To evaluate the safety and efficacy of interventional care in pediatric hemoptysis for anomalous bronchial arteries (BAs) and to identify the potential factors resulting in hemoptysis recurrence. 20 children complained of hemoptysis were diagnosed with anomalous BAs. All patients received transcatheter plug occlusion in Department of Cardiology, Children's Hospital of Chongqing Medical University. The safety and efficacy were evaluated according to clinical symptoms and images monitoring of enrolled subjects grouped as recurrence group and nonrecurrence group. The potential factors causing hemoptysis recurrence were reviewed and summarized.P=0.049), mycoplasma pneumonia particle assays (MP-PA) titers (P=0.030), and number of anomalous BAs (P=0.020). Meanwhile, 50% recurrent scenarios were associated with a respiratory infection by microbiological assessment before transcatheter plug occlusion. The repeat occlusion was applied for unclosed BAs leading to visual recurrent hemoptysis, the average interval time of which was 5.4 ± 3.6 mon. No deaths were recorded in a follow-up. Otherwise, hemoptysis recurrence was found in 8 subjects for 14 times, accounting for about 40%. Compared with nonrecurrence group, it indicated a statistical significance in hemoglobin levels ( The data from this retrospective study have shown that transcatheter plug occlusion is a relatively safe procedure with a low mortality. The number of abnormal BAs has been identified as a highly significant predictor of recurrence, and the role of MP and other potential factors should be verified in a multicenter, larger sample size, and randomized controlled trial. A remorrhage . BAs mosmorrhage . Since bmorrhage . Data frmorrhage . CurrentThis study retrospectively reviewed the clinical data of 20 pediatric patients, who were complained of hemoptysis and received interventional care for the first time in the latest five years (from Nov. 2012 to Oct. 2017). The enrolled subjects comprised 9 males and 11 females aged 9.3 ± 3.0 years of age. All patients were hospitalized and registered in the Department of Cardiology, at the Children's Hospital of Chongqing Medical University (CHCMU). The diagnosis of pediatric hemoptysis was carried out according to a published Chinese standard in 2014. According to clinical records, laboratory examinations, and diagnostic images, the patients were diagnosed with aberrant BAs. Additionally, subjects with the following characteristics were excluded: (1) presence of congenital heart disease (CHD) history; (2) with an imaging confirmation of bronchiectasis; (3) with evident outcomes for tuberculosis infection; (4) highly suspected of idiopathic pulmonary hemosiderosis (IPH) through fiber bronchoscope; (5) did not display an accurate image indicative of bronchial anomalies; (6) had not undertaken an interventional management course of clinical care. This study was approved by the local Ethics Committee of CHCMU. Informed and written consent was obtained from the parents of each patient.According to the estimated standard , 11, we DSA was performed using the transfemoral route using angiographic contrast injected at a dose of 1 ml/kilogram per second from a 5F angiographic catheter . Thoracic aortography was then performed to achieve an approximate view of aberrant BAs or collateral arteries. In addition, selective angiograms identified the anomalous arteries that were characterized by dilation and tortuosity, which clearly paralleled the bronchi and the bleeding sites of target vessels. The vascular plug  was used in each patient (size of the plug selected was in accord with the diameter of targeted vessels) using a transition system comprising 4F/5F catheters, of which an actual picture was displayed in Hospital stay  was recorded to assess the safety of procedure. After the discharge, the clinical conditions were then tracked by telephone interview, and each patient was commenced with a regular hospital visit for recording of any clinical manifestations, chest radiography, and echocardiogram in the outpatient department of our hospital. When necessary, a repeat DSA could be recommended. A primary endpoint of each hemoptysis scenario has been defined. For every scenario of recurrent hemoptysis, the blood loss, routine blood examination, microbiology, interval time from the last closure, and targeted anomalous vessels were recorded.Continuous variables were tested for normal distribution and described as mean ± standard deviation (SD) about the mean. For an abnormal distribution, data were displayed as median , and the nonparametric Mann-Whitney U test was applied. The Fisher exact test was calculated for frequency. An p<0.05 was considered a statistically significant difference. The Cox regression analysis has been applied to identify the predictors of the hemoptysis recurrence, additionally, and furthermore, the cumulative risk of the occlusion was censored by Kaplan-Meier analysis . The analysis of DSA presentation was used to identify the factor(s) that led to recurrence of hemoptysis after occlusion via catheterization.∗109/L. For microbiological analyses, 15 (77%) patients were found to be associated with increased serum antibody for respiratory MP. For imaging features , among the 20 pediatric patients, we found diagnostic fields in 18 cases (exudative lesions/inflammation/consolidation) and a negative outcome in two cases. On imaging, right-sided lesions were detected in 15 (75%) patients and left-sided diagnostic fields were involved in two (10%) of the patients, and bilateral involvement was found in one (5%) patient. The most frequent zone was centralized to the right inferior lobe. CTA indicated a rough graph of abnormal BAs in 80% of the patients, which predominantly targeted the bronchial vessel (n = 1) originating from the descending aorta at the T4-T6 level . In addition, a BA was detected in the aorta of one case. In two cases, there was evidence of tortuous blood vessels (n≥2). Combined with CTA outcomes, DSA was taken prior to transcatheter plug closure on an individual basis.Among all hemoptysis admissions, three (15%) children presented with a mild hemoptysis, six (30%) children presented with a moderate hemoptysis, and the remaining eleven (55%) children had a massive hemoptysis, among whom 3 patients had a life-threatening condition. Reviewing the history and laboratory analyses of 20 patients revealed that 18 patients (approx. 90%) suffered from a prior respiratory infection with symptomatic hemoptysis, and 75% of WBC counts were more than 10.0All cases were confirmed with aberrant BAs by preoperative DSA. In addition, the origin and number of abnormal BAs were identified by aortography. Moreover, the site of vascular rupture was detected by “smoking phenomenon” , 13. In P=0.535) and age (P=0.578). Though patients presented with a less than obvious blood loss, the hemoglobin levels indicated a decreased outcome in children that suffered from a recurrence (P = 0.049). At initial, we detected and treated the BAs with obvious anomalies, and yet the neglectful BAs were involved in further recurrent bleeding. Eventually, the number of abnormal BAs in the recurrence group was significantly greater than that found in the nonrecurrence group (P = 0.020). It was likely that these arteries that supplied local tissues caused a vascular rupture, which led to subsequent high losses in blood. For each recurrence scenario, the mean volume of blood loss was 102.5 ± 89.7 ml/24 h and ranged from 20 ml/24 h to 350 ml/24h (IQR = 170 ml/24h). Before recurrent hemorrhage, no obvious symptoms in 42.9% of the case scenarios were evident; however, respiratory infectious symptoms were found in eight (about 60%) scenarios within one week and mainly included evidence of cough (28.6%) and fever (28.6%). With regard pathogenic microbiological analyses, MP-PA ≥ 1:320 was detected in 75% patients with a recurrent hemoptysis, which indicated MP acted as an important predictor, possibly.At the endpoint, in sum, hemoptysis recurrence was found in 8 patients on 14 occasions, and the interval time from every occlusion was 5.4 ± 3.6 mon. Two patients suffered from a first recurrent hemoptysis within one month, four patients within 6 months, and the remaining two patients no more than 12 months. Three out of eight patients suffered from recurrent hemoptysis on three occasions. The eight patients, including four females and four males, were mainly centralized at school age that ranged from 8.0 to 10.0 years, with a medium age of 9.2 years. The characteristics in the initial hemoptysis scenario were recorded and compared among the recurrence groups and nonrecurrence groups for a detection of potential factors . No sign P=0.028).Eventually, Cox regression analysis has been performed to identify the factors of the recurrence after the procedure for hemoptysis subjects, shown in In all recurrent conditions, a repeat angiograph nearly indicated a “smoking phenomenon” in the initial rupture site, while none of the older occluded arteries were found to be involved that led to the reappearance of bleeding. Other unprocessed abnormal arteries initially, associated with some newborn tortuous routes originating from different sites including the descending aorta (above T4-T7), the abdominal aorta, the aortic arch, the subclavian artery, and the thyroid artery, jointly flowed to the segmental pulmonary tissues . These BTherapeutically, repeat plug closure was required. Following the second occlusion, a cessation of hemoptysis was achieved in five patients. In addition, three patients underwent the procedure four times. In sum, 34 occlusions were taken in all patients and it presents as a cumulative risk of recurrence in No death and serious adverse events were observed.Anomalous bronchial circulation plays an important role in pediatric hemoptysis . Since tWe identified eight recurrent cases who had several corresponding arteries with different routes and unequal diameters, originating from the thoracic aorta, the abdominal aorta, the subclavian artery, and the aortic arch. To evaluate the imbalanced distribution of blood flow, the use of conventional angiographic contrast medium can easily reveal the morphology of the main BA trunks, which are responsible for a large share of the blood perfusion. Consequently, a tiny-diameter artery that is inconspicuous can easily be overlooked. In addition, surgeons become accustomed to paying less attention to abnormal arteries of an uncommon origin. This has commonly been demonstrated in the imaging windows in aortic angiography used for observing the anatomy of the aortic arch and the thoracic aorta. Hence, it is important to enlarge the image windows to display every slice, including those of the base of the neck and the upper abdomen, particularly for patients that suffer from bleeding in the lower lobes of the lung, as this bleeding most likely originates from the lower thoracic aorta or the upper ventral aorta. Otherwise, after the bronchial trunks have been occluded, the underlying small arteries and the preexisting tiny BAs will have to accommodate greater blood perfusion to the ruptured vessel , 17. ThiP=0.028). So, for targeted vessels, a relatively complete occlusion, not only of the main trunk, but also of the visually tortuous and smaller vessels, is employed to lower the risk of recurrent hemoptysis. In a severe condition with above factors, a segmental lung resection should be taken into consideration to decrease the risk of rebleeding episodes [Hence, the number of abnormal BAs, anemia manifestation, hemoptysis severity evaluation , respiratory tract infections, etc. are likely the risk factors for recurrent hemoptysis following transcatheter occlusion. Moreover, a late childhood group probably suffers more from this condition according to our data. For the above reasons, recurrent hemoptysis is not rare, and yet it presents as a potential life-threatening condition in children. Eventually, a Cox regression analysis showed that the number of abnormal BAs was an independent prognostic factor for hemoptysis recurrence after an occlusion (episodes , 30.The transcatheter plug closure is a safe and efficient procedure for hemoptysis caused by anomalous BAs, however, which could be influenced by many potential factors. Based on the outcomes of this single-center study with a relatively small sample size, our study calls for a multicenter, larger sample size, and randomized controlled trial to verify our observations reported herein.


