S-1000 Vector Laboratories (for NeuN slides) transferred to the primary antibody  diluted in PBST 0.3% (1:500) and incubated for 12h at 4°C with gentle and continuous agitation. Washed sections (PBST 1%) were then incubated overnight in the secondary antibody  and Biotinylated Goat Anti-Mouse IgG Antibody, BA-9200, Vector Laboratories (for NeuN), 1:400 in PBST 0.3%) followed by 0.3% hydrogen peroxide for 15 minutes, washed three times in PBST for 2 min each time, then immersed in avidin-biotin-peroxidase complex (ABC) solution  for 60 minutes. After a 2 min wash in PBS, sections were reacted to visualize DCX immunolabeled neurons using the glucose-oxidase-DAB-nickel method. As a control of the immunohistochemical labeling patterns we omitted the primary antibody and confirmed that the secondary antibody did not produce any unspecific labeling .We defined the sandpiper hippocampal formation as comprising the hippocampus proper and the parahippocampal area . To measAfter selective DCX and NeuN immunolabeling, we determined the number of neurons. We did not distinguish between migratory (elongate morphology) and recruited  phenotypes , 53. We We used a digital camera  coupled to a NIKON Eclipse Ci microscope to capture digital images which were processed with Adobe Photoshop software for illustrations shown in Figs Calidris pusilla. Doublecortin was broadly expressed in the telencephalon of adult C. pusilla wintering individuals showed on average, 2.4 times more DCX immunolabeled cells than C.pusilla migrating birds ; two-tailed t-test, df = 7 t = 2.36 p<0.00). As expected the density values of DCX immunolabeled cells showed that wintering birds had 1.73 more DCX positive cells/mm3 than migrating ones ; two-tailed t-test, df = 7; t = 2.01; p<0.00) (3 ± 0.3 vs Migrating 4.46 mm3 ± 0.7 (mean ± SD); two-tailed t-test, df = 5; t = 2.01, p<0.00) shown in Table F in We found a major difference between migrating and wintering birds in the number of DCX-immunolabeled hippocampal cells and in hippocampal volume . C. pusi p<0.00) . Total nNeuN counts showed on average, no significant differences between migrating and wintering sandpipers. Indeed, NeuN total counts were 946,247 ± 139,352 neurons on migrating and 909,540 ± 138,470 (mean ± SD) on wintering sandpipers (data extracted from )  and non-migrating  white-crowned sparrows showed a greater number of doublecortin-positive neurons in the hippocampus of the migratory subspecies [It is not clear why hippocampal neurogenesis might differ between wintering and actively migrating birds. Cognitive activity, environmental enrichment, diet, and stress are known to affect levels of hippocampal neurogenesis. It is difficult to distinguish their relative contributions to neurogenesis. Indeed, it has been previously described that migration, as noted above, probably engages several cognitive processes. Increased spatial processing is associated with an increase in the number of new neurons in the hippocampus , 64–67 abspecies .In addition, long distance migration might also be considered a kind of environmental enrichment. The journey may be visuo-spatially enriched and may engage perceptual processes involved in celestial, olfactory and geomagnetic navigation in ways that do not occur outside the period of migration. Long distance migrants may be exposed to more diverse spatial information, compared to short distance migrants, resulting in greater recruitment of new neurons . From maIf increased cognitive activity and environmental enrichment are indeed the causes of increased hippocampal volume and neurogenesis in semipalmated sandpipers, their effects however, are seen not during migration, but during the wintering period that follows.Long distance flight also involves intense exercise, and exercise reliably increases hippocampal neurogenesis . EuropeaCorophium volutator in which 45% of total lipids are in the form of PUFAs. This diet may therefore depress hippocampal neurogenesis during stopover.The same study of starling wind tunnel flight also showed that a diet high in polyunsaturated fatty acids (PUFA) led to less hippocampal neurogenesis than a diet low in PUFAs . SemipalStress and elevated glucocorticoid levels reduce hippocampal neurogenesis , 46, 47.Finally, there is an alternative interpretation connected to a reduced use of the hippocampus during fall migration. In that case, long distance migration in the sandpipers may rely on compass direction whereas local navigation in the wintering home range may rely on constant use of the hippocampus stimulating neurogenesis in higher proportion. To support this hypothesis, previous findings demonstrated that pigeons with hippocampal ablation usually find their way back home . If simiTo find out if DCX-positive cells survived and were integrated into existing hippocampal circuits, or disappear, we checked how neuronal numbers compare in the two groups. The absence of significant difference between migrating and wintering sandpipers with NeuN labeling indicates that hippocampal neuronal number does not increase after each winter, and the newly generated neurons seem to compensate for the loss that might occur during the transatlantic flight. Thus, the difference in hippocampal volumes may be related to other modifications that do not come from neuronal number changes. We suggest that other cell numerical and/or morphological changes and/or expansion of extracellular matrix may contribute to the hippocampal volume differences.Thus, if long distance migration does act to upregulate neurogenesis its effects are seen not during migration but during the wintering period that follows. Alternatively, there may be diet and glucocorticoid effects that reduce hippocampal neurogenesis during migration. In addition, there may be effects specific to the wintering period that result in elevated neurogenesis, perhaps in preparation for spring migration. Because all these effects and environmental inputs are associated with migration, and because all of them may have an influence on neurogenesis, it would be important in near future to compare more groups of birds at different stages of their annual cycle. Indeed, our study was limited to a group caught in Canada at the beginning of migration and a second group in Brazil during wintering. It would be very informative if there were two more groups of birds, one caught in Canada before migration, when the birds were settled in the region, and one in Brazil just as the return migration started. This design would allow to partially account for dietary, environmental, social (including reproduction) or other factors and stress.We hypothesized that neurogenesis in the hippocampal formation of migrating birds would be higher than that of wintering birds. We found higher levels of adult hippocampal neurogenesis and a larger hippocampal formation in wintering birds, suggesting that these changes may be late occurring effects of long distance migratory flight or the result of conditions the birds experienced while wintering. We also detected no differences in NeuN immunolabeled cells in migrating and wintering birds suggesting at least for this time window that neurogenesis just replaced neurons that might be lost during the transatlantic flight. The clear differences we observed between migrating and wintering birds indicate that long distance shorebird migrants provide an opportunity to investigate many questions about the natural control and function of adult hippocampal neurogenesis.S1 FileTables A to G.(DOCX)Click here for additional data file.S2 FileARRIVE guidelines form.(PDF)Click here for additional data file.


