Anti-seizure medication (ASM) treatment is one of the significant risk factors associated with abnormal vitamin D status in epilepsy patients. Multiple studies have shown that adult epilepsy patients can exhibit vitamin D deficiency. However, there are few reports investigating pediatric epilepsy patients. In this study, we aimed to identify risk factors related to hypovitaminosis D in pediatric epilepsy patients in Thailand.A cross-sectional retrospective cohort study was conducted in 138 pediatric epilepsy patients who received anticonvulsants from April 2018 to January 2019. Demographic data, seizure types, puberty status, physical activity, duration, and types of anti-seizure medications were analyzed. Patients with abnormal liver function, abnormal renal function, and who received vitamin D supplements or ketogenic diet containing vitamin D were excluded. Levels of serum vitamin D (25(OH)D) were measured.All 138 subjects were enrolled, the age ranged from 1.04 – 19.96 years; (mean = 9.65 ± 5.09), the mean serum 25(OH) D level was 26.56 ± 9.67 ng/ml. The prevalence of vitamin D deficiency was 23.2% and insufficiency was 47.8% respectively. Two risk factors—puberty status  and non-enzyme-inhibiting ASMs therapy —were significantly associated with hypovitaminosis D, as shown by multivariate analyses.Our study reports the high prevalence of hypovitaminosis D in pediatric epilepsy patients in Thailand despite being located in the tropical zone. These findings can guide clinicians to measure vitamin D status in pediatric epilepsy patients particularly when they reach puberty and/or are using non-enzyme-inhibiting ASMs therapy. Early detection of vitamin D status and prompt vitamin D supplementation can prevent fractures and osteoporosis later in life.http://www.clinicaltrials.in.th/).TCTR20210215005 ( Epilepsy is one of the most common neurological disorders, globally as well as in Thailand. The prevalence rate of epilepsy in Thailand was approximately 7.2 per 1000 population D), a prehormone that is the major circulating form of vitamin D and is used to determine an individual’s vitamin status. Circulating 25(OH) D is eventually metabolized in the kidneys to a more biologically active form known as calcitriol 2D), which serves various functions such as increasing calcium and phosphorus absorption in the intestines, inhibiting the secretion of parathyroid hormone, and modulating the formation and development of bones and teeth [Additionally, vitamin D deficiency is another known adverse effect of ASMs  Essentiand teeth .Anti-seizure medications can reduce vitamin D levels, increase parathyroid hormone levels, and cause hypocalcemia and hypophosphatemia, resulting in an increased risk of bone fracture in patients with long-term ASM use . In addiThe prevalence rate of vitamin D deficiency in epileptic patients in the United States is reportedly 11.9% in adults and 25% in children aged between 3 and 17 years . ComparaIn previous studies, risk factors for vitamin D deficiency are found to be female sex, adolescents over 12 years of age, high BMI, developmental delay and limited physical activity, enzyme-inducing ASMs, polytherapy, using drugs over a long period of time, Indian race, and exposure to sunshine less than 60 min per day –10.However, there are few reports of the prevalence of hypovitaminosis D, including vitamin D insufficiency and deficiency, in pediatric epilepsy patients treated with different ASMs. We aim to determine the prevalence and risk factors of hypovitaminosis D in children with epilepsy.This study was designed as both a cross-sectional study to evaluate the prevalence of vitamin D deficiency and a retrospective cohort study to investigate the factors affecting vitamin D levels in children with epilepsy who were treated with ASMs. Ethical approval was obtained from the Institutional Review Board of the Royal Thai Army Medical Department (R227h/60). Informed consent was obtained from the patients or their parents/guardians.We enrolled 138 pediatric epilepsy patients aged 1–20 years at the Neurology pediatric clinic at Phramongkutklao Hospital from April 2018 to January 2019. Patients that had abnormal kidney or liver function, received vitamin D supplements, or were on a ketogenic diet containing vitamin D were excluded.The data collected from the patients included age, gender, weight, height, BMI, types of seizures, and cause of epilepsy. Puberty status was assessed using Tanner staging. Daily activity abilities were divided into two categories; normal activity and limited activity, such as walking with aid, using a wheelchair, or bedridden. Developmental status was initially evaluated using the Denver II test. If developmental delay was suspected, patients under 6 years of age were assessed with Mullen Scales of Early Learning (MSEL), and the children older than 6 years of age were tested with the Wechsler Intelligence Scale for Children-Third Edition. The duration of sun exposure between 10 am and 2 pm per day were assessed through an interview and recorded.The name, number, and duration of the ASM treatments were recorded. Participants were divided into the enzyme-inducing ASM , valproate, and benzodiazepine (BZD) and newer ASMs  groups based on the type of ASM used.Serum 25(OH) D levels were used to categorize the vitamin D status, which was defined as vitamin D sufficiency: 25(OH) D level >  30 ng/ml (75 nmol/L), vitamin D insufficiency: 25(OH) D level 20–30 ng/ml (50–75 nmol/L), and vitamin D deficiency. 25(OH) D level < 20 ng/ml (75 nmol/L). In this study, we combine the insufficiency and deficiency groups into the hypovitaminosis D group for the purpose of our analyses.nmol/L). In thp-value < 0.05 was considered statistically significant.Statistical analyses were performed using IBM SPSS Statistics 25.0 for Mac . Fisher’s exact and the chi-square test were used to analyze categorical data. The Mann–Whitney U test was used to analyze continuous data. Logistic Regression was used to predict risk factors of hypovitaminosis D. A n = 32) and that of Vitamin D insufficiency was 47.8% (n = 66). Thus, 71% (n = 98) of the patients had hypovitaminosis D.The prevalence of vitamin D deficiency was 23.2% .Univariate logistic regression analyses revealed that the potential risk factors of hypovitaminosis D were puberty and ASM use of more than 2 years, with a 4.9- and 2.7-times increased risk of hypovitaminosis D, respectively  higher risk of hypovitaminosis D than pre-pubescent patients. Patients who had received non-enzyme-inhibiting ASMs had a 3.5-times  higher risk of hypovitaminosis D than those who received enzyme-inhibiting ASMs .Which role the drugs play for the vitamin D deficiency is still uncertain , 11, 12.We found that the prevalence rate of vitamin D deficiency