Ascaris suum (A. suum) infection, which could interfere with the progression of pulmonary fibrosis. In addition, we evaluated whether a previous lung fibrosis could interfere with the pulmonary cycle of A. suum in mice. The most important findings related to comorbidity in which A. suum infection exacerbated pulmonary and liver injury, inflammation and dysfunction, but did not promote excessive fibrosis in mice during the investigated comorbidity period. Interestingly, we found that pulmonary fibrosis did not alter the parasite cycle that transmigrated preferentially through preserved but not fibrotic areas of the lungs. Collectively, our results demonstrate that A. suum infection leads to comorbidity, and contributes to the aggravation of pulmonary dysfunction during pulmonary fibrosis, which also leads to significant liver injury and inflammation, without changing the A. suum cycle in the lungs.Ascariasis is considered the most neglected tropical disease, and is a major problem for the public health system. However, idiopathic pulmonary fibrosis (IPF) is a result of chronic extracellular deposition of matrix in the pulmonary parenchyma, and thickening of the alveolar septa, which reduces alveolar gas exchange. Considering the high rates of ascariasis and pulmonary fibrosis, we believe that these two diseases may co-exist and possibly lead to comorbidities. We therefore investigated the mechanisms involved in comorbidity of  Ascaris, there is no evidence relating to pulmonary fibrosis progression, possibly because Ascariasis is considered a neglected disease. We evaluated the role of Ascaris during pulmonary fibrosis. We considered two simple questions: (1) Whether Ascaris infection could protect or aggravate fibrosis (comorbidities) and (2) whether pulmonary fibrosis could change the cycle of Ascaris as a result of increased alveolar thickening, larvae retention, and the limitation of influx into airways. We answered both questions as follows: (1) Ascaris infection exacerbates pulmonary and liver injury and inflammation, but not fibrosis; and (2) Pulmonary fibrosis did not alter the course of Ascaris cycle in lungs during transmigration into airways, because Ascaris preferentially seeks and penetrates into the lung areas, which are thought to be preserved, but not into fibrotic areas.Ascariasis is considered a major problem for the public health system, which has an estimated 800 million infected people worldwide. It occurs in the United States, Africa, Asia, and Latin America, and is generally associated with poverty and precarious health conditions. Pulmonary fibrosis affects 14–63 people per 100,000 habitants/year, and is characterized by collagen deposition and alveolar wall thickening. The comorbidities caused by infections are commonly associated with pulmonary fibrosis exacerbations, poor prognosis, and high mortality. Despite the comorbidities caused by helminth infections, which display a pulmonary parasitic cycle such as that of  Idicollagen ,7,8, whicollagen . In thiscollagen ,10,11.Streptococcus pneumoniae, Streptococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa [Ascaris or Strongyloides, there is no evidence during the pulmonary fibrosis, because helminthiasis are considered neglected diseases [The etiology of IPF is unknown, and some patients exhibit distinct patterns of disease progression, which display a rapid and progressive clinical course related to periods of acute exacerbations during the IPF development ,12. Therruginosa ; althougdiseases ,17.Ascaris [Ascaris genus may be divided into two distinct phases after infection: (i) migration of parasitic larval stages through several tissues , namely larval ascariasis; and (ii) after larval ingestion, the establishment of adult worms in the lumen of the small intestine, which characterizes chronic infection. After the egg eclosion in host, they are hatched in the lumen of the small intestine by L3 larvae that penetrate the intestinal mucosa through the cecum, they gain access to the blood circulation and the portal space of the liver, and then they migrate through the liver, where the larvae gain circulation again, and finally migrate to the lungs (Looss Cycle). The larvae migrate from the alveolar capillaries through the pulmonary parenchyma, causing disruption of the alveolar walls. At this stage, larvae L3 change to L4 and ascend the bronchial tree, where they can be expectorated or swallowed; at this last stage, the larvae returns to the gastrointestinal tract, and reach sexual maturity to become male and female adult worms [A. suum migration through the tissues during the parasitic cycle; the damage triggered a mixed local and systemic Th2 and Th17 immune response, in which triple consecutive infections with A. suum reduced parasite burden related to immune specialization and memory [A. suum infection depends on a humoral-dependent response, mostly by induction of IgG antibodies, which is associated with the production of IL-5 and IL-10 after vaccination in mice [Ascariasis is the most prevalent helminthic infection in the United States, and one of the most prevalent tropical diseases caused by Ascaris ,18. ThisAscaris ,19,20. Tlt worms ,22,23. Rd memory . In fact in mice ,25.Ascaris infection in association with other diseases are still controversial. Co-infection by A. suum and Vaccinia virus promotes increased pathology associated with the virus titer, by downmodulation of the frequency of circulating CD4+T cells producing IFN-γ, which are important to control the Vaccinia virus [A. suum infection leads to immunomodulatory effects during natural infection, and it depends on the geographic distribution pattern as well as the population affected. It has been demonstrated that children from areas with high endemicity of infections with Ascaris lumbricoides, have low parasitic load of Ascaris lumbricoides, which becomes a protective factor against asthma manifestation and its symptoms. However, a high parasitic load is a risk factor, and contributes to high prevalence of severe asthma and its symptoms among these children [Recent studies have shown the direct role of helminth infections in association with other pulmonary diseases, such as virus infection and allergy. However, the immunomodulatory or immunopathogenic effects of ia virus . Howeverchildren . Regardichildren ,29, but A. suum infection could modulate pulmonary fibrosis, and whether pulmonary fibrosis could change the course of the parasite cycle through the airways during A. suum infection in mice. Our results suggested that comorbidity during A. suum infection exacerbated pulmonary and liver injury, inflammation, and dysfunction, without modification in tissue fibrosis. Finally, we demonstrated that pulmonary fibrosis induced by bleomycin did not change the pulmonary cycle of A. suum, which still transmigrated preferentially into the airways through preserved and non-fibrotic areas of the lungs.Therefore, we assumed that it is necessary to better understand the mechanisms involved in these two diseases separately and in comorbidity, to evaluate the development of lung lesions. In this context, the aim of our study was to investigate whether All experimental procedures described here were conducted according to the Brazilian Guidelines on Animal Work, and approved by the local Animal Ethics Committee (CEUA) of the Federal University of Minas Gerais, Protocol number 200/2018.Mus musculus) C57BL/6 mice, aged approximately 8 weeks, obtained from the Biotério Central of the Federal University of Minas Gerais. During the experimental period, the mice were fed with filtered water and commercial chow  ad libitum. They were kept in cages (50 × 60 × 22 cm) with sterile sawdust shavings, and the cages were changed and cleaned once a week. Mice were maintained at the Animal Facility of the Universidade Federal de Minas Gerais (UFMG) under controlled conditions of temperature (24 ± 1° C) on a 12 h light cycle (lights on at 07:00 h).For the experiments, we used female , and taken to the Laboratory of Immunology and Genomics of Parasites of the Federal University of Minas Gerais to be processed. The eggs were isolated from the uteri of female adult worms by mechanical maceration, purified through filtration on 100 μm nylon strainers, placed in culture bottles with 50 mL of 0.2 M sulfuric acid at a concentration of 25 eggs/μL, and maintained in a BOD incubator at 26 ºC. At the 150th day of culture, the peak of larvae infectivity, the fully embryonated eggs were used for the experimental infections, as we previously described [escribed .For induction of pulmonary fibrosis, the mice were injected intranasally with bleomycin , at a dose of 3 mg/Kg or with PBS as control, as previously described .Ascaris suum, and euthanized after 7 days (As); or submitted to a single infection of Ascaris suum after 21 days of fibrosis induction, and subsequently euthanized on day 28 (BL+As) ; or submitted to a single bleomycin induction, and further euthanized at 7, 21, and 28 days after instillation (BL) ; submitt (BL+As) . ExperimA. suum, a gavage needle was used, where the mice belonging to the As and BL+As groups received orally 2,500 embryonated eggs in 200 μL of PBS. The group C received only 200 μL of PBS via the same route. Parasite burden was evaluated by recovery of lungs and bronchoalveolar lavage (BAL). Tissues were collected, sliced with scissors, and placed in a Baermann apparatus for 4 h in the presence of PBS at 37°C. The recovered larvae were then fixed , and counted under an optical microscope as previously described [To infect mice with escribed ,25.g for 10 min at 4°C. Supernatants were used for total protein and hemoglobin quantification, according to the manufacturer’s instructions as we previously described [Mice were euthanized with a lethal injection of xylazine/ketamine. The bronchoalveolar lavage (BAL) was performed inserting a 1.7 mm catheter into the trachea of mice, and washing the lungs twice with 1 mL of sterile PBS three times. BAL samples (±2.7–3.0 mL each) were centrifuged at 600 × escribed . Subsequescribed . The lefescribed . In otheescribed .Pressure-Volume maneuver was performed, which inflates the lungs to a standard pressure of +30 cm H2O, and then slowly exhales until a negative pressure of -30 cm H2O, thereby measuring the volume in each point of application in the lungs. Fast-flow volume maneuver was performed, and the lungs were first inflated to +30 cm H2O, and immediately after wards connected to a highly negative pressure in order to enforce expiration until -30 cm H2O. The forced vital capacity (FVC), forced expiratory volume at 50 milliseconds (FEV50), and the flow-volume curves were recorded during this maneuver. Suboptimal maneuvers were rejected, and for each test in every single mouse, at least three acceptable maneuvers were conducted to obtain a reliable mean for all numeric parameters.Pulmonary function in mice was performed as previously described ,25,30. B2. The larvae were recovered in the pellet of the apparatus, fixed in 4% formalin, and quantified by light microscopy.Parasite burden from immunized and non-immunized mice was evaluated by the total number of larvae recovered from the lungs at day 7 post infection, as previously described ,25. BrieThe left lobes of the lungs and liver were removed from the mice of each group. The organs were fixed in 4% formalin solution, and gradually dehydrated in ethanol to be diaphanized in xylol, and included in paraffin blocks that were cut at 4–5 microns thick and fixed on the microscopy slide ,25. Slidg for 10 min at 4°C, and the supernatant used for cytokine quantification as we previously described [For evaluation of the cytokine content in lung tissue, 100 mg of lung tissue was homogenized  in 1,000 μL of phosphate buffered PBS (0.4 M NaCl and 10 mM NaPO4) supplemented with 0.05% Tween 20, 0.5% bovine serum albumin, and protease inhibitors . The homogenates were centrifuged at 8,000 x escribed ,24,25. Lg for 10 min at 4°C, and the resulting pellet was examined for EPO and MPO. The extent of neutrophil accumulation in the lung tissue was quantified by assaying myeloperoxidase (MPO) activity, as previously described [The evaluation of neutrophil and eosinophil accumulation in the lungs, MPO, and EPO from tissue homogenates were measured according to a method described previously ,11,24,25escribed ,24,25. E1, IL-10, and type I collagen, we performed immunohistochemistry as previously described [2O2 40 v/v solution at 0.2% PBS, and unspecific binding was blocked by incubation with goat serum diluted at 1:40. Next, sections were incubated with monoclonal anti-TGF-β1, IL-10, and type I collagen antibody , diluted at 1:150, 1:100, 1:50, respectively for 18 h each, then incubated with biotinylated goat anti-mouse IgG at 1:100  and peroxidase-conjugated streptavidin at 1:100 for 1 h . Color reaction was developed using DAB 0.05% in H2O2 40 v/v at 0.2%. As negative control, PBS-treated sections were used  of the histological sections per animal. All measurements were taken with the Carl Zeiss image analyzer KS300 software .The quantification of the positive areas for TGF-βescribed ,11,31,32Alanine aminotransferase (ALT) determination was performed using a kinetic test for the evaluation of hepatocellular lesion, as previously described . BrieflyThe quantification of the number and area of the inflammatory foci present in the hepatic parenchyma