The climatic conditions, consisting of strongly contrasting wet and dry seasons, make the SEO a unique hydrogeological setting, where water-rock reactions are enhanced by large storm events (up to 200 mm in a single storm). Previous work on hyperalkaline spring fluids collected within the SEO has identified the presence of microorganisms potentially involved in hydrogen, methane, and methanol oxidation , as well as the presence of methanogenic Archaea (such as Methanobacterium). Similar organisms have also been documented at other serpentinizing sites, however their functions have not been confirmed. SEO's hyperalkaline springs have elevated methane concentrations, ranging from 145 to 900 μM, in comparison to the background concentrations (<0.3 μM). The presence and potential activity of microorganisms involved in methane cycling in serpentinization-influenced fluids from different sites within the SEO were investigated using molecular, geochemical, and modeling approaches. These results were combined to elucidate the bioenergetically favorable methane production and/or oxidation reactions in this tropical serpentinizing environment. The hyperalkaline springs at SEO contain a greater proportion of Archaea and methanogens than has been detected in any terrestrial serpentinizing system. Archaea involved in methanogenesis and anaerobic methane oxidation accounted from 40 to 90% of total archaeal sequences. Genes involved in methanogenic metabolisms were detected from the metagenome of one of the alkaline springs. Methanogenic activities are likely to be facilitated by the movement of nutrients, including dissolved inorganic carbon (DIC), from surface water and their infiltration into serpentinizing groundwater. These data provide new insight into methane cycle in tropical serpentinizing environments.Uplifted ultramafic rocks represent an important vector for the transfer of carbon and reducing power from the deep subsurface into the biosphere and potentially support microbial life through serpentinization. This process has a strong influence upon the production of hydrogen and methane, which can be subsequently consumed by microbial communities. The Santa Elena Ophiolite (SEO) on the northwestern Pacific coast of Costa Rica comprises ~250 km Danta) located in the Potrero Grande watershed , total dissolved solids, and salinity) were recorded using handheld probes (Oakton PC Testr 35 and Oakton Testr 10), which were calibrated twice a day using standard solutions.2, a 10 mL sample was collected following the same protocol and transferred to a 14 mL serum vial. For dissolved inorganic carbon (DIC), a 14 mL serum vial, containing one NaOH pellet, was filled to the top, capped with a butyl rubber stopper, and microbial activity was arrested using saturated ZnCl2 . All samples were collected in triplicate and were sent for analysis as a contracted service to JBL Analytical Services .Water samples for dissolved methane concentration and isotopic composition analyses were collected from the spring source using a bubble-free 60 mL syringe with Tygon tubing attached. The samples were carefully transferred to a He-purged 160 mL serum bottle and crimp sealed immediately. For HThe filtrate from the 0.2 μm Sterivex filter cartridges  used for molecular sampling (see methods below) was collected into an acid-cleaned 30 mL Nalgene bottle . The secondary standards were: Moscow Tap Water, MTW , Deep Ocean Water, DOW , and Commercial Bottled Water, CAS . MTW and DOW standards were used to normalize the results to the VSMOW2-SLAP2 scale, while CAS was used as a quality control and drift control standard. The analytical long-term precision was: ±0.5‰ (1 s) for δ2H and ± 0.1‰ (1 s) for δ18O.Samples for water stable isotope analysis were collected in 30 mL glass E-C borosilicate bottles with tetrafluoroethylene (TFE)-lined caps . Bottles were filled completely, leaving no head space, covered with parafilm  avoiding exchange with atmospheric moisture, and stored upside down at 5°C until analysis. Water stable isotope analyses were conducted at the Stable Isotope Research Group facilities of the National University  using a Cavity Ring Down Spectroscopy (CRDS) water isotope analyzer L2120-Fluids were preserved in 3.7% (v/v) formaldehyde  for enumeration of cell abundances and stored at 4°C until processing. The preserved fluids were filtered through a 0.2 μm black polycarbonate filters , and cells were stained with 1 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) and counted by epifluorescence microscopy using appropriate filter sets according to previously published protocols  using a Masterflex® E/S portable sampler . Filters were covered with ice during the filtration process, then capped and flash frozen in liquid nitrogen, and stored at −80°C until processing. Extraction of total genomic DNA followed previously described protocols  on a BioRad C-1000 thermo-cycler with a q-PCR module using the SsoAdvanced SybrGreen Assay . Domain-specific primers, 958F and 1048R for Archaea and 967F and 1064R for Bacteria, targeted the V6 hypervariable region of the 16S rRNA gene  for amplicon sequencing of archaeal and bacterial 16S rRNA genes with an Illumina MiSeq instrument with domain-specific primers targeting the V4-V5 hypervariable regions of the 16S rRNA gene (518F-926R for bacteria and 517F-958R for archaea), following the methods described by Nelson et al.  and the Ca. genus Methylomirabilis of the candidate phylum NC10  was submitted to the Josephine Bay Paul Center, Marine Biological Laboratory for shotgun metagenomic sequencing. Metagenomic libraries were constructed with the Nugen Ultralow Ovation kit according to the manufacturer's instructions. Paired-end sequencing with a 100 cycle Illumina HiSeq run generated partial ~30 bp overlaps, and six libraries were multiplexed per lane. The raw metagenomic sequence data are publically available in the NCBI SRA database under the BioProject accession number Quality control of the metagenomic reads was performed to remove low quality and sequencing artifact reads. Cutadapt v.1.9 Martin,  was used2. The default thermo.com.V8.R6+.tdat database supplied with the program was used for the calculations. Since data were available for only some components of the fluid, nominal amounts of Na and Cl (10–15 mM for Na and Cl) were included in the calculations to account for the measured salinity and achieve charge balance since these are typically the major dissolved ion components at sites of terrestrial serpentinization  to estimate activities of dissolved species involved in the metabolic reactions, such as dissolved COQ the activity quotient of the compounds involved in the reaction. The latter factor, Q, takes into account the contribution of the fluid composition to the Gibbs energy of each reaction, and was calculated using the activities determined by the fluid speciation models. Values of the ΔG° were calculated using SUPCRT92  and δ2H (−45.1 to −50.4‰) composition. The sampling site upstream from Q. Danta spring was more enriched (δ18O = −4.33‰and δ2H = −33.4‰).Fluids from SEO's springs are highly alkaline with pH ranging between 11.2 and 11.6 and highly reducing, with an oxidation-reduction potential (ORP) ranging from −380 to −251 mV. Overall, surface water from upstream sites and groundwater in proximity of SEO are relatively alkaline with pH values ranging from 7.54 up to 8.90. Upstream sites and groundwater wells exhibited oxidizing conditions with ORP values ranging from +66 up to +97 mV. These conditions are consistent with springs sampled at other terrestrial serpentinization sites   and cell count data (cells/ml) Table , and sliBased upon tag sequencing of 16S rRNA marker genes, sequences related to known methanotrophic bacteria comprise < 2% of the total bacterial 16S rRNA gene sequences in all the samples, except in the fluids from P. Aguas Calientes, where sequences related to these microorganisms represent ~3–7% of the total sequences Figure .On the other hand, archaeal sequences related to the methane cycling (methanogenesis and AOM), accounted for ~40 to 90% of the archaeal 16S rRNA gene sequences in spring water samples, 15 to 40% in the upstream samples, and 0–15% in the control river sample  and in groundwater well samples Figure .pmoA) or methanol dehydrogenase (mxfA) were not detected in the metagenomic assemblies  from acetoclastic methanogenesis than hydrogenotophic methanogenesis  in methane from Q. Danta may represent contributions from acetoclastic methanogenesis  was dominated by ANME-1b. The detection of ANME -1 archaea in methanogenic sediments from White Oak River estuary, however, suggests that these organisms are not exclusively methanotrophs and that they might be capable of methanogenesis  yields more energy than anaerobic methanotrophy, but the sample with the most depleted δ13C-CH4 in these samples is very enriched, might be explained by the hydrology of the system. Spring 8 and Spring 9 are located at the bottom of Murciélago watershed, where water flow paths from the recharge zone (~700 m a.s.l.) to the hyperalkaline springs outflow (78 m a.s.l.) are expected to be longer. Q. Danta is located in a steeper watershed but closer to the recharge zone, resulting in potentially shorter flow paths. In terms of water mean residence time (MRT), the expected longer MRT in Spring 8 and 9 might result in a stronger abiotic methane signature, by allowing abiogenic methane to accumulate, which in turn, may mask the biological methane signature. While in Q. Danta, having shorter water flow paths from the recharge zone to the spring outflow and consequently shorter MRT, biological methanogenesis likely impacts the bulk methane signal.The fact that methanogenesis is a relatively favorable bioenergetic process and that methanogens and ANME-1 were abundant at Spring 8 and Spring 9, even though the δThe hyperalkaline springs at SEO contain a greater abundance of Archaea and a higher proportion of methanogens than has been detected in any terrestrial serpentinizing system to date. The conditions at SEO appear to sustain a predominantly methanogenic ecosystem, where biological methanogenesis is sustained by acetoclastic and hydrogenotrophic processes, in concert with abiogenic methane formation. Methanogenic activities are likely to be facilitated by the dynamic flux of nutrients, including DIC, from surface water and their infiltration into serpentinizing groundwater during intense seasonal cycles of rainfall and recharge. However, in addition to methanogenic process, anaerobic methane oxidation may be superimposed and influence the resulting gas flux. These data provide new insight into methane cycle in a tropical serpentinizing environment, and context to the patterns in methane occurrence in terrestrial serpentinites in various hydrological and climatic settings.MC and KT contributed equally to this study. MC, RS, and MS designed the project and planned the field campaign. MC and RS were in charge of sample collection. MC and MS were in charge of geochemical characterization of the fluids, RS of isotopic analysis, KT and WB of sequence analysis, TM of the bioenergetics calculations. All authors contributed to the writing and editing of the manuscript.Metagenomic analysis was funded by the Census of Deep Life (CoDL) project PRJNA340462 to MC and RS. RS would like to thank the support of the Research Office of the National University of Costa Rica through grants SIA-0482-13, SIA-0378-14, and SIA-0101-14. Travel support, geochemical, and 16S rDNA sequence analyses were supported by MSU new faculty startup funds to MS. Postdoctoral salary support was provided to MC through a grant by the Deep Carbon Observatory (Alfred P. Sloan Foundation).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.


