In Latin America, there is scarce information about severe asthma (SA) according to the ERS/ATS 2014 criteria. This study aimed to compare the demographic, socio, clinical characteristics, treatment, and use of healthcare resources between SA and non-severe asthma (NSA) patients in Argentina, Colombia, Chile and Mexico.A cross-sectional study was conducted including 594 asthma patients from outpatient specialized sites. A descriptive analysis was performed comparing SA patients and NSA. Chi-square and Mann Whitney tests were used to assess associations between asthma severity and outcome variables.p < 0.001) and had higher proportion of uncontrolled asthma than the NSA patients . SA patients reported a significantly higher proportion of both hospital admission and emergency room (ER) visits due to asthma in the last year, compared with NSA patients, 8.7% vs. 3.7% (p = 0.011) and 37.0% vs. 21.7% (p < 0.001), respectively.Using ERS/ATS 2014 criteria, 31.0% of the patients were identified as SA. SA patients were older at diagnosis , there was still a higher proportion of uncontrolled disease.The online version of this article (10.1186/s12890-019-0871-1) contains supplementary material, which is available to authorized users. Asthma is a chronic inflammatory disorder of the airways  and its Approximately 2–10% have some form of severe asthma (SA) , 7. AccoIn Latin America, there is a lack of information related to SA epidemiology and the burden of SA following implementation of the ERS/ATS 2014 criteria. Essentially, most of the Latin American studies that have assessed the SA population in the region had also included untreated patients , 11 or fThe Asthma Control in Latin America (ASLA) study was a cross-sectional multisite study. Patients were consecutively enrolled between December 2013 and December 2015. The 16 recruiting sites were located in Argentina (five sites); Chile (five sites), Colombia (three sites), and Mexico (three sites) .The study included 594 diagnosed and treated asthma patients who were recruited during routine care at specialized outpatient sites, aged over 12 years old, under pneumologists follow up, with at least one prescription of asthma medication and one medical visit for asthma within the last 6 months. Individuals were excluded if they were participating in a clinical trial at the time of the study or were unable or not willing to comply with the study requirements. This study was conducted in accordance with the amended Declaration of Helsinki . WrittenDuring the course of a scheduled medical visit, the participating patients were asked to complete the Asthma Control Test (ACT) , which wAsthma severity was operationally defined using an algorithm based on answers to questions relating to the ERS/ATS 2014 criteria. SA was defined as asthma that required step four or five treatment  regardless of ICS doses). Four treatment categories did not fulfil the ERS/ATS 2014 criteria for SA and NSA and were reviewed by a panel of three pneumologist experts on asthma to allocate a group  was calculated in adults as weight divided by the square of height, and the nutritional status categorized as: underweight < 18.5 Kg/mlsewhere  and the A total of 594 asthma patients were studied: 154 subjects from Argentina, 154 from Chile, 163 from Mexico and 123 from Colombia. Overall 72.7% were women and 50.8% white, with a median age of 47 years old at the time of the study visit and an age of 25 at the time of asthma diagnosis. According to the ERS/ATS 2014 criteria, 31.0% of the patients had SA (Table p < 0.001).Differences in socio-demographic and clinical factors by SA and NSA are shown in Table p = 0.013).Figure p = 0.011) and almost two times higher proportion for ER visits due to asthma ) in the last year, compared with NSA patients.Table p = 0.015 and 14.1% vs 7.3%, p = 0.009). The same pattern was observed when considering the mean number of clinically significant asthma exacerbations .Regarding the hospital admission and emergency visits due to other causes, SA patients also had a significant higher proportion of both events than NSA patients


Plasmodium vivax transmission in West Africa, dominant for the Duffy-negative blood group, has been increasingly recognized from both local residents as well as international travelers who contracted P. vivax malaria there. However, the relapsing pattern and sensitivity to antimalarial treatment of P. vivax strains originated from this region are largely unknown. There is evidence that the efficacy of primaquine for radical cure of relapsing malaria depends on host factors such as the hepatic enzyme cytochrome P450 (CYP) 2D6.P. vivax malaria. Treatment included 3 days of intravenous artesunate , and 3 days of chloroquine , and 8 days of primaquine . Although parasites and symptoms were cleared rapidly and he was malaria-negative for almost two months, he suffered four relapses with relapse intervals ranging from 58 to 232 days. The last relapse occurred at 491 days from his first vivax attack. For the first three relapses, he was treated similarly with chloroquine and primaquine, sometimes supplemented with additional artemisinin combination therapies (ACTs). For the last relapse, he was treated with intravenous artesunate, 3 days of an ACT, and 7 days of azithromycin, and had remained healthy for 330 days. Molecular studies confirmed P. vivax infections for all the episodes. Although this patient was diagnosed to have normal glucose-6-phosphate dehydrogenase (G6PD) activity, his CYP2D6 genotype corresponded to a *2A/*36 allele variant suggesting of an impaired primaquine metabolizer phenotype.A 49-year-old Chinese man was admitted to the Shanglin County Hospital in Guangxi Province, China, on December 19, 2016, 39 days after he returned from Ghana, where he stayed for one and a half years. He was diagnosed by microscopy as having uncomplicated P. vivax malaria originating from West Africa may produce multiple relapses extending beyond one year. The failures of primaquine as an anti-relapse therapy may be attributed to the patient’s impaired metabolizer phenotype of the CYP2D6. This highlights the importance of knowing the host G6PD and CYP2D6 activities for effective radical cure of relapsing malaria by primaquine.This clinical case suggests that  Plasmodium vivax is geographically the most widely distributed malaria parasite, and was responsible for about 14.3 million malaria cases in 2017 in the world [P. vivax; in Africa, P. vivax is much less common than the overwhelming number of P. falciparum cases. Historically, it has been considered extremely rare or “absent” in Central and West Africa because of the dominance of the Duffy-negative blood group, which was regarded as a receptor required for the invasion of the erythrocytes by P. vivax [P. vivax infections in local residents of Central and West Africa as well as international travelers returning from the regions to malaria-free countries [P. vivax cases in 2012, 122 originated in 21 Central and West African countries [P. vivax transmission in Africa. Notably, the identification of P. vivax strains able to infect Duffy-negative individuals [P. vivax malaria in these regions. The considerable number of vivax cases routinely diagnosed among Duffy-positive international travelers acquired in West and Central Africa implies hidden local transmission of P. vivax malaria in these regions despite predominance of Duffy negativity.ountries . These rividuals –8 demonsP. vivax malaria has been called a “benign tertian malaria,” but this is a misnomer, given that recent studies have demonstrated severe and fatal pathologies associated with P. vivax malaria [Historically,  malaria , 10. Rel malaria .P. vivax-endemic areas, chloroquine/primaquine (CQ/PQ), offering blood schizontocidal and hypnozoitocidal therapy, has been used for more than 60 years and remains the frontline radical cure for uncomplicated vivax cases in most regions (excepting Southeast Asia where resistance to chloroquine commonly occurs) [http://www.chinacdc.cn/tzgg/201109/t20110906_52137.htm). Although the 8-day PQ regimen has not been rigorously tested in clinical trials, earlier studies indicate that the temperate P. vivax strains from Korea are completely susceptible to a total dose of 3.5 mg/kg [In most  occurs) . The sta occurs) . To impr.5 mg/kg .P. vivax patients experienced relapses [Another problem that affects the effectiveness of PQ for radical cure of vivax malaria involves the host hepatic enzyme cytochrome P450 (CYP) 2D6 , which mrelapses , 24. Usirelapses .Here we describe multiple relapses of a case of vivax malaria acquired in West Africa that also appears to be associated with impaired CYP2D6 metabolism of PQ.P. vivax parasites. Blood tests showed increases in white blood cells , neutrophil ratio , and C-reactive protein (142.29 mg/L). He was conscious and oriented to time, place, and person. He was not dehydrated, pale, or in respiratory distress. Since he did not have any severe symptoms, he was diagnosed as having uncomplicated vivax malaria. He spent three days in the county hospital and was given three days of oral CQ therapy . To help resolve the symptoms faster, he was also given intravenous (IV) injections of artesunate . Meanwhile, after confirming that he is G6PD normal, an 8-day course of PQ (22.5 mg/day) was begun. Fever was cleared within one day and parasitemia was cleared within two days. The patient was discharged on day four with instructions for follow-up visits if symptoms reappear. Administration of the remaining five days of PQ was directly observed therapy (DOT) by local Center for Disease Control (CDC) personnel to ensure compliance.A 49-year-old man who had experienced fever and chills for half a day was admitted to the Shanglin County Hospital in Guangxi Province, China, on December 19, 2016. He showed additional symptoms including headache, body aches and cough , artesunate-amodiaquine, which has a different aminoquinoline drug. Both ACT and PQ were administered as DOT by local CDC staff.The second attack of febrile paroxysm occurred 58 days later on February 15, 2017, and he was admitted to the county hospital again with similar symptoms as the first attack and diagnosed with P. vivax malaria and hospitalized at the county hospital for six days. He received the same therapy as for the second attack including DOT of 8-day PQ. At home, he was further treated with three days of a different ACT, dihydroartemisinin-piperaquine.One hundred and thirteen days later, on June 8, 2017, he suffered a third attack of confirmed Eighty-eight days later, on September 4, 2017, he suffered a fourth attack of confirmed vivax malaria. This time he was not hospitalized, while the same CQ/PQ regimen together with three days of oral therapy of dihydroartemisinin-piperaquine was prescribed. All treatments were taken at home and supervised by local CDC staff.Despite the fact that, after returning from Ghana, this patient lived the entire time in a malaria-free area, he had the fifth attack of vivax malaria 232 days later, on April 24, 2018, 491 days from the first attack. He was admitted to the county hospital for three days and was given IV injection of artesunate six times at a 12 h interval . PQ was not prescribed since it was judged not effective. Instead, he was treated with azithromycin (500 mg/day) for seven days. At discharge, he was also given three additional days of dihydroartemisinin-piperaquine. At the time of this interview, he had remained healthy for 330 days after this