Mycobacterium tuberculosis (MTB) by culture and smear microscopy prevent early diagnosis of tuberculosis (TB) in children. Therefore, developing rapid and accurate diagnostic techniques are critical to achieving the global aim of minimizing childhood TB. The present study was performed to evaluate the diagnostic effectiveness of the novel cross-priming amplification-based EasyNAT MTB complex assay (EasyNAT) in childhood TB. Five hundred and six children with suspected TB were enrolled from January 2018 to October 2021. Gastric aspirate (GA) samples were tested by bacterial culture, acid-fast bacillus microscopy, EasyNAT, Xpert MTB/RIF (Xpert), or Xpert MTB/RIF Ultra (Xpert Ultra). Among 239 children simultaneously tested by EasyNAT and Xpert methods, both assays showed similar sensitivities in total active TB cases  and in bacteriologically confirmed TB cases [both 60.0% (9/15)]. The two assays presented similar specificities of 98.0% (100/102) and 99.0% (101/102), respectively (p = 1.000). Among 267 children who were simultaneously tested with EasyNAT and Xpert Ultra, Xpert Ultra demonstrated higher sensitivity than EasyNAT in total active TB cases . EasyNAT and Xpert Ultra yielded similar specificities, at 97.8% (90/92) and 100.0% (92/92), respectively (p = 0.155). These findings indicated that Xpert Ultra was superior to EasyNAT despite its higher cost and EasyNAT was not inferior to Xpert in the diagnosis of childhood TB using GA samples. EasyNAT may therefore be a suitable alternative diagnostic method for childhood TB based on its cost-effectiveness, speed, and accuracy.Low detection rates of  Mycobacterium tuberculosis (MTB) culture and smear microscopy detects low positivity rates in children, reducing the early diagnosis and treatment of childhood TB. Thus, developing rapid and accurate diagnostic techniques are critical to achieving the global aim of minimizing TB in children.According to the latest global tuberculosis (TB) report, there were 9.9 million new TB cases in 2020, with children accounting for 11% of these and 16% of TB-associated deaths globally. The higher proportion of child deaths and lower disease detection rate indicates that children have a poorer chance of receiving effective disease prevention and control than adults. In 2020, the estimated number of new TB cases in China was 842,000, but children comprised only 1% . The diaXpert MTB/RIF (Xpert) and its next-generation product, Xpert MTB/RIF Ultra (Xpert Ultra), are both recommended by the WHO for use as initial diagnostic tests for TB in children with signs and symptoms of pulmonary TB PTB; . Xpert aStaphylococcus aureus  symptoms suggestive of TB, including, but not limited to, long-lasting fever, night sweats, and weight loss, and (2) positive chest radiograph changes.The enrolled children were categorized into three groups  based onThis study was approved by the Medical Ethics Committee of Beijing Children’s Hospital, Capital Medical University. Written informed consent was obtained from the guardians of the patients.GA samples were collected early in the morning after an overnight fast of at least 4 h. Children without the contraindications  can be collected GA using nasogastric tube. The nasogastric tube entered the stomach through the nose, and 2–8 ml of GA was drawn with a syringe. Each specimen was transported to the lab within 6 h of collection and stored at −80°C after aliquoting. The samples were then subjected to mycobacteria growth indicator tube (MGIT) 960 culture, microscopy testing, EasyNAT, Xpert, and Xpert Ultra assays.According to clinical practice standards, MTB culture and acid-fast bacilli microscopy were tested on three consecutive days, while the molecular tests were performed once using the samples collected on the first day. Therefore, the children with bacteriologically confirmed TB were defined as those who were tested positively by MTB culture or acid-fast bacilli microscopy using the first samples collected.g for 15 min at 4°C. The pellet was resuspended in 1 ml of PBS and inoculated into the MGIT 960 system  and Lowenstein-Jensen solid medium.Two milliliters of GA sample were decontaminated with 2 ml N-acetyl-L-cysteine 2% sodium hydroxide for 15–20 min after sufficient vortexing. The mixture was then neutralized with sterile saline phosphate buffer (PBS) to a final volume of 45 ml and centrifuged at 3,000 × The pellet was smeared on a slide for Ziehl-Neelsen acid-fast staining and examined by microscopy directly.g for 15 min. The pellet was resuspended in 2 ml of the sample processing reagent and the mixture was transferred into Xpert or Xpert Ultra cartridges then loaded into the GeneXpert instrument. Both assays presented results in the same semiquantitative categories of high, medium, low, and very low. Additionally, a semiquantitative category of “trace” was introduced in the Xpert Ultra assay, which was designed to identify samples with the lowest number of targets for MTB. Invalid results were repeated. When a valid result was produced, the semiquantitative scale from each test was recorded.Two milliliters of GA sample were added to sodium hydroxide  and vortexed at 5-min intervals for 15 min, followed by centrifugation at 4,000 × g for 10 min and then 12,000 rpm for 3 min, the pellet was resuspended with DNA extraction liquid premixed with the internal control before being added to the sample chamber of the cartridge. This assay evaluated the specimens as invalid, negative, positive, and no result. Invalid results were repeated and the valid results (negative or positive) were recorded.Two milliliters of GA sample were homogenized and digested using 3–4 ml 4% sodium hydroxide solution for 15 min at room temperature until fully liquified. After centrifugation at 4,000 × p < 0.05 was considered as statistically significant.SPSS version 25.0  was used for statistical analysis. Sensitivity and specificity were compared using the Chi-square test, while concordance between the different diagnostic tests was assessed using the Kappa test. Correlation of the semiquantitative scale of the tests was presented using Spearman’s rank correlation coefficients. In total, 506 children with suspected PTB were recruited. The median (interquartile range) age was 6.9 (2.4–11.1) years, with 199 (39.3%) cases under 5 years of age, and 328 (64.8%) cases under 10 years of age. All 506 enrolled children were classified into two groups: 239 children (including 137 children with TB and 102 children with RTIs) tested simultaneously with both EasyNAT and Xpert methods were enrolled in group 1 and 267 children (including 175 children with TB and 92 children RTIs) tested simultaneously by both EasyNAT and Xpert Ultra methods were enrolled in group 2. p = 0.441]. Among 137 children with PTB, 15 (10.9%) were classified as bacteriologically confirmed TB and 122 (89.1%) were classified as probable TB. EasyNAT yielded identical sensitivity to Xpert for MTB detection in children with bacteriologically confirmed TB, with both assays demonstrating sensitivity of 60.0% (9/15). In children with probable TB, the sensitivity of Xpert was observed to be similar with that of EasyNAT, the difference was not significant . Both EasyNAT and Xpert methods showed high specificity in children with RTIs, demonstrating 98.0% (100/102) and 99.0% (101/102) specificity, respectively  tested simultaneously with EasyNAT and Xpert, similar sensitivities were observed for both methods  and in the 39 children with bacteriologically confirmed TB , which indicated that Xpert Ultra was superior to EasyNAT despite its higher cost. If “trace” results in Xpert Ultra were not included, then the sensitivities of Xpert Ultra and EasyNAT were similar . Furthermore, similar specificities were observed in EasyNAT and Xpert Ultra assays, at 97.8% (90/92) and 100.0% (92/92), respectively (p = 0.155).The detection outcomes of EasyNAT and Xpert Ultra assays among 267 children (including 175 children with PTB and 92 with RTIs) are presented in p = 0.083). Additionally, we found that among 28 Xpert Ultra trace cases, five were EasyNAT+.Agreement between EasyNAT and Xpert Ultra was further analyzed among 267 children, and concordant results were obtained for 217 children . Discordant results were found in 48 children with TB (42 with EasyNAT− Xpert Ultra+ and 6 with EasyNAT+ Xpert Ultra- results) and 2 children with RTIs .Concordance of the semiquantitative scales for EasyNAT and Xpert Ultra was analyzed in 47 PTB children with both double positive results . The resAmong all the enrolled children, none had an invalid Ultra test result. Three “invalid” results were initially obtained from the EasyNAT test; after repeated testing two were “positive” and one had “negative” results. Two “invalid” results were initially obtained from the Xpert test and the repeated resulted showed “positive” results.Since 2010, the WHO has recommended that national health authorities employ the Xpert MTB/RIF assay in the management of TB, multi-drug resistant TB, and HIV-associated TB . The gloThe first aim of the present study was to compare diagnostic accuracy between EasyNAT and Xpert or Xpert Ultra. Similar sensitivities and specificities were observed between the two assays. Furthermore, the semiquantitative scale of Xpert was also concordant with the cycle threshold generated by EasyNAT, indicating an equal detection efficiency in childhood TB using GA specimens. The sensitivities of EasyNAT and Xpert in bacteriologically confirmed TB were both 61.5%, which is similar to previously reported data from a meta-analysis 66.0%; . Considep < 0.001) and Xpert . The relative higher sensitivity of Xpert Ultra compared with Xpert in children has been reported in our previous studies, at 80 and 67%, respectively, using bronchoalveolar lavage fluid (6110 and IS1081) are used in Xpert Ultra. Because of the paucibacillary specimens obtained from children, 31.4% (28/89) of Xpert Ultra positive results were classified as “trace” in the semiquantitative scale in the present study, suggesting the value of Xpert Ultra in samples with low bacillary burden for MTB detection  were confirmed by MTB culture, and the rest of the TB cases (48/75) were clinically diagnosed based their clinical manifestations, chest X-ray results, clinical presentation improvement after anti-TB treatment, and positive results of tuberculin skin test or interferon-γ release assay. Second, Xpert Ultra is considered to be the most sensitive nucleic acid detection test, with the lowest limit of detection of 15.6 CFU/ml. So the negative results of the tests in children with discordant results suggested a underdiagnosis in these cases.We also found some discordant results between EasyNAT and the Xpert or Xpert Ultra. In children with RTIs, four with EasyNAT-positive and one with Xpert-positive results presented a very low load of MTB. All five children have no symptoms or signs or X-ray abnormalities suggestive of TB, no contact history of active TB, and positive responded to antibiotic treatment rather than anti-TB treatment. Among them, four children had negative