Aspergillus-specific DNA, galactomannan (GM), and retrospectively for (1→3)-β-D-glucan (BDG). Three probable IA and two possible invasive fungal disease (IFD) cases were identified using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSGERC) 2019 consensus definitions. Sensitivity and specificity for diagnosis of probable IA and possible IFD was 80% : 28–99%) and 55% (95% CI: 32–77%) for BDG, 40% (95% CI: 5–85%) and 100% (95% CI: 83–100%) for GM, and 60% (95% CI: 15–95%) and 95% (95% CI: 75–100%) for Aspergillus-specific real-time PCR. However, sensitivities have to be interpreted with great caution due to the limited number of IA cases. Interestingly, the low specificity of BDG was largely caused by false-positive BDG results that clustered around the date of alloSCT. The following strategies were able to increase BDG specificity: two consecutive positive BDG tests for diagnosis (specificity 80% (95% CI: 56–94%)); using an optimized cutoff value of 306 pg/mL (specificity 90% (95% CI: 68–99%)) and testing BDG only after the acute posttransplant phase. In summary, BDG can help to diagnose IA in pediatric alloSCT recipients. However, due to the poor specificity either an increased cutoff value should be utilized or BDG results should be confirmed by an alternative Aspergillus assay.Data on biomarker-assisted diagnosis of invasive aspergillosis (IA) in pediatric patients is scarce. Therefore, we conducted a cohort study over two years including 404 serum specimens of 26 pediatric patients after allogeneic hematopoietic stem cell transplantation . Sera were tested prospectively twice weekly for  Aspergillus infection, and could therefore be used as a broad spectrum fungal biomarker keeping in mind that it is not detectable in IFD caused by mucormycetes, Cryptococcus or Blastomyces dermatitidis [1→3)-beta-D-Glucan (BDG) is a fungal cell wall component released into patients’ blood during invasive fungal disease (IFD). BDG is not specific for →3-beta-Datitidis . Confounatitidis .® assay, which requires only five microliters of serum for a single BDG measurement. In addition to being used as a single biomarker, the combination with other assays may prove beneficial for an accurate and timely diagnosis of IA [Aspergillus real-time PCR assay.BDG was included as microbiological criterion in the revised consensus definitions of IFD from the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) 2008 , but excis of IA ,13,14,15n = 26) were selected for retrospective BDG analysis. Patients ; 14 females, median age of 8.5 years ) were all eligible and in preparation for alloSCT. Patients suffered from the following diseases: acute lymphoblastic leukemia , acute myeloid leukemia , and other diseases, including neuroblastoma (n = 2), lymphoma, sarcoma, and immunodeficiency (n = 1 each). In total, 404 blood samples  were analysed. Routinely, all pediatric patients received liposomal amphothericin B as prophylaxis.Patients. From April 2016 to March 2018 (24 months), 41 pediatric patients treated at the University Children’s Hospital Würzburg were screened twice weekly by GM and PCR-based diagnostic assays. All available patients with more than two serum samples and existence of frozen backup samples . This lAspergillus real-time PCR without the risk of incorporation bias.For analysis of clinical test performance probable IA cases and “not classified” patients (serving as negative controls) were used including PCR and GM as microbiological criteria. Not classified patients comprise all patients except those with proven, probable and possible IFD. In a second approach also cases of possible IFD were included as true IA cases. The reason to do so was that the entity of probable IA and possible IFD cases is defined by a host factor and a clinical feature without the requirement for mycological evidence. This enabled us to compare the diagnostic performance of BDG with GM and One not classified patient  was excluded from further analysis due to a missing HRCT scan . Data of this patient, especially in the context of BDG detection, were analysed separately.TMAspergillus Ag assay; Bio-Rad, Munich, Germany), BDG measurements and in-house fungal PCR testing. BDG measurement was performed retrospectively from serum samples that were frozen at −20 °C until testing. No additional freeze–thaw cycles occurred between GM and BDG testing. No ethical approval was necessary because BDG testing was performed retrospectively and no clinical management decisions were based on BDG results.Diagnostic molecular assays. Patients were tested within a routine diagnostic surveillance program using GM ELISA  [Aspergillus species, but cannot differentiate between them. In a second reaction, Bacillus DNA used as extraction and PCR inhibition control was detected. This internal control was tested independently of the Aspergillus target (monoplex), but within the same PCR run. The reaction mixtures  contained 0.12 μM primer 16S S (5′-ggTCTT gAC ATC CTC TgA cAA tCC TA-3′), 0.12 μM primer 16S A (5′-AAC TgA ATg CTg gCA ACT AAg ATC A-3′), 0.07 μM hydrolysis probe 16S TM (5′-FAM-AgA gTg ACA ggT ggT gCA Tgg TTg TC-TAMRA-3′), 10 μL of Takyon ROX Probe 2X MasterMix UNG, and 5 μL of template DNA. Amplification was carried out with a StepOnePlus machine (Applied Biosystems), with the following steps: 50 °C for 2 min, 95 °C for 3 min, and 60 cycles of 95 °C for 5 s, 54 °C for 15 s (detection step), and 72 °C for 1 s. Negative and positive PCR controls were included in each run, and were analysed in duplicate (Aspergillus). The limit of detection was below two copies. Two positive results (non-reproducible or reproducible positive duplicates) from two independent samples within 15 days were defined as positive PCR assay for EORTC/MSGERC classification.For the Germany) . The proGermany) . Real-tiGermany) . Briefly® assay  was used according to the manufacturer´s instructions. Briefly, 5 µL of serum were added to 20 µL of pretreatment reagent in a glucan-free 96-well plate. After 10 min of incubation at 37 °C, 100 µL of Fungitell® reagent were added to the pretreated sample. The Limulus amebocyte lysate reaction was then monitored at 37 °C for 40 min in an ELx808™ microplate reader . The mean optical density change over time was calculated and compared to a standard curve to determine the BDG concentration. Samples with indeterminate (60–79 pg/mL) or positive results (≥ 80 pg/mL) were retested to confirm the elevated BDG value where possible. BDG levels below 31 pg/mL  were calculated by extrapolation.For BDG measurement the FungitellStatistics. The clinical performance, sensitivity, specificity, positive and negative predictive value , and odds ratio of the different assays were calculated using Prism 5 (Graph-Pad Software). Diagnostic odds ratio (OR) is a global measure for diagnostic accuracy. OR depends significantly on the sensitivity and specificity of a test and does not depend on disease prevalence. A test with high specificity and sensitivity with low rate of false positives and false negatives has high OR . A confiThe relations among BDG, GM, and PCR were evaluated. Grouping continuous variables by binary variables, descriptive distribution indexes were calculated. For all evaluable patients the maximum value of BDG, GM, and PCR was calculated. The following variables were created: maxbdg (continuous), maxbdgpos (binary), maxgm (binary), maxgmindex (continuous), maxgmpos2019 (binary), and maxpcrpos2019 (binary). The Kruskal–Wallis test was performed on “maxbdg” (continuous) vs. “maxgmpos2019” (binary), on “maxbdg” vs. “maxpcrpos2019” (binary), and on “maxgmindex” (continuous) vs. “maxpcrpos2019”. Maxgmpos2019 = 0 is low level , maxgm = 1 is high (outside the norm). Maxpcrpos2019 = 0 is no target detection, maxpcrpos2019 = 1 is the opposite.Aspergillus-specific real-time PCR (n = 404), GM (n = 403) and BDG assay . EORTC/MSGERC classification resulted in 3 cases of probable IA (11.5%), 2 cases of possible IFD (7.7%) and 21 not classified patients without specific high-resolution CT (HRCT) scan signs of IFD (80.8%). Due to a missing HRCT scan in the time period of suspected IA one not classified patient (K207) was excluded. However, this patient was analysed separately, because he perfectly illustrates the complexity and insufficient specificity of BDG kinetics in pediatric SCT patients.A set of 404 serum samples from 26 pediatric patients with high-risk for IFD were collected shortly before, during and after alloSCT. These specimens were screened for the presence of fungi by using three diagnostic assays, namely, The sensitivity, specificity, PPV and NPV of a single positive BDG measurement (cut-off ≥ 80 pg/mL) in patients with probable IA was 100% (95% CI: 29–100%), 55% (95% CI: 32–77%), 25% (95% CI: 5–57%), and 100% (95% CI: 72–100), respectively . If two Aspergillus PCR and with combinations of these biomarkers we calculated the diagnostic parameters for probable IA and possible IFD cases versus not classified patients  of all three biomarkers in general. Best sensitivity was shown for BDG , as one possible IFD patient was detected by BDG only, followed by PCR  and GM  was shown for the combination of BDG with PCR followed by the remaining combinations with 40% each (95% CI: 5–85). By this approach, the specificity was increased to 100% (95% CI: 83–100) for all combinations with PCR and to 95% (95% CI: 75–100) for the combination of BDG with GM.p-value = 0.0212). The calculated values for PCR/BDG were close to significance , but not for PCR/GM . The lack of statistical significance was likely attributable to the limited number of patients under investigation. In conclusion, all three diagnostic tests showed a trend towards higher values in cases of probable IA.The Kruskal–Wallis analyses showed that higher mean BDG values were associated with higher GM and PCR values, and that higher mean GM values were associated with positive PCR . HoweverThe specificity of one single or two consecutive positive BDG measurements in our cohort was poor. In order to improve specificity, several previous studies have discussed adaptations of BDG cutoff values ,24,25,26In order to find reasons for the poor specificity of BDG we analysed the clinical data of the false-positive patients. Interestingly, 8 of 9 (89%) not classified patients with positive BDG showed condensed positivity around the date of SCT (range: 11 days before (d −11) to 49 days after SCT (d 49); All these patients were affected by ALL, whereas seven out of eight (88%) received a T cell depleted stem cell graft. Median age was 11 years (range: 2–17). In contrast, in a control cohort  only three patients were affected by ALL (50%) and only two received a T cell depleted graft (33%). Median age was 4.5 years (range: 2–18). In order to investigate depleted stem cells