were performed using morphometric analysis as previously described ,34. The A. suum larvae, slides with histological sections of the lung stained with Gomori trichrome, were visualized under a light field optical microscope coupled to a digital image capture system (Motic 2.0), and subsequently we obtained images of the entire pulmonary parenchyma of each mouse with 10X magnification. Then the captured images were visualized in the same digital image capture system (Motic 2.0) to count all the larval fragments. The observed larval fragments were grouped in the following parameters: (1) Preserved area; when found in preserved regions of the pulmonary parenchyma with absence of fibrous connective tissue, (2) Fibrosis area; when found in fibrous regions of the pulmonary parenchyma, and (3) Airways; when found in bronchi and bronchioles.To evaluate whether fibrosis interferes with the migration of the GraphPad Prism 6  was used for the statistical analysis. The Grubb's test was used to detect the outliers in the samples and non-parametric analysis of all the results were performed. To verify the distribution of the data, the D'Agostino & Pearson omnibus normality test was used. For the comparison between the parasitic burden and the number of larval fragments found in histological sections of the lung, we used the Student’s t-test. For the comparison of the production of tissue cytokines, type I collagen, total cell count, comparison of the activity of neutrophils and eosinophils, level of alanin aminotransferase, number of inflammatory foci, mean of inflammatory foci, mean of total inflammatory area, and the fibrosis score, the One-way ANOVA test was used. Kruskal-Walis test followed by Dunn's test were used for comparison of pulmonary mechanics, differential cell count, hemoglobin and protein levels, and analysis of the fibrosis area. The Chi square test was used for the survival and Flow–volume curves. All tests were considered significant when the p value ≤ 0.05. The number of mice used per group varied according to each experiment and it was obtained from statistical sample calculation . Sample calculation was performed based on previous published data from the group.A. suum infection, they were evaluated three-time points at days 7, 21, and 28 after intranasal instillation of bleomycin , protein , and leukocytes (inflammation) in the BAL, when compared to those in the control mice or mice submitted to pulmonary fibrosis , as well as in chronic pulmonary fibrosis (28 d). Although comorbidity showed a significant increase in mice compared to A. suum infection or bleomycin-induced chronic pulmonary fibrosis, no significant differences were observed , but was induced at then final phase of fibrosis that counterbalanced the chronic activation of TGF-β1. As we observed previously about IL-17A, mice with comorbidity also showed a significant increase in IFN-γ like A. suum infection, but was not reduced or increased in relation of bleomycin-induced pulmonary fibrosis  Whether A. suum infection could immuno-modulate (down-modulate) fibrogenic response or exacerbate pulmonary fibrosis leading to comorbidities, and (ii) whether the lung parenchyma fibrogenesis, which is the preferential loci of larvae influx into airways, could interfere in the pulmonary cycle of A. suum as result of increased alveolar thickening, with possible larvae retention [A. suum infection led to exacerbation of pulmonary and liver injury, inflammation, and dysfunction without modification during tissue fibrosis. Moreover, we demonstrated that pulmonary fibrosis induced by bleomycin did not change the pulmonary cycle of A. suum that transmigrated into the airways preferentially through preserved but not fibrotic lung areas. In order to clarify the discussion of our data, we divided the main findings of our investigation into three topics, as follows: pulmonary inflammation and fibrosis, liver injury and inflammation, and lastly the pulmonary A. suum burden and cycle.Considering epidemiologic data from ascariasis, it has been estimated that approximately 800 million people are infected by per year ,17, and per year . We beli disease ,17. Thisetention  limitingA. suum infection and pulmonary fibrosis. Generally, during tissue infection, damage, stress or malfunction, the immune response, which has a physiological purpose of maintaining the host’s defense against infections and tissue repair, is triggered; this type of tissue adaptation could generate a new pulmonary homeostasis during parasite control or tissue healing [A. suum infection exacerbated tissue damage and inflammation, with marked hemorrhage and exudation, which was getting worse by the pre-established chronic inflammatory disease fibrosis. Moreover, we found that comorbidity led to increased leukocytes, mostly macrophages/monocytes/dendritic cells, neutrophils, lymphocytes, and eosinophils. These results are consistent with the histopathological findings that demonstrated the increase hemorrhage, as well as, increased MPO and EPO activity in lungs, which were mostly related to increased neutrophils and eosinophils localized in pulmonary parenchyma. In fact, increased number of leukocytes in BAL is a worse prognosis of fibrosis. Eosinophils are involved in the inflammatory process of pulmonary fibrosis, which released other cytotoxic eosinophil products, and may contribute to the exacerbation of lung injury and fibrosis during IPF [A. suum infection, in addition to the initial influx of monocytes and their consequent differentiation into macrophages [A. suum and comorbidity infection. Collectively, our data suggested that the major effect of Ascaris suum infection during pulmonary fibrosis was the maintenance of a chronic granulocyte inflammation and hemorrhage caused by physical damage during larval migration. Indeed, most of the pulmonary mechanical dysfunctions were detected during comorbidity, such as loss of IC, TV, FEV50, Flow–Volume curve, compliance and increased resistance were very similar to that found in A. suum infection, suggesting that inflammatory response induced by infection ascribes the worsening of pulmonary injury and dysfunction during pulmonary fibrosis induced by bleomycin in mice.Inflammation is an important physiological harm of tissue defense and repair , in our  healing ,47. On t healing ,47. Our ring IPF . Macrophring IPF . Moreovering IPF  and pulmring IPF . We obserophages , a macrorophages ,53 and iA. suum after 8 d of infection caused large lesions in the pulmonary parenchyma, as shown by the increase in multifocal hemorrhagic areas, with edema and marked inflammation of the parenchyma, perivascular, and airway, which are directly related to the acute lesion triggered by larval migration. The lack of significant differences on pulmonary fibrosis might be related to the time of infection/induction. Possibly, a later assessment in the post-infection period might render a collagen deposition in order to repair tissue damage. In fact, previous data where a long exposition to the parasite were assessed , a significant increase in collagen deposition  was observed [A. suum infection. These results demonstrate that comorbidity was able to alter the immune response, which provides a profibrogenic environment, and allows us to elucidate and assume as a shortcoming of our study that if it were maintained longer, elevated levels of IL-17A and TGF-β1 could also have been observed, as well as increased collagen deposition, thereby contributing to pulmonary fibrosis. Another interesting finding was in relation to IL-12 only in the comorbidity; it was possible to verify high levels as far as it has not been related to the profibrogenic process, and at the same time, there are no studies in the literature showing its involvement with A. suum infection. Taken together, our results demonstrate that although there were no major histopathological changes in relation to increased fibrosis, comorbidity effectively contributed to alteration of the immune response during the study period. Given these results, we can also emphasize that comorbidity did not alter the response pattern involved in A. suum infection, since cytokines such as IL-4, IL-5 and IL-33 [A. suum infection did not alter the anti-fibrogenic cytokine profile of IFN-γ [A. suum infection did not induce an immunomodulatory or a protective response by cytokines, and consequently no interference occurred when associated with fibrosis. Finally, pulmonary mechanical dysfunction observed in comorbidity in relation to the A. suum-infected group might have been caused mainly by changes in the immune response, and consequent inflammatory events, but not by fibrogenic histopathological changes, which demonstrates that larval migration after 7 d of infection did not exacerbates the development of pulmonary fibrosis.Despite the exuberant inflammatory response during comorbidity, we did not find differences between the patterns of pulmonary fibrosis, as depicted by Gomori’s trichrome and Collagen I immuno-staining in both groups: bleomycin-induced pulmonary fibrosis and comorbidity. Histopathological analysis revealed that exposure to observed . Althougobserved ,10,24,29observed ,36,37,40nd IL-33 ,39, whicof IFN-γ ,36, suggA. suum infection or comorbidity, since this organ is also related to larval migration [A. suum infected mice. It is also important to emphasize that mice submitted to fibrosis induction showed moderate levels of ALT in relation to the mice in the control group. ALT is a specific marker of liver damage [A. suum infection combined with pulmonary fibrosis in mice. We believe that liver changes in our experimental model indicate that local inflammation in the lungs produce inflammatory mediators that can gain blood circulation, and modulate the immune response in other organs. The liver, which is an organ with high metabolism and exposed to systemic circulation acts as a metabolite filter [Subsequently, we proposed to evaluate the liver damage to analyze the systemic manifestations caused by igration . Few stuigration  or damagigration . Furtherigration . Here, wr damage , suggeste filter , and migA. suum infection, since that infection depends of lung integrity and pulmonary fibrosis could delay or prevent larval migration from alveolar capillaries into the airway space by retention, a common phenomenon during Ascaris infection [A. suum infected and comorbidity mice. There were no differences observed in the numbers of larvae fragments recovered from the lungs or airways in mice submitted to comorbidity, when compared to mice infected only with A. suum. Therefore, these results confirm that comorbidity did not change the parasite burden and consequently did not affect the A. suum pulmonary cycle. We also checked, by identification and counting of larvae fragments, whether the larvae were present in the fibrotic areas, preserved areas, or bronchial and bronchioles lumen. Few larval fragments were present in the dense fibrotic areas from the lung parenchyma of comorbidity mice. However, when we evaluated the number of larvae fragments present in the preserved regions of the lung parenchyma, we found a larger number of larval fragments. Together these results suggested that, in relation to larval migration, the burden of A. suum larvae are not controlled by fibrogenesis from lung parenchyma, but A. suum larvae search for preserved areas without fibrosis, and they normally perform the transmigration following the sequencing of the biological cycle. Finally, this migration preferentially occurs through preserved areas, and may increase the lung injury and inflammation, thereby ascribing the pulmonary dysfunction to the comorbidity group.Another central point of our study was to verify whether pulmonary tissue fibrosis could interfere in the course of the cycle of nfection . AssessiA. suum infection. The comorbidity contributed to worsening lung damage, due to the increased hemorrhage induced by larval migration. Additionally, the comorbidity directed the immune response to a profibrogenic profile, but did not affect collagen deposition during the study period and did not alter the response to A. suum. Moreover, the comorbidity also contributed to greater liver damage, without change in mice survival. The condition of previous fibrosis in the pulmonary parenchyma did not alter the parasitic load, did not impair the larval migration, and consequently did not alter the pulmonary A. suum cycle. The present study highlights pathological changes in lungs during comorbidities related to infectious agents, which may affect the pathology progression and morbidity. These observations indicate that parasitic transmigration through the pulmonary tissue is directly involved in the exacerbation of damage, leading to comorbidity and clinical progression. Finally, we can mention that the impact of the present study highlights the need for more exploratory research to elucidate possible pathological changes in comorbidities involving parasitic diseases, which may affect the progression of the associated morbidity. Taking into consideration the epidemiology and high rates of ascariasis in the world population, experimental models of comorbidities involving Ascaris sp. are assets for future research.In conclusion, our study described the pathophysiological manifestation of pulmonary fibrosis comorbidity associated with S1 Fig(TIF)Click here for additional data file.