IGRAs results, and one child was diagnosed as latent TB infection and The first generation EasyNAT TB Isothermal Amplification Diagnostic Kit (EasyNAT TB IAD) and the next-generation EasyNAT MTC assay used here have been evaluated in the diagnosis of TB in adults . One stuSecondly, the detection rates of molecular tests vary between different specimen types. Sputum, the main specimen type in adult PTB patients, is difficult to obtain in children. However, GA is an invasive but well-tolerated procedure that can be used in children. WHO Guidance for the management of tuberculosis in children suggested to use early-morning GA for pediatric TB diagnosis as children swallow respiratory secretions . TherefoIn conclusion, the EasyNAT assay had a similar diagnostic capacity for MTB in GA samples compared with the Xpert assay in children but demonstrated a lower sensitivity than Xpert Ultra. EasyNAT may therefore be useful as a suitable alternative method of childhood TB diagnosis based on its cost-effectiveness, speed, and accuracy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Ethics Committee of Beijing Children’s Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SQ, TJ, WJ, YW, and LS performed the tests. YZ, QL, YL, MF, YS, LD, XT, and XS enrolled the subjects and collected data and samples. SQ and LS analyzed the data and drafted the initial manuscript. CW, LS, and AS conceptualized and designed the study and reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was partly supported by the Capital’s Funds for Health Improvement and Research (2020-1-2091), Key Project of the Department of Science and Technology Beijing, China (D181100000418003), and National Science and Technology Major Project of China (2018ZX10103001-003).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Anaplastic oligodendrogliomas are high-grade gliomas defined molecularly by 1p19q co-deletion. There is no curative therapy, and standard of care includes surgical resection followed by radiation and chemotherapy. However, the benefit of up-front radiation with chemotherapy compared to chemotherapy alone has not been demonstrated in a randomized control trial. Given the potential long-term consequences of radiation therapy, such as cognitive impairment, arteriopathy, endocrinopathy, and hearing/visual impairment, there is an effort to balance longevity with radiation toxicity.We performed a retrospective single institution analysis of survival of patients with anaplastic oligodendroglioma over 20 years.159 patients were identified as diagnosed with an anaplastic oligodendroglioma between 1996 and 2016. Of those, 40 patients were found to have AO at original diagnosis and had documented 1p19q co-deletion with a median of 7.1 years of follow-up (range: 0.6–16.7 years). After surgery, 45 % of patients were treated with radiation and chemotherapy at diagnosis, and 50 % were treated with adjuvant chemotherapy alone. The group treated with chemotherapy alone had a trend of receiving more cycles of chemotherapy than patients treated with radiation and chemotherapy upfront (p = 0.051). Median overall survival has not yet been reached. The related risk of progression in the upfront, adjuvant chemotherapy only group was almost 5-fold higher than the patients who received radiation and chemotherapy (hazard ratio = 4.85 (1.74–13.49), p = 0.002). However, there was no significant difference in overall survival in patients treated with upfront chemotherapy compared to patients treated upfront with chemotherapy and radiation (p = 0.8). Univariate analysis of age, KPS, extent of resection, or upfront versus delayed radiation was not associated with improved survival.Initial treatment with adjuvant chemotherapy alone, rather than radiation and chemotherapy, may be an option for some patients with anaplastic oligodendroglioma, as it is associated with similar overall survival despite shorter progression free survival. Anaplastic oligodendrogliomas (AO) are grade III tumors which harbor the 1p/19q co-deletion, and are known to be chemosensitive . Currenthttp://www.r-project.org/). Hazard ratios with corresponding 95 % confidence intervals (CIs) for overall survival (OS) and progression free survival (PFS) were calculated. Data collection was closed on October 10th 2019. Patients received serial surveillance MRI scans and clinical examinations approximately every 2–6 months.This study was approved by the University of California, San Francisco IRB. Between 1996 and 2016, patients with histologically proven AO at UCSF were identified. A diagnosis of anaplastic oligodendroglioma was made by the pathologist when there was confirmation of a 1p/19q codeletion and either an elevated MIB index or the presence of anaplastic features, such as pleomorphic nuclei, hypercellularity, microvascular proliferation, or necrosis. Of these patients, those that did not follow-up at UCSF after surgery and cases where 1p19q co-deletion was either not tested or found to be negative were not included in the analysis. Only patients with AO at time of initial diagnosis were included and patients who progressed from Grade II oligodendrogliomas were not included. Demographic data, performance status, location, extent of resection , treatment modalities and pathology, including 1p19q co-deletion florescence in situ hybridization (FISH) testing were collected from the medical record. Patients were categorized as either receiving RT alone, RT plus chemotherapy (TMZ), chemotherapy alone (TMZ), or refused treatment. Pre-operative and post-operative tumor volumes were quantified by using BrainLab Smartbrush software . Manual segmentation of tumor volume was performed with region-of-interest analysis based on T1 post-gadolinium sequences and the T2 FLAIR sequences from pre- and post-operative MRI scans, respectively. EOR was calculated as: (pre-operative tumor volume – post-operative tumor volume)/pre-operative tumor volume x 100 %. Overall survival and progression free survival were assessed from time of initial diagnosis to time of death or last follow-up with Kaplan-Meier curves using the statistical software R . The remaining patients did not have sufficient tissue available for 1p19q testing. The median age at diagnosis was 44 (range: 22.7–83.9 years), and there was male predominance with 63 % male and 37 % female  were alive at the time of data collection. Median progression free survival (mPFS) was 56.3 months (95 % CI: 32.1 months-NA) and median overall survival (mOS) was not yet reached . 17.5 % of cases had no contrast enhancement, and 32.5 % of patients had very minimal or wispy enhancement. Accordingly, the extent of resection of contrast enhancing disease, when present, was 94.1 %, although this was not significant . There was no association with residual FLAIR or enhancing disease or EOR and overall survival.There was no significant difference in OS in patients who were treated with TMZ upfront compared to patients treated with TMZ and radiation at initial diagnosis (p = 0.8) Fig. . Of the Here we report extended survival for anaplastic oligodendrogliomas patients, which in the past have been thought to have poor prognosis. As with other low and high grade gliomas, minimizing residual T2 FLAIR disease was associated with increased progression free survival , 12. We One limitation of this study, similar to prior published reports, is its relatively small sample size, and more advanced multivariate analysis could not be performed. However, we restricted the analysis to patients with confirmed 1p19q co-deletion as most prior studies have looked at a heterogeneous population of either all anaplastic tumors, or a combination of anaplastic oligodendroglioma with anaplastic oligoastrocytoma which may significantly impact overall survival. Additionally, the study looks specifically at TMZ mainly due to institutional preference and therefore it remains unclear if these results will translate to other chemotherapy regimes including CCNU and PCV. Furthermore, this is a retrospective study, and there may have been some selection bias in the patients who did and did not receive radiation therapy. For example, there was a higher number of patients with bilateral tumors in the upfront chemotherapy group which may represent preference to limit radiation in cases that would require a large radiation field given the concern for cognitive decline. Finally, as our practice does not to routinely assess executive function or cognitive processing speed, we are unable to directly evaluate for cognitive changes between the two cohorts, which remains an important area of future investigation.Given the responsiveness of these co-deleted tumors to chemotherapy, some reports have questioned the benefit that surgical extent of resection has on overall survival and malignant transformation for patients with oligodendroglial tumors , 14. NevAlternatively, upfront radiation may result in fewer cycles of chemotherapy and prolong progression free survival, two potentially desirable outcomes for patients. A reduction in the number of chemotherapy cycles could improve patient quality of life, minimize treatment related toxicities, and reduce costs. While these observations need to be investigated further in randomized trials, we believe this data warrants a discussion with patients regarding the utility of early radiation therapy versus the potential side-effects.


Methods and Results: A total of 7,958,538 subjects aged over 19 years without baseline HF  were included. Anthropometric and biochemical measurements were evaluated. FLI scores were calculated and FLI ≥ 60 was considered as having NAFLD. Hazard ratios (HRs) and 95% confidence intervals (CIs) for HF incidence were analysed using multivariable time-dependent Cox proportional hazard models. During a mean follow up of 8.26 years, 17,104 participants developed HF. The FLI components associated with the incidence of HF and FLI showed a causal relationship with HF; the FLI ≥ 60 group had a higher HR for HF  than the FLI < 30 group. Subgroup analysis showed that fatty liver (FLI ≥ 60) with age ≥ 65 years or women displayed higher HR for HF than fatty liver with age < 65 or men, respectively. An increase in FLI score significantly increased the HR for HF except for those with a FLI score change from <30 to 30–60. Conclusion: NAFLD defined by FLI and increase in FLI score were associated with the incidence of HF. Further detailed prospective studies are needed.Fatty liver index (FLI) is a simple and useful index that evaluates non-alcoholic fatty liver disease (NAFLD), particularly in large epidemiologic studies. Heart failure (HF) is becoming a burden to public health as the global trend toward an aging society continues. Thus, we investigated the effect of FLI on the incidence of HF using large cohort data from the Korean National Health Insurance health database.  