as source for BDG positivity, they were tested for the presence of BDG. The supernatant of the depleted stem cells contained 134 pg/mL BDG. However, only a very small amount is administered during SCT and consequently this cannot be responsible for systemically elevated BDG levels. Additionally, patients’ records were screened for known confounding factors of BDG positivity. Treatment with albumin or immunoglobulins and mucositis as possible sources were found in five of eight patients. Additionally, one of these patients showed signs of liver dysfunction during the observation period due to a beginning sinusoidal obstruction syndrome. Due to prophylaxis most of the patients received at least one antimicrobial drug described to be contaminated with traces of BDG [Biomarker positivity in probable IA cases was correlated to the time span of positive HRCT scans . For twoStenotrophomonas maltophilia was repeatedly detected in sputum and blood, together with other microorganisms like Candida species (in sputum and BAL) and viruses such as CMV and adenovirus in blood, sputum, and BAL. Although the patient partially stabilized under intensified antimicrobial treatment, the further course was characterized by an on-off situation with respiratory deterioration and partial stabilization on an intermediate care unit (IMC). On day 115 he developed an acute septicemia which necessitated mechanical ventilation and circulatory support on an ICU and treatment with voriconazole, meropenem, cefepime, and piperacillin/tazobactam (amongst other antibiotics). Pseudomonas aeruginosa was cultured from tracheal secretion. Under intensified regimen, the patient recovered partially, but could not be weaned entirely from mechanical ventilation support. On day 219, he died from refractory respiratory distress and multiorgan failure. Interestingly, GM was constantly negative, whereas PCR detected A. fumigatus at later stages of the disease between day 114 and 196 post SCT . A. fumigatus was repeatedly cultured in tracheal secretion from day 194 onwards. HRCT scans were stopped after day 134 as they continued to show severe pulmonary alterations; however, chest X-rays repeatedly documented infectious infiltrates. Due to the lack of HRCT scans in the later phase, the patient was probably wrongly classified as “not classified”, and excluded from overall patient analysis. BDG showed positive results from the first available sample at day 65 post SCT onwards and was intermittently positive until day 114 and positive again at day 196 (next available sample). Multiple infections with Stenotrophomonas maltophilia and Pseudomonas aeruginosa as pathogens and additionally A. fumigatus in the terminal phase are very likely. BDG cross-reactivity with Stenotrophomonas maltophilia and Pseudomonas aeruginosa [Aspergillus fumigatus can be regarded as the responsible agent, as this fungus was repeatedly cultured directly from tracheal secretion after day 194 until the death of the patient.Patient K207  was a 12ruginosa ,28,29 coruginosa . For theAspergillus PCR and galactomannan for detection of IA in high-risk pediatric patients. In total, three molecular biomarkers  were analysed in 404 blood samples of 26 pediatric patients shortly before, during, and early after alloSCT. Three cases of probable IA and 2 cases of possible IFD were classified according to EORTC/MSGERC criteria. One not classified patient was excluded and analysed separately.The aim of this study was to evaluate a standardized BDG screening (twice weekly) alone and in combination with During the two-year study period only three patients suffered from probable IA. This is most likely a result of the routinely administered prophylaxis with liposomal amphotericin B. The small number of IA cases in our cohort is responsible for the wide confidence intervals that flaw the results of sensitivity. Consequently, it is not feasible to compare the sensitivities of the biomarkers either alone or in combination. However, it may be mentioned that BDG was elevated in all patients with probable IA. Additionally, our results are in line with previously published results showing a very wide sensitivity range of the Fungitell assay (27% to 100%) .In contrast to sensitivity, the results for specificity are based on 23 patients and more than 380 serum samples. The specificity of a single positive BDG value was only moderate but again falls into the previously published specificity range of 0% to 100% . So far,The low specificity of BDG is well known from adult patients and can be caused by several confounding factors including mucositis, bacterial infections or treatment with albumin, immunoglobulins or particular antimicrobial drugs ,4,30. AtWhat are ways to increase specificity in pediatric patients after SCT? One possibility is the requirement of a second consecutive positive BDG test. In our cohort the specificity increased from 55 to 80% by this measure. Even stronger is the gain in specificity if BDG is combined with PCR and both biomarkers have to be positive. Specificity is 100% with this approach and three out of five patients with probable IA and possible IFD are detected (sensitivity 60%). Another possibility is to increase the cutoff. Adapting the BDG threshold for IFD was discussed for adults  and veryAspergillus-specific PCR is able to detect probable IA and possible IFD with excellent specificity  and PPV . Therefore, BDG or GM and Aspergillus-specific PCR could be used during the SCT phase.Another approach is to exclude BDG completely from screening around the day of SCT. This would increase the specificity of the whole assay tremendously. During this time, clinical signs together with biomarker combinations could be used to screen for IFD. According to our data a combination of BDG or GM with Aspergillus, the test can be used to detect other fungi, but cross-reacts also with some bacteria [Aspergillus-specific assays GM or PCR had not detected this patient. Another case of false-positivity is intensively described above (K207), as this patient suffered from different bacterial infections showing intermittently numerous positive BDG results.As BDG is not specific for bacteria ,28,29. CA clear limitation of our study is the limited number of patients in our study cohort resulting in a low number of probable IA cases. Due to a limited number of probable IA cases the CI of sensitivity was quite wide for all biomarkers making conclusions difficult. In contrast, results for specificity and NPV showed a relatively narrow CI confirming the high value of NPV. Future multicenter studies are necessary to finally solve this issue in pediatric patients. As this is a single-center study and only pediatric patients were included, our data represent another brick in the wall of evidences about the applicability of BDG, especially in respect to the pediatric field where data is quite scarce. Our results are in accordance with other published data describing low PPV for BDG screening if used as sole biomarker and therefore limited usefulness as screening tool . On the BDG can help to diagnose IA in pediatric SCT recipients when used in combination with PCR and as a sole biomarker in the surveillance phase of alloSCT patients after mucositis has resolved and intense antimicrobial treatments are not required any more. Specificity can be increased significantly by raising the cutoff and by the requirement for two consecutive positive BDG results.


Thyrotoxicosis is the state of thyroid hormone excess. But, in sub-Saharan Africa (SSA), specifically Northern Ethiopia, scientific evidence about thyrotoxicosis and its cardiac complications like dilated cardiomyopathy is limited. Therefore, this study aimed to explore the thyrotoxicosis presentation and management and identify factors associated with dilated cardiomyopathy in a tertiary hospital in Northern Ethiopia.p < 0.05.An institution-based cross-sectional study was conducted in Ayder Comprehensive Specialized Hospital from 2017 to 2018. Data from 200 thyrotoxicosis cases were collected using a structured questionnaire. After describing variables, logistic regression was conducted to identify independent predictors of dilated cardiomyopathy. Statistical significance was declared at p = 0.001) and tachycardia . All patients took propylthiouracil and 13.0 % of them experienced its side effects. Concerning β-blockers, propranolol was the most commonly (78.5 % of the cases) used drug followed by atenolol (15.0 %). Six patients underwent surgery.Mean age at presentation of thyrotoxicosis was 45 years and females accounted for 89 % of the cases. The most frequent etiology was multinodular toxic goiter (51.5 %). As well, the most common symptoms and signs were palpitation and goiter respectively. Thyroid storm occurred in 6 % of the cases. Out of 89 patients subjected to echocardiography, 35 (39.3 %) of them had dilated cardiomyopathy. And, the odds of dilated cardiomyopathy were higher in patients who had atrial fibrillation  and side effects of propylthiouracil. Therefore, we recommend that patients should get adequate health information about thyrotoxicosis and anti-thyroid drugs including their side effects. Additionally, hospitals and other concerned bodies should also avail of TSH tests and methimazole at an affordable cost. Furthermore, community awareness about iodized salt and iodine-rich foods should be enhanced. The term thyrotoxicosis refers to the hypermetabolic clinical syndrome resulting from serum elevations in thyroid hormone levels, specifically free thyroxine (T4) and/or triiodothyronine T3) , 2. The  , 2. TheThe usual manifestations of thyrotoxicosis are hyperactivity, irritability, dysphoria, heat intolerance, sweating, palpitation, fatigue, weight loss with increased appetite, diarrhea, polyuria, oligomenorrhea, and loss of libido , 5. AlsoThe thionamide drugs (propylthiouracil (PTU), methimazole, and Carbimazole), beta-blockers, radioactive iodine, and surgery are the treatment modalities used in the management of thyrotoxicosis and thyroid storm each with different success rates. Patients with thyroid storm will need further treatment with glucocorticoids and Lugol’s solution , 12–17.However, in sub-Saharan Africa (SSA), specifically Northern Ethiopia, scientific evidence about thyrotoxicosis and its complications like dilated cardiomyopathy is limited. Therefore, this study aims to explore the pattern, clinical manifestation, complication, and management of patients with thyrotoxicosis including factors associated with dilated cardiomyopathy in Ayder Comprehensive Specialized Hospital which is the only tertiary hospital in Tigrai, Northern Ethiopia. An institution-based cross-sectional study was conducted in Ayder Comprehensive Specialized Hospital (ACSH) which is located in Mekelle, Tigray