last episode of vivax malaria.Plasmodium species were identified by nested PCR targeting the 18S rRNA genes using genus-specific and species-specific primers for P. falciparum, P. vivax, P. malariae and P. ovale [P. vivax (data not shown).Venous blood was collected at the time of diagnosis at the first, second, third, and fifth attacks. Blood samples were used for molecular diagnosis and genotyping at the Kunming Medical University laboratory. For each sample, total DNA was extracted from 0.2 mL of venous blood using the High Pure PCR Template Preparation Kit  following the manufacturer’s instruction and eluted in 100 μl of water. P. ovale . The PCRP. vivax merozoite surface protein (PvMSP) 3α gene by the nested PCR and restriction fragment length polymorphism (PCR/RFLP) methods described earlier [PvMSP3α alone detected a similar band size for the first three attacks, but the PCR product from the fifth attack was smaller , the patient’s CYP2D6 genotype corresponds to a *2A/*36 allele variant. CYP2D6*2A is predicted to be functionally normal (score 1), but *36 is non-functional (score 0). Thus, the overall genotype activity score was 1, suggesting that this patient’s CYP2D6 was an impaired PQ metabolizer phenotype [CYP2D6 gene copy number using a previously described method [CYP2D6 gene in this patient was a single copy.Since the effectiveness of PQ for radical cure of vivax malaria is influenced by host CYP2D6 activity, we wanted to determine whether the failure of PQ in this case might be linked to CYP2D6 genotypes suggestive of poor metabolizer of PQ. The single nucleotide polymorphisms (SNPs) in  earlier . PrimaryP. vivax infection acquired in West Africa producing four relapses with the last relapse occurring at 491 days after the first attack. This study suggests that although vivax malaria is considered to be exceedingly rare in West Africa because of predominance of Duffy-negativity in the local human population, there is a considerable hazard for Duffy-positive travelers to acquire relapsing malaria parasites in this region. This malaria case provides evidence indicating that P. vivax strains from West Africa may produce multiple relapses extending beyond one year but less than two. The relapse patterns of P. vivax strains from different geographical regions can vary significantly, from the tropical ‘frequent-relapse phenotype’ to the temperate “long-latency” phenotype’ [P. vivax strains from the tropical Pacific region such as the Chesson strain, which relapse quickly but typically do not extend beyond one year [P. vivax occurred. Furthermore, genotyping of the parasites by PCR-RFLP of PvMsp3a gene indicates that the first two relapses had the same genotype as the primary infection, although the parasite from the fourth relapse had a different genotype. This suggests that the initial inoculum might include more than one strain of P. vivax as sporozoites from one oocyst in the mosquito may comprise sibling progeny of a single gametocyte mating event, or that he had multiple infections during his stay in Ghana. It is not uncommon that relapses and primary infections are genotypically different and relapses often result from activation of heterologous hypnozoites [We documented a enotype’ . The relone year . This paone year , thus thnozoites , 33.P. vivax infection, PQ therapy failed to prevent subsequent relapses. The excessive blood schizonticides given to this patient were not rational and did not prevent relapses. There are a number of possible reasons for the PQ treatment failures. One possibility is that the 8-day PQ treatment is able to prevent relapses for the temperate P. vivax strain [P. vivax. It is noteworthy that although the daily adult dose for the 8-day PQ regimen (22.5 mg) is higher than for the standard 14-day regimen (15 mg/day), the total PQ dosage for the 8-day course (180 mg) is lower than for the 14-day course (210 mg). In addition, since the 8-day PQ regimen (0.375 mg/kg/day) is calculated for adults weighing 60 kg, this patient was also slightly under-dosed (0.32 mg/kg/day). Currently, there are no clinical studies testing the effectiveness of PQ treatment against relapses of West African P. vivax strains. In Shanglin County Hospital, of 105 P. vivax patients who received the 8-day PQ regimen between 2013 and May 2019, four patients (3.8%) had relapses, suggesting that the 8-day PQ regimen worked at least partially for preventing relapses of P. vivax parasites. Another possibility for the PQ failure lies in the patient’s CYP2D6 enzyme, which is known to metabolize PQ to its active metabolite(s) for the hypnozoitocidal activity of PQ [P. vivax; among the 7 patients evaluated by Bennett et al., none relapsed, whereas among the 22 patients evaluated by Baird et al., 12 relapsed [P. vivax malaria acquired in West Africa adds further support to the importance of the CYP2D6 polymorphisms for the effectiveness of the PQ anti-relapse therapy. With the demonstrated efficacy of a single-dose tafenoquine in preventing relapses of vivax malaria [Despite the fact that this Chinese patient received PQ treatment each time he had acute x strain  and the x strain  in Chinaty of PQ . This pa regimen  mg/day, ty of PQ . Subject regimen  mg/day, relapsed . Althougrelapsed . The cur malaria , tafenoq


Hecogenin is a steroidal sapogenin largely drawn from the plants of the genus Agave, commonly known as ‘sisal’, and is one of the important precursors used by the pharmaceutical industry for the synthesis of steroid hormones. Hecogenin acetate (HA) is a steroidal sapogenin-acetylated that produces antinociceptive activity. Thus, we evaluate the antihyperalgesic profile of HA in mice in inflammatory models, as well as its possible involvement with c-fos expression on spinal cord area and cytokines to produces analgesic profile. Acute pretreatment with HA  inhibited the development of mechanical hyperalgesia induced by carrageenan, TNF-α, dopamine and PGE2. Additionally, the immunofluorescence data demonstrated that acute pretreatment with HA, at all doses tested, significantly inhibited Fos-like expression in the spinal cord dorsal horn normally observed after carrageenan-inflammation. Moreover, HA did not affect the motor performance of the mice as tested in the Rota rod test. This antinociceptive profile seems to be related, at least in part, to a reduction of pro-inflammatory cytokines, as IL-1β. The present results suggest that HA attenuates mechanical hyperalgesia by blocking the neural transmission of pain at the spinal cord levels and by cytokines-inhibitory mechanisms. Pain management remains one of the greatest challenges of contemporary medicine, nevertheless, there are a relatively limited list of treatment options for pain . Althoug®), a peptide toxin obtained from Conus magus (marine gastropod mollusk in the family Conidae), and it is the first member in the new drug class of selective N-type voltage-sensitive calcium-channel blockers [Although the expansion of synthetic medicinal chemistry in the last decades caused the proportion of new drugs based on natural products to drop to ~50%, 13 natural product–derived drugs were approved in the U.S. Food and Drug Administration (FDA) between 2005 and 2007, with five of them being the first members of new classes . For theblockers ,7,8.Agave , and belongs to family Agavaceae, widely distributed in tropical and subtropical regions throughout the world [etc.), sexual hormones (progesterone), and protein anabolic hormones  [et al. [et al. [Hecogenin is a steroidal sapogenin largely drawn from the plants of the genus he world . ‘Sisal’he world ,11. The dienone) ,13,14. H [et al.  demonstr [et al.  proposedRecently, our group reported that hecogenin acetate (HA) , a steroThe current study shows that systemic administration of hecogenin acetate (HA) could able to reduced inflammatory hyperalgesia in a dose-dependent manner and its reduce fos expression in the dorsal horn of the spinal cord normally produced by carrageenan suggesting it reduces central excitability.As showed in The inhibitory effect of HA  on the mechanical hyperalgesia induced by TNF-α is shown in et al. [Hyperalgesia induced by i.pl. injection of carrageenan is widely used for evaluating new anti-hyperalgesic drugs in rodents . In expeet al. , this caet al. .Cytokines produced after i.pl. injection of carrageenan may exert in direct cytotoxic effects by the release of NO, reactive oxygen species, eicosanoids and excitatory amino acids (EAA - glutamate and aspartate) EAA . AlternaIt is well know that cytokine TNF-α is recognized as a potent pro-inflammatory endogenous substance, which is rapidly produced in large quantities by macrophages in response to inflammatory stimuli such as bacterial infection . TNF-α iet al. [et al. [Taking those findings into account, we evaluated whether the antinociceptive activity of hecogenin acetate involves the blockade of sensitization or activation of the nociceptor through evaluation of its effect on hyperalgesia induced by PGE2 and dopamine . As the et al.  and Gama [et al. . et al. [et al. [et al. [In fact, the acute pretreatment with hecogenin acetate increased threshold sensitivity towards mechanical stimuli in carrageenan-, TNF-α, PGE2- or dopamine-induced mechanical hyperalgesia, this compound is suggested to have produced an inhibition of the inflammatory cascade. Previously, Peana et al.  and Cerq [et al.  have sho [et al.  proposedAs it is well known, peripheral or central opioid receptor activation could lead to the decrease of the pain sensation towards an inflammatory stimulus, through the NO-cGMP-K+ATP channel pathway activation . Based op < 0.05) reduced , is a major projection site of small-diameter afferent nerve fibers that predominantly transmit nociceptive signals. SG neurons also receive descending inputs from the brainstem . Baba etet al.  demonstrThe protooncogene c-fos, when activated, makes the immunologically detectable nuclear protein Fos . A strikIn addition, CNS-depressant drugs, such as gabapentin, frequently used as analgesic in some types of painful conditions, inhibit glutamatergic excitatory neurotransmission at the spinal dorsal horn and decrease Fos expression when they produce analgesic effect . Thus, oet al. [Earlier studies suggested that the CNS depression and the non-specific muscle relaxation effects can reduce the response of motor coordination and might invalidate the behavioral test results, including mechanical hyperalgesic tests ,39. Sincet al.  showed tSwiss mice (32–39 g), 2–3 months of age, were used throughout this study. The animals were randomly housed in appropriate cages at 21 ± 2 °C on a 12 h light/dark cycle (lights on from 6:00 a.m. to 6:00 p.m.) with free access to food  and water. Before the experiments, the animals were acclimatized to the laboratory for at least 1h. Mice were used only once in each test. Experiments were carried out between 9:00 a.m. and 2:00 p.m. in a quiet room. All experiments involving the behavioral analysis were carried out by the same visual observer and in a double-blind manner.All experimental protocols were approved by the Animal Care and Use Committee (CEPA/UFS # 04/12) at the Federal University of Sergipe, and handling procedures were in accordance with the Guide for the Care and Use of Laboratory Animals  for the use of animals in pain research . Male Swet al. [®, Ribeirão Preto-SP, Brazil) adapted with a polypropylene tip. The investigator was trained to apply the tip perpendicularly to the central area of the hindpaw