The prevalence of heart failure (HF) is about 1% to 2% in developed countries and increases to more than 10% in individuals over 85 years of age . Known rNon-alcoholic fatty liver disease (NAFLD) is one of the common chronic liver diseases and has increased and affected a quarter of the whole global population . In Asian = 10,422 and 13 years of follow-up) [n = 308,578 and 5.4 years of median follow-up), Roh et al. demonstrated that FLI > 30 was related to higher risk of incident HF risk in healthy Koreans [Interestingly, a cohort study from Simon et al. demonstrated that biopsy-proven NAFLD in the Swedish population was correlated with a 65% increase in the risk of incident major adverse cardiac events, which includes HF, regardless of cardiometabolic risk factors (llow-up) . In addi Koreans .Evidence supporting the association between NAFLD and HF is available. However, the use of FLI in analysing the relationship between NAFLD and HF with a comprehensive population database in Korea has been limited in the past. We hypothesize that NAFLD, defined by FLI, is an independent risk factor for incident HF in the general population. Therefore, we examined the incidence of HF in subjects with or without NAFLD as defined by FLI using Korean National Health Insurance Service (NHIS) health check-up data.The NHIS is an insurer that manages the National Health Insurance (NHI) program, which contains medical information that covers approximately 50 million Koreans . Patientn = 877,389), subjects who were diagnosed and claimed for HF prior to the index date , subjects with a history of liver cirrhosis , and subjects with missing data . Finally, 7,958,538 subjects were included in this study , and mean observation time was 8.26 ± 0.78 years. This study was approved by the Institutional Review Board of the Korea University Anam Hospital (IRB No. 2019AN0001), and permission for the use of health check-up data was granted by the NHIS (NHIS-2018-1-049).We used the NHIS health check-up database from 2002 to 2017 and selected subjects aged ≥20 years old and those who had undergone the health check-ups in 2009. The year 2009 was selected because waist circumference measurements, a requisite for FLI calculation, was available starting from 2009. Additionally, we aimed to confirm that the participants were free of HF from 2002 and 2009, while maximizing the follow-up period. A total of 10,490,491 subjects were followed up to 31 December 2017. The following participants were excluded: subjects who drank heavily (drinking ≥30 g/day in men and ≥20 g/day in women) , alcohol drinking , and regular exercise, defined as vigorous physical activity for at least 20 min/day. The frequency of exercise was categorized into 0, 1–4, ≥5 times/week.2) was calculated using weight (kg) divided by the square of the height (m). We defined obesity as a BMI ≥ 25 kg/m2, according to the World Health Organization recommendation for Asians [Anthropometric measurements such as weight (kg), height (cm), waist circumference (WC) (cm), systolic blood pressure (SBP) (mmHg), and diastolic blood pressure (DBP) (mmHg) were measured by trained examiners. Body mass index (BMI) , total cholesterol (mg/dL), triglycerides , high-density lipoprotein cholesterol (mg/dL), aspartate aminotransferase , alanine aminotransferase , gamma-glutamyl transferase , serum creatinine levels (mg/dL), and urine analyses. The Korean Association of Laboratory Quality Control warranted the quality of the laboratory tests, and the health check-up performing hospitals were certified by the NHIS.0.953 × loge (triglycerides) + 0.139 × BMI + 0.718 × loge (ggt) + 0.053 × waist circumference − 15.745)/(1 + e 0.953 × loge (triglycerides) + 0.139 × BMI + 0.718 × loge (ggt) + 0.053 × waist circumference − 15.745) × 100 [We used the FLI, a surrogate marker of NAFLD, which was calculated based on a report by Bedogni et al., as follows: FLI = (e 5) × 100 . We cate5) × 100 . An FLI 5) × 100 .Incidence of HF was defined according to the International Classification of Diseases (ICD-10) code I50 and an admission history with the HF code from 1 January 2009 to 31 December 2017 for each participant.Diabetes mellitus (DM) was defined as a fasting plasma glucose ≥126 mg/dL or at least one claim for antidiabetic medication prescription with the ICD-10 codes E11–E14. Hypertension was defined as an SBP ≥ 140 mmHg or a DBP ≥ 90 mmHg, or having at least one claim for antihypertensive medication prescription with ICD-10 codes I10–I15. Dyslipidaemia was defined by a total serum cholesterol level ≥ 240 mg/dL or having at least one claim for an anti-dyslipidaemic medication prescription with ICD-10 code E78. Cardiovascular disease (CVD) was identified when the subject gave an affirmative answer to the following question: “Do you have a history of acute myocardial infarction?”.p for each subgroup was calculated for subgroup analysis. We also calculated the HR for the incidence of HF according to the change in FLI level during the four-year follow-up with stable FLI < 30 (no change in FLI < 30 during the four-year follow up) as the reference, using multivariable Cox proportional hazard models after adjusting for all covariables.Subject general characteristics are expressed as mean ± standard deviation (SD) for continuous variables and percentages (SD) for categorical variables. Variables which did not have a normal distribution  are expressed as geometric means and interquartile ranges. The hazard ratios (HRs) and 95% confidence intervals (CIs) for the incidence of HF according to FLI group were assessed by multivariable Cox proportional hazard models using the FLI < 60 group as the reference group. Other variables were adjusted: age, sex, smoking, alcohol, and exercise in model 1 and age, sex, smoking, alcohol, exercise, HTN, dyslipidaemia, DM, and BMI in model 2. The HR and 95% CI for the incidence of HF according to each subgroup was also obtained by constructing a multivariable Cox model: age ≥ 65 years old, sex, history of cardiovascular diseases, and obesity. Interaction p-value less than 0.05 was considered statistically significant.All statistical analyses were performed using SAS version 9.4 , and a two-tailed p < 0.001). The proportion of men, current smoking, mild alcohol drinking, DM, HTN, dyslipidaemia, and history of CVD was higher in the subjects with fatty liver, but the proportion of income (Q1) and regular exercise was lower in the subjects with fatty liver than subjects without fatty liver .2) had the highest HR for HF (HR = 1.718 and 95% CI = 1.678–1.758), and overweight (23 < BMI < 25 kg/m2) subjects had the lowest HR for HF (HR = 0.884 and 95% CI = 0.838–0.933) among the five BMI categories, and the HR for HF had a U-shaped curve (ed curve b.p for interaction = 0.001). The subjects who were older than 65 years also showed an increased HR compared with the subjects who were younger than 65 years . However, fatty liver without the history of CVD or without obesity had higher HRs for HF than those with a history of CVD or with obesity   showed a higher HR for HF than men  .In In this study, NAFLD as defined by FLI was associated with an increased HR for HF in the Korean population independent of conventional CVD risk factors, confirming the initial research hypothesis. The subgroup analysis revealed that subjects aged ≥65 years and the female sex had a higher increase in HR of HF than subjects aged <65 years and the male sex, respectively, by FLI group. Increase in FLI score correlated significantly with an increased HR for HF except for FLI score change from <30 to 30–60.There has been a controversy over the relationship between NAFLD and CVD—namely, is NAFLD a precursor for future CVD or is it a passenger biomarker in the overall course of CVD development? The confusion arises partly because most NAFLD patients are also diabetic patients , and theA growing body of evidence is suggesting a strong association between NAFLD and cardiac complications, which may lead to HF development. Simon et al. have provided important evidence that biopsy-proven NALFD may act as a risk factor for new-onset HF . MoreoveCardiac remodelling is a well-understood and crucial component in the disease course of HF. Accumulating evidence suggests a link between NAFLD and new-onset HF with respect to cardiac remodelling. Many studies have reported an association between NAFLD and LV diastolic dysfunction or greater LV hypertrophy, regardless of metabolic risk factors ,32,33. NPossible mechanisms underlying the increased risk of HF in NAFLD may involve hepatic and extrahepatic pathways . The hepp for interaction 0.001). Many epidemiological studies have confirmed that women are more likely to suffer from HFpEF than men. Researchers suspect that the female-specific pathway is involved; however, the precise mechanism is yet to be found [p for interaction 0.001). The obesity paradox may contribute to higher incidence of HF in the obese subgroup. In contrast, the “lean paradox” may explain the higher HR related to the non-obese subgroup. The lean paradox proposes that low BMI is associated with less physiologic reserve against cardiac mass loss, or cachexia, and therefore low body fat and low BMI are associated with more CVD outcomes [Subgroup analyses with those with FLI ≥ 60 showed that both men and women exhibited increased risk of HF (1.28 and 1.48, respectively, be found ,48. Addioutcomes ,50.This present study displays how NAFLD defined by FLI was associated with the increased risk of HF. Hence it is crucial that future studies focus on NAFLD treatment options, especially on how, and to what degree, NAFLD treatment can prevent HF and/or CVD. Current NAFLD treatment includes lifestyle modification, vitamin E, pioglitazone, and weight loss, including metabolic surgery . HoweverThere are several limitations in our study. First, this study only included Korean participants, and results cannot be generalized to other ethnicities. Second, FLI is an indirect indicator of NAFLD, and NAFLD was not histologically confirmed in this study. Third, biomarkers that mediate FLI and HF—such as adiponectin, leptin, and dimethyl arginine—were not investigated ,54. FourHowever, to the best of our knowledge, this is the first study to examine the relationship between NAFLD defined by FLI and incidence of HF in a comprehensive database of the Korean adult population. Second, we believe this is the first study to present how HF risk increases as NAFLD severity changes in the Korean population. Third, we conducted a nationwide study that involved a homogeneous participant group. Furthermore, we analysed various subgroups while adjusting for covariates that potentially affect HF incidence.In conclusion, FLI was associated with increased HF incidence in national-scale epidemiological data. Although the FLI does not reflect the status of liver fibrosis, FLI could be used as one of the risk factors that predicts the risk of HF. Further detailed prospective studies that can confirm fatty liver by biopsy or at least with ultrasonography are needed to examine the relationship between fatty liver and HF. In addition, change in FLI was associated with increased HR for HF, thus developing medication or managing risk factors for NAFLD may be helpful in decreasing the incidence of HF in subjects with NAFLD.