Previously, it has been reported that nanoparticles synthetized from this polymer are able to decrease the viability of breast and colon tumor cells. In addition, it is well known that the size of therapeutic particles plays an essential role in their effect. As a consequence, the influence of this parameter on the cytotoxicity of polydopamine nanoparticles was studied in this work. For this purpose, polydopamine nanoparticles with three different diameters  were synthetized and characterized. Their effect on the viability of distinct sorts of human carcinomas  and stromal cells was investigated, as well as the possible mechanisms that could be responsible for such cytotoxicity. Moreover, polydopamine nanoparticles were also loaded with doxorubicin and the therapeutic action of the resulting nanosystem was analyzed. As a result, it was demonstrated that a smaller nanoparticle size is related to a more enhanced antiproliferative activity, which may be a consequence of polydopamine’s affinity for iron ions. Smaller nanoparticles would be able to adsorb more lysosomal Fe Nanomedicine has acquired an essential role in recent decades due to the urgent need for developing novel therapeutic strategies for cancer treatment. This multidisciplinary field, by taking advantage of nanotechnology, aims to overcome the pharmaceutical limitations of conventional chemotherapeutics. Thus, the improvement of their stability, aqueous solubility and dose limiting toxicity, as well as avoiding the apparition of multidrug resistances (MDR), are some of the objectives that are highly relevant for the scientific community today ,2. MoreoIn this manner, a considerable number of nanomaterials have been developed recently ,4,5. Amo2+ and Fe3+ [Thus, there are only a few existing works that report nanoparticle systems devoid of drugs with an antineoplastic behavior in the literature ,16. Howeand Fe3+ ,22,23. Tand Fe3+ , their mand Fe3+ ,26,27,28and Fe3+ .On the other hand, it is well accepted for the design of therapeutic nanosystems that size may determine their endocytosis rate and, therefore, their antiproliferative activity ,30,31. CFor all these reasons, the size-dependent cytotoxicity effect of PD NPs was studied in the present work. PD NPs with three different sizes  were synthetized and characterized. Their antiproliferative activity was analyzed and compared in four distinct types of human carcinomas  and in healthy stromal cells. Moreover, it was again proven that such antiproliferative effects may be related to their metal-loading ability. To do that, their cytotoxicity was studied in co-treatment experiments of PD with either DFO (iron chelator) and GSH (antioxidant compound), conducted with all of the previous mentioned cell lines ,33,34. FPD NPs of different sizes were synthetized through dopamine oxidative polymerization in a basic aqueous medium ,18,37. ISince it was demonstrated that size of NPs plays an important role in their endocytosis rate and therapeutic effect ,30,31, aAs shown in In the case of the treatment of colon and liver carcinoma cells (HTC116 and HEPG2 cell lines) , it was Likewise, a similar trend in the results obtained after the treatment of lung carcinoma cells (H460 cell line) can be observed in Positively, in the same manner, stromal cells’ viability was not reduced as much as the carcinoma cell lines’ viabilities when they were treated with PD NPs, neither those PD NPs with a size of 100 nm nor those with a size of 200 nm . TreatmeTherefore, in brief, it was found that the cytotoxicity of PD NPs was more noticeable, at all administered concentrations, for the HTC116, HEPG2 and, especially, for the BT474 cell line. The lowest concentration (0.0074 mg/mL) was barely toxic during the studied time for all treated cell lines. However, the two highest concentrations (0.029 and 0.042 mg/mL) achieved a notable decrease in cancer cell viability 72 h after a treatment of 35–40%, except for H460 viability, which was not so remarkably reduced. Furthermore, PD NPs did not decrease the viability of stromal cells in the same manner as the viability of cancer cells. In fact, the two lowest concentrations employed (0.0074 and 0.015 mg/mL) of PD NPs showed pretty specific results. Treatment with them almost had no affect on HS5 cellular viability, possibly because tumor cells present high intracellular iron levels ,26. FinaNext, since it was corroborated that size affected the therapeutic effect of PD NPs, MTT assays were again performed with synthetized 420 nm PD NPs . For sucFinally, in order to visually corroborate the cellular viability results obtained in the MTT assays, a live/dead assay with confocal laser scanning microscopy (CLSM) was performed. For this purpose, BT474 cells were again chosen and treated for 72 h with 0.042 mg/mL of 115 nm PD NPs, which were the NPs and the concentration that had previously reduced the survival of the breast carcinoma cell line the furthest. 24, 48 and 72 h after treatment, these cells were stained with calcein AM and propidium iodide (PI), and the resulting images are shown below in 3+ has been demonstrated in previous studies, as well as the fact that when these NPs are endocyted [PD NPs’ ability to adsorb Fendocyted ,42, theyndocyted ,24  were tested in BT474 cells, which were again chosen because the best MTT assay results had been achieved with this breast carcinoma cell line. Likewise, treatment with equivalent DOX concentrations , which were pretty low in comparison to those employed in other works ,44, was The results of these experiments indicated that, as shown in Since nanomedicine’s occupation of a fundamental role in the development of novel therapeutic systems, the scientific community has been aware of the importance possessed by size in determining their effect ,30,31,47Thus, PD NPs with three different diameters  were synthetized. Once they had been characterized by TEM and DLS, the cytotoxicity of four different concentrations of the two smallest types of PD NPs (115 and 200 nm) was studied in different types  of human carcinomas and in stromal cells. Furthermore, the largest PD NPs’ (420 nm) effect on cellular viability was also tested with the breast carcinoma cell line, BT474. The results obtained showed that, in all cases, the smaller the size of the NPs, the more noticeable their antiproliferative activity, with this being even more noticeable in BT474 cells. In addition, with such experiments, it could be noticed that the lowest tested concentrations of PD NPs had a tumor-selective effect. In this manner, employing low doses of these NPs could be a possible strategy for achieving greater treatment specificity and overcoming the any undesirable side effects. So far, most developed therapeutic nanosystems are vehicles for the delivery of cytotoxic drugs and, in this way, the employment of PD NPs could represent an awesome strategy for reducing the administration of chemotherapeutics [3+ cations once they have been endocyted could explain the death of cells treated with them by means of Fenton chemistry [On the other hand, it was demonstrated that the adsorption of iron could be responsible for the cytotoxic effect of such PD NPs. Their affinity for different metallic cations has been described in numerous studies, and the ability of PD NPs to load lysosomal Fehemistry ,18,24,34Finally, 115 nm PD NPs were loaded with DOX. This drug was chosen because it is one of the most commonly used chemotherapeutic drugs, and because it has also been associated with ferroptosis processes in recent toxicity-related studies ,36. The 4OH), Phosphate Buffered Saline , Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum , Thiazolyl Blue Tetrazolium Bromide (MTT reagent), L-glutathione Reduced (GSH) and Deferoxamine Mesilate CRS (DFO) were all supplied by Sigma-Aldrich . Ethanol absolute was purchased from VWR International Eurolab S.L. . Penicillin-Streptomycin (5000 U/mL), Calcein AM, Propidium Iodide (PI) ReadyProbesTM reagent and Doxorubicin Hydrochloride Solid (DOX) were obtained from Thermo Fisher Scientific .Dopamine Hydrochoride, Ammonium Hydroxide  with ethanol (40 mL) and deionized water (90 mL). In all cases, the obtained mixture was left under magnetic stirring at room temperature for 30 min, and a dopamine hydrochloride aqueous solution  was later added. The reaction was allowed to react for 24 h. PD NPs were isolated and purified by several centrifugation-redispersion cycles in deionized water (35 mL). Finally, NPs were re-suspended in PBS at final concentrations of 2, 3.2 and 1 mg/mL, respectively [PD NPs with average diameters of 115, 200 and 420 nm were synthetized by mixing distinct volumes of NHectively ,18,35.To characterize them, TEM images  were taken, employing a voltage acceleration of 120 kV. The synthetized PD NPs were dispersed in deionized water (pH = 6.0) in a concentration of less than 0.01% (WT), and a drop of this dispersion was deposited on a copper grid with a collodium membrane and dried for 24 h. Images were recorded after this time, and histograms exhibiting the size ranges were obtained. To this end, the size of a minimum of 300 PD NPs in different images was determined  in order to build each histogram. Moreover, the hydrodynamic diameter of the PD NPs was analyzed using DLS (on the basis of their intensity–average size distribution)  when they were re-suspended in a trizma base solution (pH = 10.0) in a concentration that was also less than 0.01% (WT).2 (5%).BT474, HTC116, HEPG2, H460 and HS5 cell lines were cultured with medium supplemented with FBS (10%) and antibiotics (penicillin-streptomycin) (1%) as instructed . All cells were cultured at 37 °C in a humidified atmosphere in the presence of COThe antitumor activity of 115 and 200 nm PD NPs was firstly studied based on MTT assays . BT474, Secondly, the reduction of viability caused by 115 nm PD NPs was corroborated in BT474 cells by CLSM  with an alive/dead cellular assay . The celTo demonstrate that the iron affinity of PD could be implicated in the cytotoxicity of cells treated with PD NPs, HTC116, HEPG2, H460, BT474 and HS5 cells were seeded in 24-well plates and cultured with supplemented medium overnight. After 24 h, the culture medium was replaced by supplemented medium containing PBS (control), DFO (0.7 μM), GSH (50 μM), 115 and 200 nm PD NPs (0.029 mg/mL) and PD NPs plus DFO and GSH at the same concentrations. Again, cell viability was analyzed every 24 h for 4 days, following the same protocol as before . The resWith the aim of adsorbing DOX onto PD NPs, an aqueous solution (1% DMSO) of the drug was prepared at a final concentration of 10 nM. PD NPs  with a diameter of 115 nm were mixed with DOX solution, and the mixture was kept under stirring overnight . Next, DTo study the reduction in tumor cells’ viability caused by DOX@PD NPs, the BT474 cell line was chosen to carry out further MTT assays. Following the steps described above, the cells were again seeded in 24-well plates and, 24 h later, they were treated with medium supplemented with PBS (control), different concentrations of 115 nm DOX@PD NPs  and equivalent adsorbed DOX concentrations . Cellular viability was measured every day for 72 h, always following the same protocol as before . The resp < 0.05.Data related to the size of the PD NPs is the mean ± SD of three different measurements. Otherwise, results of the different MTT assays are represented as the mean ± SD of three replicates for each treatment of three different experiments, and the results were considered statistically significant where In this work, it has been demonstrated that size plays an important role in the cytotoxic effect of PD NPs. This was demonstrated on the basis of viability assays carried out with different human carcinoma cell lines and with stroma cells, in which the therapeutic action of two or three PD NPs of different sizes was analyzed . In all cell lines, and with all of the tested PD NP concentrations, it was shown that the smaller the diameter of the NPs, the more enhanced their antiproliferative activity. Therefore, the size of PD NPs could be tailored depending on the desired application.3+ in lysosomes, and this fact could originate an abnormal cellular ROS production. However, when either an iron chelator (DFO) or a potent antioxidant (GSH) were employed, their antiproliferative ability was notably decreased. For this reason, when PD NPs were loaded with DOX, their antiproliferative effect was enhanced. Apart from triggering DNA damage, DOX cytotoxicity is also a consequence of a process of ferroptosis which, in addition to the one potentially produced by PD NPs, may result in the achievement of a synergist effect.With respect to the cytotoxic effect of PD NPs, it was observed that this may be related to a process of ferroptosis. PD NPs are able to load Fe