with a gradual increase in pressure. The end point was characterized by the paw withdrawal followed by clear flinching movements. After this response, the intensity of the pressure was automatically recorded. The intensity of stimulus was obtained by averaging four measurements performed with minimal intervals of 3 min. The animals were tested before and after treatments.Mechanical hyperalgesia was tested in mice as reported by Cunha et al. , with alet al. . In a qun = 8, per group), which were treated with vehicle , hecogenin acetate , indomethacin  or dipyrone . Thirty min after treatment, 20 μL of carrageenan (300 µg/paw), PGE2 (100 ng/paw), DA (30 μg/paw) or TNF-α (100 pg/paw) were injected subcutaneously into the subplantar region of the hindpaw, as described by Cunha et al. [et al. [Mice were divided into five groups  according to Quintans-Junior et al. [To evaluate the possible non-specific muscle-relaxant or sedative effects of hecogenin acetate in the doses used, mice were submitted to the rota-rod test  were perfused with phosphate buffer (0.01 M) saline isotonic (PBS) followed by 10% buffered formalin (100 mM). The spinal cord (L4-L6) were removed and stored at −80 °C for the imunofluorescence against Fos protein.Ninety minutes after the intraperitoneal injection of HA at doses of 5, 10 and 20 mg/Kg or vehicle , the animals  using a plug-in (written by the authors) that uses the same level of label intensity to select and count the Fos-positive cells.Light level photomicrographs of spinal cord sections were acquired for each animal with an Axioskop 2 plus, Carl ZeissMice were slightly anesthetized with halothane and received 10 µL of complete Freund’s adjuvant  subcutaneously in the plantar region of the right hind paw, according to a method reported previously . The tra® 488, a Donkey Anti-Rabbit IgG (H+L), was obtained from Life technology .λ-Carrageenan, tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), dopamine (DA), ethylenediamine tetraacetic acid (EDTA), Tween 80, fluoromount G, glycine and bovine serum albumin (BSA) and hecogenin acetate  were purchased from Sigma . Indomethacin and dipyrone were purchased from União Quimica (Brazil). c-Fos Antibody, a rabbit polyclonal IgG, was obtained from Santa Cruz Biotechnology  and Alexa Fluorp < 0.05. All statistical analyses were carried out using Graph Pad Prism 5.0 .Data are presented as means ± standard error of the mean (SEM) of measurements made on 6–8 animals in each group. Comparisons between three or more treatments were made using two-way analysis of variance (ANOVA) followed by Tukey’s test. In all cases, differences were considered significant if Together, our results clearly indicate that HA displays significant anti-hyperalgesic effect in animal protocols. The precise mechanism by which HA promotes its effects is not clear, but the compound’s ability to modulate spinal cord dorsal horn and inhibit IL-1β expression appear to be likely one of the possible mechanisms. However, it is possible that another central or peripheral mechanism, not studied in present work, may be related to the analgesic effect of hecogenin acetate. Therefore, this steroidal sapogenin may be of potential interest in the development of new clinically relevant drugs for the management of painful conditions.


Ginsenosides are one of major types of bioactive compounds in American ginseng (AG) and utilized to assess the quality of various AG samples. The contents of ginsenosides showed cultivation region-related variation, which is possibly associated with AG’s pharmacological effect difference. Therefore, to reveal the quality difference of AGs in different cultivation regions, AG samples from seven cultivation regions were evaluated via analyzing their contents of nine ginsenosides and the biochemical parameters in AG-treated irradiated mice. Pre-administration of AG decoctions could reversely modulate the irradiation-induced changes of antioxidant enzymatic activity, cytokine level and hormone level in irradiated mice, which demonstrated that AG had the radioprotective effects due to its antioxidative, immunomodulatory and anti-inflammatory properties. However, this radioprotection effect varied among different cultivation regions of AGs. Collectively, Beijing and Canada-cultivated AGs had the best radioprotection. Heilongjiang and Jilin-originated AGs had the similar pharmacological effects while USA, Shandong and Shaanxi-grown AGs had closer pharmacological effects. This biochemical measurements-based PCA and heatmap clustering of AGs from seven cultivation regions was nearly consistent with ginsencoside content- and the previous serum metabolome-based analyses. However, the pearson correlation analysis revealed that only Rb3 and Rd were significantly correlated with some of assayed biochemical parameters in irradiated mice pretreated with different cultivation regions of AG extracts. Fukushima Nuclear Leak. Although the localised irradiation is usually adopted to reduce radiation risk in the radiotherapy, ionizing radiation had adverse effects on neighbor or even distant unirradiated cells due to its bystander and abscopal effects5. Meantime, ionizing radiation caused injuries to almost all the organs/tissues in the living organism such as spleen, liver, skin, brain and the gonads, although the probability and severity of ionizing radiation-induced risk often depends on various factors such as radiation dose and time, the health condition and age of the exposed person. To the current radiobiological knowledge, the radiation-induced injuries at the cellular or tissue level are mainly attributed to the oxidative damage on macromolecules DNA, lipids and proteins via the generation of free radicals and reactive oxygen species (ROS)6. Concurrently, immune and inflammation responses are also induced by irradiation to be against or adaptive to IR-induced oxidative stress7. Therefore, compounds or natural herbal products with antioxidative, immunomodulatory and (or) anti-inflammatory properties were investigated and appraised for their radioprotective performance in counteraction or alleviation of IR-induced side effects10.While ionizing radiation (IR) is increasingly used in the successful diagnosis of many human health problems and alone or combinational therapy of human cancers such as breast cancer14. Like other two precious Panax species, American ginseng  is widely used not only in folk supplementary diet as tonics and food additives but also in clinic to treat cancers, various fatigues, cardiovascular and metabolic diseases and so on. In addition, American ginseng was also prescribed in clinic by some Chinese doctors to improve irradiation-induced active syndromes such as oral mucosa inflammation and ulcer during or post radiotherapy of cancers17. Ex-vivo experiments showed that American ginseng extract could reduce irradiation-induced oxidative stress and DNA damage20, indicating that it had the radio-protective effects.Many preclinical and clinical studies revealed that AG extract and its active compounds such as ginsenosides and polysaccharides possessed the antioxidative, immunomodulatory and anti-inflammatory properties31, which are one of its major active compounds. A couple of experimental evidences indicated that AGs with the different metabolic profiles had somehow different pharmacological effects, which was possibly related to ginsenoside variation34. Studies from Panax ginseng Meyer and Panax notoginseng demonstrated that their extracts and ginsenosides  showed radioprotective effects via attenuation of radiation-induced DNA damage, oxidative stress, and inflammation etc.39. Panax ginseng extract (PGE) could markedly prevent the increases of irradiation-induced hepatic proinflammatory cytokines IL-6 and TNF-α40. Therefore, in this paper, we first investigated the variation of nine ginsenosides among AG samples from seven cultivation places: American, Canada and five provinces of China including Heilongjiang, Jilin, Shandong, Shaanxi and Beijing. Then, we investigated and evaluated the radioprotective effects of AGs from these cultivation regions via assays of antioxidative, immune function and hormone parameters in various AG-treated irradiated mice. Finally, the correlation of ginsenoside variation among different cultivation regions of AGs and the assayed individual radioprotective parameters was analyzed to evaluate the potential roles of the varying ginsenoside contents of AGs from different cultivation regions contributing to the radioprotection difference among them.American ginseng is now cultivated mainly in its native countries American and Canada and in north provinces of China. A number of chemometric studies revealed that AGs from different cultivation regions had the distinct metabolic compositions and profiles, for example, ginsenosides41. These ginsenosides were highly related to AG’s antioxidant, neuroprotective, cardioprotective, antidiabetic and anticancer properties13. These ginsenosides or most of them were simultaneously quantified by researchers to study the influence of cultivation region and year, extraction and processing on AG quality or ginsenoside content or to distinguish panax species, cultivated and wild AGs43. Rb1, Re and Rg1 were abundant in AG roots. The ratios of Rb1: Rg1 (>5), Rg1: Re (<1.0) and protopanaxadiol (PPD)-type to protopanaxatriol (PPT)-type ginsenosides (>2) were used to distinguish American ginseng from Asian ginseng (Panax ginseng C. A. Meyer)13. Therefore, the contents of these representative ginsenosides in AG samples from seven cultivation regions were determined using the optimized UPLC-UV method  to 46.73 ± 7.35 mg/g (Canada), which indicated that AGs that we collected from seven cultivation regions all met the least percentage quality requirement of AGs as qualified medicine (2%)45. The contents of Rg2 and Rg3 in AG roots are less studied compared to the other seven ginsenosides. As the reported content of Rg2 and Rg3 in AG roots from Canada or Jilin province of China47, much less Rg2 and Rg3 were present in roots of our AG samples.Ginsenosides are one of major bioactive compounds in American ginseng. Among the isolated ginsenosides, Rb1, Rb2, Rb3, Rc, Rg1, Re and Rd accounted for the majority of the total saponin content in AG rootset al.25 and Wang et al.30. Heatmap analysis of Huang et al.25 demonstrated that domestic AGs formed two chemoecotypes: inside Shanhaiguan group containing Beijing and Shandong provinces and outside Shanhaiguan group including Jilin, Heilongjiang and Liaoning provinces. PCA result of Wang et al.30 showed that ginsenosides in the roots of P. quinquefolius in Beijing, Jilin, and Heilongjiang regions were more similar than those from United States, Shandong and Shaanxi.Among nine analytes, the contents of Rg3, Rb2 and Rb3 showed no variation among seven cultivation regions. Our results revealed that the concentrations of the remaining six quantified ginsenosides differed just between some of seven cultivation regions. For example, Rg1 was significantly lower only in AGs from Shandong province, China than from Canada. Rc content showed no significant difference among the domestic AGs; its content was only found significantly different between Heilongjiang province (1.05 ± 0.19 mg/g) and the originating countries Canada (1.62 ± 0.41 mg/g) and USA (1.80 ± 0.49 mg/g). In addition, AGs from Shaanxi province of China had the lowest differential ginsenosides including Rg2, Rb1, Rd and Re, however, which were present in a relatively high