Cyanophages are viruses that target cyanobacteria and directly control their abundance via viral lysis. Cyanobacteria are known to cause large blooms in water bodies, substantially contributing to oxygen depletion in bottom waters resulting in areas called dead zones. 2 fixation, which are known to form large blooms as well as resistant resting cells known as akinetes. Here, we investigated cyanophage diversity and community structure plus cyanobacteria in dead zone sediments. We sampled surface sediments and sequenced DNA and RNA, along an oxygen gradient—representing oxic, hypoxic, and anoxic conditions—in one of the world’s largest dead zones located in the Baltic Sea. Cyanophages were detected at all stations and, based on partial genome contigs, had a higher alpha diversity and different beta diversity in the hypoxic-anoxic sediments, suggesting that cyanobacteria in dead zone sediments and/or environmental conditions select for specific cyanophages. Some of these cyanophages can infect cyanobacteria with potential consequences for gene expression related to their photosystem and phosphate regulation. Top cyanobacterial genera detected in the anoxic sediment included Dolichospermum/Anabaena, Synechococcus, and Cyanobium. RNA transcripts classified to cyanobacteria were associated with numerous pathways, including anaerobic carbon metabolism and N2 fixation. Cyanobacterial blooms are known to fuel oxygen-depleted ecosystems with phosphorus , and our cyanophage data indicate the potential for viral lysis of cyanobacteria which might explain the high nutrient turnover in these environments.Up to 20% of prokaryotic organisms in the oceans are estimated to die every day due to viral infection and lysis. Viruses can therefore alter microbial diversity, community structure, and biogeochemical processes driven by these organisms. Cyanophages are viruses that infect and lyse cyanobacterial cells, adding bioavailable carbon and nutrients into the environment. Cyanobacteria are photosynthesizing bacteria, with some species capable of NIMPORTANCE Cyanophages are viruses that target cyanobacteria and directly control their abundance via viral lysis. Cyanobacteria are known to cause large blooms in water bodies, substantially contributing to oxygen depletion in bottom waters resulting in areas called dead zones. Our knowledge of cyanophages in dead zones is very scarce, and so far, no studies have assembled partial cyanophage genomes and investigated their associated cyanobacteria in these dark and anoxic sediments. Here, we present the first study using DNA and RNA sequencing to investigate in situ diversity of cyanophages and cyanobacteria in dead zones. Our study shows that dead zone sediments contain different cyanophages compared to oxic sediments and suggest that these viruses are able to affect cyanobacterial photosystem and phosphate regulation. Furthermore, cyanophage-controlled lysis of cyanobacteria might also increase the turnover of carbon, phosphorus, and nitrogen in these oxygen-free environments at the bottom of the sea. As e formed  and conse formed , 26 and e formed  and mighOne of the largest dead zones in the world is located in the Baltic Sea , which hin situ cyanophages and cyanobacteria in dead zone sediments. To investigate this aim, we sampled the top 0- to 2-cm sediment surface from four stations along a 60- to 210-m water depth gradient in one of the largest dead zones in the world . These stations included oxic (station A), hypoxic (D), near-anoxic (E), and anoxic (F) sediment (2S), and station E higher concentrations of nitrous oxide (N2O)  . DNA andSiphoviridae cyanophages (x), Podoviridae cyanophages (y), and Myoviridae cyanophages (z) (according to the cyanophage taxonomic classifications)  were assembled from the coassembly metagenome data with an average genome size of 5,076 bp . These partial cyanophage genome sequences were used in network analyses using vConTACT , which ccations) . See Fig10.1128/mSphere.00208-21.1FIG S1FIG S1, PDF file, 1.3 MB.Network showing all virus reference nodes (noncyanophages as black and cyanophages as green) alongside identified cyanophage contigs (blue nodes). Download Copyright © 2021 Broman et al.2021Broman et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00208-21.5DATA SET S1Data Set S1, XLSX file, 0.2 MB.Constructed cyanophage contigs classified by first detecting virus contigs using VirSorter and then classified further as cyanophages based on protein content using vConTACT. The values in the table show normalized mapped bp values: bp mapped on contig/contig length (in kb)/metagenome bases (in Mb). The NCBI NT classifications of the contigs are also shown in the table. Download Copyright © 2021 Broman et al.2021Broman et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P = 0.081), 3.51 ± 0.43 , and 3.11 ± 0.39  compared to 2.37 ± 0.11 at station A .Cyanobacterial alpha diversity (Shannon’s H) in the sediment was significantly higher in the oxic station A (Shannon’s H 4.17 ± 0.02) than in the hypoxic-anoxic stations D, E, and F ) compared to the hypoxic-anoxic stations D, E, and F , while the photosystem II had a different number of RNA transcripts for each station  The first sheet shows GeTMM values for proteins classified to reference cyanobacterial species in the UniProtKB/Swiss-Prot database. (2) The second sheet shows GeTMM values for proteins and their KEGG KO and KEGG modules classifications. (3) The third sheet shows the results of the edgeR analysis. Only statistically significant proteins are shown (FDR < 0.05). The table shows logFC (fold change) values for pairwise comparisons (sample 1 versus sample 2), and values of >0 denote more RNA transcripts in sample 1. Values higher in sample 1 are denoted by red cells, while values higher in sample 2 are denoted by green cells. Note that there were no differences between stations D and E. Download Copyright © 2021 Broman et al.2021Broman et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the RNA polymerase was present at all stations in the cyanobacterial RNA data ; also, t2 fixation (genes nifDEHKNU) were present at all stations (nifU RNA transcripts was higher at station A than at D and F (FDR < 0.05), and nifH RNA transcripts were higher at station E than at station A (FDR < 0.05). Genes related to assimilatory nitrate reduction and uptake (nitrate reductase narB and nitrate transporters nrtABCDP) had together more RNA transcripts at the oxic station A than at the hypoxic-anoxic stations  than at stations D, E, and F  were correlated with the relative abundance of several cyanophages, possibly due to viral infection. On the bacterial genome, CRISPR  loci consist of virus-specific DNA repeats with adjacent cas genes. Upon viral infection, the virus DNA is sampled and inserted into a CRISPR locus and transcribed into crRNA, which is then used to guide Cas endonuclease proteins to cleave invading virus DNA . In more detail, the cyanobacterial photosynthesis data set was dominated by psbA genes at all stations except the anoxic station F. It is therefore possible that changes in cyanobacterial photosynthesis regulation could have partly been due to viral infection by cyanophages. Our results indicate that prokaryote-virus interactions are an important factor to consider in future studies looking at prokaryotic photosystem genes or protein counts. Similarly, cyanophages were also found to carry genes for the phosphorus regulon phoH. Even though the function of the translated protein is still not fully understood   in our study indicated that akinetes or fermentative metabolism could have been possible. Interestingly, the relative proportion of classified reads attributed to Anabaena was higher in the RNA-seq than in the DNA data, and this might be explained by the following: (i) Anabaena is known to produce akinetes  . The preakinetes  and (ii)nditions . The preediments , 46, resxogenous ) or akinbstrates . In contakinetes . Howeverentation . Our mRN2-fixation genes are being transcribed by cyanobacteria in dead zones. We detected the highest number of N2-fixation-related RNA transcripts in the oxic station A, possibly due to there being a higher relative abundance of cyanobacteria at that station than at the hypoxic-anoxic stations. The sampled hypoxic-anoxic stations have low or no nitrate and nitrite  where ammonium might have been scarce. However, further controlled studies are needed to investigate if cyanobacterial N2 fixation is an important metabolic process in dead zone sediments. We were also able to detect RNA transcripts classified to genes coding for photosynthesis at all sampled stations. The highest number of photosynthesis RNA transcripts was detected at the oxic station A, possibly due to cyanobacteria having the highest relative abundance at that station. Accumulation of RNA transcripts attributed to the photosystem in darkness has previously been observed for the strain Synechocystis sp. strain PCC 6803  and akinete-forming Dolichospermum/Anabaena. Cyanobacterial RNA transcripts in sediment were classified to, e.g., anaerobic glycolysis and N2 fixation. Considering that large amounts of cyanobacteria sinking to dead zone sediments are known to fuel the benthic ecosystems with phosphorus , our cyanophage data indicate the potential for viral lysis of cyanobacteria, and this might sustain the high nutrient turnover in these heavily eutrophied environments.We have here shown that cyanophages and cyanobacteria are present in one of the largest dead zones in the world. Cyanophages were detected at all stations and had a different beta diversity in the oxic sediment than in the hypoxic and anoxic sediments, suggesting that cyanobacteria and/or the environment select for specific cyanophages. Moreover, our results show that these cyanophages can infect cyanobacteria, affecting the photosystem and phosphate regulation. Top prevalent cyanobacteria detected include genera not capable of forming akinetes (2 until being transferred and stored at −80°C.Sediment was collected using a modified box corer  during 2DNA and RNA were extracted from thawed and homogenized sediment with the DNeasy PowerMax soil kit (Qiagen) and RNeasy PowerSoil kit (Qiagen), with 10 g and 2 g input material, respectively. Extracted RNA was DNase treated with the Turbo DNA-free kit (Invitrogen) and rRNA depletion using the RiboMinus transcriptome isolation kit . Multiplexed libraries were prepared with the ThruPLEX DNA-seq (Rubicon Genomics) and TruSeq RNA Library Prep v2  kits for DNA and RNA, respectively. Paired-end 2- by 150-bp sequencing was conducted at the Science for Life Laboratory, Stockholm, Sweden, on the Illumina NovaSeq platform . The sequencing yielded on average 41 million (range 32 to 52) and 82 million (range 74 to 89) read-pairs for each DNA and RNA sample, respectively.NC_001422.1). Reads were quality trimmed using Trimmomatic 0.36  were classified against the NCBI RefSeq genome database (downloaded 1 March 2019) using Kraken2 2.0.7 with a paired-end setup . The DNAgene length corrected trimmed mean of M-values (GeTMM)  . Unique  (GeTMM)  by linkihttps://de.cyverse.org). Contigs classified as “Pretty sure” and “Quite sure” by VirSorter were used for further analyses. The contigs were classified for taxonomy using BLAST 2.10.1+  using the software PAST. Differences in RNA transcripts for annotated proteins between stations were tested with the R package edgeR 3.24.3  using the software PAST 3.25 . ShannonR 3.24.3  by usingR 3.24.3 . False-dPRJNA531756 (The raw sequencing data supporting the conclusions of this article are available in the NCBI BioProject repository, NA531756 .