regional state, Northern Ethiopia. This hospital provides both referral and non-referral services to a 9 million population in its catchment areas of the Tigray, Afar, and North-eastern parts of the Amhara Regional States including the Eritrean refugees. It offers a broad range of medical services to both inpatients and outpatients of all age groups . One of All patients with thyrotoxicosis who had follow-up at the endocrine clinic at ACSH and received anti-thyroid drugs.All patients with thyrotoxicosis who had follow-up at the endocrine clinic at ACSH during the study period and are on anti-thyroid drugs.Patients above 18 years of age with thyrotoxicosis who had follow-up at the endocrine referral clinic of ACSH were involved in this study. All patients included in this study were treated for thyrotoxicosis.All patients who visited the endocrinology clinic and received antithyroid drugs during the study period were included.The dependent variable is dilated cardiomyopathy.Socio-demographic factors , clinical features (symptoms and signs), comorbidity, level of T4, and frequency of TSH follow-up were considered as independent variables.Data were collected by two trained internal medicine residents through an interviewer-administered structured questionnaire. The questionnaire was designed to collect data related to sociodemographic characteristics, the clinical presentation of thyrotoxicosis, investigation results like biochemical thyroid hormone assessment and fine-needle aspiration cytology (FNAC), management modality like the type of antithyroid and type of beta-blocker used, electrocardiography results, and echocardiography findings.Our laboratory uses a VIDAS® thyroid panel with a single dose, ready-to-use reagents to perform thyroid-stimulating hormone (TSH), free T4, and free T3 assays. Blood is taken using a Gold tube. It will be allowed to clot for 30 min in a vertical position and will be centrifuged within 2 h. Then, the extracted serum will be analyzed based on the Enzyme-Linked Fluorescent Assay technique. However, our laboratory does not do Anti-TPO and Anti-Tg assays that are used to diagnose autoimmune disorders of the thyroid gland like Graves’ disease. As a result, the diagnosis of Graves’ disease was made clinically without an antibody test. Graves’ disease was considered If a patient has symptoms and signs of thyrotoxicosis, diffusely enlarged thyroid gland which has rubbery consistency without nodularity, and FNAC result that supports Graves’s disease. Our laboratory’s normal range of thyroid function tests is as follows.TSH: 0.5-5 Pmol/L.FT4: 10.6–19.4 Pmol/L.FT3: 4-8.3 Pmo/LTherefore, thyrotoxicosis will be diagnosed if a patient has symptoms of thyrotoxicosis and her/his.TSH is < 0.05 Pmol/L, FT4 is > 19.4 Pmol/L, and/or FT3 > 8.3 Pmol/L.p-value < 0.05.Data entry and analysis were performed using Statistical Package for Social Science (SPSS) version 23. Categorical variables were described using frequencies and percentages. Continuous variables were also described using an appropriate combination of measure of central tendency and measure of dispersion. Odds ratio with its 95 % confidence interval and p-value were calculated by running logistic regression to identify factors associated with dilated cardiomyopathy. Variables with a p-value less than 0.05 during bivariate analysis were selected for multivariable analysis. Statistical significance was declared at  Ethical clearance was obtained from the Ethical Review Committee of Mekelle University, College of health sciences with ethical clearance number ERC: 1408/2018. After explaining the purpose of the study, informed consent was obtained from the participants. Confidentiality of information was also assured by removing medical record numbers and replacing them with codes.A total of 200 patients with thyrotoxicosis who fulfilled the inclusion criteria were included in this study. Those accounted for 82.3 % (200/243) of patients with a thyroid disorder who visited the endocrine clinic.There were 178 (89 %) females with a male to female ratio of 1:8.1. The age of patients ranged from 18 to 77 years. The mean age at presentation was 45.2 ± 14.3 years. More than half (58.5 %) of patients were unable to read and write. Toxic multinodular goiter accounted for 51.5 % of the cases  patients were subjected to fine-needle aspiration cytology (FNAC). The most common FNAC finding was colloid Goiter 104 (80.6 %) followed by atypia of undetermined significance (AUS) 8 (6.2 %) and autoimmune thyroid disease like Graves’ disease 13 (10.1 %)  of patients followed by fatigue 138 (69.0 %) and heat intolerance 118 (59.0 %) while the most common sign was anterior neck swelling 186 (93.0 %) followed by tachycardia 86 (43.0 %), exophthalmos 35 (17.5 %), and tremor 35 (17.5 %)  patients had thyroid storm. The most common precipitating factor was infection (41.7 %) followed by drug discontinuation (8.3 %) and diabetic ketoacidosis (DKA) (8.3 %). The rest had no identified precipitating factor.Echocardiography was done for 89 (44.5 %) patients. Dilated cardiomyopathy 39.3 %), normal (22.5 %), Pulmonary hypertension (19.1 %), and degenerative valvular heart disease (10.1 %) were the most common findings. In addition, the most common arrhythmiaS detected on electrocardiography (ECG) were sinus tachycardia (47.7 %) and atrial fibrillation (28.9 %)  and tachycardia  than their respective counterparts , only atrial fibrillation and tachycardia were found to have a significant association with dilated cardiomyopathy. Accordingly, the odds of dilated cardiomyopathy were higher in patients who had atrial fibrillation . The mean dose of PTU given was 90 ± 20 mg, three times a day. Usually, patients are started on a 100 mg dose of PTU at a frequency of three times a day. Propranolol was the most commonly (78.5 % of the cases) used beta-blocker followed by atenolol (15.0 %) and metoprolol (2.5 %). The rest did not take any beta-blocker due to normal Free T4 level and/or bradycardia. Six (3 %) of the patients underwent surgery. Because of the unavailability of radioiodine therapy, none of the participants received radioiodine treatment. Health information was offered to 48 % of patients. Regarding the TSH follow-up interval, 56 % of patients had a follow-up interval greater than 3 months. After treatment, 51.5 % of the patients had their symptoms resolved within 3 months.This study found nearly all patients with thyrotoxicosis are females and in the age of mid-forties. The most predominant cause of thyrotoxicosis was a multinodular toxic goiter. The most common symptoms and most common sign were palpitation and palpable thyroid, respectively. The most common arrhythmias detected on electrocardiography was sinus tachycardia and atrial fibrillation. Among those who had an echocardiographic examination, 40 % of them were diagnosed to have dilated cardiomyopathy. Regarding management, all patients received PTU as an anti-thyroid drug and almost all (96 %) of them also took beta-blockers.In this study, the majority (89 %) of patients were females. The mean age at presentation was 45 years and those who were above 40 years accounted for 58.5 % of the cases. This is similar to a study done in Addis Ababa, in which 90 % of patients were females and patients above 40 years of age accounted for 59 % of the cases with a mean age of 43.1 years .In this study, the leading cause of thyrotoxicosis was a multinodular toxic goiter. This finding was similar to a study done in the North-Western part of Ethiopia, which reported 54.9 % of thyrotoxicosis cases due to toxic multinodular goiter . This isThe most common symptom of patients with thyrotoxicosis was palpitation which was present in 85.5 % of the patients followed by fatigue (69.0 %) and heat intolerance (59.0 %). Similarly, Addis Ababa’s study revealed palpitation and heat intolerance as the most common symptoms of patients with thyrotoxicosis . As wellThe most common sign in our study was palpable thyroid (93.0 %) followed by tachycardia (43.0 %), exophthalmos (17.5 %), and tremor (17.5 %). According to Addis Ababa’s study, palpable thyroid (goiter) was also the commonest sign of thyrotoxicosis which was detected in 99 % of the cases. Congruently, the most common sign in Nigerian study was palpable thyroid and found in 97 % of the cases . On contThe most common arrhythmia detected on electrocardiography (ECG) was sinus tachycardia followed by atrial fibrillation and this finding goes parallel with several case studies on thyrotoxic cardiomyopathy –29. ThisIn our study, PTU was the only thionamide used to treat thyrotoxicosis. This is similar to the study done in Addis Ababa, Ethiopia where almost all of the patients received PTU . This fiThe interval of TSH follow up was > 3 months in 112(56 %) of the patients and it was against the recommendations by the American Association of Clinical Endocrinologists and the American Thyroid Association which recommend TSH determination every 4 to 6 weeks for all patients till normalization of TSH and T4 , 37. TheIt is the first study to assess the pattern, clinical presentation, and management of thyrotoxicosis in Tigray and can serve as a baseline for future studies related to the current topic. It has also tried to assess many aspects of thyrotoxicosis by collecting primary data using a pre-tested and comprehensive questionnaire.Echocardiography, electrocardiography, and cytology were not done for all patients. As a result, this study may not show the true prevalence of cytologic types and cardiac complications in patients with thyrotoxicosis. Due to infrequent TSH follow-up, this study did not address time to normalization of free T4 and TSH.This study has shown that most of the patients with thyrotoxicosis were females and more than half of the thyrotoxicosis cases happened in patients with multinodular goiter. The most common symptom and most common sign of thyrotoxicosis were palpitation and goiter, respectively. All patients took PTU as an anti-thyroid drug. Dilated cardiomyopathy was the commonest abnormal echocardiography finding in patients with thyrotoxicosis and it was significantly associated with atrial fibrillation and tachycardia. The prevalence of thyrocardiac disease was higher even taking all participants as denominator which may be attributed to the longstanding thyrotoxicosis. Overall, the findings of this study complement and strengthen studies done in another part of Ethiopia and other African countries.Patients should be given adequate health information about thyrotoxicosis and anti-thyroid drugs including their side effects. Hospitals and other concerned bodies should also avail of TSH tests and methimazole at an affordable cost. In addition, community awareness about iodized salt and iodine-rich foods should be enhanced.