amount in AGs from Canada. It was difficult to directly rank and compare the quality of AGs from different cultivation regions simultaneously based on these differential ginsenosides. Therefore, an unsupervised principal component analysis (PCA) on the contents of nine ginsenosides was further conducted to reveal the overall quality difference and relationship of AGs from different cultivation regions  significantly increased MDA content and decreased the spleen weight index  and the SOD and GSH-PX activities in irradiated model mice  are two important enzymatic members in antioxidant defense system against the formation of reactive oxygen species (ROS) and free radicals in living beings. Malonyldialdehyde (MDA) is produced during the peroxidation of polyunsaturated fatty acids and used as an evaluation marker of various oxidative stresses. Ionizing radiation led to the significant alterations of living beings in activities of antioxidant defense enzymes such as SOD and GSH-PX and MDA level. It was observed that administration of American Ginseng Capsule (AGC) suspension could significantly decrease liver MDA level while increase the contents of liver SOD and GSH in rats exposed to 900 MHz cell phone electromagnetic radiation7. Cytokines play important roles in radiation-induced immune and inflammatory responses and are modulated by ionizing radiation. Proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were elevated by irradiation and regarded as the early responsive biomarkers of radiation injuries51. They were also observed in our study to be significantly enhanced, respectively in the spleen and serum of irradiated mice, compared to the unirradiated control group , follicle stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in development, growth, pubertal maturation, gonadal maturation and fertility of the body. GnRH is synthesized and released from GnRH neurons within the hypothalamus. Gonadotropins FSH and LH are synthesized and released from the anterior pituitary, which are modulated by neuropeptides such as GnRH. Radiation induces hypothalamic–pituitary axis (HPA) dysfunction especially in the treatment of nasopharyngeal carcinoma and intracranial neoplasmssed Figs . General57. However, the radioprotection effects varied among different cultivation regions of AGs. Generally, Beijing and Canada-cultivated AGs had the best radioprotection in irradiated mice. Heilongjiang and Jilin-originated AGs showed the similar pharmacological effects whereras USA, Shandong and Shaanxi-grown AGs had the closer pharmacological effects. This biochemical parameters-based classification of AGs from seven cultivation regions was nearly consistent with serum metabolome-based analysis58, even though it demonstrated that AG had no effect in modulation of the radiation-altered metabolites in serum.Although our above comparisons of individual biochemical parameters revealed the relative radioprotective effects of AGs from different cultivation regions, however, it is hard and impossible to make a clear and consistent conclusion just based on some or few of the obtained measurements, for example, testicular estradiol and brain hormones. Therefore, to obtain a better overall pharmacological effect difference of AGs from different cultivation regions, we further performed principal component analysis and hierarchical clustering analysis on these data Figs  and 8. P13. In-vitro and in-vivo Panax ginseng experiments of individual ginsenosides showed that ginsenosides Rg1, Rg2, Rg3, Rb1, Rb2, Rb3, Rc, Rd and Re had the radioprotection against radiation-induced ROS, oxidative stress, aging, apoptosis and other side effects66. Pure ginsenoside experiments revealed that Rg160, Rb262, Rb364 and Rg265 significantly attenuated irradiation-induced changes of SOD, MDA and GSH, which indicated that the varying content of some ginsenosides in AG extracts was possibly correlated with the varying values of the assayed biochemical parameters among AG treatments. Therefore, we further performed the pearson correlation analysis of AG ginsenoside contents and mice biochemical measurements to evaluate the contributions of AG bioactive compounds in AG extract on AG radioprotection effects  effect difference presented among AGs of seven cultivation regions was nearly similar to ginsenoside content- and serum metabolome-based results, which provided the scientific basis that we could evaluate the pharmacological difference of AG samples based on measurements of some important multiple bioactive compounds in plants. AG polysaccharides are another important compounds with antioxidant and immunoregulatory activities and scarcely studied among different AG samples. Thus, it would be interesting to study its quality difference among different origins of AGs and its contribution to pharmacological difference of different origins of AGs.Thirty-eight four-year-old main root samples of American ginseng (AG) were collected from American, Canada and five provinces of China including Heilongjiang, Jilin, Shandong, Shaanxi and Beijing Table . The domhttp://cdmust.guidechem.com/). The purities of all the standards were higher than 98%. The purified water from Wahaha was used throughout the experiment. Other reagents of analytical grade were obtained from Beijing Chemical Industry Inc., China.HPLC-grade acetonitrile (ACN) and methanol were purchased from Thermo Fisher (USA) and Honeywell Co.Ltd., separately. Standards of nine ginsenosides including Rb1 (GR-16021907), Rb2 (GR-16032211), Rb3 (GR-16032211), Rc (GR-15111902), Rd (GR-16012503), Re (GR-16012701), Rg1 (GR-16022407), Rg2 (GR-16011604) and Rg3 (GR-16030711) were purchased from Chengdu Must Biotechnology Co. Ltd., China  were, separately, 1.756 mg/ml, 0.029 mg/ml, 0.024 mg/ml, 0.16 mg/ml, 0.178 mg/ml, 1.36 mg/ml, 0.206 mg/ml, 0.0117 mg/ml and 0.02 mg/ml in 5 mL of methanol. To make calibration curves, seven series concentrations were then separately prepared by diluting the working stock solution s1 with 0.7 fold difference. That is, 3.5 mL of former standard solution and 1.5 mL of methanol were taken to make 5 mL of standard solution.0.5 gram of each root powder sample was accurately weighed and ultra-sonicated once at the 100 Hz with 10 mL of chromatographic grade methanol at room temperature for 30 min. The extract was made up to the same volume with methanol and then centrifuged at 10000 rpm for 10 min at 4 °C. The supernatant was filtered with a 0.22 μm millipore membrane filter. One mL of the filtrate was transferred into a sample vial for the below UPLC-UV determination of the above nine ginsenosides. Three replicates of each location of root sample were prepared.Two microliters of ginsenoside standards and filtered AG root extracts were run at 35 °C on a Waters ACQUITY ultra performance liquid chromatography  system coupled with a Waters BEH C18 column . The mobile phase consisted of acetonitrile (A) and H2O (B). The optimized gradient conditions were set as: 0–3 min, 19% A; 3–5 min, 19–21% A; 5–10 min, 21–24% A; 10–12 min, 24–29.3% A; 12–14 min, 29.3% A; 14–16 min, 29.3–32% A; 16–18 min, 32%A; 18–20 min, 32–43% A; 20–23 min, 43–60% A; 23–24 min, 60% A; 24-25 min, 60-19% A. The flow rate was 0.4 mL/min for the first five minutes and then 0.3 mL/min for the rest time. The UV wavelength of the detector was set at 203 nm. Each extract was run twice. The contents of these nine ginsenosides in AG root samples were calculated using their respective standard curves , Jilin (JL), Shandong (SD), Shaanxi (SX) and Beijing (BJ) provinces of China. Then, 20 grams of the well-mixed root powder was weighed and extracted for three times. Each time, the root powder or residue was soaked in 500 mL of distilled water for one hour and boiled for 45 min. The pooled filtrate from three extractions was concentrated to 100 mL and kept at 4 °C till the commencement of the below mice administration.53. 108 ICR male mice weighing 20 ± 2 g were obtained from Peking University Health Science Center, Beijing, China (No. SCXK (Jing) 2014-0006) and housed at the animal center of IMPLAD, Beijing, China. The housing conditions were set as: 22–23 °C, 55 ± 5% humidity and 12-h light/dark cycle. The mice were acclimatized for one week and then randomly divided into normal control (NC) group, model control (MC) group and seven AG-administered groups. The seven administered mice groups were separately injected with seven cultivation regions of AG decoctions for 28 days prior to irradiation. The daily intragastrical injection dose for each experimental mouse was 0.2 mL AG decoction per 10 g of body weight. NC and MC mice received the same amount of distilled water daily as AG-administered groups. The total injection volume of the individual mouse was weekly adjusted based on its weekly body weight. After 28 days’ chronic administration, MC and AG-administered groups were daily whole body irradiated with 1.0 Gy 60Cobalt γ-rays for five consecutive days. The Guidelines of National Health Institutes of China for the Care and Use of Laboratory Animals (Certificate No. SYXK2013-0023 (Jing)) was obeyed throughout the animal experiment. The protocols were approved by our institutional ethics review board .The experiments including AG administration and irradiating mice were performed in accordance with Qin’sAn enzyme-linked immunosorbent assay (ELISA) method was employed to measure the levels of the following antioxidant, cytokine and hormone parameters.60Cobalt radiation, all the mice were sacrificed by cervical dislocation. Blood was collected through eye vessels and centrifuged at 4 °C at 3500 rpm for 10 min to obtain serum supernatant. Sera were then kept at −80 °C until the measurement of cytokines: interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α).On the next day of the last The activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and the concentrations of malondialdehyde (MDA) and interleukins  were assayed in the spleen. The levels of gonadotrophin-releasing hormone (GnRH), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured in the brain. The sex steroid hormones including Testosterone (T) and Estradiol (E2) were quantified in the testis. On the sampling day, spleen, brain and testis were separately collected, rinsed with physiological saline, dried on tissue paper and immediately stored at −80 °C until use. The organ tissues were thawed and homogenized in the ice-cold physiological saline to make 10% (g/ml) homogenates. The homogenate was centrifuged at 3,000 rpm at  4 °C for 10 min and then the supernatant was collected for the quantitation of total protein and ELISA assays. The protein concentrations of homogenate supernatants were determined using BCA method and subsequently normalized to the equal content before ELISA analysis.http://www.njjcbio.com/), all other ELISA kits were purchased from Beijing Donggeboye company (http://www.dg-reagent.com/). The procedures for all measurements were conducted according to the protocols of the corresponding ELISA kits.Except that the mouse ELISA kits for antioxidant activities were obtained from NanJing JianCheng Bioengineering Institute, China . Pearson correlation analyses of AG ginsenosides and AG pharmacological effects were conducted in SPSS (version 22.0) for accessing the influence of AG bioactive compounds on AG pharmacological effects.All data were represented as mean ± standard deviation (SD). The significance analysis was assessed with one-way ANOVA in excel or SPSS software. P < 0.05 was considered significant between the pairwised comparison. To reveal the relationships of AGs from different cultivation regions, principal component analyses based on AG ginsenoside contents and mouse biochemical measurements were conducted in SIMCA-P software . The unit variance scaling was applied to process the data before PCA. Meantime, the mouse physiological data were subjected to the heatmap analysis, which was implemented in MetaboAnalyst (Supplementary dataset 1 2