We hypothesized that VDR polymorphisms are associated with infertility and COS responses. Thus, we evaluated the association between the TaqI, BsmI, and FokI VDR polymorphisms and ovarian responses in women undergoing COS.Calcitriol, or 1,25-hydroxycholecalciferol, is the active form of vitamin D. It binds and activates vitamin D receptor (VDR). Infertility and defective folliculogenesis have been observed in female n = 121) comprising volunteers with a history of natural conception and a second group of women undergoing COS (n = 70). TaqI, BsmI, and FokI genotyping was performed via restriction fragment length polymorphism analysis or TaqMan qPCR and Sanger sequencing. Intrafollicular 25(OH)D contents were measured in follicular fluid collected from COS patients during oocyte retrieval. Ovarian response parameters were obtained from patient medical records.In this study, we recruited a control group  between the control and COS groups. However, the allele frequency of TaqI  was significantly lower in the COS group than in the control group (p = 0.02). Follicle number but not oocyte number was lower in patients with TaqI polymorphic (TC/CC) genotypes (p = 0.03). Importantly, the ratio between the number of follicles retrieved and intrafollicular estradiol concentrations was higher in patients with the TC/CC TaqI genotypes (p < 0.02).There were no significant differences in the genotype frequencies of VDR TaqI polymorphism and reduced follicle number in women undergoing COS, suggesting that VDR signaling affects the ovarian response to stimulation via unknown mechanisms.We identified an association between the  Calcitriol, or 1,25-hydroxycholecalciferol 2D), is the active form of vitamin D, a steroid hormone that exerts classical functions in calcium and phosphorus homeostasis and bone mineralization , and repVDR expression has been reported in different central  and peripheral reproductive organs  , and associated infertility  and follicle-stimulating hormone (FSH) concentrations, indicating a peripheral rather than a central defect (oviduct) , 15, 16.ertility . Moreovel defect  and suggVDR gene may lead to important defects in gene activation. Alterations were reported to affect calcium metabolism, cell proliferation, and immune function (VDR single-nucleotide polymorphisms (SNPs) may contribute to a genetic predisposition to certain diseases. SNPs present in the VDR gene alter receptor length and decrease its activation in target cells  and BsmI  are present in the 3′ untranslated region (3′ UTR) of the VDR gene and are related to modulation of gene and protein expression of the receptor. The FokI polymorphism , in turn, is present in a translated region and effects functional activity by generating a longer VDR protein with reduced transcriptional activity  (TaqI polymorphism) (FokI polymorphism)  , specifiorphism) , 23, andorphism) . Conversorphism) , osteopoorphism) , and astorphism) . Howeveroporosis , colorecoporosis , and metoporosis . In the etriosis , 31–38, in vitro fertilization (IVF) through stimulation of folliculogenesis, revealed a decrease in the pregnancy (25(OH)D deficiency is now recognized as a pandemic condition . In Brazregnancy  and fertregnancy  in womenin vitro and animal model studies strongly support a significant role of calcitriol in orchestrating reproductive processes and IVF outcomes D  or at leoutcomes . HoweverVDR polymorphisms are associated with infertility and response to COS. The identification of specific VDR polymorphisms that can be shown to be related to infertility and response to COS may help clarify the causes underlying female infertility and poor ovarian response.In the present study, we hypothesized that TaqI polymorphism analysis, 57 controls and 49 COS-treated women in the FokI analysis, and 86 controls and 54 patients in the BsmI analysis.Two groups of patients were enrolled for each polymorphism analysis. The control group comprised volunteer women with no history of reproductive disorders. To be included in the control group, volunteers had to declare that they had become pregnant through natural conception at least once and had never experienced any difficulties in conceiving. The COS group consisted of women who underwent COS for intracytoplasmic sperm injection (ICSI) at the Fertipraxis Center for Human Reproduction, a clinic certified by the Brazilian Health Surveillance Authority (ANVISA) and the Latin American Network of Assisted Reproduction (REDLARA). We enrolled 62 controls and 47 COS-treated women in the VDR polymorphism genotyping.This study was approved by the local Ethics Committee and was registered on the Brazilian platform of research under the number 02213812.4.0000.5275. All the enrolled subjects (volunteers and patients) provided written informed consent before joining the study. In the COS group, clinical data, including hormone concentrations  and indicators of ovarian response (number of follicles and oocytes retrieved), were obtained from patient medical records. Clinical data for the control group were obtained during patient enrollment and interviews. All patients underwent blood collection for further ® 0.25 mg, Merck-Serono, Italy) was administered to induce hypophysis suppression, and on the second day of menstruation, ovarian stimulation was initiated with synthetic FSH alone  or FSH and LH  treatments. FSH dosage varied from 150 to 300 IU/day, and LH dosage ranged from 75 to 300 IU/day.Controlled ovarian stimulation protocols were performed according to the specific clinical requirements of the patients. Briefly, the gonadotropin-releasing hormone antagonist analog cetrorelix acetate  was administered to mimic LH. Thirty-five hours post-Ovidrel® administration, the oocytes were retrieved, and FF was obtained during the follicular aspiration procedure. In addition, blood samples were collected for VDR genotyping following FF isolation.When at least one follicle had reached 18 mm or at least two follicles had reached 16 mm (assessed by ultrasound), human chorionic gonadotropin (hCG) . After the extraction, DNA quantity and quality were examined using a NanoPhotometer .Blood (1 ml) was submitted for genomic DNA extraction from peripheral leukocytes echnique  using a TaqI (rs731236) and FokI (rs2228570) polymorphisms was performed using the restriction fragment length polymorphism (RFLP) technique. Table VDR polymorphism analysis, which were validated through the Primer Blast program to ensure PCR quality; intron-spanning primers were used to avoid contamination with external genomic DNA. To perform the PCR reactions, a commercial kit  was used and the conditions were as follows: 95°C for 4 min, 35 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 1 min, and 72°C for 7 min. DNA samples were digested by TaqI and BseGI (Btscl isoschizomers that recognize the same sequence recognized by the FokI) endonucleases . The mixtures were incubated at 65°C and 55°C, respectively, to promote cleavage. The samples were then subjected to electrophoresis on 2–4% agarose gels to determine the lengths of the fragments and genotyping results  polymorphism genotyping was performed using TaqMan Genotyping Master Mix  and a TaqMan® SNP Genotyping Assay  in a ViiA™ 7 Real-Time PCR System . Allele discrimination was analyzed using the ViiA™ 7 software, and genotyping was performed with Genotyping version 3.1 from Thermo Fisher Cloud. Furthermore, Sanger sequencing (Big Dye® Terminator v 3.1 Cycle Sequencing Kit) was performed on the four amplified products for which real-time PCR did not achieve accurate results to confirm the genotyping assay results  and a 17G oocyte aspiration needle  connected to a closed vacuum system under 90 mmHg of negative pressure, which was used to empty the follicles. The follicle exhibiting the largest diameter, greater than 16 mm, was selected, captured and placed in a sterile container. FF was extracted after oocyte detection and subsequently frozen in liquid nitrogen. This technique allowed the collection of fluid from a single follicle and decreased the chance of blood contamination.Follicular fluid collection was performed during oocyte capture, as previously described , 49. BriFF 25(OH)D concentrations were assessed using an electrochemiluminescence fixation assay . The range of measurements was 3–70 ng/ml. Inter- and intra-assay variations were 5.9 and 5.2%, respectively. This technique is based on competition, and a vitamin D-binding protein binds 25-hydroxycholecalciferol (25(OH)D3) and 25-hyroxyergocalciferol (25(OH)D2).2 test. If the χ2 test resulted in a p value greater than 0.05, the population was considered to be in HWE. The influence of each VDR polymorphism on COS variation was assessed by an odds ratio (OR) analysis. We performed χ2 tests to analyze heterogeneity, and a value of p < 0.05 was considered to indicate statistical significance. The dominant model, in which heterozygous and homozygous minor alleles were grouped, was analyzed.Genotype and allele frequencies were calculated based on the observed genotypes. Departure from Hardy–Weinberg equilibrium (HWE) in the distribution of the genotypes was estimated with the χp value <0.05 was considered statistically significant. All comparisons were performed using SPSS (version 22) software. Graphics were generated using Prism (version 6) software.The Mann–Whitney test was used to investigate possible associations between polymorphisms and ovarian response variables and to test associations between polymorphisms and FF concentrations of 25(OH)D. A VDR gene affected the ovarian response of women undergoing COS, we extracted clinical data from control volunteers who had declared a history of natural conception and from women who underwent the COS protocol and ICSI treatment. The demographic parameters of the control and COS groups are depicted in Table To determine whether the presence of polymorphisms in the The COS infertility diagnoses in our group were as follows: unexplained (37%), tubal factors (18%), ovarian failure (13%), endometriosis (13%), female anatomical causes (6%), and other causes of infertility (13%), including hypogonadism, colonic surgery, ovarian failure