Chronic urticaria (CU) is comprised of diverse phenotypes, and thus, a shift towards a precision medical approach is warranted in its management. 2 blockers, and low-histamine diet.  This study enrolled 78 patients with CU. Serum erythrocyte sedimentation rate, hemoglobin, hematocrit, eosinophil count, IgE, antinuclear antibody (ANA), and serum diamine oxidase (DAO) levels of the patients were measured and were compared according to the patient's response to second-generation antihistamines (sgAH), corticosteroids, leukotriene receptor antagonist (LTRA), H2 blocker response. Furthermore, concomitant angioedema (aOR = 10.56), multiple food triggers (aOR = 11.69), and a DAO level < 5.4 U/mL (aOR = 3.78) were significantly associated with a low-histamine diet response. Conversely, dermatographic urticaria and a hematocrit level < 36% were significantly and inversely associated with low-histamine diet response.  Age- and sex-adjusted logistic regression analysis showed that patients with duration of CU > 3 years (adjusted odd ratio [aOR] = 4.39) and a DAO level < 10 U/mL (aOR = 3.90) were significantly associated with a good sgAH response. Age > 50 years (aOR = 0.02), duration of chronic urticaria > 3 years (aOR =0.06), and an ANA titer ≥ 1 : 80 (aOR = 0.03) were significantly and inversely associated with corticosteroid response. A low-histamine diet response was significantly associated with LTRA response (aOR = 67.29). In addition, a DAO level < 5.4 U/mL (aOR = 71.95) was significantly associated with H1 and H2 receptors. Several promising biomarkers were identified in this study to predict the efficacy of chronic urticaria treatment. DAO could be a novel biomarker for predicting the efficacy not only of dietary intervention but also for antagonists of H Chronic urticaria (CU) has traditionally been defined as episodes of urticaria rash that occur almost daily for more than six weeks. CU is a common disease and is prevalent in approximately 1% of the general population. However, treatment options are limited. The recommended treatment outlined in the CU guidelines includes second-generation H1 antihistamines (sgAH), omalizumab, and cyclosporine . Due to r = −0.335, p = 0.001) between DAO and serum histamine level was reported by Cho et al. [Diamine oxidase (DAO) is the main enzyme involved in histamine catabolism, and DAO deficiency has been suspected as a probable cause of histamine intolerance. Beyond a critical level, histamine can induce a variety of symptoms in individuals with histamine intolerance, including urticaria, migraine, and gastrointestinal upset. A significant inverse correlation  or Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (No. 09-C-095).Adult patients 18 years of age or older with CU were recruited from two regional hospitals in Taiwan. The diagnosis of CU was based on the definition given in current CU guideline . All parThe following laboratory tests were performed as follows: serum erythrocyte sedimentation rate (ESR), hemoglobin (Hb) and hematocrit (Hct) levels, eosinophil count, IgE, and antinuclear antibody (ANA) and DAO levels. The serum was separated and kept at −20°C until needed. Laboratory blood parameters and serum DAO levels were measured in the following way. Briefly, total and specific IgE were analyzed by fluorimetric enzyme-linked assay (FEIA) . DAO levels were measured using the DAO ELISA kit , according to the manufacturer's protocol. A dose-response curve of optical density versus a standard concentration was generated, using the values obtained from the standard. The reference values from the manufacturer's specification for DAO are as follows: histamine intolerance is highly likely in patients with DAO activity < 3 U/mL, likely in patients with DAO activity < 10 U/mL, and improbable in patients with DAO activity > 10 U/mL. Other index data were obtained from the routine laboratory examinations.t-test for continuous variables. Pearson's correlations were performed to analyze the correlation between continuous variables. To identify significant predictors of treatment efficacy in patients with CU, five separate age- and sex-adjusted logistic regression analyses were performed with the following dependent variables: good response to sgAH, corticosteroid response, leukotriene receptor antagonist response, H2 blockers, and low-histamine diet response. When the logistic regression model failed to converge for binary outcomes, firth logistic regression was used [p value < 0.05 was considered statistically significant. All statistical analyses were conducted using IBM SPSS version 28.0 .Comparative analyses were conducted using chi-square tests for categorical variables and independent was used . The prop = 0.004). They were significantly lower in patients with CU who were >50 years of age compared with those ≤ 50 years of age (p = 0.033). Patients with dermatographic urticaria had a significantly lower DAO compared to those with chronic spontaneous urticaria (p = 0.006). Moreover, the mean ESR level was significantly lower in patients with dermatographic urticaria compared to those with chronic spontaneous urticaria . DAO was also significantly correlated to ESR (p = 0.004) and inversely correlated with Hb (p = 0.022) and Hct (p = 0.016).The basic characteristics of the patients with CU are shown in p = 0.032) and a DAO level < 10 U/mL .Of the 76 patients with available sgAH treatment response, 44 (57.9%) reported having a good response, 30 reported having a partial response (39.5%), and 2 reported no response (2.6%). The results of the multiple logistic regression analysis showed that a significantly favorable response to sgAH was observed in patients who had CU > 3 years , > 50 years of age , and duration of CU > 3 years .Of the 20 patients who needed oral corticosteroid treatment for symptomatic relief, 14 (70%) reported a good response, and 6 (30%) reported a poor response. The results of the multiple logistic regression analysis showed that a significantly reduced risk of corticosteroid response was observed in patients with CU who had a ANA titer ≥ 1 : 80 .Of 11 patients with available montelukast response, six reported having a good response (54.5%), and 5 reported having a poor response (45.5%). The results of the multiple logistic regression analysis showed that a significantly higher risk of montelukast response was observed in patients with CU who benefited from a low-histamine diet  reported having a good response, and 5 (62.5%) reported a poor response. The results of the multiple logistic regression analysis showed that patients with CU who had a DAO level < 5.4 U/mL (the median of DAO levels) were significantly more likely to respond to H2 blockers .Of eight patients with available Hp = 0.024). In addition, patients with multiple food triggers were more likely to benefit from a low-histamine diet . Moreover, patients with CU who had a DAO level < 5.4 U/mL, which was the median of DAO levels, were significantly more likely to respond to a low-histamine diet . Conversely, patients with dermatographic urticaria were less likely to significantly benefit from a low-histamine diet . Patients with CU who had an Hct level < 36% were less likely to benefit from a low-histamine diet   reported improvement of CU symptoms. The results of the multiple logistic regression analysis showed that a significantly higher risk of low-histamine diet response was observed among patients with concomitant angioedema  .n = 12), followed by alcohol (n = 6), peanuts (n = 5), and processed food with food additives (n = 5). These foods were included in this study due to intermittent eruptions despite the elimination of culprit foods. Four individuals with a shrimp trigger had specific IgE (sIgE) to shrimp detected. No individuals with the peanut trigger had sIgE to peanut detected. Patients with exacerbations after consuming shrimp were more likely to benefit from a low-histamine diet . Patients with exacerbation after pumpkin consumption were less likely to respond to a low-histamine diet . In the subgroup with DAO levels below 5.4 U/mL, patients who had reactions to processed foods with food additives had significantly lower DAO levels . The prevalence of angioedema was significantly higher in patients who had exacerbation of symptoms after consuming peanuts . Patients with exacerbations after consuming plant-derived foods were more likely to have contact dermatitis with culprit foods or herbal medicines . The prevalence of eczema was higher among patients who had exacerbations with alcohol consumption . Patients with eruptions triggered predominantly by heat were more likely to have exacerbations of symptoms when eating spicy foods .Among the 78 participants, the most common food trigger reported was shrimp . Meanwhile, an association of high IgE levels with an insufficient sgAH response was observed in patients with CU who had an IgE level > 100 U/mL , suggesting that there may be a U-shaped relationship between IgE and insufficient sgAH response. In the present study, no association of insufficient sgAH response with elevated ESR levels or eosinopenia was observed.In clinical practice, the response to sgAH in patients with CU varies, even though it is the first-line treatment recommended in all phenotypes of CU. Consequently, up to 50% of patients with CU need other therapeutic options –8. Increp = 0.010). However, the association of resistance to corticosteroid with ANA titers ≥ 1 : 80 was also observed. In the meantime, we have not observed the association of responders to corticosteroid with a long duration of wheals or elevated ESR. A possible explanation could be that ANA may be another marker of CU severity that results in resistance to corticosteroids at a lower dose prescribed. Unlike allergic diseases, an increased eosinophil count may not be helpful in selecting patients with CU who would be good candidates for corticosteroids.Corticosteroids, potent immunosuppressants, can be the only rescue for some severe eruptions. However, resistance to corticosteroids can be seen in some patients with CU. In our study, older age seems to be a predictor of less favorable response to corticosteroid in patients with CU. In this respect, corticosteroid should be cautiously used in patients with CU, particularly older adults, due to not only the susceptibility of potential adverse effects but also because of less favorable efficacy. In clinical practice, when a patient with CU has wheals for a long time, elevated ESR, or positive ANA levels, underlying autoimmune diseases usually come to mind, which can be an indication for a corticosteroid. In fact, we found that patients with CU who had an ANA titer ≥ 1 : 80 were more likely to have a long duration of wheals . However, patients with CU were excluded from the study [2 blockers was also observed in patients with CU, suggesting antagonists of H1 and H2 receptors might be helpful in dealing with excessive histamine when histamine metabolism is impaired. However, their efficacy may be insufficient when the capacity of histamine catabolism is excessively impaired, because the association of insufficient sgAH response with low DAO was observed in patients with DAO levels < 10 U/mL .Adherence is often a major issue in dietary intervention. A biomarker for the prediction of efficacy would be required to prompt patients with CU to adhere to the dietary recommendations. In comparison with healthy controls, significantly lower DAO levels in individuals with histamine intolerance benefiting from a histamine-limited diet have been reported  [Hemoglobin and Hct level analysis were included in this study because polycythemia could result in increased histamine formation. In the present study, an association of low Hct level with histamine tolerance was observed. Based on the inverse correlation between DAO and Hb, DAO consumption by excessive histamine in the process of erythrocyte metabolism might be considered. Additionally, a positive correlation was also observed between ESR, an acute phase reactant affected by erythrocyte number, and the level of DAO. Daschner et al. reported a significant correlation between IL-6, a proinflammatory cytokine, and DAO levels in patients with CU who were Anisakis sensitization Rho = 0.57; = 0.003) , which c; = 0.003= 0.003) .Theoretically, pseudoallergic reactions are not evident immediately after consuming histamine-rich foods because the symptoms principally result from a buildup of histamine. However, exacerbation of CU followed by specific food intake has been reported by a number of patients. In the study of Magerl et al., fewer than 10% of patients with chronic spontaneous urticaria had reported food as a trigger . In our p = 0.065) and vice versa. While the association of concomitant angioedema with benefit from a low-histamine diet was observed, patients with dermatographism were less likely to benefit from a low-histamine diet.Instead of chronic spontaneous urticaria (CSU), dermatographic urticaria was classified as chronic inducible urticaria (CindU). While there is no identified trigger in patients of CSU, CindU can be triggered by physical stimuli. The differences observed in the efficacy of diet intervention between CSU and dermatographic urticaria suggested that a low-histamine diet may be less likely to be beneficial in CindU compared to CSU. In addition, we observed a trend for a lower prevalence of angioedema in patients with dermatographic urticaria (0/13; 0% vs 14/65; 21.54%; 2 blocker, and a low-histamine diet. This is not surprising because DAO is the key enzyme in histamine metabolism. Furthermore, with appropriate patient selection, the efficacy of LTRA, H2 blockers, and a low-histamine diet in patients with CU should be evaluated with additional studies.A number of biomarkers identified in our study can be helpful to achieve better CU control. DAO appears to be a novel biomarker in CU, predicting the efficacy of sgAH, H