The main scope was the assessment of vinegar antioxidant and metabolic profiles and the identification of the most appropriate features influencing their type and subtypes. Red grape balsamic vinegars exhibited the strongest antioxidant profile. High total phenolic content and radical scavenging-antioxidant activity of vinegars was strongly correlated with high hue-angle and colour density values and low lightness and a* values. FT-IR spectra analysis confirmed the presence of organic acids and carbohydrates and, in combination with Gas Chromatography-Mass Spectrometry (GC-MS), the occurrence of phenolic compounds. NMR spectroscopy enabled the identification of 27 characteristic metabolites in each type of vinegar. The combination of all applied techniques provides critical information on compositional differences among the vinegars and could serve as an application tool for similar fermentation products.Commercially available common and balsamic vinegars were examined, using a combination of spectrophotometric, chromatographic, colorimetric and spectroscopic methods. Total phenolic content, antioxidant activity, radical scavenging capacity, phenolic profile, colour parameters, Fourier Transform Infrared (FT-IR) absorbance spectra and Nuclear Magnetic Resonance ( Vinegar is a widespread ingredient of the diet and used as acidic seasoning in salads, sauces and cooking, to improve the taste and the flavour of foods. In Ancient Greece vinegars were used as medicine and antiseptic agents. Vinegar is a fermentation product which can be produced by conventional and rapid methods from various fruits and vegetables, as grape, apple, pomegranate, sugarcane and rice . VinegarRecent studies have shown that the particular composition of vinegar, especially regarding organic acids, polyphenols and other phenolic compounds, affect the final characteristics and, hence, the quality of a vinegar and may have a positive effect for human health due to their antioxidant activity ,2. There1Η-NMR has been utilized for the differentiation of various types of cereal and grape vinegars in relation to their metabolic profiles  from Tokyo Chemical Industry Co. Ltd  and 3,4,5-trihydroxybenzoic acid from Alfa Aesar . All reagents and solvents used were of analytical grade and they were purchased from Mallinckrodt Chemical Works , Alfa Aesar GmbH & Co  and Sigma-Aldrich Chemie GmbH . NMR solvents and standards DTwenty-three common and twenty balsamic vinegars from different producers were purchased. Vinegars were further grouped according to their colour (red or white) and origin (grape or other fruit). The information provided on the bottle label is listed in The total phenolic content of each sample was determined applying a modified micro method of Folin–Ciocalteu’s colorimetric assay according to Andreou et al. . The absThe radical scavenging activity of vinegar samples was determined according to the method described by Lantzouraki et al. . Absorba4·7H2O per L of vinegar.The ferric reducing antioxidant power assay, based on the reduction of a ferric-2,4,6-tripyridyl-s-triazine complex to the ferrous form, was carried out according to the method described by Lantzouraki et al. . The absN,O–bis-trimethylsilyl-trifluoroacetamide (BSTFA). Simple phenolics in the form of trimethylsilyl (TMS) ethers, were quantitated by selective ion monitoring GC-MS employing 3-(4-hydroxyphenyl)-1-propanol as internal standard [Simple phenolic compounds were isolated from vinegar samples by solid phase extraction (SPE) on C8 Isolute columns, essentially as described by Soleas et al. . Columnsstandard . The tarColour values such as L* (lightness), a* (redness/greenness), b* (yellowness/blueness) and h (hue angle in degrees) were measured with a tristimulus chromatometer  calibrated with a white standard plate . Three random readings per sample were taken and averaged.The colour characteristics of vinegars were determined by measuring the absorbance spectrophotometrically at 420 nm (yellow) and 520 nm (red), using a 1 cm path length cuvette. The absorbance was measured on a double-beam UV–vis spectrophotometer Hitachi U-3210 . Colour density and tint were calculated using the following equations:−1 and the average of 64 scans at a resolution of 8 cm−1 with a scanner velocity of 7.5 kHz was recorded. One background measurement was taken before each sample measurement. The ALPHA Wine Analyser comes with a starter calibration that was assembled by the accredited (DAkkS) Institute Heidger . The organic acid and sugar contents were measured for each vinegar sample using the “ALPHA wine analyser” apparatus. FT-IR spectra were collected with an Alpha- P spectrometer, the Alpha FT-IR wine analyser (Bruker Optics Inc.)  on a diamond ATR crystal covered with a flow through cell, facilitating sample injection. The Alpha-P instrument has a potassium bromide (KBr) beam splitter and a 2 × 2 mm temperature controllable ATR diamond crystal sample plate, which was set at 40 °C. The instrument was fitted with OPUS software . No further sample preparation was done for spectral analysis and volumes of 5 mL were used. The spectrum of each sample and background were obtained from 4000 to 375 cm2O, then mixed with 50 μL of buffer (pH 5.6 in D2O containing 0.1% of TSP and 0.013% of sodium azide) and transferred into a 5 mm-NMR sample tube.300 μL of each vinegar sample were diluted with 150 μL D1H NMR spectra were acquired with the 1D NOESYPRESAT pulse sequence. The receiver gain was set at 30; two hundred and fifty-six transients were collected with 72 K data points using a spectral width of 9615.4 Hz with a relaxation delay of 2 s, mixing time of 100 ms and an acquisition time of 4.00 s.1H NMR metabolic database and literature data [A series of 2D experiments, gCOSY, zTOCSY, gHMBCad, gHSQCad were recorded at 25 °C and permitted the assignment of the existing metabolites. The interpretation of 2D spectra was performed with the use of MestReNova v.10.1 software. The identification procedure was based on 2D NMR spectra, the Chenomx Suite 7.0 reference ure data ,13. 2O (4.6–4.8 ppm), acetic peak (1.9–2.1 ppm). The spectra were then normalized to the standardized area of the reference compound and converted to ASCII format. The ASCII format files were imported into MATLAB  and all spectra were aligned using the Correlation Optimized Warping (COW) method [The NMR spectral data was reduced into spectral buckets of 0.0001 ppm. The following regions were removed: DAll the IR spectroscopic, the spectrophotometric assays and colorimetric measurements were carried out in triplicate for all samples. The colour values were treated as dependent variables and analysed using one way analysis of variance. The values for total phenolic content, radical scavenging and antioxidant activity did not meet the ANOVA assumptions , thus the Kruskal–Wallis non-parametric test was performed. The normality was tested by performing the Kolmogorov–Smirnov test. Correlations were performed using the Spearman’s rank-order correlation coefficient, which is a nonparametric measure of the strength and direction of association that exists between variables. In addition, one-way ANOVA was used to compare statistically test the NMR data in terms of the vinegar type. All statistical calculations were performed with the SPSS package  for Windows. 2 (0 ≤ R2 ≤ 1) and the predictive ability Q2 (0 ≤ Q2 ≤1) values [The IR data set were imported into the SIMCA-P version 14.0  for statistical analysis. The exploratory principal component analysis (PCA) was applied to acquire a general insight and visualize any relation  among the observations (samples) . Loading) values .The data concerning the total phenolic content (TPC), radical scavenging and antioxidant activity of the forty-three studied vinegars as well as their descriptive statistics are reported in p < 0.05), radical scavenging and antioxidant activity compared to the white grape balsamic (WGBV), red grape (RGV) white grape (WGV) and fruit (FV) vinegars (p < 0.05) than red grape balsamic vinegars with honey (RGBVH), no significant variation in radical scavenging and antioxidant activity was observed. According to Almaraz-Abarca et al. [p < 0.05) TPC, scavenging and antioxidant activity than the white grape (WGV) and fruit (FV) vinegars. This result could be correlated with the total phenolic content of the raw materials  since their phenolic composition contributes to the phenolic profile of vinegar. Additionally, RGV exhibited higher (p < 0.05) TPC and scavenging activity than WGBV, whereas no significant variation in antioxidant activity was observed (p < 0.05) TPC , scavenging activity (496.49 ± 13.84 mg Trolox E/L) and ferric reducing power (2409.87 ± 154.55 mg FeSO4·7H2O/L), compared to all white wine, apple and pomegranate common vinegars (p < 0.05) than red wine vinegars, it did not exhibit a similar result regarding the radical scavenging and antioxidant activities (p < 0.05) TPC , radical scavenging (4417.44 ± 52.22 mg Trolox E/L) and antioxidant activity (26293.23 ± 128.99 mg FeSO4·7H2O/L), compared to all samples, followed by the ageing balsamic vinegar Aceto Botanico Adriani Gold Seal from Lazaridi (BR10) (p < 0.05) radical scavenging and antioxidant activity, whereas the pomegranate vinegar from Ayanoglou S.A. (sample F4) exhibited the lowest (p < 0.05) TPC, compared to all samples. The apple vinegars (samples F1–F3)  . The wins F1–F3)  revealedGenerally, the considerably higher fluctuations of antioxidant activity values observed when compared to the respective TPC and radical scavenging activity values, could be possibly attributed to the different phenolic compounds’ profile of the samples, which is in accordance to Dávalos et al.  and to ONevertheless, high positive Spearman correlation values were observed between TPC and radical scavenging activity, TPC and antioxidant activity as well as among radical scavenging and antioxidant activity , confirmp-hydroxybenzoic, p-hydroxyphenylacetic and syringic acids were detected in almost all balsamic and common vinegars. According to Gil-Muñoz [Vitis and displays high antioxidant, antimicrobial, anti-inflammatory, anticancer and anti-anxiety activity. The presence of chrysin, quercetin and kaempferol was considerably higher in the common vinegars compared to the balsamic ones. Naringenin was detected only in the samples WW4-WW7 and F5 and may be attributed to their enrichment with oregano, basil, rosemary and thyme as well as sea buckthorn. Apple cider vinegars were characterized by higher proportion of phloretic acid and lower proportion of gallic acid compared to the grape ones. Regarding phenolic aldehydes, only vanillin was detected in low proportion in all common and in most of balsamic vinegars, in accordance with previous findings [The main phenolic compounds determined in the studied vinegar samples were phenolic acids and flavonoids . In partil-Muñoz  the extril-Muñoz . Interesil-Muñoz  reportedfindings .p < 0.05) L* (lightness) and a* values, were the darkest and the greenest in appearance, compared to the all other studied