and tubal factors, female endocrine factors, breast cancer, tubal factors and endometriosis, or polycystic ovarian syndrome and endometriosis.VDR polymorphisms studied in the control versus COS groups. No differences were found, and the FokI polymorphism was in HWE . Table VDR polymorphisms studied in the control versus COS groups. No differences were observed in the BsmI and FokI allele frequencies between the control and COS groups. However, the TaqI polymorphism exhibited a higher frequency of the C allele and a lower frequency of the T allele in the COS group . We then applied the dominant model and identified a considerable trend in the genotype distribution for the TaqI polymorphism .Table TaqI alleles, we next examined whether the TaqI polymorphism is associated with variables related to the COS protocol. We, therefore, sorted the COS group according to genotype based on the dominant model . The duration of COS and the FSH dose administered did not differ according to the TaqI genotype . However, we found no significant differences in the number of oocytes retrieved. We also analyzed the serum concentrations of E2 and P4 on the day of hCG administration as well as the ratio of intrafollicular E2 to follicles retrieved according to TaqI genotype  , suggesting that these polymorphisms do not alter FF 25(OH)D concentrations. Importantly, while serum concentration of 25(OH)D were not evaluated in this study, recent findings reported by our (We did not find an association between follicular 25(OH)D concentrations and any specific d by our  and othed by our , 44, 51 d by our , 44 in bTaqI polymorphism does have a role in this context. In contrast, some studies have demonstrated an association between the TaqI C allele and decreased serum 25(OH)D concentrations in women with colorectal cancer D.The above results indicate a lack of a direct relationship between FF concentrations of 25(OH)D and infertility and suggest that the l cancer , whereasl cancer  demonstrTaqI polymorphic genotypes  and 25(OH)D deficiency. This finding is in line with our results, given that we did not found any associations between TaqI polymorphism genotypes and intrafollicular 25(OH)D concentrations. Altogether, these data highlight the relevance of the TaqI polymorphism under different conditions and suggest the need for further studies investigating the relationship between VDR polymorphisms and 25(OH)D serum concentrations in different pathologies, including infertility disorders.However, a study of polycystic ovarian syndrome in Caucasian women  found noVDR polymorphisms in all samples we evaluated. We also observed that there was a lack of HWE in the control population due to the exclusion criteria. This decreased the size of the study population and may have contributed to the observed imbalance in genotype and allele frequencies, resulting in a lack of HWE in the study populations. However, the COS population was under HWE and exhibited an association between the TaqI TC/CC polymorphic genotypes and the production of fewer ovarian follicles. These results suggest a possible role of the C allele in determining the number of pre-ovulatory follicles.Our study has some limitations, including the relatively low number of included patients and the fact that we did not genotype all three TaqI polymorphism on follicular development and hormone secretory function.The above observation is supported by our data showing a higher ratio of retrieved follicles to intrafollicular E2 in women with TC/CC genotypes than in women carrying the TT genotype, i.e., women who have the TC/TT genotypes exhibited lower E2 availability in pre-ovulatory follicles. This finding demonstrates an important impact of the VDR mRNA and 1-α hydroxylase enzyme are expressed in the ovary  (Recent studies have shown that 25(OH)D is present in FF , 19. Whiultures) , 38, indVDR TaqI polymorphism allele and infertility. This association is likely mediated by impaired calcitriol signaling, which may impact the number of follicles in women undergoing COS via mechanisms that are yet to be described.In conclusion, this study revealed an association between the presence of the C This study was approved by the local Ethics Committee of the Maternidade Escola of the Federal University of Rio de Janeiro, which was registered on the Brazilian platform of research under the number 02213812.4.0000.5275. All of the enrolled subjects (volunteers and patients) provided written informed consent before joining the study.Conceptualization of the experiments. Formal analysis. Performed experiments. Writing review and editing.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 handling Editor and reviewer PP declared their involvement as co-editors in the Research Topic, and confirm the absence of any other collaboration.


Outdoor pollen grain and fungal spore concentrations have been associated with severe asthma exacerbations at the population level. The specific impact of each taxon and the concomitant effect of air pollution on these symptoms have, however, still to be better characterized. This study aimed to investigate the short-term associations between ambient concentrations of various aeroallergens and hospitalizations related to asthma in the Brussels-Capital Region (Belgium), an area recording especially high rates of admissions.2, O3) was tested.Based on administrative records of asthma hospitalizations and regular monitoring of 11 tree/herbaceous pollen taxa and 2 fungal spore taxa, daily time series analyses covering the 2008–2013 period were performed. Effects up to 6 days after exposure were captured by combining quasi-Poisson regression with distributed lag models, adjusting for seasonal and long-term trends, day of the week, public holidays, mean temperature and relative humidity. Effect modification by age and air pollution , birch  and hornbeam  pollen concentrations. For several taxa including grasses, an age modification effect was notable, the hospitalization risk tending to be higher in individuals younger than 60 years. Air pollutants impacted the relationships too: the risk appeared to be stronger for grass and birch pollen concentrations in case of high PMThese findings suggest that airborne grass, birch and hornbeam pollen are associated with severe asthma exacerbations in the Brussels region. These compounds appear to act in synergy with air pollution and to more specifically affect young and intermediate age groups. Most of these life-threatening events could theoretically be prevented with improved disease diagnosis/management and targeted communication actions.The online version of this article (10.1186/s12940-018-0378-x) contains supplementary material, which is available to authorized users. Asthma represents a complex and heterogeneous respiratory disorder. It is characterized by a chronic inflammation of the lower airways, leading to variable and recurring respiratory difficulties such as wheezing, chest tightness, coughing, shortness of breath, etc. . It seveThe prevalence of this disease, and its associated burden on global health care systems, greatly increased over the last decades. Nowadays, around 334 million people are estimated to be affected worldwide, making this disease a major public health concern . At regiFor several years, research has attempted to identify the triggers contributing to severe asthma exacerbations. A number of environmental factors appear to be involved, especially for the allergic form of the disease. Among outdoor parameters, air pollution and weather conditions have been widely recognized as (in)directly responsible for day-to-day variation in asthma hospitalizations –9. Also,Green spaces represent a significant part of the Brussels territory   and the This study aimed to analyse the short-term relationships of airborne pollen and fungal spore concentrations with hospital admissions for asthma, considering the potential modifying effects of age and air pollution.To do so, an ecological time series approach was adopted, capitalizing on routinely collected data provided by the federal health services and other public monitoring institutions. This protocol was approved by the Belgian Commission for the Protection of Privacy.2 during the studied time period. This study covered six outdoor aeroallergen seasons, from 2008 to 2013 included.The research focused on the Brussels-Capital Region in Belgium. This urban area includes the country capital and represents the largest agglomeration of the state with 1.0–1.1 million inhabitants spread over 161 kmThe primary health outcome investigated for this study was the daily number of hospital admissions with a main/first diagnosis of asthma  that were registered in the Brussels-Capital Region between the 1st January 2008 and the 31st December 2013. These figures were provided by the Federal Public Service Health, food chain safety and environment. They were derived from the Belgian Minimal Hospital Summary database: any Belgian general hospital is required to systematically generate a standardized summary of the medical record of every inpatient (with a few exceptions). This record includes information on diagnosis, medical services involved, dates of admission and discharge, patient’s demographic characteristics, etc.Alnus spp.), hazel (Corylus avellana), yew (Taxus spp.) and Cupressaceae , ash (Fraxinus excelsior), hornbeam (Carpinus betulus), birch (Betula spp.), oak (Quercus spp.), plantain (Plantago spp.), grasses (Poaceae) and mugwort (Artemisia spp.). Two fungal spore taxa belonging to the Ascomycetes group were also considered: Alternaria spp. and Cladosporium spp. Their mean daily concentrations (grains or spores/m3 of air) for the area of interest were supplied by the Mycology and Aerobiology unit of the Belgian Scientific Institute of Public Health. This unit is responsible for the national aerobiological surveillance network [Based on their local presence and their relevance for allergy, 11 pollen taxa were selected a priori for this study: alder  for the Brussels-Capital Region were made available by the Belgian Interregional Environment Agency. They were derived from a monitoring network