To investigate whether the administration of hydrogen/oxygen mixture was superior to oxygen in improving symptoms in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD).This prospective, randomized, double-blind, controlled clinical trial in 10 centres enrolled patient with AECOPD and a Breathlessness, Cough, and Sputum Scale (BCSS) score of at least 6 points. Eligible patients were randomly assigned (in a 1:1 ratio) to receive either hydrogen/oxygen mixture or oxygen therapy. Primary endpoint was the change from baseline in BCSS score at day 7. Adverse events (AEs) were recorded to evaluate safety.p < 0.001). Changes in pulmonary function, arterial blood gas and noninvasive oxygen saturation did not differ significantly between groups as well as other endpoints. AEs were reported in 34 (63.0%) patients in Hydrogen/oxygen group and 42 (77.8%) in Oxygen group. No death and equipment defects were reported during study period.Change of BCSS score in Hydrogen/oxygen group was larger than that in Oxygen group . Similar results were observed in other time points from day 2 through day 6. There was a significant reduction of Cough Assessment Test score in Hydrogen/oxygen group compared to control (− 11.00 vs. − 6.00, Trial registration: Name of the registry: U.S National Library of Medicine Clinical Trials; Trial registration number: NCT04000451; Date of registration: June 27, 2019-Retrospectively registered; URL of trial registry record: https://www.clinicaltrials.gov/ct2/show/study/NCT04000451?term=04000451&draw=2&rank=1.The trial demonstrated that hydrogen/oxygen therapy is superior to oxygen therapy in patient with AECOPD with acceptable safety and tolerability profile. The online version contains supplementary material available at 10.1186/s12931-021-01740-w. Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by chronically poor airflow, which now has been a global disease with an estimated 63 million people worldwide . UnfortuTherapeutic medical gas as pharmaceutical gaseous molecules are emerging as a novel and innovative therapeutic tool for COPD, including oxygen, nitrous oxide and helium , 13. A pHere, we proposed a hypothesis that hydrogen/oxygen mixture may be superior to oxygen therapy in the improving respiratory symptoms for patient with AECOPD. Thus, the primary purpose of present study was to compare the efficacy of hydrogen/oxygen mixture produced by this novel Hydrogen/Oxygen Generator and oxygen alone in patients with AECOPD. The secondary objective was to assess its safety and tolerability.This was a prospective, multicentre, double-blind, randomized, controlled trial (registration number: NCT04000451) comparing the hydrogen/oxygen mixture therapy and oxygen alone therapy in patients with AECOPD. Patients were recruited from 10 centres in China. The trial was approved by the local ethics committee of all participating centres. All recruited patients provided written informed consent before participating in the trial. The study was conducted in accordance with Declaration of Helsinki.1) less than 80% and FEV1/forced vital capacity (FVC) less than 70% in pulmonary function test. The AECOPD was defined as an increase in or new onset of at least two major COPD symptoms , or one major COPD symptom plus at least one minor COPD symptom  during at least 2 days consecutively and requiring any change of pharmacological treatment , additional oxygen inhalation, additional NIV, and instrument performance evaluation. The CAT score, pulmonary function, and arterial blood gas were measured at baseline and day 8. The SpO2, additional oxygen inhalation and NIV, instrument performance evaluation were recorded from baseline to each visit time point. Safety were assessed by the incidence of adverse events (AEs) or death, the changes of physical examinations, vital signs, laboratory data, and the incidence of equipment defects . Any AEs were recorded during trial period. Severity of AEs was classified using the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03 ) or median (interquartile range [IQR]) as appropriate. Comparison of mean change in BCSS score and SpOOf the 142 subjects screened, 108 were eligible and randomly assigned to treatment  for the Hydrogen/oxygen group and 6.4 days (range 1–7 days) for the Oxygen group, with no significant between-group differences. The mean treatment period ranged from 364.9 to 375.4 min/day in Hydrogen/oxygen group and 366.0–378.5 min/day in Oxygen group, without significant between-group differences. The compliance between two groups were generally balanced.p < 0.0001), and the time by group interaction effect was also significant (p < 0.0001). But, no plateau period was observed in the tendency of symptom improvement during the entire study .For the primary endpoint in FAS population, the change from baseline in BCSS score was − 5.3 (range − 10 to − 1) in the Hydrogen/oxygen group and − 2.4 (range − 6 to 0) in Oxygen group ] compared to the control [− 6.00 ] in FAS population  or group (p = 0.805) on changes from baseline in SpO2. However, there were time by group interactions with respect to the changes from baseline in SpO2 (p < 0.0001). Similar results were found in PPS population.The changes from baseline in SpOp = 0.309), FEV1 (p = 0.769) and FEV1/FVC (p = 0.536). In addition, the arterial blood gas was measured at day 8 after initial treatment to evaluate the arterial oxygenation. The patients in both treatment groups did not differ in terms of the arterial oxygenation parameters (Table p = 0.700), PaO2 (p = 0.461), PaCO2 (p = 0.160), and HCO3− (p = 0.136). The consistent results of pulmonary function and arterial oxygenation parameters were observed in the PPS population . The common AEs in two groups included upper respiratory infection (Hydrogen/oxygen: 5.6% vs. Oxygen: 5.6%) and hypokalemia (3.7% vs. 5.6%). The majority of AEs in two groups were mild or moderate in severity (Grade 1 or 2), and only a small percentage of patients experienced severe AEs . In the Hydrogen/oxygen group, device-related AEs occurred in 2 patients (3 events), including dizziness (mild), nasal mucosa injury (mild), and aggravated wheezing (moderate). In the Oxygen group, device-related AEs occurred in 3 patients (3 events), including agrypnia (moderate), abdominal distention (mild) and aggravated wheezing (moderate). In addition, two groups respectively reported 2 cases of AEs leading to withdrawal , without statistic difference. All AEs were resolved with treatment interruption and symptomatic treatment. No notable changes were observed in physical examinations, vital signs, liver and kidney functions. No death and equipment defects were reported during the study period.Summary of adverse events was shown in Table To our knowledge, this is the first multicenter, randomized, controlled trial investigating the efficacy of hydrogen/oxygen mixture in patients with AECOPD. Our direct comparison with the oxygen therapy which used widely in AECOPD has established the better efficacy of hydrogen/oxygen therapy in the improvement of respiratory function for the treatment of AECOPD. Meanwhile, the safety analysis demonstrated that hydrogen/oxygen therapy has acceptable tolerability.p < 0.001, Additional file Long-term home oxygen therapy has been proved to improve survival in patients with COPD , 26. HowIn addition, CAT as a cough-specific quality-of-life questionnaire was used to assessing and monitoring cough in patients . The bet2O2) in the airways and that oxidant production increases further during exacerbations [2O2 could convert to very reactive ·OH in the presence of catalytically active metals [2·− and H2O2, but significantly decreased levels of ·OH, this means that hydrogen is mild enough neither to disturb metabolic redox reactions nor to affect ROS that function in cellular signaling [The most striking finding of present study was the superiority of hydrogen/oxygen therapy in patient with AECOPD, accompanied by acceptable safety and tolerability profile. The molecular properties of hydrogen might explain why hydrogen/oxygen mixture is superior to oxygen alone in the therapy of AECOPD. We speculated that its superiority in the improvements of key symptoms of AECOPD may be attributable to its physical properties, anti-inflammatory, antioxidant and rapid cellular diffusion features . Primarirbations . H2O2 coe metals , while ·e metals . Previouignaling . Hydrogeignaling , 43. Aniignaling , 45. Colignaling , which mThe treatment-related AEs are clearly an area of main concern when considering the development of a new therapeutic regimen. In this trial, we found that both hydrogen/oxygen and oxygen therapy regimens present acceptable safety and tolerability profile. Although approximately more than 60% patients experienced AEs in two groups, the majority of AEs were mild or moderate in severity. The most common AEs from hydrogen/oxygen therapy were upper respiratory infection and hypokalemia, which were also resulted from oxygen alone therapy. Similarly, most of these AEs were known and have also been reported in study on helium/oxygen therapy . In factAlthough this study demonstrated important findings, it also has several limitations. First, the lack of a control design with helium/oxygen for this trial is a potential criticism. In fact, the use of oxygen as control was considered because the helium-producing devices have not been used in AECOPD therapy in China. Meanwhile, the voice alteration caused by helium inhalation would break the double-blind design. Thus, given that the definite effect of oxygen in COPD , 51, a mIn the first trial to date on hydrogen/oxygen therapy for AECOPD treatment, compared with oxygen, the inhalation of hydrogen/oxygen mixture resulted in a more significant improvement of AECOPD symptoms, including breathlessness, cough and sputum, with acceptable safety and tolerability profile. The findings suggested that the hydrogen/oxygen therapy was feasible and well tolerated for patients with AECOPD, having potential to be used as an alternative emergency management for AECOPD.Additional file 1:Table S1. Complete eligibility criteria. Table S2. The included patients in each centre.Additional file 2: Figure S1. Forest plot of the multivariate analysis. Change from baseline in BCSS score >1 was identified as substantial improvement and ≤1 as non-substantial improvement, which were used as outcomes in logistics regression analysis.