vinegars  having the lowest (vinegars .p < 0.05) L* and b* (yellowness) values compared to the respective ones of RGBV, probably due to their enrichment with honey. Interestingly, balsamic vinegars were characterized by negative a* values, instead of positive a* values for the common vinegars. Li et al. [p < 0.05) brightness and lower yellowness than the vinegars from red grapes. The colour density is the sum of the absorbance, measured spectrophotometrically, at 420 and 520 nm. High values of colour density denote vinegars with dark or brown red colour, whereas white or light yellow vinegars possess a low colour density (p < 0.05) tint values than the dark or red ones. In accordance to the aforementioned results, De la Haba et al. [p < 0.05) b* values compared to all the other samples, as well as substantially (p < 0.05) higher redness and colour density and lower colour tint, compared to all white or light yellow vinegars. Taking into consideration that pomegranate and sea buckthorn are rich sources of natural anthocyanins [p < 0.05) hue-angle values, followed by RGBVH, pomegranate and sea buckthorn vinegars, all corresponding to the yellow-green region of the colour wheel. Additionally, white grape balsamic, red grape, white grape and apple vinegars exhibited hue-angle values corresponding to the yellow region of the colour wheel. The red grape balsamic vinegars with honey (RGBVH) showed higher  of the studied vinegars.Fourier Transform Infrared (FT-IR) spectroscopy provides structural information on molecular features of a large range of compounds. −1, around the wavenumber of 1700 cm−1 and from 1450 to 770 cm−1. Most of these bands are associated with organic acids, phenolic compounds and carbohydrates. According to all spectra, the most characteristic bands in order to assess the vinegars’ composition and any differentiations between them are located from 3100 to 2800 cm−1, corresponds to aryl or vinyl C–H stretching vibrations [−1 is assigned to C–H stretching of methyl- and methylene groups of carbohydrates or carboxylic acids [p < 0.05) intensities for both the aforementioned bands, followed by red and white grape balsamic vinegars , while the common vinegars gave the lowest intensities  intensities at 1721 cm−1 compared to those of red vinegars, regardless if they were balsamic or common. Interestingly, this finding was also in accordance with the lower organic acid content of the specific vinegars  higher intensities (over than 1.5 fold higher) in the region 1080–1040 cm−1, compared to the rest red and white grape balsamic vinegars, whereas the RGBV and the WGBV higher (over than 13 fold higher) intensities than the common vinegars (p < 0.05) high fructose and sucrose content, as expected. The band at 1425–1380 cmompounds ,46 and tavonoids . Accordiavonoids . Moreoveavonoids  that brovinegars . This revinegars , as oppo−1 could be assigned to C–H out of plane bending vibrations of phenyl ring of polyphenols [−1 could be assigned to aromatic ring vibrations [p < 0.05) signal in RGBVH, followed by RGBV and WGBV , the intensity values at 1300–1260 cm−1  and the intensity values at 825–770 cm−1 . This high correlation confirms that the vibrations in the aforementioned areas are associated with substances  with significant radical scavenging and antioxidant activity.Interestingly, high positive Spearman correlation values were observed among the TPC, ABTS and FRAP results and the intensity values at 1425–1380 cm−1 and increasing intensity at 3140–2840 cm−1 and 1430–770 cm−1  of identified compounds for the six categories of vinegars are presented in −1, 1300–1260 cm−1 and 825–770 cm−1, mainly attributed to the vibrations of aromatic compounds, polyphenols and hydroxyflavonoids. NMR spectroscopy enabled the identification of 27 metabolites in each type of vinegar. Concentration box plots of characteristic compounds exhibited similar trends with the FT-IR results especially in respect to the organic acid and sugar content.In the present study, forty three samples of balsamic and common vinegars were examined. Spectrophotometric, chromatographic, colorimetric and spectroscopic methods were synergistically applied in order to assess the vinegar’s compositional profile and to pinpoint the features that influence their type. The strongest antioxidant and radical scavenging capacity was exhibited by the red grape balsamic vinegars; a finding which was also correlated to the highest level of total phenolic content as well as to highest hue-angle and colour density values and lowest lightness and a* values. FT-IR spectra analysis determined specific organic acids and carbohydrates, as well as the occurrence of phenolic compounds. High positive correlation values were observed among the TPC, ABTS and FRAP values and IR intensities at 1425–1380 cm


Traditionally, surgical pathology reports are narrative. These report types are prone to error and missing data; therefore, structured standardized reporting was introduced. However, the effect of synoptic reporting on the completeness of esophageal and gastric carcinoma pathology reports is not yet established.A population-based retrospective nationwide cohort study in the Netherlands was conducted over a period of 2012–2016, utilizing the Netherlands Cancer Registry for patient data and the nationwide network and registry of histology for pathology data.p < 0.01). Out of 21 standard items, 15 were significantly more frequently reported in synoptic reports.In total, 1148 narrative and 1311 synoptic pathology reports were included. Completeness was achieved in 56.4% of the narrative reports versus 97.0% of the synoptic reports  contains supplementary material, which is available to authorized users. Traditionally, surgical pathology reports are narrative, meaning they are written without a fixed form or structured outline . NarratiIn 1991, the idea of a synoptic pathology format was introduced, meaning that completing a standardized structured form based on relevant and up-to-date guidelines will create a pathology report . Since iThe main objective of this study is to assess the effect of SSR on the completeness of esophageal and gastric carcinoma pathology reports. We hypothesize that SSR will improve the completeness of reporting .Pathologisch-Anatomisch Geautomatiseerd Landelijk Archief; PALGA) [A population-based retrospective nationwide cohort study was conducted including surgically treated patients with primary gastric or esophageal cancer, diagnosed between January 2012 and January 2016. Patient data, as registered by the Netherlands Cancer Registry (NCR), were linked to corresponding pathology report data from the nationwide network and registry of histo- and cytopathology in the Netherlands  . Since nn = 45) were not included in further analyses.Surgical pathology reports concerning EC and GC types 15.0 to 15.9 and 16.0 to 16.9 respectively, according to the 10th edition of International Classification of Disease (ICD), were included in the study. The period 2014–2016 was selected, since both SSR and NR were used during that period, with SSR for EC and GC being gradually introduced in March 2014 in the Netherlands. Narrative pathology reports from the cohorts 2012 and 2013 were added as a separate reference group. The small number of SSR in this period of introduction , comparable to the guidelines of the College of American Pathologists’ (CAP) [The primary outcome was completeness, which was defined as the inclusion of all items in the pathology report as recommended by Dutch guidelines (s’ (CAP) , 8. Stans’ (CAP) , 12. StaDifferentiation grade is applied for grading squamous cell carcinomas and intestinal adenocarcinomas according to the WHO classification system . The iteCompleteness was also compared when pathology reports were further grouped into academic and non-academic centers regarding SSR.p values less than 0.05 were considered to be statistically significant.Statistical analysis was performed with the SPSS software package, version 24.0 . For dichotomous or ordinal variables, the Pearson’s chi-squared test or Fisher’s exact test was used if appropriate. All tests were two-sided, and p = 0.80).In total, 4838 pathology reports were included: 2379 (49.2%) reference, 1311 (27.1%) synoptic and 1148 (23.7%) narrative reports . Besides “number of lymph nodes” and “T stage,” which were comparable, all standard items were significantly more frequently reported in SSR. The difference in completeness between SSR and NR did not affect the distribution of reported histological tumor types in the EC group (p = 0.46): adenocarcinoma 890 (80.8%), squamous cell carcinoma 154 (14.0%), and other types 57 (5.2%). Regarding optional items, SSR resulted in a much higher percentage of reported “lymphovascular invasion” and “perineural invasion”.In total, 2858 EC pathology reports were included, 685 narrative and 806 synoptic and 1367 in the reference cohort. Completion was achieved in 450 (65.7%) narrative versus 789 (97.9%) synoptic reports (p < 0.01). “Differentiation grade” was significantly more reported in SSR compared with NR , whereas “invasion depth,” “number of lymph nodes,” and “histological tumor type” showed no significant difference between NR and SSR. Reporting of GC histological tumor type, consisting mainly of adenocarcinoma (864 (96.0%)), did not differ between SSR and NR; however, “subtype of AC” was less frequently reported with NR compared with SSR . The optional items, “lymphovascular invasion” and “perineural invasion” were much more frequently reported with SSR.In total, 1980 GC pathology reports were included, of which 463 narrative, 505 synoptic and 1012 in the reference cohort. In total, 197 (42.5%) NR were complete versus 483 (95.6%) SSR (Missing data in SSR was caused on one hand by software-related issues in the early stages of implementation (teething problems). For EC, 20%, and GC, 19% of the omitted items were due to software-related factors. Also, in the SSR software, differentiation grade is considered optional when neoadjuvant therapy was given (since grading is not possible in (sub-)total regression). On the other hand, the option of manually turning off several features, including standard items, remained through all development stages. It remains up to the pathologist whether he uses the complete protocol and/or the standard pathology conclusion or not.This study shows that SSR increases the completeness of surgical esophageal and gastric cancer resection pathology reports, in particular for items as tumor size, grade, location, tumor regression after neoadjuvant therapy. SSR also increases lymphovascular invasion and perineural invasion reporting. Essential items for staging including depth of invasion, number of (metastasized) lymph nodes and tumor type were present in almost all narrative reports, and only minimal improvements could be achieved by SSR. For GC reporting especially, SSR leads to an increase in the reporting of histological subtype of adenocarcinomas. This is clinically relevant, since poorly cohesive adenocarcinomas (WHO) show a different biological behavior resulting in different clinical patient outcomes .all pathologists with their histopathology reports. Although improved report completeness does not seem to have a direct impact on adjuvant treatment in GC and EC, as is the case in colorectal cancer—since the presence of lymph node metastases and pT4 tumor stage necessitate adjuvant therapy—this might very well change in the future. We do not claim that the introduction of SSR is the only or main factor for improved pathology reporting. Other factors, that may have influenced awareness among Dutch pathologists for EC/GC pathology reporting, are increased centralization of gastro-esophageal oncological surgery, comparative feedback by the Dutch Upper GI Cancer Audit (DUCA) (https://dica.nl/duca/) and monitoring during the multicenter gastric cancer CRITICS trial [The differences between NR and SSR, regarding pathology report completeness, are in line with the systematic review by Sluijter et al. . In accoCS trial .To our knowledge, this is the first report on a nationwide level with roughly 5000 pathology reports describing the influence of SSR on the completeness of EC/GC surgical pathology reports. The data originate from a nationwide database, which increases the power of the overall study, despite its retrospective nature.Furthermore, PALGA’s SSR software is considered to be level 6 on the 1–6 reporting level and quality scale developed by Shrigley et al., which scores pathology reporting on format, content and several other quality indicators . Level 1Despite comparing reporting type on academic and non-academic level, inter- and intra-pathologist reporting differences were not accounted for, which possibly could have affected the completeness of reports .In conclusion, our study has shown that SSR definitely improves the quality of surgical pathology reports of EC/GC and, therefore, should be part of standard patient care.ESM 1(PDF 354 kb)