of fixed stations spread over the whole region (10 stations during the study period), augmented by a land-use regression model (RIO-CORINE) [The role of four air pollutants as confounders or effect modifiers was investigated: PM-CORINE) .The influence of temperature (°C) and relative humidity (%) parameters was considered. The data were supplied by the Royal Meteorological Institute of Belgium as 24-h averages measured within the study area .Lastly, potential confounding by influenza episodes and general respiratory infections was taken into account. Data on weekly consultation rates for influenza-like illnesses were obtained from the representative Belgian Sentinel General Practitioner network, coordinated by the Scientific Institute of Public Health . InfluenAlternaria (January–November) and Cladosporium (January–November). Potential delayed effects of aeroallergens on asthma hospitalizations up to 6 days after the exposure were allowed by combining quasi-Poisson regression with distributed lag models [The associations between outdoor aeroallergen concentrations and hospitalizations for asthma were investigated using daily time series. Analyses were limited to the months of the year for which the pollen or fungal spore taxa under study were present in the air (i.e. months for which the probability that the concentration is higher than 1% of the maximum concentration is different from zero): alder (January–April), hazel (January–April), yew & Cupressaceae (February–April), ash (February–May), hornbeam (March–May), birch (March–May), oak (April–May), plantain (April–September), grasses (April–September), mugwort (June–September), g models . A distrg models .To capture the  effects of meteorological factors on asthma hospitalizations, cross-bases for mean temperature and for mean relative humidity were also included in the model. As for aeroallergens, a maximum lag of 6 days with 4 df was used for the lag-response function. The exposure-response functions were modelled using natural cubic splines with 5 df for temperature and 3 df for humidity, placing knots at equally spaced values of the actual temperature/humidity ranges to allow enough flexibility in the two ends of the distributions. Seasonality and long-term trends were modelled using natural cubic splines with equally spaced knots every 30 days of observation. Models were additionally adjusted for indicator variables for day of the week and public holidays.In secondary analyses, confounding by air pollution was investigated by adding a cross-basis for each air pollutant in separate models. A maximum lag of 6 days and 4 df were used for the lag-response function and a linear exposure-response function was assumed. Confounding by influenza and by general respiratory infections was also assessed by including a binary variable for influenza epidemics and daily counts of hospitalizations for general respiratory infections in the model respectively. Effect modification by age group  and by air pollution (below or above the median and the 85th percentile) were investigated through an interaction between the cross-basis for the aeroallergen and indicator variables for age group and air pollution respectively . Effect In sensitivity analyses, the robustness of results was assessed with respect to the adjustment for temporal trends (by testing knots every 15 or 60 days of observation instead of every 30 days) and with respect to the specification of the lag structure (by using an unconstrained lag model ).Reported estimates represent the cumulative (lag 0–6 days) percentage change  in asthma hospital admissions for an interquartile range increase in aeroallergen concentration. All analyses were performed with the statistical software R  using the “dlnm” package .Five thousand ninety-four hospitalizations for asthma involving individuals from the Brussels-Capital Region were registered between 2008 and 2013. 35% of asthma hospitalizations were in the age group 0–14 years, 41% in the age group 15–59 years and 24% in the age group 60 years and more. The daily number of admissions ranged between 0 and 11 with an average (± standard deviation) of 2.3 (± 1.7). The average daily number was the highest in September (3.5) and the lowest in July and August (around 1.4). Besides, admissions were the most frequent on Mondays (2.8) and the least frequent on Saturdays (1.9).3 for plantain and 82 grains/m3 for birch. Exposure to fungal spores took place from January to November, the highest daily concentrations being registered for Cladosporium (daily mean: 2731 spores/m3). Daily concentrations of some aeroallergens were correlated. Spearman coefficients ranged from 0.01 between alder and birch to 0.78 between Alternaria and Cladosporium  , grasses (P = 0.05), mugwort (P < 0.01), Alternaria (P = 0.10) and Cladosporium (P = 0.13) . Exclusion of the patients aged between 0 and 4 years did not significantly affect the conclusions  Table . The est10 and PM2.5 was found for Alternaria: effect estimates appeared close to null for PM levels below the median and significantly negative for PM levels above the median. An interaction with PM10 was also observed for grasses, estimates being significantly positive for concentrations above the median , but not for levels below the median . Ozone seemed, for its part, to modify the associations for hazel, hornbeam, birch and mugwort. For hazel and mugwort, ozone levels above the 85th percentile showed (non-significant) negative estimates, whereas estimates were closer to zero (and positive) for ozone levels below the 85th percentile. For hornbeam, birch and grasses, significant risks were only observed for ozone concentrations above the median  or for ozone levels above the 85th percentile , although effect modification by this pollutant was only significant for hornbeam (ozone categorization according to the median) and for birch (ozone categorization according to the 85th percentile). Lastly, effect modification by NO2 was found for oak pollen, with a significant negative relationship for levels below the median .Potential effect modification by air pollutants was tested in separate analyses Table . A signiAlternaria  and Cladosporium .The use of unconstrained distributed lag models gave similar results  in asthma admissions for an interquartile range increase in pollen concentration. This relationship has also been observed in the United Kingdom , 29, SpaBetula pendula syn.: B. verrucosa and B. alba) or downy birch (B. pubescens). It constitutes the principal source of tree pollen grains [A significant increase in asthma hospitalizations with increasing pollen concentrations was also observed for birch . This result agrees with findings from the United States   and the n grains , whose cn grains , 21.Asthma hospitalizations were significantly associated with hornbeam pollen concentrations too . To the best of our knowledge, only one similar study investigated the individual contribution of this taxon . This woAlternaria and Cladosporium.Contrary to previous similar works , 37, 38 Alternaria and Cladosporium too).Overall, conclusions appear consistent with a previous study which investigated the short-term associations between allergy medication sales and outdoor aeroallergen concentrations in the Brussels-Capital Region . These aAlternaria and Cladosporium. For all of these taxa except plantain, a trend of lower effect estimates in the group 60 or more years compared to younger age groups was demonstrated. Two previous works from North America showed for various tree species the strongest associations for the age group 5–17 years [Significant effect modification by age was observed for plantain, grasses, mugwort, 17 years , 15. Als17 years . These f17 years . Also, a10 and ozone concentrations seemed to potentiate the risk of hospitalizations associated with the grass and birch taxa respectively. These interactions have been observed in other population studies [Consistent with other studies, no confounding effect of air pollution was demonstrated here , 15, 17. studies , 42 but  studies . In Cana studies . The bio studies . This co studies –47. Thes studies , 44, 47. studies , 33, 35.We also considered potential confounding by influenza epidemics and respiratory viral infections. The latter are recognized as a major cause of asthma symptoms aggravation around September. It is probably behind 50–80% of exacerbation episodes, with an apparent higher risk for individuals suffering from allergic asthma compared with individuals having a non-allergic form of the disease –51. In CAlternaria and Cladosporium whose prevalence of sensitization among the general population does not exceed 2.5% in Belgium (Antwerp) [More generally, even in case of similar aeroallergen concentrations, allergy and symptom exacerbation risks can vary due to a wide range of factors: other aeroallergens simultaneously present, air pollution levels, weather conditions, season or difference with regard to population sensitization or susceptibility patterns. In this framework, strong geographical variations in skin test results have been observed worldwide. Differences are notably high for Antwerp)  while itAntwerp)  (where aAntwerp) , 18). BeAntwerp) , confounFew time series studies have investigated the short-term relationships between ambient aeroallergen concentrations and asthma hospitalizations in the ecological context of Northern Europe. To our knowledge, all of them focused on the United Kingdom , 33, 38.Some limitations should however be highlighted. First, because of the ecological and observational nature of this study, results should be interpreted at the population level and cannot be read as causal associations. Unmeasured confounders may still influence the estimates even if time-series design controls for important individual risk factors that do not vary with time . Second and following on from this, one should highlight approximations made regarding exposure measurement. Indeed, aeroallergens data were derived from a unique spore sampler, located on the top of a building and potentially influenced by local environmental characteristics. Analyses assumed an even exposure to these