Staphylococcus aureus and Enterococcus faecalis. Pseudomonas spp. were more sensitive to red skin pomace extracts rather than white skin pomace extracts. Given these results, both red and white pomace extracts could be exploited for future application in food, pharmaceutical and cosmetic industry.Wine pomace has attracted the attention of the food industry, due to its high content in bioactive compounds, and its multiple healthy activities. In this work, whole and separated skin pomaces from fermented (red) and un-fermented (white) grape by-products were characterized for their antioxidant and antimicrobial activities in order to exploit them as functional food ingredient. Antioxidant activity, measured by both ORAC and TEAC assays, was higher in whole than in skin pomace extracts. The characterization of phenolic composition in whole and skin pomace extracts confirmed the peculiarity of some compounds such as anthocyanins (107.84 + 10.3 mg/g TP) in red skin pomace and a great amount of flavanols (80.73 + 4.04 mg/g TP) in white skin pomace. Whole and skin pomace extracts displayed the same antibacterial activity at 250 µg gallic acid equivalents (GAE)/mL. Red and white skin pomace extracts showed a Minimum Inhibitory Concentration (MIC) of 31.25–62.5 GAE/mL against  The health-promoting activities of plant polyphenols have been widely ascertained by hundreds scientific publications. Studies on plant extracts and phytochemicals showed that polyphenols could play an anti-inflammatory and antibacterial action ,2,3,4. GHowever, the bioactivities of grape pomace extracts are the result of their polyphenolic profile that is affected by the grape variety, the geographical origin and the winemaking process ,8,9. TheStaphylococcus aureus and Escherichia coli, resulting particularly useful to control the growth of multi-drug resistant clinical isolates , acetonitrile, formic acid, ethanol,  from Sigma-Aldrich . Milli-Q water  was used for the preparation of reagents and antioxidant assays.Reagents were acquired from various suppliers: authentic standards of oenin , (achieved after fermentation for red wine making), and Fiano (F)  were obtained from a commercial winemaking facilitylocated in Salento . Pomace samples were dried in an oven at 50 °C, until constant weight. Grape skins, manually separated from whole pomaces, were stored at −20 °C until further assays.Two batches of wine pomace (both obtained in the late summer of 2019), v:v) at room temperature for 16 h in the dark under continuous stirring. Extraction mixtures were centrifuged (4000× g) for 5 min and the supernatants were dealchoolized by Rotavapor  and freeze-dried by a Freezone® 2.5 model 76530 lyophilizer  and stored at −20 °C until biological activity analysis. The extracts of whole and skin pomace (WP and SP respectively), of grape cultivars Negramaro (N) and Fiano (F) were lyophilized and suspended in phosphate-buffered saline  and then analysed for their antioxidant activity (TEAC and ORAC) and the total phenol content (TP). The powder obtained was weighed and suspended in PBS, at a concentration of 500 µg/mL total polyphenols . This stock solution was used for antibacterial assays.Phenol compounds were extracted from whole and skin pomaces. Whole grape pomace and skins separated from dried grape pomace were freezed in liquid nitrogen and grinded with a blender until a fine powder was obtained. In order to evaluate the antibacterial activity of pomaces, whole and skin samples (1 g) were extracted in 10 mL of ethanol:water  equipped with a DAD detector, the separation was performed on C18 column , as described by Gerardi et al. [The TEAC assay of whole and skin pomace samples was performed trough the method reported by Gerardi et al. . The ABTThe ORAC procedure was carried out as per Gerardi et al. . The reav/v) (50 μL) was placed in each well, and then 100 μL of sodium hydroxide solution (0.35 M) was added. The absorbance value at 760 nm was recorder after 5 min of incubation. Gallic acid was used to obtain a calibration curve in the range from 2.5 to 40.0 mg/L (R ≥ 0.9997). Gallic acid equivalents (GAE) were used to express the total phenol content of different samples.A rapid method  was usedhttp://server.ispa.cnr.it/ITEM/Collection/; accessed on 10 May 2021). The three food spoilage bacterial strains were Pseudomonas fluorescens NCPPB 1964T, P. chicorii ITEM 17296, P. putida ITEM 17297 [P. aeruginosa DSM 939, Staphylococcus aureus DSM 799, and Enterococcus faecalis ATCC 47077. Spoiler strains were cultivated in mPlate Count Broth  for 24 h at 30 °C, whereas pathogens were cultivated in Brain Heart Infusion  for 24 h at 37 °C. After the incubation, bacterial cell density was adjusted to 0.3 ± 0.05 (ca. 108 cfu/mL). Then, cell suspensions were diluted to 105 cfu/mL in mPCB amended with SP or WP at 250 µg/mL total polyphenols. Then, the antibacterial activity of SP extract was further assayed at the final concentration of 250, 125, 62.5, and 31.25 µg/mL total polyphenols. Cell suspensions in mPCB without any grape skin pomace extract were used as controls. Bacterial growth was monitored by measuring optical density every 10 min with the Varioskan Flash  spectrofluorimeter at a wavelength of 600 nm up to 24 h at 30 °C. Each antimicrobial assay was performed in triplicate. WP and SP extracts from different cultivars were assayed independently in different plates. The Minimum Inhibitory Concentration (MIC) was defined as the lowest SP concentration inhibiting the bacterial growth during the incubation. MICs are expressed in µg of gallic acid equivalents (GAE) per mL of growth medium.The antibacterial activity of the whole and skin grape pomace extracts (WP and SP) cv. Fiano (WPF and SPF) and cv. Negramaro (WPN and SPN) was determined against six strains of bacteria purchased from international microbial collections or included in the Agri-Food Toxigenic Fungi Culture Collection (ITEM) of the Institute of Sciences of Food Production , maximum optical density (ABSmax), and lag time .The doubling time , defined as the time needed to doubling the OD value starting from the begin of the exponential growth [Growth curves were analyzed through the SkanIt™ software (Thermo Fischer Scientific) to calculate the following parameters: maximum growth rate . For microbiological tests, growth parameters were compared, for each strain and each SP, through the Tukey test (p ≤ 0.05) with the SPSS software .The experiments with pomace extracts were conducted in three independent tests, and data were presented as mean ± standard deviation (SD). For chemical analyses, multiple comparisons were carried out by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc comparison tests to establish differences between means (The comparison of antioxidant and antimicrobial activity of lyophilized whole and skin pomace extracts from red and white grape varieties highlighted differences in antioxidant capacity, phenolic composition, and specific antimicrobial activity against pathogenic and spoilage bacteria. The whole and skin pomace extracts of the Negramaro cv and Fiano cv showed antioxidant and antibacterial activities. Red skin pomace extract of Negramaro cv showed higher antioxidant and antibacterial activity than white skin pomace extract of Fiano cv. Lyophilized grape pomace extracts could be used as natural preservatives in the food industry and as additives in the pharmaceutical/cosmetic sector. Phenols such as flavonoids and soluble phenolic acids can be produced by renewable and low-cost source, exploiting the nutritional and biological value of winery by-products.


Nitrosomonas and Rhodocyclaceae, nitrogen-sensing dissimilative nitrate respiration regulator that responds to nitrogen species (DNR) in Rhodocyclaceae, and nitrogen-sensing nitrite and nitric oxide reductase regulator that responds to nitrogen species (NnrR) in Nitrospira MAGs. Our data also predict that CRP/FNR homologs in Ignavibacteria, Flavobacteriales, and Saprospiraceae MAGs sense carbon availability. In addition, a CRP/FNR homolog in a Brocadia MAG was most closely related to CRP TFs known to sense carbon sources in well-studied organisms. However, we predict that in autotrophic Brocadia, this TF most likely regulates a diverse set of functions, including a response to stress during the cyclic aerobic/anoxic conditions. Overall, this analysis allowed us to define a meta-regulon of the PNA microbiome that explains functions and interactions of the most active community members.Microorganisms must respond to environmental changes to survive, often by controlling transcription initiation. Intermittent aeration during wastewater treatment presents a cyclically changing environment to which microorganisms must react. We used an intermittently aerated bioreactor performing partial nitritation and anammox (PNA) to investigate how the microbiome responds to recurring change. Meta-transcriptomic analysis revealed a dramatic disconnect between the relative DNA abundance and gene expression within the metagenome-assembled genomes (MAGs) of community members, suggesting the importance of transcriptional regulation in this microbiome. To explore how community members responded to cyclic aeration via transcriptional regulation, we searched for homologs of the catabolite repressor protein/fumarate and nitrate reductase regulatory protein (CRP/FNR) family of transcription factors (TFs) within the MAGs. Using phylogenetic analyses, evaluation of sequence conservation in important amino acid residues, and prediction of genes regulated by TFs in the MAGs, we identified homologs of the oxygen-sensing FNR in IMPORTANCE Microbiomes are important contributors to many ecosystems, including ones where nutrient cycling is stimulated by aeration control. Optimizing cyclic aeration helps reduce energy needs and maximize microbiome performance during wastewater treatment; however, little is known about how most microbial community members respond to these alternating conditions. We defined the meta-regulon of a PNA microbiome by combining existing knowledge of how the CRP/FNR family of bacterial TFs respond to stimuli, with metatranscriptomic analyses to characterize gene expression changes during aeration cycles. Our results indicated that, for some members of the community, prior knowledge is sufficient for high-confidence assignments of TF function, whereas other community members have CRP/FNR TFs for which inferences of function are limited by lack of prior knowledge. This study provides a framework to begin elucidating meta-regulons in microbiomes, where pure cultures are not available for traditional transcriptional regulation studies. Defining the meta-regulon can help in optimizing microbiome performance. In natural ecosystems, microbiomes often experience cyclical variations in environmental conditions such as presence or absence of light , warm and cold temperatures , and presence or absence of oxygen . Microbiomes are used in advanced wastewater treatment processes for the removal of nutrients, where controlling the presence and absence of oxygen creates a dynamic environment that allows bacteria to carry out important functions such as enhanced biological phosphorus removal, nitrification, and denitrification , 2. LikeMicrobial adaptation is critical for survival in changing environments like those experienced by wastewater treatment microbiomes. Transcription factors (TFs) often regulate this adaptation by binding to DNA and increasing or decreasing transcription initiation in response to specific environmental stimuli. Examples of microbial adaptation to changing environments by transcriptional regulation include the expression of genes that encode proteins involved in alternative metabolic pathways in response to changes in carbon source, oxygen, or alternative electron acceptors . Bacteri7–One particular TF, the fumarate and nitrate reductase regulatory protein (FNR), is among the most widely studied global regulators, activating and repressing transcription of a wide variety of functional genes. FNR is widely conserved across bacteria and is often required for the shift between aerobic and anaerobic metabolism . FNR bel13–Although the environmental stimuli and the function of genes regulated by CRP/FNR family TFs have been well studied in pure cultures of several bacterial species, little work has been done to study how homologs of these proteins may function in a microbiome. Given the conservation of members of the CRP/FNR family across bacteria , 16, we This study utilized a metagenomic and metatranscriptomic approach to identify the predicted CRP/FNR family meta-regulon in an intermittently aerated deammonification bioreactor performing partial nitritation and anammox (PNA). Out of 43 high-quality metagenome-assembled genomes (MAGs) reconstructed from the microbiome, CRP/FNR homologs were detected in 29, including 11 of the top 16 MAGs as determined by RNA abundance in metatranscriptomic data sets. We predicted potential DNA binding sites for CRP/FNR family TFs and correlated these predictions with metatranscriptomic changes to predict which genes were regulated during cyclic bioreactor conditions. This analysis provides a first glimpse of how a microbiome collectively responds to external environmental factors. This information, and the methodology described here, both improves our understanding of uncultured microorganisms and their function in changing environmental conditions and generates models that could enhance our capacity to harness microbiomes for specific functions, such as nitrogen removal from wastewater.4+-N/liter and variable organic carbon concentrations ranging primarily between 100 and 400 mg chemical oxygen demand (COD)/liter, with a few weeks exceeding 1,000 mg COD/liter  for metagenomic sequencing. DNA from each sample was sequenced twice, resulting in eight separate metagenomic DNA read data