Proteomic studies of follicular fluid (FF) exist for several species, including the horse; however, the seasonal influence on FF proteome has not been explored in livestock. The application of high-throughput proteomics of FF in horse has the potential to identify seasonal variations of proteins involved in follicle and oocyte growth.This study (i) profiles the proteomes of equine FF collected from dominant growing follicles during the spring anovulatory season (SAN), and spring (SOV), summer (SUM), and fall (FOV) ovulatory seasons; and (ii) identifies season-dependent regulatory networks and associated key proteins.Regardless of season, a total of 90 proteins were identified in FF, corresponding to 63, 72, 69, and 78 proteins detected in the SAN, SOV, SUM, and FOV seasons, respectively. Fifty-two proteins were common to all seasons, a total of 13 were unique to either season, and 25 were shared between two seasons or more. Protein-to-protein interaction (PPI) analysis indicated the likely critical roles of plasminogen in the SAN season, the prothrombin/plasminogen combination in SUM, and plasminogen/complement C3 in both SOV and FOV seasons. The apolipoprotein A1 appeared crucial in all seasons. The present findings show that FF proteome of SUM differs from other seasons, with FF having high fluidity (low viscosity).The balance between the FF contents in prothrombin, plasminogen, and coagulation factor XII proteins favoring FF fluidity may be crucial at the peak of the ovulatory season (SUM) and may explain the reported lower incidence of hemorrhagic anovulatory follicles during the SUM season.The online version of this article (10.1186/s12958-019-0473-z) contains supplementary material, which is available to authorized users. Within the existing efforts to improve fertility in livestock and companion animals, a different knowledge is sought after to achieve a greater reproduction rate, especially for females. A lack of high-quality oocytes and reliable reproductive biomarkers , 2 repreThe ovarian activity of non-pregnant mares is continuously changing throughout the year, presenting periods of intense activity during summer, low activity during winter (deep anestrus phase), and irregular activity during spring and fall transitional seasons –7. ResulHigh-throughput technologies  allow for in-depth investigations of complex samples such as FF, with potential for new biomarker discoveries, or strategies for intrafollicular treatment. Large-scale proteomics approaches (gel-based and gel-free) were applied to either profile or compare global proteomes of FF in cows , 22, humThe aims of the present study were to (i) use the shotgun (gel-free) approach to evaluate the proteome profiles of equine FF collected from ovarian follicles (30–34 mm in diameter) at different seasons of the year ; and (ii) apply comparative bioinformatics analyses to identify potential regulatory network differences.Seventeen Quarter horse mares, 8 to 14 years old and weighing 400 to 600 kg, were housed on pasture in the northern hemisphere ,https://grants.nih.Gov /grants/olaw /references/phspol.htm #US GovPrinciples). This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Southern Illinois University.under natural light conditions, with free access to fresh water and trace-mineralized salt. Animals were handled in accordance with the US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training  representing the transition season when dominant anovulatory follicles are found after the deep anestrus season and before the spring ovulatory season; − Between April and May, as Spring Ovulatory (SOV) representing the beginning of the ovulatory season, with regular cycling of mares; − Between June and July, as Summer (SUM) representing the middle of the ovulatory season, with maximum of ovarian cyclic activity expected; − and September, as Fall Ovulatory (FOV) representing the final period of the ovulatory season befor the transition to the fall anovulatory and deep anestrus seasons. In all four seasons, follicles ≥6 mm in diameter were ablated, as previously described [n = 6 follicles) were collected when the follicles reached 30–34 mm in diameter. During SOV, SUM, and FOV, mares were monitored daily with ultrasonography until ovulation; thereafter, follicle ablation was performed on day 10–12 after ovulation (day 0 = ovulation) and follicle tracking of the new induced wave was performed daily to collect FF when a dominant follicle reached 30–34 mm in diameter. Samples of FF were aspirated during SOV, SUM, and FOV seasons . In all seasons, the presence of uterine edema (estrus-like) and the absence of a corpus luteum detected through ultrasonography at the moment of FF collection did qualify the animal for the procedure.Follicular fluids were collected from dominant growing follicles during various seasons of the same year: – escribed , to induSamples of FF were collected using transvaginal ultrasound-guided follicle aspiration as recently reported . Samplesv/v - FF:Acetone), precipitated overnight at 4 °C, and washed twice with acetone by successive centrifugations . Final protein samples were quantified  and aliquots of each sample were mixed with sample buffer and loaded into wells of a 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gels were run as previously described [Optimal isolation of frozen-thawed equine FF proteins was tested through various equine FF:Acetone:Trichloroacetic Acid (TCA) mixture ratios . Mixtures were incubated , centrifuged , and supernatants were discarded. Cold acetone  was added to each pellet and sample mixtures were vortexed (20 min), centrifuged , and resulting supernatants (acetone) were discarded. After three repetitions, pellets were dried under the fume hood and resuspended in the nanopure water. All protein samples were subjected to albumin depletion according to the manufacturer’s instruction . Depleted protein samples were mixed with acetone in a 1:4 ratio  formic acid, 2.0% (v/v) acetonitrile. Aliquots of peptides representing two micrograms of protein were subjected to LC-MS analysis as described previously [Extracted FF proteins of each mare were determined  and equal amounts of proteins of two to four mares were pooled for each season . For proteomic analyses, three independent pools (100 μg protein each) were constituted for each season. Pooled samples were precipitated overnight with 100% acetone (1:5 ratio), washed two times with 100% acetone, air-dried, and stored at − 20 °C. Prior to in-solution digestion, protein precipitates were dissolved in 100 μl of 100 mM ammonium/5% acetonitrile, reduced with 1/10 volume of 100 mM dithiothreitol (DTT) for 15 min at 65 °C, and alkylated with 1/10 volume of 10 mM iodoacetamide (IAA) for 30 min at room temperature in dark. Digestion was carried out with Trypsin/Lys-C Mix  at 37 °C overnight. Samples were freeze-dried and protein tryptic digest was resuspended in 0.1% (eviously . Brieflywww.ncbi.nlm.nih.gov) Equus caballus taxonomy referenced protein database  served as the target database, while its reversed copy  served as a decoy database. The search results were filtered by FDR < 1% for high-confidence protein identification. Proteins were functionally annotated  using the online tools of Agbase (http://agbase.arizona.edu/), DAVID , and STRING (https://string-db.org/cgi/input.pl?sessionId=LyvanBxDO3QN&input_page_show_search=on) using the default settings.The raw files were searched using the SEQUEST algorithm of the Proteome Discoverer 1.1.0 software (Thermo Fisher Scientific) as described previously . VariablThe protein concentrations of pure FF derived from all seasons  averaged 39.2 ± 0.4, 38 ± 0.3, 38.3 ± 0.4, and 39 ± 0.4 μg/μl, respectively. The use of pure FF samples (33.2 ± 0.4 μg/μl) for protein precipitation tests  resulted in decreased protein concentrations , while the additional albumin depletion procedure led to lesser protein concentrations in all tested FF groups . Representative electrophoresis gels of both precipitated  indicates 52 proteins shared across all seasons, 25 proteins detected in two or three different seasons, and 13 unique proteins identified in a specific season , and all seasonal proteome datasets with full protein annotations are provided as supplementary data , molecular functions (MF), and biological processes (BP). Regardless of season, proteins were distributed within 9–10, 12, and 20 GO terms associated with CC, MF, and BP, respectively. The functional categorization of shared proteins and the observed quantitative variations in GO terms constituting each functional category are shown Fig. .Fig. 3FuP < 10− 5, FDR < 0.01). Other GO names such as “blood microparticle” and “fibrinogen complex” were significantly enriched  in our datasets. Similarly, the GO terms associated with binding  were highly represented (~ 49%) within the MF category . Nonetheless, several GO terms associated with binding  and enzymatic activity  were significantly enriched , but with higher FDR (> 0.01). As for the BP category across seasons, approximately 70, 8, 5, and 4% of total annotations were respectively associated with “processes”, “response to stimulus”, “transport”, and “cell communication” , and the “blood coagulation” GO term was substantially enriched in the SUM samples only . Finally, the “complement and coagulation cascades” KEGG pathway was significantly enriched , regardless of the reproductive season.Irrespective of the season, GO terms associated with extracellular components  represented approximately 54% of the total annotations within the CC category Table . These s1Qualitative and quantitative differences in GO terms constituting each functional category were found across seasons Tables -5. GO teP < 10− 16). A representative PPI network generated from shared dataset is shown , SOV (n = 14), FOV (n = 13), and shared (n = 12) protein datasets, while the combination of F2 (n = 13) with PLG  appeared as the main players in the SUM dataset. In cluster 2 , while the combination of both plasminogen (n = 