in pediatric epilepsy patients in Bangkok, Thailand was 23.2%, which was similar to the rates reported in previous studies conducted in Thailand  and MalaSimilar to the previous reports, we also found puberty status to be a key risk factor of hypovitaminosis D. This may be because puberty represents a critical period of skeletal bone mineralization, which results in a high rate of vitamin D utilization and increased need for vitamin D, leading to high rates of deficiency .The other factors included in our analyses, namely BMI, cause of epilepsy, physical activity status, developmental status, ASMs used for monotherapy or polytherapy, and duration of sun exposure, were not significantly associated with hypovitaminosis D.More than 2 years of ASM use was significantly associated with hypovitaminosis D in the univariate analysis but not in the multivariate analysis. This may be because various drugs were simultaneously studied, and they may affect vitamin D levels through different mechanisms.Interestingly, 53 of the 138 patients (38%) were receiving an enzyme-inhibiting ASM . The multivariate analysis revealed that those treated with non-enzyme-inhibiting ASMs had a 3.5-times higher risk of hypovitaminosis D than those treated with enzyme-inhibiting ASMs. This finding is similar to those of previous studies that found normal levels of active vitamin D in patients receiving valproate therapy –16. One Among our participants, the most commonly used newer ASMs were levetiracetam (57.9%) and topiramate (38.4%). As seen in previous studies, we found no statistically significant association between newer ASM use and vitamin D level. This may be because newer ASMs, except for topiramate and oxcarbazepine, do not affect CYP450, the enzyme that regulates the metabolism of vitamin D in the liver , 20. Addp = 0.092, Fig. Among newer ASMs, topiramate has unique pharmacokinetics in that it acts as an enzyme-inhibiting agent (metabolized via CYP2C19 in the liver) at low doses (< 200 mg/day in adults) and as an enzyme-inducing agent (metabolized via CYP3A4) at high doses (≥ 200 mg/day) . Hence, We recognize that our study has a limitation of a small sample size. A multicenter study with a large number of participants should be conducted in the future to confirm our results.Our study shows a relatively high prevalence rate of vitamin D deficiency and vitamin D insufficiency of 23.2 and 47.8%, respectively, even though Thailand is located in the tropical zone. The key risk factors of hypovitaminosis D are puberty status and non-enzyme-inhibiting ASM use. There is currently a lack of guidelines on the appropriate time to assess the vitamin D status of epilepsy patients. Our findings suggest that clinicians who treat pediatric epilepsy patients, especially those entering puberty and being prescribed non-enzyme-inhibiting ASMs should consider regular monitoring of vitamin D adequacy. In cases of hypovitaminosis D, clinicians should recommend prompt vitamin D supplementation as it is essential to prevent pathological fractures and osteoporosis for the patients in later life.


To investigate the retinal changes in choroideremia (CHM) patients to determine correlations between age, structure and function.n = 8) and ≥50-year (n = 5) old groups. Patients were seen at baseline, 6-month, and 1-year visits. Optical coherence tomography (OCT), OCT angiography, and fundus autofluorescence were performed to measure central foveal (CFT) and subfoveal choroidal thickness (SCT), as well as areas of preserved choriocapillaris (CC), ellipsoid zone (EZ), and autofluorescence (PAF). Patients also underwent functional investigations including visual acuity (VA), contrast sensitivity (CS), colour testing, microperimetry, dark adaptometry, and handheld electroretinogram (ERG). Vision-related quality-of-life was assessed by using the NEI-VFQ-25 questionnaire.Twenty-six eyes from 13 male CHM patients were included in this prospective longitudinal study. Participants were divided into <50-year . CS and VA did not change over the year, but CS was significantly correlated with age . Delayed to unmeasurable dark adaptation, decreased colour discrimination and no detectable ERG activity were observed in all patients. Minimal functional deterioration was observed over one year with a general trend of slower progression in the ≥50-years group.Over the 1-year follow-up period, progressive loss was detected in SCT, EZ, CC, PAF, and CFT. Those ≥50-years exhibited more structural and functional defects with SCT, EZ, CC, and PAF showing strong correlation with patient age (Quantitative structural parameters including SCT, CC, EZ, and PAF are most useful for disease monitoring in CHM. Extended follow-up studies are required to determine longitudinal functional changes. CHM gene (OMIM 303390) encoding Rab-escort protein 1 (REP1), which is involved in lipid prenylation and intracellular protein trafficking. The primary site of pathogenesis remains unknown, but dysfunctional REP1 results in accumulated unprenylated Rabs, with abnormal photoreceptor outer segment phagocytosis and ineffective melanosome transport within the RPE leading to cell death . The. The8]. Central retinal sensitivity was assessed using the Macular Integrity Assessment (MAIA) microperimetry system (CenterVue SpA). The default “Expert Exam” mesopic and scotopic protocols were used. Mean sensitivity was recorded, and the colour-coded sensitivity map was exported Figs.  and 3e.FAutomated static perimetry was performed using the Octopus 900 perimeter (Haag-Streit Diagnostics). Sensitivity was measured at a custom 148-point grid covering 60° area. Tests with “Reliability Factor” greater than 33% were excluded. Mean sensitivity (MS), mean defect (MD), vertical and horizontal extent of the residual visual field (in degrees) parameters were collected for the purpose of statistical analysis Fig. .-3 cd/m2 (3 log units). Rod intercept time longer than 6.5 min indicated dark adaptation dysfunction [The AdaptDx dark adaptometer (MacuLogix) was used to measure dark adaptation. A bleaching photoflash (80% rod bleaching level) was used and testing duration was 13 min Figs.  and 3, Gfunction .