sets. Of ∼206 million metagenomic reads, ∼201 million passed quality control criteria and were used as input for one metagenomic coassembly .10.1128/mSystems.00906-21.3TABLE S1Table S1, XLSX file, 0.03 MB.DNA and mRNA alignment statistics. Download Copyright © 2021 Beach et al.2021Beach et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00906-21.4TABLE S2Table S2, XLSX file, 0.02 MB.Metagenome and metatranscriptome statistics. Download Copyright © 2021 Beach et al.2021Beach et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Twenty-four samples were collected at day 454 of bioreactor operation for metatranscriptomic analysis (these reads are referred to here as mRNA reads). Approximately 306 million mRNA reads aligned to the 43 MAGs (out of ∼411 million mRNA reads that mapped to the assembly) , and S3,10.1128/mSystems.00906-21.5TABLE S3Table S3, XLSX file, 18.4 MB.Metatranscriptomic linear RPKM values. Download Copyright © 2021 Beach et al.2021Beach et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Nitrosomonas europaea (GenBank accession no. AL954747), which was also the main AOB in the Olburgen PNA bioreactor , which has been reported in wastewater treatment systems operated under low-DO conditions  Nitrospira, since representatives of comammox  formed a different cluster within Nitrospirae , AMX_44 (Brocadia), and NSP_46 (Nitrospira), accounted for just 2.1%, 1.3%, and 1.3% relative DNA abundance (Ignavibacteria (IGV_58) MAG dominated in both categories, with relative DNA abundance and relative gene expression at ∼32% and ∼40%, respectively. Other MAGs within the 16 most active groups had variable levels of relative DNA abundance (from 0.4% to 7.2%) but less than 3% relative gene expression.To calculate relative DNA and mRNA abundance , we normalized the respective nucleic acid read counts for each MAG by its predicted genome size and then divided this quantity by the total DNA or mRNA read counts for all 43 MAGs, respectively. The direct comparison of these two metrics revealed that relative DNA abundance and gene expression did not always correlate for each MAG. For example, MAGs associated with nitrogen cycling such as NSM_48  and two Rhodocyclaceae MAGs (RDC_57 and RDC_54R) clustered within known FNR TFs  contained a predicted CRP/FNR homolog that also clustered near the FNR proteins in the phylogenetic tree  clustered near known DNR TFs  contains both an FNR homolog and a DNR homolog, raising the possibility that it can respond to both oxygen and NO concentrations. We also found that a CRP/FNR homolog in the Nitrospira MAG (NSP_46) clustered with proteins that have been identified as the TF NnrR (Rhodobacter sphaeroides shows conservation of the tyrosine residue at position 93 (in vivo regulatory activity (Nitrospira (NSP_46) uses NnrR to sense and respond to changes in nitrogen concentration during the cyclic operation of the bioreactor. Another CRP/FNR TF from NSP_46 clustered near FNRBAC. FNRBAC is distinct from FNR from Escherichia coli in that it is activated by a two-component regulatory system (ResDE) and has the sensor domain at the C terminus , an intermediate in nitrogen cycling metabolism , 29. Onl DNR TFs . Comparition 139 , which htion 139 , suggest TF NnrR  31, 32), 32Rhodoition 93 , which hterminus  , 35Ignav cluster . Flp hasike MalR . Indeed,n IGV_58 , suggestBrocadia MAG (AMX_44) did not cluster within a group of previously studied TFs , and its phylogenetic distance from the main CRP cluster  and Nitrosomonas (NSM_48 and NSM_41) MAGs. Using a PWM from binding sites in E. coli . Interestingly, when comparing the FNR-controlled gene expression patterns from the Nitrosomonas and Rhodocyclaceae MAGs, most genes with higher expression in the anoxic conditions came from the Nitrosomonas MAGs , a high-efficiency Cbb3-type cytochrome c oxidase (EFKGCFLK_01956), and an Na+ pump that may be required for nutrient uptake (EFKGCFLK_01284)  . AdditioK_01284)  44), an, anRhodoNitrospira NSP_46 MAG, we used the binding site analysis from a PWM constructed from sites in R. sphaeroides and enterobacteria , a Flavobacteriales MAG (FLB_49), and a Saprospiraceae MAG (SPS_50R)  . This anSPS_50R) . The genSPS_50R) . HoweverSPS_50R)  and a geSPS_50R) , suggestSPS_50R) , purine SPS_50R) , cell diSPS_50R) , and vitBrocadia MAG (AMX_44) showed a high percentage of amino acid identity with CRP from E. coli , we, weBroca conditions. Microbial community members were identified using metagenomic analysis, and the connections between relative abundances based on DNA and mRNA levels were analyzed. Similar differences in relative abundance of organism and transcript based on DNA and mRNA analysis of the PNA bioreactor to those found in this study have been described in other microbiomes , 51, incThis disconnect between relative DNA abundance and gene expression levels prompted us to develop a CRP/FNR family meta-regulon of the PNA bioreactor community since the CRP/FNR family of TFs is widely conserved across bacteria and because individual members of this group of proteins are known to control transcriptional responses to the presence of oxygen, alternative electron acceptors, and carbon sources . The ideN. europaea was identified as the bacterium performing aerobic ammonia oxidation, represented by the NSM_48 and NSM_41 MAGs, whereas Brocadia (AMX_44) was identified as performing deammonification. An FNR TF was identified in the N. europaea MAGs, indicating that this organism directly senses and responds to O2 availability. The predicted FNR regulon did not include any of the core enzymes for ammonia oxidation  or CO2 fixation , which are core pathways for which different transcript abundances have been detected under ammonia-limited or oxygen-limited conditions  in the PNA bioreactor for which the predicted FNR regulon included genes that encode nitrite reductase and nitrous oxide reductase. The expression of these genes was found to be higher under anoxic conditions, indicating these MAGs are responding to O2 levels and carrying out a conventional denitrification function in the PNA bioreactor Nitrospirechanism . Thus, ttoxicity .Nitrospira MAG NSP_46 (Nitrospira MAGs from other studies (nirK), which catalyzes the reduction of nitrite to nitric oxide, an NO reductase gene has not been identified in Nitrospira  generated by anammox provided an organic carbon source needed to support growth of the Ignavibacteria. In addition, the previous analysis indicated that Ignavibacteria did not have complete pathways for vitamin and amino acid biosynthesis, and hypothesized that vitamin B12 generated by anammox could be assimilated by the Ignavibacteria   . The prec carbon –68. Rece carbon –. In suppbacteria . HoweverThis study provides a first glimpse of the coordinated response of members of a PNA microbiome to changes in environmental stimuli brought about by establishing aeration cycles. Our hypothesis that CRP/FNR family TFs would play a central role in the transcriptional response of community members was confirmed by finding TFs of this family in 11 of the most active 16 MAGs in this PNA microbiome. Although the remaining 5 most active MAGs   did not The methodology we used, which relied on using PWMs obtained from well-studied bacterial TFs to search for DNA binding sites upstream of genes in MAGs, predicted the regulon of different CRP/FNR family TFs and, in many cases, supported hypotheses for the role of PNA community members that were inferred from either the physiology of the individual community members, the presence/absence of specific genes, or transcript abundance measurements , 74. Conin silico predictions of the regulon of a specific TF could be tested experimentally by genetic and other approaches. However, it is often difficult or impossible to study the transcriptional networks of a microbiome since most of the major organisms are usually not culturable. Further research is needed to develop strategies that allow experimental verification of DNA binding sites of TFs in specific community members. Thus, adapting high-throughput approaches such as chromatin immunoprecipitation (ChIP) combined with DNA sequencing (ChIP-seq) (in vitro DNA affinity purification sequencing (DAP-seq)   or in viDAP-seq)  to studyFinally, the strategies and methodologies used in this research could be used to begin elucidating transcriptional regulatory networks in other microbiomes. From the perspective of wastewater treatment, knowing how key members of the communities are wired to respond to cyclic environments could help identify optimal conditions for creating the desired aeration cycles and simplify the trial-and-error basis on which current practices and new treatment processes are developed and optimized.A 3-liter lab-scale bioreactor was originally inoculated with biomass obtained from the full-scale York River Treatment Plant , which uses a PNA deammonification process (DEMON) to treat reject water from the solids dewatering facility . The labThe bioreactor was a sequencing batch reactor operated with three 8-h cycles per day. Each cycle consisted of a 4-h fill period, where approximately 1 liter of feed was pumped into the bioreactor, followed by a 3.5-h postfill period. Subsequently, the biomass settled for 3 min before 1 liter of supernatant was decanted over a 12-min period. Then, the reactor remained idle for 15 min before beginning the next 8-h cycle. With one-third of the reactor filled and decanted at each cycle, the hydraulic retention time (HRT) was 24 h. The solids retention time (SRT) was not controlled, with biomass leaving the reactor only during sampling or with the decanted supernatant. Intermittent aeration  was maintained during the 7.5-h fill/postfill periods. During aeration, air was supplied with an aquarium pump at a flow rate between 0.1 and 0.15 liter/min. During the 10-min aerated period, microaerobic conditions were maintained using a LabVIEW control program  to turn off air delivery if DO was above 0.2 mg/liter and turn it on when DO was below 0.2 mg/liter. The bioreactor was heated to keep the water temperature between 30 and 35°C. The pH was uncontrolled but remained at an average of 7.5 ± 0.3. A magnetic stir bar and stir plate were used to mix the culture at 250 to 300 rpm in the 7.5-h fill/postfill periods.4+-N plus NH3-N), TSS, and VSS were conducted following Standard Methods for the Examination of Water and Wastewater (2-N) and nitrate (NO3-N) were measured with high-performance liquid chromatography (HPLC) using a RestekUltra Aqueous C18 column  and detection by UV at 214 nm in a Shimadzu HPLC system .To evaluate reactor efficiency, tests of total chemical oxygen demand (tCOD), soluble chemical oxygen demand (sCOD), ammonium-N  at the University of Wisconsin Biotechnology Center. For DNA, an average insertion size of 550 bp was used, and 2 × 250-bp reads were generated. For cDNA, 1 × 100-bp reads were generated. Raw DNA and cDNA (mRNA) read data were deposited as a BioProject at National Center for Biotechnology Information (NCBI) (see “Data availability” below).DNA samples from the four sampling days were sequenced twice, resulting in 8 DNA sequencing read files. Low-quality DNA sequencing reads were removed using Sickle , with a http://merenlab.org/2016/06/22/anvio-tutorial-v2). Briefly, after simplifying the assembly scaffolds headers with anvi-script-reformat-fasta, the reads that passed quality control criteria were mapped back to the assembly using the bbwrap option of BBMap v38.22  using the gene annotations and searching for FNR, CRP, DNR, NnrR, and MalR descriptors . For eacE. coli , 40–42, li 40–43–  study accession number SRP235198. RNA sequencing reads are available through the NCBI SRA accession number SAMN14405149.MAGs and annotations can be found on the National Center for Biotechnology Information (NCBI) website under BioProject accession number


Background: Heart valve disease is very common, with approximately 5 million people diagnosed with this disease annually in the United States. There has been substantial innovation in the technologies and techniques of surgical repair and replacement over the past decade. However, there is little information that allows the potential time savings associated with these technologies and techniques to be quantified from the hospital perspective.Objectives: The study objective was to estimate the variable cost per operating room (OR) minute in valvular procedures – aortic valve replacement (AVR), mitral valve replacement (MVR) and mitral valve repair (MVRepair) – and determine if there is a difference in OR cost per minute between traditional sternal versus less invasive right thoracotomy surgical techniques.Methods: The Premier database was queried from 2007 to 2011 for patients undergoing AVR, MVR, or MVRepair. Patients were identified using the International Classification of Diseases, 9th Revision (ICD-9) procedure codes. Propensity score matching created cohorts adjusted for patient differences and surgical approach –any sternal incision  or right thoracotomy (RT). Regression analysis was performed to estimate the OR cost per minute based on heart valve procedure.Results: There were 2,656 heart valve procedures – 1,604 AVR, 434 MVR and 618 MVRepair – that met the inclusion criteria. The mean OR cost per minute for AVR procedures was $26.49 and $25.16 (p <0.01) for Sternal and RT, respectively. MVR procedures by surgical approach had a mean OR cost per minute of $25.66 and $25.00 and (p NS) for Sternal and RT, respectively. MVRepair procedures by surgical approach had a mean OR cost per minute of $25.17 and $24.40 and (p NS) for Sternal and RT, respectively. The overall estimate of the OR cost per minute for valvular procedures was $25.99.Conclusions: Quantifying the variable cost of an OR minute from a multi-institution database provides researchers with an important benchmark to use in economic evaluations of valvular procedures.