Dietary records indicated that children living in urban Bangkok tended to consume modern high-fat diets, whereas children in rural Buriram tended to consume traditional vegetable-based diets. Sequencing of 16S rRNA genes amplified from stool samples showed that children in Bangkok have less Clostridiales and more Bacteroidales and Selenomonadales compared to children in Buriram and bacterial diversity is significantly less in Bangkok children than in Buriram children. In addition, fecal butyrate and propionate levels decreased in Bangkok children in association with changes in their gut microbial communities. Stool samples of these Thai children were classified into five metabolotypes (MTs) based on their metabolome profiles, each characterized by high concentrations of short and middle chain fatty acids , amino acids , arginine , amino acids, and amines , or an overall low level of metabolites . MT1 and MT4 mainly consisted of samples from Buriram, and MT2 and MT3 mainly consisted of samples from Bangkok, whereas MT5 contained three samples from Bangkok and five from Buriram samples. According to the profiles of microbiota and diets, MT1 and MT2 are characteristic of children in Buriram and Bangkok, respectively. Predicted metagenomics indicated the underrepresentation in MT2 of eight genes involved in pathways of butyrate biosynthesis, notably including paths from glutamate as well as pyruvate. Taken together, this study shows the benefit of high-vegetable Thai traditional diets on gut microbiota and suggests that high-fat and less-vegetable urban dietary habits alter gut microbial communities in Thai children, which resulted in the reduction of colonic short chain fatty acid fermentation.Loss of traditional diets by food globalization may have adverse impact on the health of human being through the alteration of gut microbial ecosystem. To address this notion, we compared the gut microbiota of urban ( The gut microbiota consists of hundreds of microbial species, collectively 100 trillion cells, that play an important role in the interface between food intake and host health. It aids in nutrient metabolism as well as conditioning the gut environment for host health. Notably, some commensals benefit the host by fermenting dietary fibers into short-chain fatty acids (SCFAs) . SCFAs pFoods are recognized as important to maintaining the microbiota and facilitating efficient production of SCFAs . HoweverDiets in Thailand are notably rich in a variety of unique and healthy foods, some of which have had a strong impact on the dietary cultures of surrounding countries. Particularly, the Thai traditional diet has been relatively high in vegetables and low in fat . A studyPrevotella-types represent the majority of children in Khon Kaen, whereas Bacteroides-type represents the majority of children in Bangkok. Furthermore, guts of adult vegetarians were mostly colonized by Prevotella-type, whereas those of non-vegetarians were mostly colonized by Bacteroides-type . Among rural cities, we chose Buriram city, located in the Isan region, which is a geographically contiguous area in North–Eastern Thailand. The population is largely Thai and consumes traditional Thai (Isan) diet. Notably, many local Isan dishes are low in fat and high in vegetable and herbal content  and sample sizes . As described in the footnotes, some samples and dietary records were missing because of failure by participants. There were statistical differences in age, gender, weight, and body mass index (BMI) between children in Bangkok and Buriram. The confounding effects of age and gender biases were examined as described below.Fresh stool samples were collected from these 45 children and were subjected to the 16S rRNA gene sequencing and metabolome analysis as described below. The parents/guardians reported 1-week dietary records as described below as well as answered a questionnaire that addressed the children’s physiological characteristics. Welch’s Diets of children participating in this study were recorded by children’s parents/guardians, using a dietary record form that asked about the menu, ingredients, and quantity of every meal, including breakfast, mid-morning snack, lunch, mid-afternoon snack, dinner, and after-dinner snack for 7 days before stool samples were collected. Using the INMUCAL-Nutrients V3 database NB1 program , recorded foods were categorized and their energy  and nutrient contents  were estimated. The energy from each food group and its fat portion were calculated per day per person and subjected to Wilcoxon rank-sum test in R ver. 3.3.2 to determine the statistical significance of differences between children in Bangkok and Buriram  for microbial genomic extraction and a 55 × 44-mm sterile container for metabolite extraction . The samples were placed on ice immediately for transfer to a -20°C freezer. For the metabolome analysis, the fecal samples were lyophilized and then stored at -80°C. Details of the stool sample collection and storage are described in Supplementary Methods.Bacterial genomic DNA was extracted from stool samples as described previously   and 2 (PC2) were subjected to the Wilcoxon rank-sum test to determine the statistical significance of differences between samples from Bangkok and Buriram.The number of observed OTUs, and ACE and Chao1 alpha diversity indices were determined at a sequence depth of 5,000 reads per sample with 10 random iterations, using the QIIME alpha_rarefaction.py script. The statistical differences of these indices between Bangkok and Buriram were examined by the Wilcoxon rank-sum test. To determine the statistical significance of differences in the Chao1 index between the five metabolotype groups, the pairwise Wilcoxon rank-sum test was performed.3. For OTUs with an average abundance in all samples that was greater than 0.1%, abundances were normalized to the sum of the values per sample in 1 million and then subjected to linear discriminant analysis (LDA). The LDA was performed using a one-against-all strategy, and OTUs showing a score higher than 2.0 were selected.The LEfSe  was calc4, version 1.0.0  analysis using the Greengenes reference sequence database (gg_13_5) . The OTUon 1.0.0 . The OTUD-camphol-10-sulfonic acid, and 2-(N-morpholino) ethanesulfonic acid as the internal standards, and then cleaned with chloroform and water extraction and ultrafiltration using Ultrafree-MC . The purified fractions were analyzed using a CE-TOF MS system consisting of an Agilent CE capillary electrophoresis system , Agilent G32500AA LC/MSD TOF system (Agilent Technologies), Agilent 1100 series binary high-performance liquid chromatography pump, G1603A Agilent CE-MS adapter, and G1607A Agilent CE-ESI-MS sprayer kit. For the analysis, metabolites were quantified using a standard curve obtained from standard samples. Concentrations below the detection limit were substituted with zero, and metabolites whose levels were below the detection limit in all of the samples were excluded. The data of CE-TOF MS is deposited in Supplementary Table Metabolites in the stool samples were analyzed as previously described . Briefly5 To find metabolites abundant or depleted in a specific metabolotype group, the concentration of each metabolite was subjected to Wilcoxon rank-sum test between one group and all other groups combined  and the effect size level determined by the mean and standard deviation for the Wilcoxon rank-sum test or the 2R for the Pearsons’ correlation analysis.A  3.1.9.2  to retroPRJDB5860, which contains links and access to the stool sampling data (BioSample SAMD00097316 to SAMD00097371).The raw sequence data were deposited in the DNA Data Bank of Japan (DDBJ) sequence read archive (DRA006238) under BioProject no. Figures 1B–D) . The statistical power for this comparison was 0.92 at p = 0.05 in the post hoc analysis, indicating sufficient power of this study to address the difference in the dietary habit between these two groups. The difference in the fat consumption ratio was a result of the higher intake of fried rice, cooked breads, and processed meats by Bangkok children . Three enterotype-like variations were observed, although they were not entirely discrete: the first one, localized in the PC1-positive region, was defined by a high abundance of Bacteroidaceae; the second one, localized in the PC1-negative region, was defined by a high abundance of Prevotellaceae; and the third one, localized in the PC2-negative region, was defined by a high abundance of Bifidobacteriaceae, Lachnospiraceae, Ruminococcaceae, and Peptostreptococcaceae. Although there was no obvious relationship between the city of residence and the enterotype-like cluster, Bangkok and Buriram samples tended to be driven in PC2-positive and -negative directions, respectively .We analyzed the bacterial composition of stool samples by amplicon sequencing of the 16S rRNA V1–V2 region. Then, we profiled the bacterial community structures of 45 samples using pairwise weighted UniFrac distances. As unanticipated, there was no statistical differences in the community structures between Bangkok and Buriram groups, even though the statistical power reached adequate level to detect a large and middle effect size such as enterotype differentiation or impact by controlled-diet feeding . TherefoFigure 3 shows the family level composition of fecal microbiota by city. There was a tendency for Bacteroidaceae to be less abundant in Buriram children, whereas some Clostridia families such as Lachnospiraceae and Peptostreptococcaceae were more abundant in Buriram children. Figure 3B shows the number of OTUs belonging to each family. More than a half of over 100 total OTUs were found to belong to class