−3 u′ v′ units for the protan and deutan vectors, and those exceeding 150 × 10−3 u′ v′ units in the tritan vector were considered abnormal.The Cambridge Colour Test v2.31 (Cambridge Research Systems Ltd) was used to assess the colour vision deficiency. The basic “Trivector” test version was performed to estimate discrimination thresholds along the protan, deutan, and tritan axes. Measurements exceeding 100 × 10Photopic flash (2 Hz) and flicker (30 Hz) ERGs were recorded using the handheld RETeval full-field ERG device . White flash stimuli were presented to natural pupils and periorbital skin surface electrodes were used to record the ERG.Vision-related quality of life was assessed by using the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25) . Patientp values for multiple comparisons. The correlation between age and structural and functional parameters were investigated using Spearman’s rank correlation coefficient (rho) and scatter plots. Pairwise partial correlations between structural and functional measurements were calculated while controlling for age (r) [Statistical analysis was performed using SPSS v. 25.0 (IBM Corporation), Microsoft Excel 2017 (Microsoft Corporation), and R. All measurements were reported as population mean ± standard deviation (SD) after filling in the missing longitudinal data using the last observation carry‐forward method . Intra- Thirteen male CHM patients  from 8 pedigrees were included in this study. Twelve patients completed the 6-month follow up visit, and eleven participants completed the 1-year follow up visit. Genetic variants for study participants are reported in Supplemental Table Sp = 0.006, GEE). Similarly, CS was significantly worse in group 2 .Central vision is generally preserved in patients with choroideremia, especially during early stages of the disease. At baseline, all eyes were able to see at least 55 letters on the ETDRS chart, except three eyes from three different patients (in group 2) which had a BCVA of 42 letters, hand motion, and light perception, with no apparent cause other than the choroideremia. In group 1, eyes were able to see on average 82.0 ± 7.8 letters on the ETDRS chart , whereas in group 2 it was 55.2 ± 31.6 letters (Snellen 6/24). The differences were found to be statistically significant between groups  but not so for VA . Longitudinally, neither visual acuity nor contrast sensitivity showed significant progression over the 1-year follow up period within group 1  or group 2 .We observed a trend of decreasing VA and CS with increasing patient’s age Fig. ; the corEn face OCTA of the choroid showed marked peripheral degeneration of CC, as well as the attenuation of the deeper large choroidal vessels  were also clearly detected at the transitional zone between the degenerated and relatively preserved photoreceptor regions in all eyes  . Meanwhile, longitudinal changes in group 2 were not statistically significant . However, the percentage rates of change in CC and EZ areas were statistically equivalent in both groups . The preserved areas of CC and EZ at baseline showed moderate negative correlation with patient’s age   Fig. .Table 2Sp = 0.36 and 0.29, respectively, GEE).Central foveal (CFT) and subfoveal choroidal thickness (SCT) measurements at baseline, 6-month, and 1-year visits are summarised in Table p = 0.18, GEE). Patients in group 2 showed marked thinning of mean SCT by 45% when compared to group 1 . Over the 1-year period, slow but relatively consistent progressive thinning was detected in the central fovea in patients of both groups . But between baseline and 1-year measurements, the 3.3% and 6.7% foveal thinning in groups 1 and 2, respectively, were found to be statistically significant . The rate of retinal thinning appeared to be slightly faster in older patients, but the difference in percentage progression between both groups was not significant .At baseline, no statistically significant difference was observed between groups 1 and 2 for CFT . Both groups showed statistically significant differences in SCT measurements between the 3 visits . The rate of choroidal loss between all visits  was statistically significant . SCT showed a strong negative correlation with patient’s age    Fig. . However16) Fig. .Patients with CHM typically show progressive concentric loss of autofluorescence, retaining an island of preserved AF (PAF) at the macula. Areas of PAF tended to be vertically expanded, and favouring the central and the temporal side of the macula Figs.  and 3c. p = 0.01, GEE), with statistically significant progressive shrinkage of PAF between scans 1-year apart  and at 6-month intervals (p < 0.05). The absolute rate of PAF area reduction in mm2 was significantly larger in group 1 when compared to group 2 . However, the percentage rate of progression was statistically similar between both . A moderate negative correlation was observed between patient’s age and the area of PAF   Fig. .Nineteen eyes from 12 patients had reliable mesopic and scotopic microperimetry testing during the baseline visit. All eyes had cone and rod system dysfunction, with the rod-mediated measurements being more severely affected.p < 0.001, GEE). Similarly, in dark-adapted conditions, group 1 had significantly higher sensitivity to cyan and red stimuli  .Group 1 had significantly better mesopic function when compared to group 2 . Meanwhile, scotopic sensitivity to cyan and red stimuli were only weekly and insignificantly associated with age at baseline .The baseline mesopic sensitivity correlated moderately with age  in our cohort had a normal rod intercept time of 5.34 min  Fig. . The DA p < 0.001, GEE) . Similarly, we did not observe significant associations between age and baseline colour sensitivities in our cohort of patients with CHM .Eleven patients 22 eyes) underwent the colour testing at baseline and/or 1-year follow-up visit. All patients showed some degree of colour vision impairment, especially for the blue-yellow spectrum. Higher chromatic discrimination thresholds were observed in group 2 compared to group 1 in the protan, deutan, and tritan axes . Additionally, the SCT showed moderate association with CC, EZ, and PAF areas , whereas, the CFT was poorly associated with preserved retinal areas .The photoreceptors, RPE and choriocapillaris layers were closely related targets for the degeneration seen in CHM. The measured preserved areas of CC, EZ, and AF correlated very strongly with one another  .CFT was the only structural parameter which correlated with visual acuity (06) Fig. . The decr = 0.87, p < 0.001) . The CFT and SCT were not associated with mesopic or scotopic sensitivity (to cyan and red stimuli) in our cohort of patients.In microperimetry, the area of EZ was the largest determinant of mesopic macular sensitivity (01) Fig. . Area ofr = 0.72 and 0.75, p = 0.007 and 0.004, respectively) . The vertical extent of the preserved VF was most strongly related to the area of the EZ  .Visual field parameters were investigated for the relationship with retinal structure. MD was significantly negatively correlated with preserved areas of CC and EZ (ly) Fig. , but not01) Fig. , and to r = −0.64 and −0.55, p = 0.016 and p = 0.04, respectively) and VA    Fig. . However07) Fig. .en face images were generated for direct visualisation with reliable delineation and measurement of areas of preserved EZ and CC. The