OBC was significantly higher among patients with an SDQ score of <11 (nondysphagic) (p=0.01), and the SDQ score was lower in patients with higher OBC requiring professional oral care (p=0.03). However, OBC was also negatively correlated with LEDD . These results indicated low self-awareness of dysphagia among the participants and an association between dysphagia and PD progression. Moreover, the oral environment could have deteriorated with swallowing dysfunction. Patients and clinicians should be aware that higher LEDD can increase xerostomia and associated deficits in oral health.This study investigated the impact of the severity and treatment of Parkinson's disease (PD) on the swallowing ability and oral environment of patients. Swallowing dysfunction increases the aspiration risk and may lead to poor oral health among patients with PD. We investigated the influences of PD progression and drug treatment on the swallowing ability and oral environment using simple noninvasive screening measurements. We recruited 87 patients with PD . The PD condition was assessed in each patient using the unified Parkinson's disease rating scale (UPDRS) part III, diet type and oropharyngeal function using the swallowing disturbances questionnaire (SDQ), maximum bite force (MBF), tongue pressure (TP), and oral bacterial count (OBC). Levodopa equivalent daily dose (LEDD) was also calculated for 56 participants. Based on an SDQ score of ≥11, 29.5% of patients were dysphagic, but almost all were still on a regular diet. The SDQ score was positively correlated with disease duration ( Swallowing function deteriorates with the progression of Parkinson's disease (PD), necessitating careful adjustment of diet and monitoring of oral health. Swallowing dysfunction (dysphagia) among patients with PD is often evaluated using video fluoroscopic swallowing study (VFSS) or fiberoptic endoscopic evaluation of swallowing (FEES), and VFSS in particular provides detailed information on the oral and pharyngeal stages of swallowing dysfunction , 2. HoweWe recruited 87 patients with PD . All patients had been diagnosed with PD and received treatment from medical institutions, excluding eight patients who had missing data in the examination. Thirty-one patients with PD  who attended the meeting of the Japan PD Association of Fukuoka City on 27 May 2017 were evaluated using UPDRS part III. They completed the SDQ and underwent measurements of occlusal condition, bite force (BF), tongue pressure (TP), and oral bacterial count (OBC) . Next, 5UPDRS part III, SDQ, BF, TP, and OBC results were collected from a total of 87 participants and LEDD from 56 participants. Physical examinations were performed for all participants, and body weight, height, and body mass index (BMI) were recorded. The regular diet type was classified into three of seven levels based on the functional oral intake scale (FOIS) [Medical history including disease duration was recorded and UPDRS part III was administered by two neurologists, generally in the “on state” after each dose of medication, to assess disease severity. Usual medications for PD were checked for the next 56 participants, and LEDD [The SDQ is a self-reported questionnaire comprising 15 items. Fourteen items are rated from 0 (no disability) to 3 (severe disability), and the final item is a “yes/no” question in which “yes” is scored 2.5 points and “no” is scored 0.5 points. The reliability and validity of the SDQ Japanese version (SDQ-J) have been confirmed . The parThe Eichner index (EI) was used as an evaluation of occlusal condition based on existing natural tooth contacts between the maxilla and mandible in the bilateral premolar and molar regions. BF was recorded using the Bite Force Analyzing System  . This meMaximum TP (MTP) was measured using a probe with a small air balloon pressurized to 19.6 kPa  . The par5; Level 2, 105–106; Level 3, 106–106.5; Level 4, 106.5–107; Level 5, 107–107.5; Level 6, 107.5–108; and Level 7, >108. The participants were divided into two groups: those > Level 5, indicative of the need for professional oral care, and those ≤ Level 5 [The number of oral bacteria, measured as OBC, was determined using a bacteria detection apparatus  2 h after lunch and regular oral care . A steri Level 5 .t-tests. All statistical analyses were performed using SPSS version 13.0 J for Windows . p < 0.05 (two-tailed) was considered statistically significant for all tests.The relationships among BF, TP, OBC, and BMI were evaluated using Pearson's correlation, and other relationships were evaluated using Spearman's rank-order correlation. Mean values of subgroups with SDQ scores of <11 or ≥11 and subgroups with OBC of >Level 5 or ≤ Level 5 were compared using independent samples rho = 0.497, p=0.016). However, the SDQ score was negatively correlated with OBC . Both BF and TP were lower in the patients than in healthy volunteers  in previous studies [r = 0.540, p=0.002) and between BF and EI  but not between TP and EI .The mean UPDRS part III score, BMI, FOIS, SDQ, BF, TP, and OBC from a total of 87 participants are summarized in  studies , 13. The2, both of which are clinical predictors of dysphagia [p=0.01; p=0.03; Among the next 56 participants, 13.8% of participants were at Hoehn and Yahr stages 4/5, and 16.9% had a BMI of <20 kg/mysphagia . Howeverrho = 0.307, p=0.007; rho = 0.228, p=0.047). Notably, OBC was not correlated with disease duration or UPDRS part III score but was negatively correlated with SDQ score  and LEDD , suggesting an effect of medication on OBC and the oral environment.As in the first 31 participants, there were significant positive correlations between swallowing dysfunction, as assessed using SDQ and UPDRS part III  FEES could provide more definitive conclusions, the measures used are relatively simple to acquire and provide valuable information for clinical dentistry. Second, we have no definitive proof that LEDD leads to xerostomia as suggested by the decreased OBC. Moreover, many participants took various medications in addition to antiparkinsonian drugs such as antihypertensive, anticoagulation, gastric coating, constipation, and anxiolytic drugs which can cause xerostomia. The information of prescribed drugs was collected based on self-enumeration, and it was difficult to confirm the accurate dosage. If we subclassify the prescribed drugs, it will be hard to perform statistical analysis due to the reduced number. Thus, quantitative measurements of salivation should be conducted to confirm or refute this notion.Although 29.5% of the participants were defined as dysphagic, most were still on normal or only slightly adjusted diets. These results indicate low self-awareness of dysphagia and underscore the need for more vigilant monitoring of diet type based on objective assessments to reduce the incidence of aspiration. Moreover, the reduction of OBC with greater SDQ score and increasing LEDD suggest that both the patients with PD and neurologists should be informed of possible xerostomia induced by dopaminergic medication. Screening for xerostomia and oral care and moisture are recommended for patients with PD suspected to have dysphagia based on SDQ scores of ≥11 and treated with high doses of levodopa.These results indicate low self-awareness of dysphagia among patients with PD and highlight the importance of careful diet-type monitoring and adjustment to prevent aspiration. Moreover, patients and clinicians should be alert for deterioration in oral health with swallowing dysfunction and be aware that higher dopaminergic medication dose (LEDD) may impair the oral environment by inducing xerostomia. Screening for xerostomia and oral care and moisture are recommended for patients with PD with dysphagia who receive high doses of levodopa.


Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti–mouse CD49b-PE (phycoerythrin) antibody. Platelet–endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [ Platelets are activated in a distinct manner during asthma compared with hemostasis, leading to migration into inflamed lungs rather than to platelet-aggregatory events. Understanding mechanisms relating to platelet function might be exploited for novel therapies and will inform clinicians of the likely effectiveness of current antiplatelet drugs.Platelets are traditionally considered as the main effector cells in hemostasis and thrombosis. However, there is accumulating evidence that platelets can participate and respond to immune and inflammatory signals , as wellin vitro and that pulmonary platelet accumulation and activation occurs directly in response to allergen exposure via IgE in a murine model of allergic airway inflammation  enzyme allergen from house dust mite (HDM) extract via the nasal route, platelet recruitment to the lungs was analyzed in fixed tissue, and the characteristics of platelet adhesion to the vasculature using a cremaster-muscle preparation with real-time intravital video microscopy were studied after local DerP1 challenge and after the administration of antagonists to CCR3, CCR4, and CXCR4 chemokine receptors. Parts of the data represented in this manuscript are in the King's College London institutional repository for the Ph.D. thesis of Dr Sajeel Shah , some of whom were from a previously reported study  were recruited and underwent fiberoptic bronchoscopy. Tumor-free lung tissues were also taken from subjects undergoing lung resection for bronchial carcinoma (REC: 08/H0407/1), processed and sectioned by Dr. Amanda Tatler and Professor Alan Knox (University of Nottingham), and labeled as controls without asthma. All lung tissue was formalin fixed and paraffin-wax embedded before sectioning. Postmortem human lung tissue from patients without asthma, patients with nonfatal asthma, and patients with fatal asthma from the Airway Disease Biobank  was also used. The demographic characteristics of patients are shown in The bronchial-biopsy component of the study was approved by the national research ethics committee at Guy’s and St. Thomas’ Hospitals (Research Ethics Committee: REC 08/H0804/67 and 10/H0807/99). Eight subjects without asthma ; 12 subjects with steroid-naive, atopic, mild asthma .Animal experiments were approved and performed under the Animals (Scientific Procedures) Act of 1986, amended UK regulations of 2012, and the Animal Welfare and Ethics Committee at King’s College London. Male BALB/c inbred mice (20–25 g) between 6 and 10 weeks of age were procured from Covance Laboratories. HDM extract containing the DerP1 allergen at 54 μg of DerP1 per 1 mg of total protein  was used for sensitization and challenge (see data supplement).To achieve visualization of platelet interactions with the vasculature, a cremaster-muscle preparation was chosen as a stable method for real-time microscopy. Hamster anti–mouse CD49b-PE (phycoerythrin)–conjugated antibody (BD Biosciences), which has been used for intravascular platelet recording in previously published work , was injsee data supplement).Sham-sensitized and HDM-sensitized mice were administered saline or HDM (100 μg/100 μl) subcutaneously (s.c.) to the scrotum on Day 13. The cremaster muscle was then dissected, and video-capture microscopy was conducted 24 hours after HDM administration before tissue was fixed with 3.7% paraformaldehyde (PFA) for processing and histological staining to detect leukocytes, eosinophils , and plain vivo experiments. SB328437, C-021, and AMD3100 are highly selective antagonists of CCR3, CCR4, and CXCR4 receptors, respectively . Probability values less than 0.05 were considered to indicate statistical significance.Data are presented as the mean ± SEM as indicated, with data having been tested for normal distribution. Raw data values were compared using a Student’s + platelets in the submucosa  and within the epithelial layer , which appeared mostly as singular events compared with their appearance in nonasthmatic tissue , patients with nonfatal asthma (n = 10), and patients with fatal asthma (n = 9) were also stained using immunohistochemistry to test for the presence of platelets. In sections taken from patients who died of asthma, platelets were observed in the alveolar walls and in the alveolar space as single, nonaggregated entities (2 vs. 48.8 ± 7.9 platelets/mm2) . The presence of airway-wall platelets occurred with insufficient frequency in lung sections to determine the localization of platelets to specific airway compartments and perform statistical analyses. However, in concordance with studies of biopsy specimens from patients with mild, atopic asthma, platelets were distributed around the airway walls in histological sections from patients with nonfatal asthma and from patients who had died of asthma but were absent in sections from patients without asthma (A second distinct set of lung biopsy sections taken postmortem from patients without asthma (entities . Plateleets/mm2) . Howevert asthma . Single t asthma . There w4 eosinophils/ml vs. 12.6 ± 8.0 × 104 eosinophils/ml, P < 0.05; 0 ± 0 neutrophils/ml vs. 5.0 ± 2.7 neutrophils/ml, P < 0.05)   . Further < 0.05) , where elatelets  also rev< 0.001) . These pP < 0.001; P < 0.001; P < 0.001; P < 0.01; To study the process of platelet recruitment and migration to allergic inflammatory sites, an allergic model of the mouse cremaster muscle was developed using intravital microscopy. This was necessary to provide tissue for stable recording of platelet-sized events because of the inherent instability of the lungs. Naive, sham-sensitized, and DerP1-sensitized mice (as above) were administered 100 μg of HDM extract containing DerP1 or saline s.c. to the scrotum. The cremaster muscle was exposed, and videos of postcapillary venules were taken for intravital analysis and histological morphometry after 24 hours. Leukocyte migration to the extravascular compartment of the cremaster muscle was used to gauge the severity of the allergic inflammatory response. A comparison between acute saline and DerP1 administration (administered s.c.) in sham-sensitized mice revealed significantly elevated numbers of extravascular leukocytes in the cremaster muscle of DerP1-challenged mice , indicaP < 0.001; P < 0.001; P < 0.001, P < 0.05; P < 0.05; P < 0.05; P < 0.0001; P < 0.05; P < 0.01; P < 0.05; in vitro  to express eotaxin. We show that eotaxin expression is significantly increased in cultured ASM derived from patients with moderate asthma compared with that derived from patients without asthma, and this appeared to be associated with the severity of asthmatic disease in the tissues biopsied and with which ASM bundles were dissected  necessary to develop in vivo models of allergen sensitization and inflammation via inhaled administration  without the use of sophisticated technology and adaptions , 63 thatTo conclude, we have demonstrated the presence of single, extravascular platelets in the lung tissue of subjects with asthma and in the lungs of allergic mice after allergen challenge. At least in mice, this extravascular recruitment in the absence of interactions with leukocytes appears to be dependent on the activation of CCR3 receptors, implicating eotaxin in this process. Given that it is now appreciated that platelets are important for allergen sensitization, leukocyte recruitment, bronchospasm, and airway-wall remodeling in animal models of allergic inflammation, the presence of activated platelets in lung tissue may contribute to these activities and further confirms the likely importance of this cell type in this disease beyond the familiar requirement of platelets in hemostasis . Underst