substances over the study area, ruling out the impact of time spent outdoor by each person, spatial heterogeneity in sources, physical barrier to transport and leading to potential exposure misclassification. Some studies have, nevertheless, concluded that despite rather poor representativeness of personal exposure, concentrations derived from stationary pollen traps correlate well with patients’ symptomology . In geneDespite these limitations, the public health impact of outdoor aeroallergens should not be underestimated. Asthma is indeed one of the leading causes of morbidity worldwide and allergens exposure is almost unavoidable . ConsideThis study suggests that increasing concentrations of airborne grass, birch and hornbeam pollen can severely exacerbate asthma symptoms, leading to hospitalizations in the Brussels-Capital Region. Despite some inconsistencies, a trend of stronger associations between aeroallergens and asthma hospitalizations in individuals younger than 60 years and on days with high air pollution levels was observed. These results highlight the importance of a regular monitoring for outdoor aeroallergen levels as well as the need for additional efforts to anticipate and reduce the health risks associated with these compounds.Additional file 1:Table S1. Spearman’s correlation coefficients among aeroallergens, Brussels-Capital Region, 2008–2013. Table S2. Confounding by air pollutants, influenza epidemics and general respiratory infections - Cumulative (lag 0–6 days) percentage change  in asthma hospitalizations associated with an interquartile range increase in pollen or fungal spore concentrations, Brussels-Capital Region, 2008–2013. Table S3. Sensitivity analyses - Cumulative (lag 0–6 days) percentage change  in asthma hospitalizations associated with an interquartile range increase in pollen or fungal spore concentrations, Brussels-Capital Region, 2008–2013. Figure S1. Cumulative (lag 0–6) exposure-response (ER) functions for the association between asthma admissions and some aeroallergen concentrations - Functions modelled using a natural cubic spline–natural cubic spline DLNM with 3 degrees of freedom (df) for the ER function and 4 df for the lag structure. Relative risks (RR) are relative to the reference value of 0 grains/m3. The vertical dotted lines represent the 75th, 95th and 99th percentiles of the pollen concentrations). (DOCX 37 kb)


The purpose of this study is to investigate the population exposure to UltraFine Particles  in outdoor environments. The particle number doses deposited into the respiratory system have been compared between healthy individuals and persons affected by Chronic Obstructive Pulmonary Disease (COPD). Measurements were performed by means of Dust Track and Nanoscan analyzers. Forty minute walking trails through areas with different traffic densities in downtown Rome have been considered. Furthermore, particle respiratory doses have been estimated for persons waiting at a bus stop, near a traffic light, or along a high-traffic road, as currently occurs in a big city. Large differences have been observed between workdays and weekdays: on workdays, UFP number concentrations are much higher due to the strong contribution of vehicular exhausts. COPD-affected individuals receive greater doses than healthy individuals due to their higher respiratory rate.Particulate matter has recently received more attention than other pollutants. PM UFPs play a significant role in PM-induced adverse effects, both for the pulmonary system and other sites such as the cardiovascular and nervous systems [1) persist for a longer period, contaminating outdoor and indoor air [Outdoor air pollution is an important health threat for humans in both developed and developing countries. The World Health Organization (WHO) estimated that in 2012 ambient air pollution caused three million premature deaths worldwide. These deaths have been mainly ascribed to exposure to particulate matter (PM) of 10 microns or less in diameter  and other pre-existing particles [Despite all this evidence, UFPs have not yet received the proper attention from environmental policy makers or consideration in air quality regulations. Thus, an important issue in this field is a better understanding of UFP sources, composition, and size distribution, as well as their routes of potential exposure and their impact on adverse outcomes . Regardiarticles . Furtherarticles .Within this context, the aim of the present study is to estimate the potential UFP exposure of people living in typical urban environments. We performed an intensive monitoring campaign in the urban areas of a big city , characterized by different levels of traffic density during the winter season in severe atmospheric conditions . Along with PM measurements, we investigated UFP number concentrations and size distributions and evaluated individual exposure. These data were used to assess the doses of particles deposited into the respiratory system for each investigated urban scenario.This paper represents the first attempt to describe the doses deposited in the respiratory tract of people commuting in a large city. Moreno et al.  comparedAerosol measurements have been carried out for some typical scenarios currently encountered in urban environments. Measurements have been performed along an urban path in Rome, including areas with different vehicular traffic densities. Furthermore, the ultrafine particle exposure of pedestrians waiting for a bus or standing close to a traffic light along a high traffic density road, waiting to cross the road, was evaluated.A 40-min walking path was considered, as, on average, this would be the time required to reach a workplace in Rome. The selected path  proceedsThe entire sampling campaign was carried out in January–February 2016 for 30 workdays and 6 holidays. During this period, the considered path was followed both on workdays and holidays. The data reported in the present study are representative of a typical daily trend in such a period.Three different areas can be identified on the selected path : the firBy using a camera, the number of vehicles circulating per unit of time was evaluated; on average, about 200 vehicles  per minute passed in zone 1, an average of 44 vehicles per minute passed in zone 2, and 2 vehicles per minute passed in zone 3 on workdays and 10 cars on holidays.It is important to point out that, during the sampling period, the weather in Rome was unfavorable to the pollutant dispersion. High barometric pressure characterized the entire campaign period and induced the Rome City Mayor to limit the car circulation on workdays and to prohibit it on holidays. The data reported as holidays refer to Sundays, when car traffic was completely halted, except emergency vehicles, buses, and taxis.Aerosol measurements were performed to evaluate the exposure of pedestrians waiting at a bus stop and at a traffic light close to a high traffic density road. In the first case, an average waiting time of about 20 min was considered. In the second case, 50 s were considered on workdays and 40 s on holidays (the duration of the red traffic light). Both operations were carried out on workdays and on holidays.Throughout the campaigns, a baseline measurement was performed in order to evaluate indirect vehicular traffic influence; the measurements were carried out in Villa Ada Park, a park considered the background area of Rome in terms of air pollution and which is quite far from direct vehicular traffic emissions.1, PM2.5, PM4, or respirable fraction, PM10, and Total PM size fractions). It uses a sheath air system that isolates the aerosol in the optics chamber to keep the optics clean for improved reliability and low maintenance. A NanoScan SMPS 3910 (TSI), which adopts a scanning mobility particle sizing technology, was also used to measure particle number concentrations in the range 10–365 nm [Different PM size fractions have been investigated along with the temporal trend of particle number concentration in the 11–365 nm size range. A backpack was equipped with a portable DustTrack system and a Nanoscan instrument. The DustTrakTM II Aerosol Monitor 8532 , a handheld battery-operated, data-logging, light-scattering laser photometer, simultaneously measures both mass and size fractions (PM0–365 nm . AerosolTwo probes (anti-electrostatic tubes) were placed on the jacket lapel to get detailed information on personal exposures, on opposite sides in order to avoid interferences and to prevent air vortices that could affect the measurement.−1 and 13.1-min−1 breathing frequency for healthy and COPD (Chronic Obstructive Pulmonary Disease) patients, respectively [tV) of 1.25 L [The methodology of particle dose calculation has been thoroughly described in previous papers ,34,35,36ectively ; and  and individuals affected by Chronic Obstructive Pulmonary Disease (COPD) [iHealthyF and iCOPDF are the fractions of the particle deposited doses in the respiratory tract of healthy people and individuals affected by COPD, respectively; i(t)C is the particle number concentration measured by NanoScan; and tV is the tidal volume, i.e., the volume of air involved in each respiratory act. The iHealthyF and iCOPDF deposition fractions were derived from Löndahl et al. [tV of 0.86 L has been assumed for both cases, whereas a respiratory frequency of 10.7 min−1 and 13.1 min−1 has been assumed for healthy people and COPD-affected people, respectively.The total doses of particles deposited in the human respiratory tract vs. time has been estimated for healthy individuals (e (COPD)  accordinl et al. ; the autThe methodology reported in the Experimental section was applied to the overall campaign. First, the different fractions of aerosol were estimated; 2.5 represented 95% of PM10 and that 94% of PM10 was made of PM1.The comparison between the fraction levels evidenced that PM1 (average 32 µg·m−3) represented 91% of the PM10 (average 35 µg·m−3).A similar situation occurred for the measurements performed during holidays , wherein1 to PM10; they ranged from 0.999 for PM2.5–PM1 to above 0.9 for other fractions below PM10. These PM fractions were so highly correlated