definition of EZ and CC slabs were based only on BM segmentation, which is easier and less laborious to segment compared to the EZ line [This longitudinal study provides detailed characterisation of structural and functional abnormalities in CHM patients. It is the first to describe the rate of structural loss in CHM within different age groups. CHM causes progressive concentric retinal and choroidal degeneration, with a characteristic preserved small island of retinal tissue at the macula until late stage of the disease. OCTA acquires simultaneous structural and angiographic data, allowing for the concurrent investigation of the outer retina and choroid –23. In t EZ line . Althoug EZ line . However EZ line , 26, whiAlthough, EZ, PAF islands and CC had very strong correlations between their area measurements, the temporal relationship between photoreceptors, RPE and CC loss is controversial. In our study, CC and EZ images were produced from the same scan, thus we were able to perform direct comparisons between these areas. But as FAF imaging covers a larger area than OCTA, direct comparison between PAF and CC/EZ area measurements was not possible. Nearly 70% of eyes showed more degeneration in the CC than the EZ. Additionally, a steady decrease in choroidal thickness was observed at the foveal centre of all patients, which long preceded any observable central changes on OCT or FAF. These findings suggest that photoreceptor and RPE loss are secondary to the vascular changes in the CC and/or deeper choroidal vessels. Histopathologic studies previously suggested a primary vascular problem in CHM due to defects in vascular endothelial cells of the choroidal vessels , 28. AddCystic macular oedema was a common feature in our cohort. The prevalence of unilateral and bilateral CMO in our cohort  are very similar to previous reports of 62.5% and 50.0%, respectively . HoweverThe preserved retinal island generally allows patients to retain good VA and CS until the degeneration reaches their fixation centre, usually after the fourth or fifth decade of life. In our cohort, patients with poorest VA and CS were older (≥ 50 years) and exhibited severe degenerative changes at the fovea, forcing them to use the relatively preserved adjacent parafoveal regions for fixation. These findings are consistent with VA findings from previous reports , 31. We Our investigations of the colour discrimination in our cohort revealed higher prevalence of tritan deficiency (blue colour) compared to protan-deutan (red-green colours). The blue colour discrimination is mediated by S-cones which are most prevalent in the parafoveal region, while the red and green colours discrimination is thought to be dependent on the L- and M-cones, with highest density at the centre of the fovea. The spatial distribution of different classes of cone photoreceptors might provide an explanation for the variation in patients’ perception sensitivity to different colours. The relative preservation of the central retinal island can retain more L- and M-cones than S-cones, maintaining the red-green discrimination until more advanced disease stages. The observed correlation between red-green sensitivity and VA also agree with the aforementioned explanation since VA is also dependent on the cones at the centre of the fovea. Meanwhile, this correlation was not observed with tritan discrimination. A recent study by Seitz et al. reported similar results by using the “ellipses” test from the Cambridge Colour Test . By contrho. However, we did not observe significant differences on the longitudinal data from microperimetry testing. Previous microperimetry reliability studies in CHM patients revealed high inter-test variability, especially at the borders of the atrophic tissue [Visual field and mesopic microperimetry showed marked constriction, with the area of preserved sensitivity closely following the extension of the retained retinal tissue, with no apparent residual function outside the central retinal island. Jolly and colleagues reported exponential relationship between mean sensitivity and age , which sc tissue . FatigueWith the advent of several therapeutic approaches for CHM , 39, 40,The main limitations of this study were a relatively small sample size with only adult participants and a relatively short period of follow-up time (1 year). Given the slow-progressive nature of CHM, longer follow-up with representation from the paediatric population would confirm the findings of this study and provide more insight into the earlier changes seen, guiding optimal intervention age. We only recruited participants retaining some degree of visual function to be able to comply with study procedures. This can potentially produce a selection bias by excluding severely affected patients. However, in our cohort, we had several eyes with advanced CHM with central degeneration and severe visual impairment. Another limitation of the study is the utility of manual segmentation of BM and areas of CC, EZ, and PAF which can be relatively time consuming and prone to intra- and inter-grader variability. The use of advanced image processing and machine learning techniques might enhance the visualisation and automated analysis of ophthalmic images –44.In conclusion, longitudinal multimodal assessment of patients with CHM provides valuable information on the natural history of structural and functional status of the degenerating eyes. Structural parameters appear more sensitive for subtle short-term changes. Detailed correlation analyses between retinochoroidal structure and function can give some insight into the pathophysiological processes. Further studies are needed for better characterisation of the progression patterns in patients with CHM.Previous cross-sectional and retrospective studies suggested progressive degeneration of the RPE and photoreceptors.Emerging clinical trials for the treatment of choroideremia require the identification of proper metrics to monitor disease progression and response to treatment.The findings of this prospective longitudinal study suggest that vascular choroidal abnormalities precede apparent photoreceptor loss in choroideremia.Structural parameters appear more sensitive for monitoring subtle short-term changes in choroideremia.Supplementary InformationSupplementary Table S1Supplementary Table S2Supplementary Figure S1