10) and ENSECAG00000000339 (complement C3) with four and five interactions may have important roles during SOV and FOV seasons, respectively. Contrarily in the SUM dataset, the F12 (coagulation factor XII) protein appeared as the main player with 12 interactions. Finally, the cluster 3  and significant PPI enrichment  during the SAN, SOV, SUM, and FOV seasons. The existence of seasonal proteins in a similar follicle class, reported herein for the first time, suggests potential critical roles of FF proteins during the folliculogenesis and maybe oogenesis in the equine species. Likewise, various biological functions, protein enrichment, and protein interaction networks are reported to have been influenced by the seasonal variations.Combination of procedures, such as protein precipitation and depletion of high-abundant proteins, are routinely used to enhance the quality of starting samples for proteomic analyses , 34, 35.The gel-free LC-MS proteomics has been successfully used in previous studies of FF of stock animals , 23, 26.Among the 90 proteins, a core set of 52 was detected across all seasons. It is expected that these proteins may have essential roles during folliculogenesis  and oogeIn contrast, keratin-10 was detected in SAN samples only and may, therefore, be associated with the non-ovulatory seasons. Although keratin is considered a common contaminant in proteomic studies, the keratin-10 family member has been reported as a negative modulator of cell cycle progression throughout the Phospho-Inositol 3 kinase (PI3 kinase) signal transduction pathway . NumerouIn this study, the interpretation of bioinformatics analyses focused only on proteins exhibiting thresholds of significance that were lower than 1% in both Benjamini-Hochberg and FDR analyses. About half of the protein annotations belonged to the Extracellular GO term, and only 10% of the total annotations were attributed to Intracellular localization, regardless of the proteome dataset. This distribution is expected, given the composition of the FF, known to contain secretions of follicle cells and blood plasma exudates. Thus, proteins attributed to Intracellular regions may be residues of the various catabolic processes and/or cell breakdown (apoptosis) of follicle cells (granulosa cells) that occur throughout the follicle growth , 49, 50.Regarding protein functions, approximately 49% of the total annotations belonged to binding (protein binding and nucleic acid binding GO terms), and 32% corresponded to other cellular and molecular activities. This specific distribution is in agreement with previous studies in humans , 51, andProteins associated with inflammatory responses , a FF signature across studies and species such as humans , goats , cattle The functional categorization indicated a higher proportion of proteins associated with the “Extracellular space” GO name during the SUM season. The increase in FF protein content found in our study during the SUM season may have been due to an increase in ovarian vascularization/blood flow , 55, 56,Proteins associated with the “complement and coagulation cascades” pathway were significantly enriched in all seasons: 25.4% in SAN, 23.6% SOV, 23.2% SUM, and 25.6% FOV. Indeed, the complement system and inflammatory processes regulate follicle development and ovulation –59. NumeThe PPI network information is one of the major fields in systems biology allowing for complex network analyses . The PPIFirstly, prothrombin or F2 is a glycoprotein and an essential component of the blood-clotting mechanism exerting effects through its mature form, thrombin, by interacting with specific receptors (or protease-activated receptors or ThRs) on the granulosa cell membrane , 64. HowIn summary, this study describes, for the first time, the proteome profile of the equine FF collected during anovulatory (SAN) and ovulatory  seasons. Functional analyses revealed differences that may be essential to better characterization of reproductive seasonality in mares. The findings show that SUM follicular fluid proteome of dominant follicles (30–34 mm) differs from other seasons and appears to be characterized by a higher fluidity. This former characteristic may allow a more efficient natural flux of biological factors to the oocyte, influencing its maturation, ovulation, and safe transport to the oviduct. While the “coagulation and complement” cascades were confirmed as the prime signatures of the FF proteome, the balance between prothrombin, plasminogen, and coagulation factor XII proteins seemed crucial for the fluidity of the FF at the peak moment of the ovulatory season.Additional file 1:Table S1. All seasonal proteome datasets, with full protein annotations. (ODS 1926 kb)


Background: There is increasing interest in screening for and diagnosing pulmonary hypertension earlier in the course of disease. However, there is limited data on cardiopulmonary abnormalities in patients with pulmonary hypertension newly diagnosed in World Health Organization Function Class (WHO FC) I.Methods: Data were retrieved from the ASPIRE registry  for consecutive treatment naïve patients diagnosed with pulmonary hypertension by cardiac catheterization between 2001 and 2010 who underwent incremental shuttle walk exercise testing.Results: Eight hundred and ninety-five patients were diagnosed with Group 1-5 pulmonary hypertension. Despite the absence of symptoms, patients in WHO FC I (n = 9) had a significant reduction in exercise capacity  65 ± 13%, Z score −1.77 ± 1.05), and modest pulmonary hypertension with a median (interquartile range) pulmonary artery pressure 31(20) mmHg and pulmonary vascular resistance 2.1(8.2) Wood Units, despite a normal diffusion of carbon monoxide adjusted for age and sex (DLco)%pred 99 ± 40%. Compared to patients in WHO FC I, patients in WHO FC II (n = 162) had a lower ISWD%pred 43 ± 22 and lower DLco%pred 65 ± 21%.Conclusion: Our results demonstrate that patients with newly diagnosed pulmonary hypertension with no or minimal symptomatic limitation have a significant reduction of exercise capacity. Despite advances in treatment, pulmonary hypertension (PH) remains a progressive life-limiting disease . StudiesAt rest the pulmonary circulation has large microcirculatory reserves. These are recruited during exercise, increasing the capillary surface area available for gas exchange and maintaining a low pulmonary artery pressure despite increased flow . Any redThe incremental shuttle walking test (ISWT) has no ceiling effect  and corrData were retrieved from the ASPIRE registry  for consecutive, treatment naïve patients diagnosed with pulmonary hypertension between 2001 and 2010 . The sysThe ISWT was performed according to the method of Singh et al. . PatientLung function tests were performed in accordance to the European Respiratory Society guidelines –25. Predt-test and Mann Whitney U-test and Kruskal-Wallis test were used to compare groups. Event (death or transplantation) free survival from date of diagnosis was estimated using the Kaplan–Meier method with comparison between groups performed by the log-rank test. Cox proportional hazards regression analysis was used to assess the effect of ISWD%pred, age, sex, BMI, mPAP and DLco on survival time. As left to right shunt is known to result in high DLco%pred .Statistical analysis was performed using IBM SPSS Statistics v19 . Data is presented as mean ± SD for parametric data and median (interquartile range) for nonparametric data. Categorical variables were presented as %. To evaluate our hypothesis that the incremental shuttle walking test will be reduced in WHO FC I, the main outcome variable was ISWD%pred. DLco%pred was a secondary outcome to be used in comparison to the ISWD. Pearson's correlation test was used to assess correlations between these parameters and WHO FC and hemodynamic parameters. The student Lco%pred , separatDuring the duration of the study 895 patients were diagnosed with pulmonary hypertension and met the entry criteria Figure . At diag2 with no significant difference compared to WHO FC II.Patients' characteristics are shown in Table p all < 0.05) with WHO FC, mRAP, mPAP, sPAP, CI, PVR, and SmVO2  score) were all significantly higher in WHO FC I compared to WHO FC II. In addition, resting oxygen saturation pre-test was significantly higher in WHO FC I compared to WHO FC II. There was no significant difference in Borg dyspnea score between these 2 groups either pre- or post-ISWT and no significant difference in highest heart rate or highest systolic blood pressure measured. ISWD%pred correlated significantly , DLco%pred (74 ± 14 vs. 69 ± 17) and FVC%pred (96 ± 12 vs. 89 ± 23) were no longer significantly different (p > 0.05). Similar patterns of heterogeneity was seen in the frequency of standardized scores.Although there were significant correlations between WHO FC and ISWD%pred and DLco%pred, there was heterogeneity in ISWD%pred and particularly DLco%pred within each WHO FC Figure . FiguresTo assess the suitability of the ISWT as a screening tool, cut- off points using %pred and z-score were investigated. Figure p < 0.0001)   Table .To our knowledge we have shown for the first time that patients with pulmonary hypertension in WHO FC I have a significant reduction in exercise capacity compared to predicted values. We have also demonstrated that exercise capacity is more sensitive than measurements of gas transfer made at rest in identifying patients with pulmonary hypertension in WHO FC I and WHO FC II. A single cut-off point is often suggested for screening as it is easy to remember and apply. We evaluated the use of a cut-off point of 80%pred looking for early changes and found that 89% of patients in WHO FCI were below this cut-off point. Whilst traditionally the normal range for a lung function parameters was considered as being between 80 and 120%pred it is known now that this could lead to misdiagnosis . We therIn addition to confirming the findings of previous studies , 29, 30 It could be possible that there is some misclassification of the patients as classification into WHO FC is limited by patient and physician subjectivity and agreement between observers is often poor . There aCurrent ESC/ERS guidelines recommend the use of Doppler echocardiography for screening for pulmonary hypertension in at risk patients. Like all screening tools echocardiography has limitations. In 10–20% of patients it is not possible to obtain interpretable results and the precision of echocardiography estimation of systolic pulmonary artery pressure can be poor . To overA number of studies have looked at the use of exercise testing including cardiopulmonary exercise testing, exercise Doppler echocardiography or diffusion capacity during exercise –39 to trThis was a retrospective, single center study. Within the studied population only 1% of patients were diagnosed in WHO FC I and 18% in WHO FC II. These percentages are small (but are very similar to those found in the Geissen Pulmonary Hypertension Registry, which has 1.0% patients in WHO FC I, and confirm the findings in other registries , 26 thatOur results demonstrate that patients with newly diagnosed pulmonary hypertension with no or minimal symptomatic limitation have a significant reduction of exercise capacity.CB, IA, RC, and DK: study design; JH, RL, CB, IAS, MA, AC, CE, RC, and DK: performance of the research; CB, AT, CE, IS, AC, AL, and DK: data analysis; CB and DK: writing of the paper; All authors: revision of 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.