Drosophila melanogaster (D.m.) is a suitable model to analyze hypoxia; however, little is known about the impacts of hypoxia and especially of the subsequent reperfusion injury on the behavior and survival of D.m. To address this knowledge gap, we subjected two wild-type D.m. strains (Canton-S and Oregon-R) to severe hypoxia (<0.3% O2) under standardized environmental conditions in a well-constructed hypoxia chamber. During posthypoxic reperfusion (21% O2), we assessed fly activity (evoked and spontaneous) and analyzed molecular characteristics  production, and metabolic activity) at various timepoints during reperfusion. First, we established standard conditions to induce hypoxia in D.m. to guarantee stable and reproducible experiments. Exposure to severe hypoxia under defined conditions impaired the climbing ability and reduced the overall activity of both D.m. strains. Furthermore, a majority of the flies died during the early reperfusion phase (up to 24 h). Interestingly, the flies that died early exhibited elevated activity before death compared to that of the flies that survived the entire reperfusion period. Additionally, we detected increases in ROS and stress marker  levels as well as reductions in metabolic activity in the reperfusion phase. Finally, we found that changes in environmental conditions impacted the mortality rate. In particular, decreasing the temperature during hypoxia or the reperfusion phase displayed a protective effect. In conclusion, our data suggest that reperfusion-dependent death might be associated with elevated temperatures, predeath activity, and oxidative stress.Hypoxia is an underlying pathophysiological condition of a variety of devastating diseases, including acute ischemic stroke (AIS). We are faced with limited therapeutic options for AIS patients, and even after successful restoration of cerebral blood flow, the poststroke mortality is still high. More basic research is needed to explain mortality after reperfusion and to develop adjunct neuroprotective therapies.  A new fruit fly model of reperfusion injury, the tissue damage commonly seen in stroke that occurs when an organ is starved of oxygen and then exposed to oxygen again, opens new possibilities for understanding stroke pathology. Pardes Habib from Aachen University, Germany, and colleagues subjected flies to extremely low oxygen levels for up to six hours before returning them to a normal oxygen environment. Most flies died after re-exposure to oxygen, but not before showing a ‘last gasp’ burst of physical activity compared to individuals that survived the treatment. The return to normal oxygen levels led to an increase in signs of cellular stress and reduced metabolic activity, especially among those flies kept at warmer temperatures. The findings lay the groundwork for better understanding of reperfusion injury and how to treat it. The latter is one of the main causes of death and disability in adults worldwide2. Although recent advances in reperfusion modalities after stroke, especially in endovascular stroke treatment (EST), have significantly improved clinical outcomes, only a small number of stroke patients actually benefit from these therapies3. Despite the high effectiveness of EST, there is a high mortality rate (29–67 %), and many stroke patients remain severely disabled; functional independence is achieved in fewer than 50% of cases after the initial 90 days6. Postischemic reperfusion injury is considered a key mechanism for the devastating clinical outcomes. One of the main causes of postischemic reperfusion injury is free radical damage due to oxidative stress7. Thus, there is a pressing need for more basic research to understand and develop effective neuroprotective/neurorestorative strategies addressing pathomechanisms such as oxidative stress during the reperfusion period after ischemic stroke.Hypoxia (low oxygen) plays crucial roles in the etiology and pathophysiology of several diseases, including obstructive sleep apnea, chronic obstructive pulmonary disease (COPD), pulmonary hypertension, heart attack (cardiac arrest/ischemia), and cerebral ischemiaDrosophila melanogaster (D.m.) is a suitable model for analysis of hypoxia-related detrimental effects and elucidation of potential protective agents and biological mechanisms for stroke treatment. The major advantages of D.m. are its short life cycle, its large numbers of progeny, its limited need for resources, and the remarkably high degree of conservation between the D.m. genome and the human genome with regard to many biological processes. Approximately 75% of human disease-associated genes and the entire hypoxia-induced cascade are conserved in flies8. The availability of various mutants, transgenic strains, and genetic manipulation techniques facilitates the study of neurodegenerative diseases, including stroke, and translation of the discoveries into humans10.Drosophila. Moreover, there is a lack of established standard operating procedures for the induction of hypoxia in D.m. to guarantee valid and reproducible experimental conditions.However, little is known about the impact of hypoxia and especially that of the subsequent reperfusion injury on the mortality and behavior of D.m. but hypoxia experiments with flies were conducted only in 88 publications (Supplementary Table Our literature search (on NCBI and Web of Science) revealed 154 publications addressing hypoxia in 11.However, some of the aforementioned environmental conditions are known to have considerable influences on the survival of stroke patients. It is widely accepted, for instance, that therapeutic hypothermia has cytoprotective and neuroprotective effectsD.m. strains (Canton-S and Oregon-R) to different durations of hypoxia followed by reperfusion for various durations and performed behavioral assessments utilizing a computer-assisted DAM system; a climbing assay (negative geotaxis); and molecular analyses of oxidative stress marker gene and protein expression, ROS production, and metabolic activity. To clarify whether the observed death occurs predominantly during hypoxia or in the reperfusion phase and to evaluate the influences of environmental conditions, we varied the latter individually or in combination and detected the resulting mortality.Hence, we aimed to establish a standardized hypoxia and reperfusion protocol utilizing a self-constructed hypoxia chamber with the ability to measure and control environmental conditions such as oxygen levels, temperature, humidity, and pressure. To assess the impacts of hypoxia and reperfusion, we subjected the two commonly used wild-type Drosophila melanogaster strains. Furthermore, the majority of the flies did not die directly from hypoxia but rather died within 24 h of reperfusion. Moreover, the latter also displayed a higher activity rate than the flies that survived the entire observation period (120 h) after hypoxia. Low humidity and high pressure led to an increased mortality rate, whereas decreasing the temperature during hypoxia and the reperfusion phase revealed a protective effect. Additionally, we provide evidence that reperfusion-dependent death might be associated with increased oxidative stress and decreased metabolic activity.We were able to demonstrate that severe hypoxia impairs the climbing ability and activity of both D.m. wild-type strains Canton-S (#64349) and Oregon-R (#5) were obtained from the Bloomington Drosophila Stock Center . The w;;ubi-EGFP-ODD/ubi-mRFP-nls stock was a gift from Professor Dr. Stefan Luschnig . All flies were raised and maintained in plastic vials containing standard cornmeal food at 25 °C under a 12 h/12 h light/dark cycle. Every five days, the flies were transferred to new vials with fresh food.The 2) in a self-constructed hypoxia chamber consisting of a heated water chamber, a gas-flow regulator, a humidifier and an airtight acrylic glass compartment with a stainless-steel bottom plate and lid , humidity, temperature (Habor Thermo-Hygrometer) and pressure (Fluke 700RG06 100 PSIG pressure gauge).Hypoxia was induced with nitrogen (N2). The mortality rate was assessed daily for the following 5 days under normoxic conditions. The temperature, pressure, and humidity inside the chamber were recorded regularly during this time .To study the impact of hypoxia on survival, male Canton-S and Oregon-R flies at 1 to 5 days of age were subjected to 1 to 6 h of severe hypoxia . Each fly was placed individually into a polycarbonate vial containing food (2% agar and 4% sucrose). Activity was monitored by recording the light beam interruptions caused by the fly passing through the light beam in the center of each vial. The flies were kept in the DAM system for a total of 5 days, during which the numbers of interruptions of the light beams were recorded every hour. The experiment was conducted 3 times in total, including 180 flies for each genotype and treatment.To evaluate activity after hypoxia, wild-type flies were transferred into a 12 with slight modifications. Climbing ability was assessed 3, 6, 24, 48, 72, 96, and 120 h after hypoxia, and each vial contained a group of 20 male flies.A negative geotaxis assay was conducted as described previouslyIn 4 individual experiments, the negative geotaxis performance was evaluated for a total of 240 flies for each genotype. The impact of hypoxia on negative geotaxis was assessed using only living flies. The dead flies were removed, and the results are therefore presented as mortality-adjusted percentages.TM Protein Assay Kit . Twenty micrograms of protein was separated via SDS-PAGE before being blotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBS-T, after which they were incubated with the primary antibodies overnight at 4 °C. Incubation with the corresponding HRP-conjugated secondary antibody was carried out for 2 h at room temperature and followed by signal detection via chemiluminescence . The following antibodies were used: mouse anti-GFP  and mouse anti-Drosophila CSP-2 .Flies were snap-frozen in liquid nitrogen, and their heads were collected and homogenized in RIPA buffer (10 µl/head) using a Speedmill P12 . The total protein concentration was measured with a DCTM , total RNA was prepared by phenol-chloroform extraction as previously described13. Complementary DNA was synthesized using an iScriptTM cDNA Synthesis Kit  and random hexanucleotide