Dear Editor,Platelet functions are well known to surpass thrombosis and hemostasis and platelets play an important role in inflammatory and profibrotic mechanisms. Thrombocytopoiesis is traditionally considered to occur in the bone marrow. However, thrombocytopoiesis was shown to occur in the lungs as well where circulating megakaryocytes release platelets and are responsible for up to 50% of total platelet production . The speU test and the Kruskal–Wallis-analysis-of-variance with the post-hoc test by Conover). Categorical variables were presented as frequencies and percentages and were analyzed using the Χ2-test. Time to event analyses for survival were based on the Kaplan–Meier method. The log-rank test and the Cox regression were used for univariate and multivariate survival analyses. Follow-up was considered from the time of hospital admission up to 114 days, including the post-discharge period . Clinical outcomes of interest , bacteriemia, venous and arterial thromboses proven by objective imaging and laboratory methods, major bleeding defined by the International-Society-on-Thrombosis-and-Hemostasis criteria) were considered during the hospitalization period and were evaluated as binary variables. The logistic regression was used for univariate and multivariate analyses for the prediction of non-survival outcomes. All analyses were done using the MedCalc statistical software, version 20.014 .We have retrospectively analyzed a cohort of 5876 consecutively hospitalized COVID-19 patients who were admitted to our tertiary-level institution  in the period from March 2020 to June 2021, had baseline complete blood count available, and did not have known prior or subsequent MPN. COVID-19 severity on admission was defined by the World-Health-Organization guidelines as mild, moderate, severe, and critical. Clinical and laboratory data used in the study are part of the hospital registry project and were obtained by analysis of written and electronic medical records. A total of 88.8% of patients received LMWH thromboprophylaxis in dose intensity per the assessment of treating physicians, 74.6% received steroids and 14.9% received remdesivir. Acetylsalicylic acid use corresponded to baseline indication for chronic use (18%). Platelet count and mean platelet volume (MPV) were obtained by Advia 2120i counter . The normality of the distribution of numerical variables was tested using the Shapiro–Wilk test. Since the majority of numerical variables did not have normal distribution they were presented as the median and interquartile range (IQR) and were analyzed using the non-parametrical tests , and median MPV was 9 fL, IQR (8.4–9.7). A total of 1120 (19.1%) patients presented with thrombocytopenia (platelets <150 × 109/L). Three-hundred-and-six (5.2%) patients had platelets <100 × 109/L and 55 (0.9%) had platelets <50 × 109/L. A total of 270 (4.6%) patients had thrombocytosis (platelets ≥450 × 109/L). Sixty-three (1.1%) patients had platelets ≥600 × 109/L.Among 5876 patients, there were 3299 (56.1%) males. The median age was 72 years, IQR (62-81), and median Charlson comorbidity index (CCI) was 4 points, IQR (3–6). COVID-19 severity on admission was mild in 528 (9%), moderate in 281 (4.8%), severe in 4168 (70.9%) and critical in 899 (15.3%) patients. The median platelet count was 221 × 10P < 0.05 for all comparisons) and of similar age, sex and comorbidity burden as patients with the normal number of platelets (differences not significant (n.s.)). Regarding specific comorbidities, arterial hypertension, diabetes mellitus, hyperlipoproteinemia, and active smoking were similarly distributed between subgroups of patients with different platelet count (n.s.) whereas patients with thrombocytosis were significantly less likely to be obese and more likely to have active malignancy  than patients with normal platelet count, and less likely to have liver cirrhosis, chronic kidney disease or active anti-cancer therapy than patients with thrombocytopenia . COVID-19 severity on admission and presence of pneumonia did not significantly differ between subgroups of patients with different platelet count. However, the duration of COVID-19 symptoms significantly gradually increased in subgroups of patients presenting with increasing platelet count, with patients with thrombocytopenia presenting earlier and patients with thrombocytosis presenting later during the disease course in comparison to patients with normal platelet count .Patients’ characteristics stratified by platelet count on admission are shown in Table P < 0.05 for all comparisons). MPV was not significantly associated with the duration of symptoms prior to admission. MPV was significantly negatively correlated with platelet count .Patients with higher MPV were more likely to be older, of the male sex, to have higher CCI and more severe COVID-19 on admission (P = 0.016) and thrombocytopenia  with in-hospital mortality rates of 24.1%, 31.5%, and 45.6%, respectively. Gradually improving overall survival could be observed in patients with higher platelets, even with further subdivision of patients with low, normal, and elevated platelet count as shown in Fig. P < 0.001 for difference and P < 0.001 for trend; platelets stratified to <50, 50–99, 100–149, 150–299, 300–449, 450–599 and ≥600 × 109/L). Similarly, patients with thrombocytosis were less likely to experience respiratory deterioration and require MV in comparison to patients with normal and low platelet count , less likely to experience bacterial sepsis  and less likely to experience major bleeding . Patients with thrombocytosis had a higher propensity to develop VTE in comparison to patients with normal and low platelet count  whereas there was no significant difference in the occurrence of arterial thrombosis . Further considering thrombotic events, platelet count was significantly higher in both patients experiencing PE vs. other  and DVT vs. other  whereas there was no significant association of platelet count with arterial thrombosis subtypes (n.s). Neither presence of active malignancy, active treatment, nor specific malignancy subtype showed statistically significant associations with VTE occurrence (n.s.). Univariate associations of MPV with clinical outcomes are summarized in Supplementary Table P < 0.001 for difference and P < 0.001 for trend; in-hospital mortality rates of 27.5%, 28.4%, 35.4%, and 45.6% for 1st–4th MPV quartile, respectively) and patients belonging to 4th in comparison to 1st MPV quartile also had a higher risk for MV  and arterial thrombosis , whereas there were no significant differences in venous thrombosis, bacteriemia and major bleeding between patients belonging to different MPV quartiles (n.s).During hospitalization a total of 1991 (33.9%) patients died, 1033 (17.4%) required MV, 609 (10.4%) experienced bacteriemia, 362 (6.2%) venous thromboembolism (VTE), 326 (5.5%) arterial thrombosis and 184 (3.1%) major bleeding. Considering VTE, a total of 135 (2.3%) patients experienced deep venous thrombosis (DVT) and 260 (4.4%) experienced pulmonary embolism (PE). Considering arterial thrombosis events, a total of 102 (1.7%) patients experienced myocardial infarction, 139 (2.4%) cerebrovascular insult, 66 (1.1%) peripheral artery thrombosis and 18 (0.3%) mesenterial thrombosis. Univariate associations of platelet count with clinical outcomes are summarized in Supplementary Table Above mentioned associations of platelets and MPV with clinical outcomes (with the exception of MPV and MV) remained statistically significant in the multivariate analyses adjusted for platelets, MPV, age, sex, COVID-19 severity, and CCI. Independent prognostic factors and associated adjusted hazard/odds ratios are shown in Supplementary Table There are several important points that we would like to emphasize. A much smaller proportion of COVID-19 patients presents with thrombocytosis 5%) than with thrombocytopenia (19%). Platelet count seems to be dominantly affected by specific comorbidities and duration of COVID-19 as opposed to its severity on presentation. Patients with higher platelets were more likely to have a longer duration of COVID-19 symptoms, stronger stimulation of myelopoiesis, and different, probably more mature inflammatory profile . Elevated platelets were accompanied by a more favorable course of the disease, with less need for MV and less deaths. Even among patients with low, normal, and elevated platelets, further subgroups of patients could be stratified over whom a linear trend of improved survival with increasing platelets exists. Mechanisms behind these observations are elusive but could be attributable to the protective role of platelets for the lung parenchyma and more effective viral clearance by a higher number of platelets. Higher platelets were also associated with a lower rate of bacterial sepsis highlighting their important role in the immune system. It is of special interest that the frequency of VTE was significantly higher among COVID-19 patients with higher platelet count. These findings highly resemble phenomena reported in MPN patients with COVID-19 where patients with essential thrombocythemia (ET) who presented with higher platelet count in comparison to other MPN subsets % than wi. Hence, Our findings are limited by single-center experience, retrospective study design, and no data on intensity and frequency of use of anticoagulant and antiplatelet therapies at the specific time of thrombotic events. Although we excluded patients with known or subsequently proven MPN, some patients could still have unrecognized MPN but their number is likely to be very low considering MPN incidence in an overall population. The main strengths of our study are a large sample size and adequate statistical power to perform presented analyses. These results provide new insights into the biology of thrombosis and hemostasis in COVID-19 patients where abnormalities in hematologic findings are common and difficult to interpret directly. The reactive thrombocytosis in COVID-19 patients seems to have important clinical correlations and deserves further research.Supplementary Tables and Figures


Scientific Reportshttps://doi.org/10.1038/s41598-021-89564-y, Published online 19 May 2021Correction to: The original version of this Article contained errors.The citations to Figure 4 and Figure 5 were interchanged in the Results and Discussion sections.In addition, in the legend of Figure 5,“”now reads:“(see Equation 2)”Finally, in the Data Availability section,https://radar.kit.edu/radar/en/dataset/XqHmFKADBYNNKqTi?token=LOkISQJRuNCAMUMwUzjC (https://doi.org/10.35097/441). Repository KITopen of the Karlsruhe Institute of Technology with DOI identifiers. Repository KITopen of the Karlsruhe Institute of Technology with DOI identifiers. Regarding the reservoir operations at the Upper Atbara Dam, the supporting data are available from Tractebel Engineering GmbH but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Tractebel Engineering GmbH. Data of the Multivariate ENSO Index Version 2 (MEI.v2) can be accessed via https://www.psl.noaa.gov/enso/mei/.”“Basin-averaged seasonal forecast and reanalysis data that support the findings of this study are available under now reads:https://radar.kit.edu/radar/en/dataset/XqHmFKADBYNNKqTi (https://doi.org/10.35097/441). Regarding the reservoir operations at the Upper Atbara Dam, the supporting data are available from Tractebel Engineering GmbH but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Tractebel Engineering GmbH. Data of the Multivariate ENSO Index Version 2 (MEI.v2) can be accessed via https://www.psl.noaa.gov/enso/mei/ (Supplementary Information 1).”“Basin-averaged seasonal forecast and reanalysis data that support the findings of this study are available under The original Article has been corrected.


Noggin and RNA-binding protein for multiple splicing 2 (RBPMS2) are known to regulate the expression of smooth muscle cells, endothelial cells, and osteoblasts. However, the prognostic role of combined Noggin and RBPMS2 expression in resected gastric cancer (GC) is unclear.A total of 163 patients with GC who underwent gastrectomy were included in this study. The expression of Noggin and RBPMS2 proteins in tumor cells at the tumor center and invasive front of resected GC was evaluated by immunohistochemistry, and in conjunction with clinicopathological parameters the patient survival was analyzed.n = 86, 52.8%) and low at the invasive front , while Noggin protein expression was high in both tumor center  and the invasive front . Noggin expression at the invasive front and tumor center was significantly decreased in advanced T stage, non-intestinal-type . RBPMS2 expression at the invasive front was significantly decreased in non-intestinal-type and positive lymphatic invasion (P <  0.001 and P = 0.013). Multivariate analysis revealed that high Noggin protein expression of the invasive front was an independent prognostic factor for overall survival , but not at the tumor center .RBPMS2 protein expression was high at the tumor center (Our study indicates that high Noggin expression is a crucial prognostic factor for favorable outcomes in patients with resected GC.The online version contains supplementary material available at 10.1186/s12885-021-08273-x. Gastric cancer (GC) is the fourth most common cancer and the third leading cause of cancer-related death globally . RecentlTumor stroma is composed of cancer-associated fibroblasts, immune cells, and other stromal cells around the cancer cells of extracellular matrix (ECM), which includes cell-adhesion molecules and ligands that play a role in tissue organization . BesidesNOGGIN and RNA-binding protein for multiple splicing 2 (RBPMS2), cathepsin F (CTSF) and CUE domain containing 1 (CUEDC1), in resected GC using machine learning method [Stromal gene expression or phenotype is one of the independent prognostic markers in various cancers including GC . Recentlg method  for effeg method . Orthotog method . RBPMS2,g method , 11. It g method . Perturbg method .However, the expression pattern and prognostic values of Noggin and RBPMS2 in GC has yet to be determined. Furthermore, the expression of stroma-related proteins were not known to have different clinical implications between the tumor center and invasive regions in GC. Therefore, we evaluated the expression of Noggin and RBPMS2 proteins in the tumor center and invasive front of resected GC and compared their relationship with clinicopathological parameters and clinical outcomes.NOGGIN and RBPMS2 and positively correlated genes, we ranked genes based on their co-expression measured via linear-by-linear association test with NOGGIN and RBPMS2. Subsequently, the ranked gene lists were used to identify the enriched KEGG pathways using Gene Set Enrichment Analysis (GSEA) via GSEA-P [Genes predicting the prognosis of patients with GC were identified using the support vector machine algorithm for the microarray analysis of three publicly available gene expression profiles  containing 822 samples of resected GC. To identify the molecular function enriched in a GSEA-P .We performed a retrospective analysis of patients with GC at Uijeongbu St. Mary’s Hospital of the Catholic Medical Center between 2001 and 2005. The inclusion criteria were: pathologically confirmed adenocarcinoma, radical resection without preoperative radiation or chemotherapy, removal of at least 15 or more lymph nodes, and available tissue specimens. Neoadjuvant chemotherapy or radiotherapy was permitted. Pathological staging was based on the 7th edition of the American Joint Committee on Cancer staging criteria.Tissue microarrays (TMA) were constructed for immunohistochemistry (IHC). The tissue cores (2 mm) were obtained from two representative paraffin block-embedded tumor regions in each case, containing invasive front, tumor cells or clusters at the perpendicularly deepest site of tumor invasion, and tumor center, the area that is equidistant for the tumor surface . BrieflyIndependent Samples T-test was conducted to determine the association between protein expression and clinicopathological parameters. To study linear trends for the proportions of positive staining for the expression level of Noggin and RBPMS2 proteins, a crosstabulation was performed using chi-square tests. Overall survival (OS) was determined from the date of surgery to the date of death due to any cause or the last follow-up visit. Disease-free survival (DFS) was calculated from the date of surgery to the date of the first disease recurrence or last follow-up.P-value < 0.10 in the univariate analysis were included in the multivariate analysis. Survival rates and hazard ratio (HR) s are reported with 95% confidence intervals (CIs). A P-value of < 0.05 is considered statistically significant. The statistical analyses were performed using R statistical programming language version 3.4.1 (https://www.r-project.org).DFS and OS rates were measured using the Kaplan-Meier method, and statistical differences between the cumulative survival curves were evaluated using the log-rank test. Cox proportional hazards regression models were used to evaluate the significance of the prognostic factors. All variables with a NOGGIN, RBPMS2, CTSF, and CUEDC1, showed a moderate performance with an area under ROC curve (AUC) of 0.745 for the prediction of 5-year survival [NOGGIN and RBPMS2, we conducted GSEA for KEGG pathways and identified the enriched pathways  and the tumor center . High expression of Noggin was observed in 72 (61.3%) patients at both tumor center and invasive front  was more frequent than in the invasive front . Low RBPMS2 protein expression at the invasive front was associated with non-intestinal type and positive lymphatic invasion . Low expression of RBPMS2 protein in the tumor center was significantly correlated with the non-intestinal type and positive lymphatic invasion (P <  0.001 and P = 0.047). Further, the investigated proteins showed significant associations between Noggin and RBPMS2 proteins in the invasive front as well as the tumor center  positivity and peritoneal seeding, was explored Table . Low NogP = 0.029, Fig. P = 0.476, Fig. P = 0.925, Fig. P = 0.446, Fig. P = 0.111, Fig. P = 0.290, Fig. We assessed the correlation between Noggin and RBPMS2 expression, and clinical prognosis using Kaplan-Meier curves with log-rank test. The elevated expression of Noggin at the invasive front was significantly associated with longer OS (P <  0 .001), advanced pathologic T stage (P <  0.001), lymph node metastasis (P <  0.001), lymphatic invasion (P <  0.001), vascular invasion (P = 0.030), no R0 resection (P <  0.001), and peritoneal seeding positivity (P <  0.001). High Noggin protein expression and EBV positivity at the invasive front were significantly correlated with better OS (P = 0.030 and P = 0.020), but not the high Noggin protein expression in tumor center lesions and RBPMS2 protein expression at the invasive front and tumor center (Table P = 0.036) was a significant independent prognostic indicator for OS (Table P = 0.054), without reaching statistical significance in multivariate analysis , suggesting its role to maintain gastric gland morphology. On the other hand, our results suggest the negative prognostic effect of Noggin protein expression in the patients with resected GC. Consistent with these results, several previous studies showed that low levels of Noggin protein in combination with high BMP expression are associated with poor prognosis in esophageal carcinoma and increased metastasis in prostate and esophageal cancer [The growth of normal human gastric epithelial cells requires Noggin, EGF, R-spondin1, and Wnt3a . Consistl cancer , 28. In l cancer . In addil cancer . Furtherl cancer . Interesl cancer , and medl cancer . These fl cancer .RBPMS family is generally represented by two paralogs in vertebrates, RBPMS and RBPMS2 , which sInterestingly, the expression of the Noggin protein at the invasive front is strongly correlated with prognostic factors, compared with those at the tumor center. Invasive cancer is a complex process that is related to cell attachment, matrix dissociation, migration, and tumor cell proliferation . Thus, tThere are some limitations to our study. Because of the retrospective nature of this study, it might not reflect the biological properties of the entire population with GC. The investigated protein expression in the tumor center and the invasive front was compared via TMA instead of whole tumor sections. However, to compensate for the limitations of TMA, the tumor tissue was obtained in the most representative portion. Further, the differences in the interpretation of IHC staining method, tissue aging effects, staining techniques, and