because, in the investigated periods, PM pollution was mainly due to fine aerosol deriving from anthropogenic sources. Correlation coefficients between total PM and the other PM fractions were lower (0.66–0.74), very likely because of the resuspension of road dust due to the nearby vehicular traffic. The PM composition of Rome’s urban air, extensively studied by Perrino et al. [1 was the most significant component of PM10 and, therefore, it is important to analyze its size distribution. To this aim, a NanoScan instrument was used. o et al.  and Avino et al. , was chao et al.  and 20%–o et al. , and heao et al. . Specifi−3 and it never dropped below 25,000 # cm−3. However, when the vehicular traffic was completely halted (no vehicles passing-by due to red traffic light), data were very different; the submicron particle concentrations were considerably lower , and the minimum result was approximately 5000 # cm−3. As reported in The first relevant finding is that during workdays the submicron particle concentration reached a value of about 120,000 # cm−3) and almost constant over the time.The area tagged “Park area”  is locatDuring workdays, the particle concentrations for size channels from 11.5 nm to 48.7 nm showed high CVs (up to 100% for the first three channels) due to the fast temporal evolution of such particles, whereas the CVs were below 50% for size fractions above 48.7 nm. During holidays, the trend was quite similar, although with lower values. All these data confirm that the different particle behavior strictly depends on the granulometric size fractions; nucleation particles are quickly generated by autovehicular exhausts, and their concentration rapidly drops due to diffusion, coagulation, and deposition mechanisms.UFP percentage contribution was about 75% and confirms previous measurements, performed in a street canyon in downtown Rome, in which their percentage ranged between 70%–95% ,50; studThe Pearson’s correlation coefficients among different size fractions  evidenceThe temporal concentration levels of each NanoScan size channel are reported in 10  was composed of 94% PM1  (data not shown), and the exposure to submicron particles is very high on workdays due to long bus-waiting times and high particle concentration. Total particle concentrations between 50,000 and 100,000 # cm−3 and peak concentrations as high 415,000 # cm−3 were measured on bus arrivals.People waiting at bus stops is a common scenario in urban areas. In such a simulated scenario, PM−3 were measured for traffic lights in red status [−3 were measured.Similar trimodal and monomodal distributions were measured upon waiting at traffic lights. Bimodal size distributions (modes at 15.4 nm and 115 nm) were also measured, depending on the engine emissions and loads. Total particle number concentrations as high as 100,000 # cmd status  due to tFor the “bus waiting” scenario, the cumulative dose for COPD-affected individuals was about 5.2% and 8.4% greater than for healthy individuals during workdays and holidays, respectively. Finally, simulations of individuals waiting at a traffic light were also performed. It should be considered that waiting for the “green” traffic light for ten minutes in most cases is an unlikely situation; nonetheless, in some other cases, such as a police officer standing outside near a traffic light for much more than ten minutes, the simulation is certainly much more significant.10 and PM2.5) [This paper deals with an important potential threat to human health, ultrafine particle exposure and the relevant dose deposited into the respiratory tract of pedestrians in big urban areas such as Rome, considering both healthy people and more susceptible individuals, such as those affected by COPD. UFPs are becoming an issue investigated by several authors, and data sets are being collected in different urban areas. However, health effects have been mainly associated with particulate matter  , whereasd PM2.5) ,61 with d PM2.5) ). The pr


BMPR2, ACVRL1, ENG and KCNA5 genes and to establish a genotype-phenotype correlation. Major genes were analysed by polymerase chain reaction (PCR) and direct sequencing. Genotype-phenotype correlation was performed. Fifty-seven  were included. Several mutations in different genes, classified as pathogenic by in silico analysis, were present in 26% of PAH patients. The most commonly involved gene was BMPR2 (12 patients) followed by ENG gene (9 patients). ACVRL1 and KCNA5 genes showed very low incidence of mutations . Genotype-phenotype correlation showed statistically significant differences for gender (p = 0.045), age at diagnosis (p = 0.035), pulmonary vascular resistance (p = 0.030), cardiac index (p = 0.035) and absence of response to treatment (p = 0.011). PAH is consequence of a heterogeneous constellation of genetic arrangements. Patients with several pathogenic mutations seem to display a more severe phenotype.Pulmonary Arterial Hypertension (PAH) is a rare and progressive disease with low incidence and prevalence, and elevated mortality. PAH is characterized by increased mean pulmonary artery pressure. The aim of this study was to analyse patients with combined mutations in  Pulmonary Arterial Hypertension  is a progressive, poorly characterized disease with low incidence and prevalence in the general population2In the V World Symposium on Pulmonary Hypertension in Nice6110BMPR2; MIM #600799), a member of the transforming growth factor (TGF-β) superfamily, was the first causal gene identified in PAH and is mutated in approximately 10 to 40% of IPAH patients and 80% of patients with HPAH. This gene is located on chromosome 2q3310121314ALK1/ACVRL1; MIM #601284), located on chromosome 12q1316ENG; MIM #601284)17KCNA5; MIM #176267), located on chromosome 12p1319ACVRL1, ENG and KCNA5 genes are less frequent than mutations in BMPR2 gene in patients with PAH121213KCNK3; MIM #603220)CAV1; MIM #601047)CBLN2; MIM #600433)TBX4; MIM #601719)2526The bone morphogenetic protein type 2 receptor gene   were inc1225BMPR2, ACVRL1, ENG and KCNA5 genes, we identified pathogenic mutations in 72% (40) patients. BMPR2 was the gene with a greater number of pathogenic mutations (44% of patients with mutations), followed by ENG (29%), ACVRL1 (17%) and, finally KCNA5 (10%) gene  gene . These rin silico analysis. Among them, some patients had several mutations in the same gene whereas others harboured several mutations in different genes , with several mutations classified as pathogenic after nt genes . Besidesectively . None ofcs tools  and 4.BMPR2, ACVRL1 and KCNA5 genes, and also in intronic junctions for ENG gene. Moreover, missense changes accounted for 86% of total, whereas nonsense mutations were only identified in 13% of patients. Synonymous changes and intronic variants were detected in 40% and 26% of these patients, respectively. These results are shown in Focusing on patients with several mutations, all the mutations identified here were located in coding region for BMPR2 gene in a 20% of patients with several mutations included in this study. In addition, we detected only one patient that showed two pathogenic mutations in ENG gene.We found several mutations in Clinical and hemodynamic parameters were compared between patients with several mutations and patients with only one pathogenic mutation. We also performed genotype-phenotype correlation between patients with several pathogenic mutations and patients without mutations. The statistical variables considered here were gender, age at diagnosis, mean pulmonary arterial pressure (mPaP), systolic pulmonary arterial pressure (sPaP), pulmonary vascular resistance (PVR), cardiac index (CI), 6 minute walking text (6MWT), PAH type (IPAH vs APAH) and response to treatment. Patients who did not respond were treated with Phosphodiesterase 5 Inhibitors. Variables were categorized according to the best cut off point by ROC curve.Regarding to the correlation between patients with several mutations and patients with a single mutation, we found statistically significant differences for gender (p = 0.045), with a greater number of women with several mutations, the age at diagnosis (p = 0.035), showing disease symptoms 11 years earlier, and a significantly higher PVR (p = 0.030) than patients with single mutation. Furthermore, patients with several mutations showed significant differences regarding CI (p = 0.035) and no response to therapy (p = 0.011) . When coBMPR2 mutations. The second one had IPAH and showed c.229A > T (p.I77L) and c.633A > G (p.R211R) mutations in BMPR2 gene and c.1272 + 6A > T mutation in ENG gene. Finally, the last deceased patient was classified as APAH (porto-pulmonary hypertension) and harboured c.1021G > A (p.V341M) mutation in BMPR2 gene and c.498G > A (p.Q166Q) mutation in ENG gene.Three out of 57 patients in our cohort died during the mean follow up period (14 months). The first deceased patient had APAH (connective tissue disease) and he was carrier of c.251G > T (p.C84F) and c.981T > C (p.P327P) BMPR2, ENG, ACVRL1 and KCNA5. Twelve of these patients harboured at least one mutation in BMPR2, reinforcing the role of this gene in the development of PAH. On the other hand, nine patients were carriers of mutations in the ENG gene, representing the second gene most frequently involved in our cohort of PAH patients with several mutations. Remarkably, eight patients showed mutations in both genes. However, only five and one patients had mutations in ACVRL1 and KCNA5 genes, respectively. As a whole, it is difficult to elucidate the role that each of the different mutations could have had in the development of disease. Thus, the molecular pathogenic mechanism of PAH is not fully understood; in fact multiple genetic and environmental factors have been related to the disease. Many of the involved genes are part of the TGF-β signalling pathway, so several mutations in one or more genes in the same pathway could explain the reduced penetrance for PAH.In this study, we have identified and characterized 15 out of 57 PAH patients carrying more than one pathogenic mutation in several genes related to PAH, such as in silico analysis, selecting only those identified