Clostridia, especially in the families Lachnospiraceae and Ruminococcaceae, whereas two dominant families, Prevotellaceae and Bacteroidaceae, consisted of less than 20 OTUs. A subdominant family, Peptostreptococcaceae, was statistically more abundant and more diverse in Buriram samples than in Bangkok samples, as shown in Figures 3A,B, respectively. Total alpha diversity tended to be higher in Buriram samples than in Bangkok samples, as indicated by Chao1 (p = 0.042) and ACE (p = 0.047) species richness estimates  and the number of total OTUs shown in Figure 3B (p = 0.07).Figure 5A, Bangkok children were more highly colonized by classes Actinobacteria and orders Selenomadales and Bacteroidales, whereas Buriram children were more highly colonized by some taxonomic groups belonging to the order Clostridiales, such as families Peptostreptococcaceae and unclassified Ruminococcaceae. In Figure 5B, the relative abundance of taxonomic groups showing an LDA score greater than 103  was summed for the Bangkok type (Negativicutes + Bacteroides+ Porphyromonadaceae + Bifidobacterium) and the Buriram type (Peptostreptococcaceae + unclassified Ruminococcaceae), and the ratio of the sum of the Buriram type to the sum of the Bangkok type was calculated for each child . The BR/BK taxa ratio was significantly different between children in Bangkok and Buriram, as shown in Figure 5C (p < 0.001 in Wilcoxon rank-sum test). The statistical power for this comparison was 0.88 at p = 0.05 in the post hoc analysis, indicating sufficient power of this study to address the alteration in the gut microbial community between Bangkok and Buriram children.To find more detailed differences in the fecal bacterial compositions between children in the two cities, a LEfSe analysis was performed using phylum to genus-level data. As shown in To examine the confounding effects of age and gender biases on city of residence, we performed a multivariate regression analysis of the BR/BK taxa ratio. The results indicated that the city of residence was strongly associated with the BR/BK taxa ratio even after the adjustment, suggesting there was no confounding effect by age or gender in the analysis of gut microbiota community structure . Further, butyrate concentrations positively correlated with BR/BK taxa ratios with sufficient statistical significance and power . To examine the confounding effect of age and gender on city of residence, we performed a multivariate regression analysis of the butyrate and propionate levels. The results indicated that the city of residence strongly associated with both the butyrate level and the propionate level even after the adjustment, suggesting there was no confounding effect by age and gender in the analysis of SCFA levels .Using fecal metabolome data, a cluster analysis was performed, with five clusters resulting , as observed in Buriram children. On the other hand, MT2 samples were characterized by high concentrations of proteinogenic amino acids . All of the MT2 samples were derived from Bangkok children and showed a low BR/BK taxa ratio . MT3 was also mainly derived from Bangkok children. The bacterial composition of this group was characterized by a high abundance of Parabacteroides, whereas the BR/BK taxa ratio was intermediate between those of MT1 and MT2. Taken together, MT1 represents the high-SCFA-producing microbiota of Buriram children, and MT2 represents the low-SCFA producing microbiota of Bangkok children.Except for one sample from a child in Bangkok, all of the MT1 samples were derived from Buriram children. MT1 samples contained middle chain fatty acids (MCFAs) such as capric and caproic acids, in addition to two SCFAs measured. The microbiota of MT1 samples was characterized by a high BR/BK taxa ratio and high alpha diversity .MT4 was characterized by the overall low level of fecal metabolites. This group comprised three samples from Buriram and one from Bangkok. The microbiota of MT4 was characterized by a high abundance of N-oxide, which is associated with metabolic syndrome, fatty liver disease, and cancer  and low alpha diversity .MT5 comprised five Buriram and three Bangkok samples. This type was characterized by high concentrations of choline, betaine, and carnitine, which are generally abundant in red meat and dairy products, and also a series of biogenic amines such as gamma-aminobutyric acid (GABA) and putrescine. A number of previous studies have shown that these meat-derived amines are converted to trimethylamine by gut bacteria and further metabolized by a liver enzyme to trimethylamine d cancer . In the d cancer . The micFigure 9). Those genes involved in the paths from glutamate are mainly encoded by bacteria in the genus Romboutsia, family Peptostreptococcaceae, whereas those in the paths from pyruvate are encoded by various genera, although Romboutsia is one of major contributors. Further, 19 genes involved in phosphotransferase system, including cellobiose phosphotransferase, were overrepresented in MT1, suggesting that higher capacity of MT1 microbiome to ferment a variety of sugars. There was no statistical difference in the total abundance of propanoate metabolism between MT1 and MT2. However, 8 genes, including propionate CoA-transferase [EC:2.8.3.1] gene, were statistically enriched in the pathway of propanote metabolism of MT1 group (data not shown).Genes and their functions encoded by the fecal bacterial community were quantitatively predicted by PICRUSt based on 16S rRNA gene profiles. The abundance of each KEGG pathway group was compared among the metabolotypes. As a result, KEGG pathways involved in “metabolism” and some other subcategories were shown to be overrepresented in MT2 group compared with other groups, particularly MT1 , suggesting the significant impact of high level fat consumption on the gut microbiota. Furthermore, we examined the association between dietary nutrient and fecal metabolotypes . Among the macronutrients, only fat consumption showed a significant association with metabolotypes. In addition, beta-carotene intake level significantly differed among the metabolotypes groups. Children in MT2 group consumed more fat and much less beta-carotene than those in other metabolotype groups, especially MT1. Altogether, the differences in dietary habits and gut environment between two cities were reflected to the two metabolotypes, MT1 and MT2.We examined the correlation between dietary nutrients and gut microbiome in Thai children. As a result, fat intake level showed a significant negative correlation with the BR/BK taxa ratio as shown in Figure 12). According to data from the National Health Examination Surveys and the National Health and Nutrition Examination in the United States, the BMI (kg/m2) of 9-year-olds with normal or healthy weight ranged from 14.0 to 18.6 and, in 11-year-olds, ranged from 14.5 to 20.2. The median BMIs in the MT2, MT3, and MT5 groups were over 20, suggesting that children in these groups tended to be overweight, whereas participants in group MT1 were mostly categorized as having normal or healthy weights. Particularly, three out of eight children in group MT5 were categorized as obese.A statistically significant difference was only found between groups MT1 and MT5  ratio. The F/B ratio is known to be positively correlated with high-fat diet-induced obesity . In thisin Italy  as well A number of studies have demonstrated that plant-based diets promote colonic fermentation of SCFAs and have a profound effect on host health . A line The cluster analysis based on the top–down metabolomics data generated MT1 representative of Buriram metabolome. Samples in the MT1 group were characterized by higher concentrations of SCFAs and higher alpha diversity. It is interesting that the MT1 samples also showed higher concentrations of MCFAs. Microbial production of MCFAs is not common in the human gastrointestinal tract. They may be because of the consumption of coconut milk or palm oil, which are common ingredients in Thai food. However, we could not find any correlation between MCFAs in stool samples and the dietary records of MT1 children.15N flux study in mice that showed bacterial urease releases ammonia by hydrolysis of host urea and allows the transfer of host-derived nitrogen to the gut microbiota as a source of amino acid biosynthesis.MT2, characterized by a high abundance of amino acids and low level of SCFAs, showed a lower BR/BK taxa ratio representative of the microbiota in Bangkok children. The dietary habits of MT2 children were characterized by high-fat and remarkably low-vegetable consumption. Although there is no direct evidence, this suggests that their urbanized dietary habit altered the gut microbial communities of Bangkok children and reduced the microbial production of SCFAs. Whereas SCFAs in the intestine benefit the host, overrepresentation of amino acids in the gut has been found to associated with gut dysbiosis in patients with Crohn’s disease (CD) . This paFaecalibacterium or Roseburia (Interestingly, the predicted metagenomics showed that Peptostreptococcaceae, which was overrepresented in Buriram children, was involved in the biosynthesis pathway from glutamate to butyrate. Although Peptostreptococcaceae is not a common butyrate producer such as oseburia , there aoseburia . The patFusobacterium and Desulfovibrionaceae, which are known to be overrepresented in patients with colon cancer and ulcerative colitis and to be involved in inflammation (Haemophilus belonging to potentially pathogenic bacteria in MT5 subjects was also common in CD patients (MT4, characterized by the overall low level of fecal metabolites, had a high abundance of ammation . It is nammation . Furtherpatients . Althougpatients , suggestThis study suggests that the transition of dietary habit from traditional to modern distorted the functionality of gut microbiota of Thai children, notably SCFA productivity, through alteration of their community structure. As a number of previous studies have demonstrated that plant-based diets promote colonic fermentation of SCFAs and have a profound effect on human health, the high-vegetable Thai traditional diets appears to benefit children in Thailand and the lower level of fecal SCFAs appears to be a risk marker of dietary urbanization in Thailand. A larger scale community-based cohort study is warranted to monitor the impact of urban diets on the health and disease of youngster in developing countries in South–East Asia.This study was approved by the ethics committee of the Faculty of Agriculture of Kyushu University under approval no. 13-005 and jointly approved by the ethics committees of the Bangkok Hospital Phuket under ethic approval no. sq. 001/2016. Written informed consent was obtained from the parents/guardians of all participants. We entered and analyzed all samples and questionnaire data anonymously and published them using participant identification numbers.JK, OL-o, MN, SF, KS, Y-KL, SP, SN, and JN conceived and designed the experiment. OL-o, MN, SP, KK, and SN provided the sample collection. JK, OL-o, MN, PT, RM, MT, SF, and JN performed the experiment. JK, OL-o, MN, SF, and JN analyzed the data. JK, OL-o, MN, PT, SF, Y-KL, SN, and JN wrote the paper.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.