primers  using 1 µg of total RNA according to the manufacturer´s protocol.Gene expression analyses were performed with tissue from fly heads after hypoxia or the corresponding normoxia. After homogenizing the snap-frozen fly heads in PeqGold2O  served as the no-template control (NTC). RT-qPCR analysis was performed using a MyIQTM RT-qPCR detection system . The expression levels of the target genes and two housekeeping genes, Actin-5C (Act5c) and Elongation factor 1-alpha 2 , were measured as cycle threshold (Ct) values, and relative quantification was performed by the ΔΔCt method using qbaseTM + software . The data are expressed as the expression levels of the target genes relative to those of Act5c and eEF1alpha2. Forward (fwd) and reverse (rev) primers were used for the following genes (5′→3′): Act5c , eEF1alpha2 , Similar (Sima) , Tango , Lactate dehydrogenase (Ldh) , inducible nitric oxide synthase (iNOS) , Catalase , Heat shock protein 70 (Hsp70) , and Superoxide dismutase (SOD) .RNase-free H® Cell Viability Assay  was used to assess metabolic activity after hypoxia. This test was performed as previously described14. In brief, 40 fly heads were homogenized using a SpeedmillTM P12  in 1 mL of 20 mM Tris buffer, pH 7.0, and centrifuged at 1600 x g for 10 min at 4 °C. The supernatant was incubated with 0.2 mg/mL resazurin for 4 h. The fluorescence of the converted resazurin was measured at an excitation wavelength of 573 nm and an emission wavelength of 584 nm. The metabolic activity of Canton-S and Oregon-R was evaluated 0, 6, and 24 h after hypoxia.A CellTiter-Blue14 with slight modifications. The fly head lysates were incubated with 5 mM DCFH-DA for 30 min and centrifuged at 400 x g for 5 min at 4 °C. The pellet was washed once and resuspended in Tris buffer. The fluorescence of DCFH-DA was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The ROS production of Canton-S and Oregon-R was evaluated 0, 6, and 24 h after hypoxia.To determine the ROS levels after hypoxia, fly head lysates were incubated with 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) as previously describedp < 0.05. Asterisks indicate significant between-group differences; “#” indicates a significant difference between hypoxia and the corresponding normoxia control. The individual data points for each experiment are given in the legends.Data analysis and visualization were performed using GraphPad Prism . The data for each experiment comprise the results of four independent experiments with three technical replicates, each including a total of 240 flies per group. Residuals were analyzed for normal distribution using the Shapiro–Wilk and D’Agostino-Pearson omnibus normality tests. Variance homogeneity was tested using the Bartlett test or Spearman’s rank correlation test for heteroscedasticity. For identification of outliers, a ROUT test was utilized. If the normality or homogeneity test result was significant, nonparametric tests were applied instead of one-way or two-way ANOVA. The data are given as arithmetic means ± SEMs. The level of significance was set at Drosophila melanogaster (D.m.), we constructed a hypoxia chamber with the ability to monitor and control oxygen levels, temperature, humidity, and pressure  and subsequent reperfusion (21% O2) on the mortality rates of the two wild-type D.m. strains Canton-S and Oregon-R and to define a half-lethal hypoxic stimulus for our later experiments, we subjected both D.m. strains to different durations of severe N2-induced hypoxia under controlled environmental conditions  followed by various reperfusion periods (0–120 h)  and Oregon-R (r2 = 0.91)  Fig. . Here, i h) Fig. . Hypoxia91) Fig. , indicatDrosophila is the bHLH/PAS domain transcription factor Similar (Sima)16. Under normoxic conditions, Sima is hydroxylated by the prolyl hydroxylase Fatiga (Fga) in its oxygen-dependent degradation (ODD) domain; subsequently, Sima is labeled for proteasomal degradation by the dVHL ubiquitin ligase  and binds to the respective hypoxia response elements (HREs) to regulate the transcription of hypoxia response genes such as Ldh and iNOS19. To visualize and quantify sufficient activation of the HIF/Sima pathways after 2.5 h of hypoxia, we used a hypoxia biosensor consisting of GFP fused to the ODD domain of Sima , as recently described by Misra and colleagues16. We detected increases in EGFP-ODD protein levels after 2.5 h of hypoxia that were further augmented over the course of 24 h of reperfusion  resulted in overall reductions in EGFP-ODD protein signals in Western blot analysis  alpha in ase Fig. 17–19. Upels Fig.  or Tangoels Fig.  after 2.Ldh Fig.  and iNOSNOS Fig.  were uprp < 0.001) compared to the normoxia control flies over the entire reperfusion period of 5 days. The greatest impairment of climbing ability in both fly strains was observed after 3 h of reperfusion following hypoxia. Bell-shaped recovery curves with peaks at 48 h of reperfusion (Oregon-R) and 72 h of reperfusion (Canton-S) were evident  and activity at various time points. The negative geotaxis response was significantly impaired in the hypoxia-exposed flies  after hypoxia than after exposure to the corresponding normoxia conditions over the whole observation period of 120 h Fig. .Fig. 4AcBased on our previous observations, we knew that the deaths of flies of both strains after hypoxia occurred primarily during the first 24 h of reperfusion. Hence, we aimed to investigate how many flies died directly after hypoxia, how many survived and how many survived after hypoxia but died during the first 24 h. Furthermore, we compared the activity of the flies that survived the 24 h after hypoxia with that of the flies that survived hypoxia but died during the 24 h of reperfusion.p < 0.01) greater activity than the alive group , 37.6% survived hypoxia but died within 24 h of reperfusion , and 33.9% never recovered from hypoxia . Interestingly, in the demise group of flies, we observed that the animals died within the first 12 h after hypoxia Fig. . In contoup Fig. , Oregon-oup Fig. .Fig. 5ImThese data suggest that the majority of the flies did not die directly from hypoxia but rather within the first 24 h of reperfusion. Furthermore, the flies that died within 24 h of reperfusion also showed higher predeath activity than the flies that survive the entire observation period after hypoxia.SOD, Catalase, and Hsp70 in the heads of flies of both wild-type fly strains after 2.5 h of severe hypoxia followed by a reperfusion period of 120 h. Both strains showed a tendency for SOD mRNA levels to increase in the initial phase after hypoxia, but the levels decreased over the course of reperfusion , whereas this tendency was not statistically significant in Oregon-R  levels and the activity of antioxidant defense system proteins, including the enzymes Catalase and Superoxide dismutase (SOD), and Heat shock protein 70 (Hsp70). This oxidative stress is considered to be a major contributor to death. To investigate this stress in flies, we first examined the mRNA levels of ion Fig. . In contn-R Fig. . In the xia Fig. . Next, wxia Fig. . Howeverxia Fig. . The pelxia Fig. . Metabolxia Fig. . In contxia Fig. . These rD.m. have monitored or controlled environmental conditions. Overall, there are few data on the influences of humidity, temperature, and pressure on the survival of animals after hypoxia. Therefore, we introduced both wild-type fly strains to hypoxia with altered environmental conditions individually or in combination and observed their survival over 5 days. We observed that a decrease in humidity (10–20%) resulted in 20–30% higher mortality after hypoxia than the control condition   Fig. . Similarre) Fig. . A hypoxity Fig. . An incrity Fig. . The lowity Fig. .Fig. 8ImD.m. strains . For the reperfusion period of 5 days, we either maintained the standard conditions or reduced the temperature to 23 or 18 °C Fig. . After 3ins Fig. . Compareins Fig. . With a ins Fig. .D.m., stressing the need to monitor/control these conditions to achieve reproducible results. Furthermore, low temperature seems to have a very strong protective effect after hypoxia. Interestingly, the observed protective effect of temperature reduction at the beginning and during hypoxia was greater than that of temperature reduction during the reperfusion phase.In conclusion, environmental conditions, such as humidity, temperature, and pressure, have effects on the mortality of Ldh, iNOS, Hsp70, and SOD in Canton-S flies after 2.5 h of hypoxia at 23 °C and 18 °C with a reperfusion temperature of 29 °C. Ldh and iNOS mRNA levels were upregulated in the initial posthypoxic phase (at 0 h of reperfusion) following hypoxia at 23 °C. Notably, the hypoxia-dependent increases in Ldh mRNA levels were lower at reduced temperatures than at 29 °C. While iNOS mRNA levels were also increased in the initial posthypoxic phase after hypoxia at 18 °C, Ldh mRNA levels after hypoxia were not significantly different from those in the normoxia control flies  after hypoxia at 23 °C but was not significantly different from that in the corresponding normoxia control flies after hypoxia at 18 °C Fig. .These results suggest that low temperatures during hypoxia mitigate hypoxia-induced increases in oxidative stress and reductions in metabolic activity. This might serve as an explanation for the protective effects observed with lower temperatures during hypoxia.2) on adult male flies of the wild-type Drosophila melanogaster (D.m.) strains Canton-S and Oregon-R. Our analysis focused on the hypoxia-induced effects within the reperfusion (21% O2) period of 120 h. We demonstrated that exposure to severe hypoxia under defined conditions  impaired the climbing ability and reduced the overall activity of both D.m. strains. Interestingly, a majority of the flies died during the early phase of reperfusion (up to 24 h), and these flies exhibited greater activity before death than the flies that survived the entire observation period (120 h). Furthermore, we detected increased reactive oxygen species (ROS) levels; increased Catalase, Superoxide dismutase (SOD) and Heat shock protein 70 (Hsp70) expression; and reduced metabolic activity in the reperfusion phase. Both low humidity and high pressure during hypoxia resulted in an enhanced mortality rate. In contrast, low temperatures, particularly during hypoxia but also during the reperfusion phase, displayed a protective effect.In this study, we analyzed the impact of severe hypoxia (<0.3% O21. In acute ischemic stroke (AIS), the timely and successful restoration of cerebral blood perfusion is currently the only approved therapeutic option. Aside from the establishment of thrombolysis with recombinant tissue plasminogen activator (rtPA), which has been implemented since 1996, seven randomized clinical trials have highlighted endovascular stroke treatment (EST) as a remarkable additional therapeutic option for patients with severe symptoms and large vessel occlusion (LVO) AIS29. The DAWN and DEFUSE-3 trials even demonstrated that EST is suitable for stroke patients with LVO up to 24 h after symptom onset, which extends the therapeutic window significantly from the narrow time window of thrombolysis, which is within 4.5 h after symptom onset31. However, despite the high recanalization rates of EST (70–80%), fewer than 50% of AIS patients achieve functional independence. In addition, poststroke mortality within 90 days remains relatively high (29–69%), even after highly effective reperfusion treatments6. To date, the cause of the high poststroke mortality rate is not fully understood, and the restricted applicability of the two abovementioned therapies for AIS patients underlines the unmet need for understanding of the pathophysiology in the reperfusion phase in order to develop promising neuroprotective therapies.Hypoxia-reperfusion injury is a crucial factor in various medical conditions, including organ transplantations, renal diseases, cardiac arrest/infarction, wound healing, and ischemic strokeTranslational gaps, especially in the field of neuroscience, are known to exist, and improper selection of models as well