as pathogenic by at least three software tools, whereas synonymous and intronic mutations were classified as pathogenic if two bioinformatic programs that analyse splice sites gave positive results. Thus, we consider this approach is stringent enough to make an accurate classification at this level. However, it is important to note that this is only a bioinformatic prediction to characterize the nature of the change, the variants do not appear in public databases, nor detected in general population so those are pieces of evidence for the pathogenic nature of the changeThe characterization of putative missense mutations was performed by et al.ENG mutations. They detected 10 patients with Hereditary Hemorrhagic Telangiectasia  with more than one mutation in ENG or with one mutation in ENG and another one in ACVLR1 gene, but after functional analysis there were not differences compared to wild-type, considering these ENG missense mutations as rare benign variantsENG, but also mutations affecting the splicing process, and interestingly a high proportion of patients with ENG mutations harbouring an additional mutation in BMPR2 gene. ENG inhibits the TGF-β pathway in endothelial cells by down-regulating the ALK5/Smad3 pathway but enhanced ALK1 signalling. As it have been described in other oligogenic diseases with a specific major gene involved in their development, mutations in other genes within the same pathway should be consideredet al.BMPR2 gene described by Wang et al.BMPR2 or in the ENG gene, respectively. They suggested that the mutation in the promoter region could explain the variable penetrance of the diseaseBMPR2.Recently, Mallet BMPR2. Approaches like next generation sequencing (NGS) analysis could give us genetic information that will help in the understanding of the molecular basis of PAH. The oligogenic inheritance might increase the risk of developing the disease and perhaps a more severe phenotype, as occurs in other diseases, like Bardet-Biedl Syndrome or Autosomal Dominant Retinitis Pigmentosa3435Total mutational load has been described for other pathologies, involving mutations in several genes that codify for proteins belonging to the same or related pathways, in the same individual34BMPR2 gene, and four of them showed two mutations in this gene. All these changes were predicted to alter the splicing process or the conservation of the protein, producing a shorter transcript or a misfolding protein susceptible of degradation, which could prevent achieving a minimum protein translation and therefore, the development of the disease29ENG gene. These mutations are located in the first exons and were predicted to affect the splicing process. Thus, mutations in ENG gene could prevent the correct anchoring of ENG protein in the cell membrane, impairing TGF-β/ALK1 signalling responsesThirteen of our patients were carriers of a mutation in BMPR2 and ENG mutations (one of them showed a third mutation in ACVRL1 gene), one patient had two mutations in ENG gene and the remaining patients showed different combination of mutated genes: one patient was double heterozygote for ENG and ACVRL1, another two for BMPR2 and ACVRL1genes and finally, one patient showed a combination of ACVRL1 with KCNA5 genes.On the other hand, eight patients were double heterozygotes for BMPR2, ACVRL1 is the gene most frequently mutated in PAH patients. However, we show that ENG was the second gene most frequent in our cohort. All of these genes have been described to be involved in the development of the disease with or without HHT, being BMPR2 the major causal gene and the others genetic modifiers modulating the penetrance of the disease3236In the last years a second hit hypothesis have been proposed that two mutations, one major and other as modulator, in the same gene or different gene take place32BMPR2 gene have been established as pathogenic, others remains indeterminate, as mutations in the cytoplasmic tail that still retain capacity for downstream signalling. The pathogenic impact of others genes in the disruption of the TGF-β pathway directly or by modulating related pathways, is still unknown394041Although almost all mutations described in None of our patients had relatives with PAH, so we could not perform segregation analysis; but none of the mutations described here were detected in 110 control chromosomes. As most of the mutations identified in PAH are private, and due to the confluence of two or more mutations in several genes, performing genotype-phenotype correlations revealed as a hard task. For this reason, the genotype-phenotype correlation has been performed grouping mutations identified on the same gene, comparing the clinical and hemodynamic parameters with patients carrying only one pathogenic mutation and also with the group of patients without pathogenic mutations.et al.BMPR2 gene, suggesting hormonal implication. Our study did not corroborate such hypothesis, but it seems that the molecular basis of this disease could be more complex in women than men. The age of diagnosis was 11 years younger in patients with several mutations as previously described by Rodríguez-Viales et al.et al.et al.The co-occurrence of several pathogenic mutations was more prevalent in women, which is in agreement with the higher prevalence of PAH in women1011Three patients died after the follow-up period. They had an early age at diagnosis and were carriers of several pathogenic mutations. In addition, these patients did not respond to treatment, achieving a gradual increase of the characteristic phenotype of PAH leading to a premature death. These patients, as well as all cases with various pathogenic mutations, had a more severe phenotype and a higher functional class at diagnosis than patients without pathogenic mutations or with only a single one, but this small number does not allow us to perform statistical analysis.Our results are consistent with those obtained by other authors, but the small number of patients can be considered a limitation. However, the extensive genetic study and monitoring of our patients add extra values to our results.BMPR2 was the more frequently affected gene, followed by ENG, ACVRL1 and KCNA5 genes. Some mutations had not been previously described. We cannot rule out that patients with a single pathogenic mutation have other mutations in genes not included in this study. There is no doubt that other genes could be involved in PAH and it will be important to understand their role in the development of the disease. Patients with several pathogenic mutations seem to show a more severe phenotype. We wonder whether these additional mutations act as a second event in the development of the disease, increasing the penetrance or simply modifying the phenotype of patients.In summary, we report a series of IPAH and APAH patients with a high percentage of them carrying more than one pathogenic mutation in several genes. Moreover, Complexo Hospitalario Universitario de Vigo . All patients are included in the CHUVI DNA Biobank . Patients signed an informed consent and the Regional Ethics Committee approved the study , following the clinical-ethical guidelines of the Spanish Government and the Helsinki Declaration.Fifty-seven patients with idiopathic or associated PAH (group 1 of the new classification of Nice)12Cardiac catheterization was performed using the latest consensus diagnostic criteria of the ERS-ESC (European Respiratory Society-European Society of Cardiology)12et al.BMPR2 gene, by Berg et al.ACVRL1 gene, by Gallione et al.ENG gene, and by Yang et al.KCNA5 gene. Amplification of exons and intronic junctions was performed with 50 ng of genomic DNA using GoTaq® Green Master Mix , according to the manufacturer’s protocol. GoTaq® Green Master Mix contained MgCl2, dNTPs, reaction buffer and Taq DNA polymerase. PCR was performed in a GeneAmp PCR System 2700 .Genomic DNA was extracted from leukocytes isolated from venous blood using the FlexiGene DNA Kit  according to the manufacturer’s protocol. We used primers described by Deng PCR products were confirmed by electrophoresis through 2% agarose gels with SYBR® Safe DNA Gel Stain  in a Sub-Cell GT . HyperLadder V was used as molecular weight marker . The PCR product was purified using the Nucleic Acid and Protein Purification NucleoSpin Extract II kit  or ExoSAP-IT kit . Purified PCR products were sequenced for both forward and reverse strands with BigDye Terminator version 3.1 Cycle Sequencing Kit . The sequencing reactions were precipitated with Agencourt CleanSEQ - Dye Terminator Removal  and analyzed in an ABI PRISM 3100 genetic analyzer . All results were confirmed by a second independent PCR.BMPR2 gene, [ENST00000388922] for ACVRL1 gene, [ENST00000344849] for ENG gene and [ENST00000252321] for KCNA5 gene, and examined for sequence variations. We use the Basic Local Alignment Search Tool (BLAST) software to align sequences and compare them with different organisms. Rare missense variants were analyzed to predict their potential pathogenicity, used combined computer algorithms: Polyphen-2PmutSIFT)MutationTaster2 softwareHSF HumanNetGene2Splice ViewNNSpliceSequence data were aligned with the reference Ensembl cDNA sequence [ENST00000374580] for We classified a missense variant as a mutation when is considered pathogenic by at least three software tools. In addition, synonymous and intronic variants were classified as pathogenic if at least two out of four bioinformatics tools used to predict alterations in mRNA processing showed a new donor/acceptor splice site or if the prediction change dramatically.We used statistical package SPSS v19 for Microsoft. A non-parametric test (U Mann-Whitney) was used for comparisons between patients and controls, but this approach was only exploratory. To compare the different genotypes with clinical and hemodynamic variables we used the Chi-square test. Values were expressed as mean ± SD (standard deviation). P-values < 0.05 were considered statistically significant.How to cite this article: Pousada, G. et al. Complex inheritance in Pulmonary Arterial Hypertension patients with several mutations. Sci. Rep.6, 33570; doi: 10.1038/srep33570 (2016).