Daucus carota L. This group of bioactive compounds has been chosen for this review since most of the scientific papers proved their antioxidant properties relevant for inflammation, stress-related disorders, cancer, or neurological and neurodegenerative diseases, such as stroke and Alzheimer’s Disease. Besides, carrots constitute one of the most popular sources of carotenoids. In the presented review emphasis has been placed on the supercritical carbon dioxide and enzyme-assisted extraction techniques for the relevant tetraterpenoids. The detailed descriptions of these methods, as well as practical examples, are provided. In addition, the pros and cons of each method and comparison with the standard solvent extraction have been discussed.Multiple reviews have been published on various aspects of carotenoid extraction. Nevertheless, none of them focused on the discussion of recent green chemistry extraction protocols, especially for the carotenoids extraction from  Carotenoids belong to the isoprenoid group of pigments that are produced by both photosynthetic plants and some non-photosynthetic fungi and bacteria. Most animals cannot synthesize carotenoids and have to obtain them from foods . CarotenMomordica charantia L.) also bear high contents of lycopene .,47.2) ang others ,49,50. TDue to a difference in the genotype and structure of food samples, there are no commonly established carotenoids extraction procedures or standard methods ,51. HoweThe high water content of food sources is considered a negative factor for an efficient carotenoid extraction, particularly when considering superfluid extraction (SFE), because of the hydrophobic nature of solvents and carotenoids ,56. TherIn general, a few points should be followed to reduce the degradation processes during carotenoids extraction: (i) a carbonate-based neutralizers such as sodium bicarbonate, calcium carbonate, or magnesium carbonate should be added to neutralize acids generated from plant samples, as the acids can hinder the extraction of carotenoids; (ii) antioxidants such as butylated hydroxytoluene, tert-butlylhydroqinone, or ascorbyl palmitate can be added to prevent oxidation during carotenoids extraction; (iii) extraction time should be minimized to avoid enzymatic oxidation and efficient extraction of carotenoids; (iv) food samples should be protected from UV light to avoid photodestruction of carotenoids; and (v) sample tubes should be cleaned with nitrogen to remove oxygen and offer an inert environment ,59.Haematococcus lacustris  [Pandalus brasiliensis and P. paulensis) residue, compared to milling and dehydration pre-treatments [Ettlia oleoabundans  [The complex and rigid cell wall present in plant structures could hinder the entry of solvents inside the cells to extract carotenoids. Also, the linkages between carotenoids and other macromolecules (proteins and fatty acids) could further reduce the efficiency of carotenoids extraction. Thus, during the extraction of relevant phytochemicals, various sample pre-treatment methods are applied. Their main objective is the breakdown of the cell wall and other physical barriers in the food samples, thus permitting an efficient carotenoids extraction . Among trophyta) . Mezzomorophyta)  studied eatments ,62. On trophyta) ,64. Highrophyta) . In thisrophyta)  studied rophyta) . Ultrasorophyta) ,68. Manyrophyta)  but diffSolvent extraction is the most widely used method due to its simplicity and having been scaled up industrially in the past. Different organic solvents such as chloroform, hexane, methanol, diethyl ether, acetone, isopropanol, and methylene chloride have been used to efficiently extract carotenoids. Moreover, a combination of these solvents has been utilized to get synergistic effects on carotenoids extraction. The selection of appropriate solvent or combination of said solvents depends on the polarity and chain length of the target carotenoids but the food matrix components and moisture contents also play an important role in solvent selection . Mostly,Still, solvent extraction requires large amounts of organic solvents and can also cause the degradation of carotenoids when heating is applied (a process necessary for some of the solvents). Thus, another method was tested for carotenoid extraction. One of them is solid-phase extraction (SPE). SPE uses solvents and a solid media to separate desired components from a liquid matrix. This method uses smaller volumes of solvents than standard solvent extraction and could have the selectivity to separate very similar compounds from each other . SPE is 2 is a gas or liquid that has been compressed and heated beyond a critical pressure and temperature [2 possesses liquid-like density and has intermediate physiochemical properties between liquids and gases  and 7.3 MPa (72.8 bar), allowing to operate near room temperature and mild pressure [2 at supercritical state could lead to rapid penetration of CO2 into the pores of complex food matrices, thereby, enhancing the efficiency of carotenoids extraction [SC-COperature ,71. At tnd gases . CO2 cri 7.3 MPa .8 bar, atraction . Additiotraction ,74.2 is a non-polar solvent that can replace one of the most commonly used non-polar solvents such as hexane. The compound’s solubility in SC-CO2 is dependent on the compound’s polarity, molecular weight, and structure [2 because they could perfectly match the solvent’s polarity at this pressure. Moderate to highly polar compounds are almost insoluble in SC-CO2 [2 extraction, especially when coupled with a modifying solvent, can be successfully used as an extraction solvent. Extracts obtained with SC-CO2 are widely used in food applications because carbon dioxide is inert, non-flammable and inexpensive. Furthermore, the extracts obtained with SC-CO2 are odorless, tasteless and “generally recognized as safe” (GRAS), because the commercial CO2 gas stream can be recycled, and SC-CO2 extraction is regarded as a green extraction  process [2 under optimized conditions is the best method among others to obtain the optimum extraction yield of environmentally safe, non-toxic and high purity carotenoids [SC-COtructure . Componen SC-CO2 . The pren SC-CO2 . SC-CO2  process ,78. Sing process  have revotenoids .2 extraction has also been the subject of much research and development over the years for the extraction of various compounds from samples derived from nature. Though SFE extraction can be performed on various sample types, the basic system for all extractions is the same [2 causes cell disruption to remove carotenoids with reduced time and labor requirements [2 extraction of polar carotenoids (xanthophylls) and non-polar carotenoids (β-carotene) require appropriate levels of temperature, CO2 density, and pressure and flow rates. In general, extraction temperature from 40 to 60 °C, pressure from 300 to 400 bar, treatment time from 30 to 120 min, appropriate CO2 density, CO2 flow rate from 1 to 5 mL/min, and concentration of entrainers from 5 to 25% v/v, are the five most important parameters during SC-CO2 extraction of carotenoids [2 extraction of carotenoids was done by using solvents such as N,N′-dimethyl formamide and methanol from the microalgae Dunaliella salina [SFE extraction has gained popularity in the last three decades because when carbon dioxide is used as a solvent, the extracts obtained are considered natural and contaminant-free . SC-CO2 the same . The fouthe same ,81,82. Rirements . SC-CO2 otenoids ,84,85. Ta salina ,87. The a salina  showed ta salina . Thus, ba salina .2 without any modifier should efficiently extract from carrots the carotenoids with non-polar nature, a low extraction rate (34%) was achieved due to the high molecular weight of the targeted compounds. Therefore, ethanol as a co-solvent may be used to enhance the recovery rate of targeted compounds. Ethanol has the ability to enhance the polarity of CO2, dissolving several polar macronutrients such as proteins, carbohydrates, and lipids. Thus, the high mass yield of extract might be obtained after using a high concentration of ethanol as a co-solvent. In addition to the concentration of the co-solvent, other parameters such as temperature and pressure used during SC-CO2 are also important factors that affect the process of carotenoids extraction. Among these factors, pressure plays the main role in increasing the solvation power of CO2, enhancing the extraction of carotenoids and other phytochemicals [2 could extract 96.2% carotenoids from carrot peel when the extraction vessel’s full capacity was used with appropriate temperature and pressure. Also, an application of the SC-CO2-based methods together with the enzyme and microbial inactivation for apples or orange juices was shown to be beneficial for carotenoids isolation [2 extraction of β-carotene from tray dried carrots at 40, 50, and 55 °C and 30, 35 and 40 MPa  [C is concentration of β-carotene in the extract (µg/g) at time t = ∞; k is extraction rate (h−1); and t is the time (h)The total carotenoid contents in carrot vary from 4.6 to 548 μg/g, depending on the different cultivars . The tothemicals . Moreovehemicals ,93. Highhemicals . Scientihemicals ,94. Somehemicals ,96,97. Themicals  revealedsolation ,50. Anot to 6 h) . They poEquation (1). The Weibull model for the description of the supercritical extraction of β-carotene from carrots.2 with pressure in the range of 10 to 40 MPa could be used for continuous non-thermal pasteurization of carrot juices. It leads to the significantly extended shelf life of the juice, with no loss in most of the nutritional, and functional properties of the raw extracts [2 was applied as a mild technology to enhance the microbial and enzymatic stability of the product, without altering its main qualitative aspects, for the pasteurization of freshly cut carrots with no modification of their texture, color and nutritional properties [Besides being used for the carotenoid’s extraction from carrot, SC-COextracts . Also, Soperties . The menoperties .As stated above, the solvent-based extractions of bioactive compounds from natural sources often suffer from low extraction yields, require long extraction times and the final product may contain trace amounts of organic solvents that have a huge negative impact on the product quality. The EAE was shown to be effective, environmentally friendly and a selective method for bioactive compound extraction. Enzymes can be perfectly matched catalysts to assist in the extraction of various bioactive compounds from natural origins. The inherent ability possessed by enzymes to catalyze reactions with high specificity, to degrade or disrupt cell walls and membranes and to operate under mild processing conditions in aqueous solutions makes the EAE an interesting alternative for the more efficient extraction of phytochemicals from biological matrices . EAE hasPalmaria palmata  with the use of proteases and carbohydrases, whereas Fernández, Vega and Aspé [® L  might be used for the pretreatment of garlic. Enzyme pretreatment of garlic resulted in a higher yield of oil (0.5%), compared to the control (0.28%), without any meaningful differences in physicochemical properties of the volatile oil [Among enzymes that could be applied in the EAE approach various enzymes, especially highly complex large polymers such as proteases, pectinase, cellulase, tannase, or carbohydrases could be mentioned . In 2010and Aspé  used pecand Aspé ,118. Othtile oil . The wattile oil .® L, pectinase and cellulase for the liquefaction ability in terms of recovery of total carotenoids, total phenolics, total flavonoids and ascorbic acid from the red capsicum extract. Obtained extracts were planned to be used as natural colorants and functional ingredients in foods. The authors studied the performance of the above-mentioned enzymes and their utility for carotenoid extraction. Viscozyme® L and pectinase were observed to cause significantly higher liquefaction that was proved by the increased extract yield (80%–87%). Also, the improvement in percentage yield of aqueous extract was determined with increasing dosage of all enzymes. To conclude, EAE significantly improved the recovery of total carotenoids in the aqueous extract [Cordyceps militaris  by evaluating the antioxidant activities of the extract [There are numerous reports on the extraction of carotenoids from vegetable sources, with the preferred source being tomato peels and carrots. But other plant matrices are also under investigation. Nath et al.  presente extract . The aut extract  showed t extract . The sci extract  demonstr extract . Also, tMai et al.  presente® Ultra SP-L  could be combined with either Cellubrix® L or Cytolase CL . The data showed that combinations of PU and CY was a more effective strategy than each of the enzyme alone and a 1:1 ratio was the most effective [Carrot tissue EAE was presented in the literature some time ago. The data revealed that treatment with a combination of pectinase, cellulase, cellobiase, and pectin lyase increased the lycopene yield by approx. 