as improper numbers of included animals are considered to be among the causes. Thus, we identified putative biases prior to our experiments and eliminated them by establishing a standard operating procedure Fig. .2 levels as well as the reported hypoxia tolerances of D.m. Fewer than one-third of the studies, for instance, monitored environmental conditions such as temperature, humidity, and pressure during hypoxia  and 100% (6 h). Since 2.5 h of a hypoxic stimulus caused a mortality rate of approximately 50%  and the accumulation of Sima upon hypoxia exposure (0.3% O2) followed by reperfusion for various durations. In addition, we detected transcriptional upregulation of known downstream targets of the Hif-1alpha pathway, such as Ldh and iNOS , we observed that the hypoxia-exposed flies exhibited significantly impaired negative geotaxis compared to that of the normoxia control flies over the entire reperfusion period of 5 days. Bell-shaped recovery curves with peaks at 48 h of reperfusion (Oregon-R) and 72 h of reperfusion (Canton-S) were evident  and endogenous antioxidant proteinsCatalase and SOD, and Hsp70 after various durations of reperfusion  used to supplant ambient air is usually distributed from high-pressure bottled gas and should ideally be humidified prior to administration. In addition, N2 should be distributed from the bottom of the hypoxia chamber to prevent constant gas flow and with accompanying dehydration of flies (see Supplementary Fig. Since very few of the published studies on the impact of hypoxia on ays Fig. . As expeays Fig. . Additioase Fig. . A possiase Fig. . We beliDrosophila melanogaster. We provide evidence that reperfusion-dependent death might be associated with elevated temperatures, predeath activity, and oxidative stress. Although there are obvious differences between flies and humans, we have shown that the molecular responses of both organisms to hypoxia are quite similar. This is highlighted not only by the fact that the oxygen-sensing HIF-1alpha pathway and its downstream cascade are conserved but also by the fact that increases in ROS, SOD, Catalase, and Hsp70 and decreases in metabolic activity seem to occur in similar manners. Our protocol and the available genetic alteration possibilities in flies enable further investigation of hypoxia-/ischemia-dependent damage in a high number of individuals with high reproducibility and reliability.In conclusion, we established a reliable standard operating protocol to study the impact of hypoxia-reperfusion injury in Supplemental Material


Two systems are available for the acquisition of dissolved inorganic carbon supply (DICS): the cytoplasmic localized carbonic anhydrase (CA) and the more recently described bicarbonate transporter MpsAB . In the pathogenic species Staphylococcus aureus, there are contradictions in the literature regarding the presence of a CA or MpsAB. Here, we address these contradictions in detail. We could demonstrate by careful BLASTp analyses with 259 finished and 4,590 unfinished S. aureus genomes that S. aureus does not contain CA and that the bicarbonate transporter MpsAB is the only DICS system in this species. This finding is further supported by two further pieces of evidence: (i) mpsAB deletion mutants in four different S. aureus strains failed to grow under atmospheric air, which should not be the case if they possess CAs, since we have previously shown that both CA and MpsAB can substitute for each other, and (ii) S. aureus is completely resistant to CA inhibitors, whereas Staphylococcus carnosus, which has been shown to have only CA, was inhibited by ethoxyzolamide (EZA). Taken together, we demonstrate beyond doubt that the species S. aureus possesses only the bicarbonate transporter MpsAB as its sole DICS system.In recent years, it became apparent that not only autotrophic but also most other bacteria require COIMPORTANCE The discrepancies in the current literature and even in NCBI database, which listed some protein sequences annotated as Staphylococcus aureus carbonic anhydrase (CA), are misleading. One of the existing problems in publicly available sequence databases is the presence of incorrectly annotated genes, especially if they originated from unfinished genomes. Here, we demonstrate that some of these unfinished genomes are of poor quality and should be interpreted with caution. In the present study, we aimed to address these discrepancies and correct the current literature about S. aureus CA, considering the medical relevance of S. aureus. If left unchecked, these misleading studies and wrongly annotated genes might lead to a continual propagation of wrong annotation and, consequently, wrong interpretations and wasted time. In addition, we also show that bicarbonate transporter MpsAB-harboring bacteria are resistant to CA inhibitor, suggesting that pathogens possessing both MpsAB and CA are not treatable with CA inhibitors. The bio3− is also equally as important even for nonautotrophic bacteria due to the fact that many metabolic pathways require either HCO3− or CO2 as the substrates or as products of metabolism (dissolved inorganic carbon supply (DICS) system  in Staphylococcus aureus  system  and the s aureus . MpsAB igradskyi , and Halolitanus  but alsocholerae , 8. MpsAcholerae .Staphylococcus carnosus harbors only a CA gene and confirmed that the protein is functional  motifs  using the database from Integrated Microbial Genomes and Microbiomes (IMG/M) (S. aureus strains do not contain any of the three CA-related Pfam motifs.To enable a quick and systematic search for the presence of CA in  (IMG/M) . We usedStaphylococcus carnosus  of the protein sequence of an experimentally confirmed CA from carnosus  against nosus CA . No signureoWiki , which iS. aureus and S. carnosus are from the same genus, they should share protein homology and more similarity with each other than with any bacteria from other genera. Therefore, the protein sequence of S. carnosus CA, which is from the class of β-CAs, was subjected to BLASTp search against all finished S. aureus genomes in IMG/M, but no similarity was found. As not all the microbial genomes might be integrated in IMG/M yet, we also performed the same BLASTp against S. aureus (taxonomy ID [taxid]: 1280) in NCBI database. We found two hits: NCBI accession numbers SPZ78436.1 and SPZ78435.1   with two finished S. sciuri genomes revealed that C0673 has 97% nucleotide identity with S. sciuri SNUDS-18 and 96% nucleotide identity with S. sciuri FDAARGOS_285. C0673 is wrongly annotated as S. aureus in NCBI database, which gave us false-positive hits in our BLASTp because S. sciuri but not S. aureus has a CA as stated in According to NCBI, C0673 is an unfinished genome with 89 contigs where the taxonomy check is inconclusive. Although this strain is annotated as PubMLST) . AccordiS. aureus genomes has a reasonable protein identity with any of the sequences annotated as S. aureus CA, we proceeded to examine the authenticity of these sequences. All of the nine sequences listed in Given the observation that not a single strain out of 4,849 mpsAB in two different S. aureus backgrounds, SA113 and HG001 (both are methicillin-susceptible S. aureus), could not grow under normal atmospheric conditions, indicating that there is no functional CA . Like with SA113 and HG001, the MRSA deletion mutants could not grow under atmospheric air, indicating that MpsAB is the only DICS system .In our previous study, we demonstrated that deletion of ional CA , 17. HerS. aureus. Acetazolamide (AZA), ethoxyzolamide (EZA), dorzolamide (DOR), and methazolamide (MEZ) are FDA-approved CA inhibitors used in the treatment of glaucoma, while celecoxib (CEL), chlorthalidone (CL), and famotidine (FAM) are a nonsteroidal anti-inflammatory agent, a thiazide diuretic, and an antiulcer drug, respectively  in mammalian heart  , all the compounds did not inhibit MpsAB-harboring g mpsABC . In CA-h473 can) . To veriS. aureus are substantial to warrant a comprehensive study to correct them, especially given that S. aureus is a clinically important pathogen. The first publication was in 1990 when Nafi et al. used a protein-binding monospecific antibody prepared against purified Neisseria sicca CA by immunoblotting method and also determined CA activity in cell extracts of various bacteria to screen for the presence of CA  were comparable to those of Helicobacter pylori , which were in the range of 200 to 300 μM for EZA. For AZA, our MIC values were >1 mM, consistent with those reported for H. pylori at 2 to 8 mM (Enterococcus faecium (VRE), which harbors CA genes, the MIC values were 0.5 μM for AZA and 1 μM for EZA, while S. aureus USA300 (MRSA) showed an MIC of >16 μM for both compounds, which was the upper limit tested  were search for the presence of Pfam00484, Pfam00194, and Pfam10563 for prokaryotic-type CAs, eukaryotic-type CAs, and putative CA-like domain, respectively , was subjected to BLASTp . In order to confirm that there is no CA present, the protein sequences annotated as S. aureus CA in NCBI were subjected to BLASTp search in all 259 finished and 4,590 unfinished genomes of S. aureus found in IMG/M  at 37°C with shaking at 150 rpm, unless otherwise specified. The BM consists of 1% soy peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose, and 0.1% K2HPO4 adjusted to pH 7.2. Bacterial cultures were cultivated in 10 ml medium using baffled 100 ml flasks. When necessary, the culture medium was supplemented with the following antibiotics at the indicated concentrations: chloramphenicol at 10 μg/ml and anhydrotetracycline at 100 ng/ml for staphylococcal strains and 100 μg/ml ampicillin for E. coli strains.All the strains used in this study are listed in Table S1. For cloning procedures, the mpsABC in S. aureus JE2  and MW2  were constructed as markerless deletions using allelic replacements as described in reference S. aureus JE2, and the subsequent deletion steps were the same as those for S. aureus MW2. For the construction of S. aureus MW2 ΔmpsABC, the up- and downstream regions of mpsABC were amplified from the chromosomal DNA of S. aureus MW2. The amplified fragments were assembled using linearized plasmid pBASE6 (SmaI restriction site) . The deletion mutant of Δon site)  via Gibsli DC10B . The clompsABC in both the strains was performed with the plasmid pRB473 carrying mpsABC along with its putative native promoter from our previous study  of 0.5. The plates were incubated overnight at 37°C in atmospheric air and 5% CO2 conditions.For growth visualization on agar, the wild type, Δ6) were added and the plates were incubated at 37°C with continuous shaking for 24 h in atmospheric air (2 conditions (Table S6). The MIC was determined as the lowest concentration that completely inhibited visible growth of the bacteria and also confirmed with a TECAN Reader (Infinite M200). Antibiotics vancomycin and oxacillin were used as standard antibiotic controls, while positive controls referred to the bacterial cells treated with DMSO or methanol at a concentration equivalent to the highest concentration used to dissolve the CA inhibitors. MHB alone was used as negative control. The MIC determinations were performed in three independent biological replicates with three technical replicates each.The CA inhibitors acetazolamide (AZA), ethoxyzolamide (EZA), dorzolamide (DOR), methazolamide (MEZ), celecoxib (CEL), chlorthalidone (CL), and famotidine (FAM) and selective sodium-bicarbonate cotransporters inhibitor S0859 ; Fig. S2eric air  and, if 578 of 0.1. Disks made of filter paper were impregnated with 10 μl of 1 mM EZA, vancomycin, and oxacillin (positive controls) and DMSO at appropriate concentration (negative control) before being placed on the agar. The agar plates were incubated overnight at 37°C in atmospheric air and 5% CO2 conditions.For visual representation of the semiquantitative results on agar, four strains that were inhibited by EZA were used . MHB agaThe main data supporting the findings of this work are available within the article and in the Supplemental Material or from the corresponding author upon reasonable request.