Observational studies have suggested that herpesvirus infection increased the risk of Alzheimer’s disease (AD), but it is unclear whether the association is causal. The aim of the present study is to evaluate the causal relationship between four herpesvirus infections and AD.We performed a two-sample Mendelian randomization analysis to investigate association of four active herpesvirus infections with AD using summary statistics from genome-wide association studies. The four herpesvirus infections  are caused by varicella-zoster virus, herpes simplex virus type 1, and Epstein-Barr virus (EBV), respectively. A large summary statistics data from International Genomics of Alzheimer’s Project was used in primary analysis, including 21,982 AD cases and 41,944 controls. Validation was further performed using family history of AD data from UK Biobank .P = 0.017, FDR-corrected P = 0.034). It has been verified in validation analysis that mononucleosis is also associated with family history of AD . Genetically predicted shingles were associated with AD risk , while genetically predicted chickenpox was suggestively associated with increased family history of AD .We found evidence of a significant association between mononucleosis (caused by EBV) and risk of AD after false discovery rates (FDR) correction (odds ratio [OR] = 1.634, 95% confidence interval [CI] = 1.092–2.446, Our findings provided evidence supporting a positive relationship between mononucleosis and AD, indicating a causal link between EBV infection and AD. Further elucidations of this association and underlying mechanisms are likely to identify feasible interventions to promote AD prevention.The online version contains supplementary material available at 10.1186/s13195-021-00905-5. The possibility of an infectious etiology for Alzheimer’s disease (AD) has long been suspected, including the roles of viruses, bacteria, and parasites. Recent meta-analyses have investigated and suggested that some herpesvirus infections were associated with a higher risk of AD , 2, espeMendelian randomization (MR) is an analytic approach using genetic variants as instrumental variables (IVs) for an exposure. Like randomized control trials, MR analyses reduce confounding and reverse causality due to the random allocation of genotypes from parents to offspring . MR analThe four herpesvirus infections involved in the present MR study have been reported to be linked with AD , 2, whicIn the present study, we adopted the two-sample MR approach to assess the causal associations between four active herpesvirus infections  and the risk of AD using summary statistics from GWAS, that is, to evaluate the effects of VZV, HSV-1, and EBV infection in AD risk.For infections, we used the GWAS summary statistics data from 23andMe cohort . IndividIn primary analysis, we used summary statistics data from a meta-analysis GWAS performed by International Genomics of Alzheimer's Project (IGAP) . IGAP isP value (P < 5 × 10−8) with a minor allele frequency greater than 0.01 were considered as potential instruments. Independent SNPs were selected at a threshold of linkage disequilibrium (LD) r2 > 0.05 and a distance of 1000 kb. For palindromic SNPs, we aligned strands using allele frequency and discarded palindromic SNP(s) that had minor allele frequency above 0.42. If any SNP was unavailable for an outcome summary data, we used LDlink (https://ldlink.nci.nih.gov/) API [r2 = 0.8. Then, exposure–outcome datasets were harmonized. We have considered the palindromic SNPs and checked original datasets to avoid reverse effects. We computed the proportion of variance in the phenotype explained by IV. The strength of the genetic instrument was judged by F-statistics, with a strong instrument defining as an F-statistic > 10 [α = 0.05 using R code provided by Burgess S [Only single nucleotide polymorphisms (SNPs) associated at genome-wide significance ov/) API , 16 to itic > 10 . Lastly,urgess S . The proI2GX statistic  [Q statistic to test the presence of heterogeneity or directional pleiotropy . Moreover, visual inspection of the forest plot, scatter plot, and leave-one-out plot were also used to assess the MR “no horizontal pleiotropy” assumption  method. The IVW method will return an unbiased estimate in the absence of horizontal pleiotropy or when horizontal pleiotropy is balanced . All cauof NOME) . We usedP values below 0.05 but not survived the FDR correction were considered as suggestive of a potential association. Analyses were conducted using R version 3.6.3, with the MR analysis performed using the “TwoSampleMR” package version 0.5.2 [To correct for multiple comparisons (four exposures), we applied a Benjamini–Hochberg false discovery rates (FDR) correction. on 0.5.2 , 28.HCP5 gene, P = 2.48 × 10−9), two SNP for chickenpox , two SNP for cold sores , 15 SNPs for shingles in primary analysis and 13 SNPs in validation . The result has been verified in UK Biobank GWAS and yielded similar patterns of effects with P value .The estimates and 95% confidence interval (CI) for the Wald ratio or IVW analysis and the numbers of SNPs are presented in Fig. P = 0.758) and was not associated with family history of AD in validation . These findings concur with the results of the previous paper on HSV [P = 0.202), but showed suggestive association with the family history of AD in validation .Using IVW method, genetically predicted cold sores was not associated with risk of AD in primary analysis . Interestingly, the weighted median and MR-PRESSO methods support this significant association with P < 0.05. However, the estimate of association between shingles and family history of AD was marginally significant in validation , and the point estimate direction was opposite to primary analysis. The Cochran Q statistic for shingles indicated no notable heterogeneity across instrument SNP effects  and MR-PRESSO global test for shingles both suggested no horizontal pleiotropy (P > 0.05) in primary analysis and in validation. Additionally, for chickenpox, cold sores, and mononucleosis, we performed pleiotropy analyses by examining previously published GWAS to identified associated traits , which is a very strict threshold. Although the proportion of variance explained by IVs was not very high, the F-statistics were all highly above the threshold of weak instruments of F-statistic < 10 [Mendelian randomization rests on three key assumptions . The reltic < 10 . The othtic < 10 , 31. AccThe exposures in our MR analysis were defined by the self-reported history of infection diseases  rather tWe found evidence that mononucleosis (mainly caused by EBV)  was assoAlthough the specific mechanism underlying the association between infection and AD has not been fully understood, studies have proposed several possible mechanisms. Some have suggested that herpesviridae infection could promote the accumulation of amyloid-β plaques in brain . CarboneThere was no clear evidence to suggest an effect of chickenpox or shingles on AD. Although primary analysis showed a significant association between shingles and risk of AD, it was not validated in independent data, and the direction of point estimates in primary analysis and in validation analysis was the opposite. The two infection diseases are caused by VZV; however, chickenpox results from primary infection of VZV, while shingles are caused by the reactivation of the latent VZV within a dorsal root ganglion. Thus, the effect of the two infectious diseases on AD may be different. Although observational studies have found that VZV DNA and sera VZV antibodies showed no positive correlations with AD risk –37, it iAPOE-ε4 allele increases AD risk [APOE genotype and other possible confounders previously, they also suggested that both carriage of and reactivated HSV-1 infection increased the risk of developing AD [Our MR results showed no significant association between cold sores, mainly caused by reactivation of herpes simplex virus type 1 (HSV-1), and AD risk. Accumulating evidence suggest HSV-1 alone does not confer an elevated risk of AD , 40, 41, AD risk , 42, 43.oping AD . A likeloping AD . HoweverThere are potential limitations to this study. First, some exposures have only one or two available SNP in our study, and the phenotypic variance tagged by SNP instruments was low . However, it is unlikely to affect the statistical power for our MR analyses. Because the primary results are validated using an independent GWAS, and the sample size of datasets in validation is large enough to give a high power. Second, a general challenge of MR is the persistent possibility of horizontal pleiotropic associations between exposure and outcome. To avoid horizontal pleiotropy, we did pleiotropy analysis and checked phenotypes of each SNP. And based on current knowledge, we found no other associated traits were confirmed to have direct effects on AD. On the other hand, our results are less likely to be affected by pleiotropy and heterogeneity due to the small number of SNPs . Third, In conclusion, we found a positive association between mononucleosis and the risk of AD, as well as an association between mononucleosis and family history of AD from MR analysis. Further elucidation of this association could provide insights into the potential biological roles of mononucleosis in AD pathogenesis.Additional file 1. Mendelian randomization analyses of the association between herpesvirus infections and Alzheimer's disease.Additional file 2. Sensitivity analysis, heterogeneity analysis and pleiotropy analysis of the association between shingles and Alzheimer's disease.Additional file 3. Summary statistics for the genetic variants used to assess the effect of herpesvirus infections on Alzheimer's disease in the present Mendelian randomization study.Additional file 4. Single SNP analysis of the association between mononucleosis, cold sores, chickenpox, shingles and Alzheimer's disease.Additional file 5. Evidence of association (p<5×10-8) of significant SNP with other traits.Additional file 6. Leave-one-out plots, forest plots, and scatter plots.