50%. Enzyme treatments lead to changes in the microstructure and extractability, while some of the carotene complexes and interactions were broken. The EAE carrot sample pretreatment resulted in increased carotene extractability. Moreover, scientists, especially those wishing to follow the “green chemistry” expectations, tried to re-use the pomace remains after the production of the carrot juice. It is commonly used as feed or fertilizer, but it could also be a valuable source of carotenes. Thus, it is of utmost importance to present novel, “green chemistry” methods aimed at recovering the carotene-rich functional food ingredient from carrot pomace. During the extraction fine grinding of the suspension of carrot pomace in water using a colloid mill and the subjection of the pomace to enzymic hydrolysis, homogenization and concentration should be carried out. For the enzymic hydrolysis, Pectinexffective . The co-ffective  the combffective .In recent years, many researchers have focused on the extraction of analyte from carrots and other vegetables by using enzymatic treatments. Usually, manual methods of carotenoids extraction could cause undesirable flavor and color changes during the cell wall disintegration to release carotenoids. EAE has been found to prevent these negative changes during carotenoid extraction, the reason being that the extracted carotenoids by enzymes are still bound to proteins, so they provide stability to the color and structure of unsaturated pigment . The utiAspergillus niger is one of the most important multi-enzymes producer microorganism to generate pectinases, hemi-cellulases, cellulases, glucoamylases, showing enzyme activities of 7.62, 15.86, 0.99 and 13.37 U mg−1 of protein respectively [Aspergillus niger, Trichoderma longibrachiatum, and Saccharomyces cerevisiae are commonly used for cellulose production [Momordica cochinchinensis Spreng.) was studied by Kha et al. [w/w) for pretreatment Kha et al. [Industrially, many microorganisms are employed for the generation of various cellulolytic enzymes . Aspergiectively . Cellulaoduction . Pectin oduction . Pectinaoduction . The coma et al. . The resa et al. . Strati a et al.  also eva2 and EAE, have been pointed out. The main benefits of using SC-CO2 for isolation of carotenoids are solvent-free products, no co-products, and low temperatures in the separation process. SC-CO2 is non-carcinogenic, non-toxic, non-mutagenic, non-flammable, and thermodynamically stable and CO2 does not usually cause oxidization of the analytes that are very important for the carotenoids. On the other side, EAE offers a faster extraction, higher recovery, reduced solvent usage, and lower energy consumption when compared to non-enzymatic methods. For the carotenoid’s extraction from carrots, EAE improves the extraction yields by disrupting the cell walls of the carrot’s tissues. The SC-CO2 method requires important technological investments, while EAE is a more convenient solution for the application in developing countries [Daucus carota L.The isolation of biologically relevant compounds from natural sources gets more attention nowadays since the quality of food is decreasing due to pollution affected by air, water, and climate changes. In the presented review, the advantages of carotenoids extraction from carrots with the use of two “green chemistry” methods, SC-COountries ,130. As


Aspergillus fumigatus and Alternaria alternata sensitization was the most common allergic sensitization among the patients with allergic disorders. According to the reviewed studies, despite the humid climate of the north of Iran, fungal sensitization has a prevalence range of 5-70% in this region. In other regions, such as central and southern Iran, which have a dry and warm climate, fungal sensitization reportedly has a prevalence range of 5-65%. The prevalence of fungal sensitizations varies in different allergic disorders due to the factors related to geographic and genetic issues, gender, sample size, test operator, and assessment method.Fungal agents account for the clinical manifestation of allergic disorders. The aim of the present study was to review the prevalence of hypersensitivity reactions to fungal aeroallergens in patients with allergic disorders, including allergic rhinitis, asthma, urticaria, and eczema, in Iran. The initial literature search resulted in the identification of 50 records, 26 cases of which met the inclusion criteria. Regarding the methods adopted for the detection of fungal allergens, serum-specific IgE and skin prick tests were used in 6 and 20 studies, respectively.  Candida species are the most common fungal allergens accounting for the development of allergic disorders. Fungal spores are broadly distributed in nature; accordingly, fungal exposure is a daily fact of human existence . Fungal Alternaria species have a role in the pathogenicity of allergic disorders , Candida and Aspergillus species have been identified as the most common sensitizing fungal allergens leading to allergic disorders  tests used as confirmatory assays , 11, 12.Allergen avoidance is the most effective way to manage allergic patients; nonetheless, it is usually difficult. Allergen-specific immunotherapy (ASIT) is an important therapeutic approach, which is administered for the patients that cannot be managed by conventional therapy . HoweverFor the purpose of the study, a comprehensive search was conducted in several databases, including PubMed, EMBASE, Google, SID, Magiran, and Irandoc, using the following keywords: “Skin prick test”, “Serum fungal-specific IgE”, “Fungal allergen”, “Aeroallergens”, “Asthma”, “Allergic rhinitis”, “Urticaria”, “Eczema”, and “Iran”. There was no time limit for searching the articles. The inclusion criteria were: 1) investigation of fungi in allergic diseases, 2) availability of the full-text version of the article, and 3) publication in English or Persian. On the other hand, the articles investigating issues other than allergic diseases or those with unclear methods were excluded from the study. The initial search process resulted in the identification of 50 records, 26 cases of which met the inclusion criteria. Regarding the methods adopted for the detection of fungal allergens, serum-specific IgE and skin prick tests were used in 6 and 20 studies, respectively. Candida species (which was positive in 28% of the patients), among allergic patients was 0-10% [The prevalence of fungal sensitization is similar in the different areas of Iran. For example, in the north of Iran, fungal sensitization has a prevalence rate of 5-70% despite the warmness and humidity of this region. In the same vein, in the other areas, such as central and southern Iran, which have a dry and warm climate, the prevalence rate of fungal sensitization is estimated at 5-65%. It seems that geographic and climatic factors play a less significant role in sensitivity to fungal allergens. In a study performed in Mazandaran, Iran, the overall prevalence of sensitivity to fungal allergens, except for the as 0-10% . In EsfaThe findings of the reviewed studies were suggestive of the higher frequency of positive reactions to mold in females and adults as compared to those in males and other age groups in Iran, respectively. The results of some studies indicated no difference between males and females in terms of fungal sensitization , 28, 36.The results of some studies indicated the reduction of mold sensitization prevalence with aging , 26, 42,Mahboubi et al. showed that fungal sensitization was more common in the summer; nonetheless, Shokohi Shormasti showed that fungal allergen sensitization was more common in the fall and winter , 22. In Alternaria species (3.7%), Aspergillus species (2.2%), and Candida species (0.7%). In a couple of reviewed articles, swamp coolers and new air conditioners were introduced as the main causes of fungal sensitization in dry and warm weather [In asthmatic patients, hypersensitivity reactions to molds had a prevalence rate of 11%, and mold sensitization was more common in the autumn and winter than in the other seasons . In a st weather , 25.Alternaria species as about 45.5% among allergic rhinitis patients by skin prick test. They also reported a significant relationship between Alternaria species and allergic rhinitis. In addition, they observed an association between Alternaria species sensitization and the isolation of Alternaria species from nasal discharge. In the mentioned study, direct examination and culture of Alternaria species were positive in 32.8% and 43.1% of the cases, respectively [Amirmajdi et al. determined the prevalence of hypersensitivity reaction to ectively . In a stectively . Aspergillus fumigatus and Alternaria alternata was more common [Aspergillus fumigatus and Alternaria alternata sensitization was more common in allergic rhinitis patients. Additionally, sensitization to Aspergillus fumigatus and Penicillium species was more common in patients with asthma disorder, while those with urticaria and atopic dermatitis showed more sensitization to Aspergillus fumigatus [In dry climates , pollen aeroallergens are more common than mold allergens. Mites were reported as the most common allergens in the different regions of Iran, except for Zahedan despite its dry and warm climate . Mites ae common -39. Furtumigatus -39.Aspergillus fumigatus sensitization was the most common allergic sensitization in allergic disorders. Based on the reviewed articles, fungal allergens that tested positive in allergic disorders included Aspergillus fumigatus, Alternaria alternata, Penicillium notatum, Candida species, Cladosporium species, and Rhizopus species. Exposure to Alternaria species is an important risk factor for the prevalence of asthma and can lead to severe and potentially fatal asthma [However, out of all fungal allergens, l asthma . Alternaria species sensitization and presentation of Alternaria species in the upper respiratory tract might induce allergic rhinitis. Nonetheless, there was no significant relationship between Alternaria species sensitization and severity of allergic rhinitis [Alternaria species sensitization had a direct association with more severe asthma [rhinitis . Severite asthma . Nonethee asthma . Mold seIn addition, mold sensitization was not different between intermittent and persistent or between mild and moderate to persistent allergic rhinitis , 42. Studies showed that mold sensitization was more common in asthmatic patients than in patients with allergic rhinitis, atopic dermatitis, and asthma plus allergic rhinitis , 27. NevThe results of a couple of studies were indicative of a significant relationship between mold sensitization and severity of allergic rhinitis , 34. A sFusarium species (23.5%), Aspergillus flavus (21.2%), Dreselera orysae (18.8%), Alternaria species (17.6%), Curvularia eragrostidis (17.6%), Penicillium species (16.5%), Pestolotriopsis gtuepini (16.5%), Rhizopus arrhizus (16.5%), Aspergillusniger (15.3%), Penicillium choy (12.9%), Aspergillus fumigatus (11.8%), and Cladosporium species (4.7%) were detected by skin prick test reactivity [The evidence revealed no significant difference between the two genders and different age groups with allergic disorders in terms of mold sensitization , 36, 38.activity . Alternaria species, is correlated with the severity of asthma. Consequently, fungal allergen should be considered in patients with allergic disorders. The prevalence of fungal sensitizations is different in allergic disorders due to the factors related to geographic and genetic issues, gender, sample size, participants’ age, climatic region, test operator, and assessment method. All fungal allergens are relatively common in the different regions of Iran. Sensitization to a few fungi, such as