differences in enzyme antibodies used between studies contribute to the study limitations. Despite our limitations, we included a homogenous population that underwent standard surgical treatment for GC and was followed-up long-term.In surgically resected GC, Noggin protein expression was strongly correlated with RBPMS2 expression. Besides, high Noggin expression in the invasive front of the tumor was independently and significantly associated with prognosis. Taken together, these results suggest that Noggin may act as a prognostic biomarker in patients with resected GC.Additional file 1: Supplementary Figure 1. The molecular function of Noggin and RBPMS2. Gene set enrichment analysis (GSEA) of positively correlated genes with Noggin and RBPMS2 in GSE62254, GSE15459, and GSE15460 datasets showed “Focal adhesion” and “ECM receptor interaction” signatures, which were enriched and positively correlated with both Noggin and RBPMS2 expression, respectively (A). KEGG pathway analysis of positively correlated genes with Noggin and RBPMS2 associated with the GO terms shown on the left side (B). Normalized Enrichment Scores are represented by the bars, where the adjusted p-values  ≤ 0.05 are presented with a green bar or FWER > 0.05 with a red bar. Supplementary Figure 2. Immunohistochemical staining of noggin  and RBPMS2  in normal gastric mucosa. Foveolar cells are negative for noggin and RBPMS2, while both proteins are weakly expressed in parietal cells. Supplementary Figure 3. Kaplan-Meier survival curves for disease-free survival were plotted according to Noggin and RBPMS2 protein expression. Noggin protein expression at the invasive front (A) and tumor center (B); RBPMS2 expression in the invasive front (C) and tumor center (D). DFS, disease-free survival; RBMPS2, RNA-binding protein for multiple splicing 2; IF, invasive front; TC, tumor center.Additional file 2: Supplementary Table 1 Clinicopathological factors and association according to RBPMS2 and NOG expression in tumor center lesions. Supplementary Table 2 Univariate and multivariate analyses of disease-free survival in all patients using Cox proportional-hazards model.


Persistent fatigue among colorectal cancer (CRC) patients might be associated with unfavorable body composition, but data are sparse and inconsistent. We studied how skeletal muscle index (SMI), skeletal muscle radiodensity (SMR), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) at diagnosis are associated with fatigue up to 24 months post-diagnosis in stage I–III CRC patients.SMI, SMR, VAT, and SAT were assessed among 646 CRC patients using pre-treatment computed tomography images. Fatigue at diagnosis, at 6, and 24 months post-diagnosis was assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire. The association of SMI, SMR, VAT, and SAT with fatigue (yes/no) was assessed using confounder-adjusted restricted cubic spline analyses.p = 0.01) non-linear association of higher levels of SAT with higher prevalence of fatigue was observed. Lower levels of SMR were linearly associated with higher prevalence of fatigue at 6 months post-diagnosis . None of the body composition parameters were significantly associated with fatigue at 24 months.Prevalence of fatigue at diagnosis was 18%, at 6 months 25%, and at 24 months 12%. At diagnosis, a significant  contains supplementary material, which is available to authorized users. Fatigue is highly prevalent among stage I–III colorectal cancer (CRC) patients. Fatigue is often already experienced before diagnosis  and can Fatigue among cancer survivors is thought to have a multi-factorial etiology. Fatigue has been associated with treatment (radio- and chemotherapy), stage of disease, presence of (multiple) comorbidities, specific medications with sedating side effects, psychological factors , decreased physical activity, and malnutrition , 5–7. SeThe aim of the current study was to investigate the association of body composition parameters , SMR, VAT, and SAT) at diagnosis with fatigue at three timepoints  in stage I–III CRC patients.Colorectal cancer: Longitudinal, Observational study on Nutritional and lifestyle factors that may influence colorectal tumor recurrence, survival, and quality of life [For this study, we used data of the ongoing COLON study:  of life . In elevThe COLON study was approved by the Committee on Research involving Human Subjects, region Arnhem–Nijmegen, the Netherlands, and all study participants provided written informed consent.Fatigue was assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 (EORTC QLQ-C30) version 3.0  at three2) of skeletal muscle, VAT, and SAT were assessed using standard radiodensity thresholds measured in Hounsfield units (HU) in preoperative CT images at the level of the 3rd lumbar vertebrae using Slice-O-Matic 5.0 . For skeletal muscle, the threshold values were between − 29 and + 150 HU, for VAT between − 150 and − 50 HU, and for SAT between − 190 and − 30 HU [2/m2) to calculate skeletal muscle index (SMI). SMR was assessed as the mean radiodensity of the total skeletal muscle cross-sectional area at the level of the 3rd lumbar vertebrae.Cross-sectional areas  .We calculated that 600 patients were needed to have 80% power to detect a prevalence ratio of larger than 1.6 or smaller than 0.6, assuming that 40% of the population would suffer from fatigue  and an aT test (continuous variables) or Pearson χ2 test .Demographic, clinical, and lifestyle characteristics are presented as mean and standard deviation (SD) for continuous variables with normal distribution, median, and interquartile range (IQR) for continuous variables without normal distribution or frequency and percentage for categorical variables. Differences in patient characteristics were analyzed using the independent Restricted cubic spline (RCS) analyses  were usep value for interaction. In none of the analyses, gender was identified as effect modifier. Potential confounders were included in the final model if they change the PR for fatigue with 10% or more when the variable was individually added to a crude model including SMI/SMR/VAT/SAT, age, stage of disease, and gender. For all analyses, potential confounders were SMI , SMR , total adipose tissue , comorbidities , tumor site (colon/rectum), and physical activity . For the analyses at 6 and 24 months, additional potential confounders were chemotherapy (yes/no), radiotherapy (yes/no), stoma placement after surgery (yes/no), and complications post-surgery (yes/no).All analyses were tested for effect modification by gender, by calculating the p < 0.05. Statistical analyses were carried out using IBM SPSS v.23 .The statistical significance level for the analyses was set at The study population consisted of 960 patients, of which we had to exclude 192 patients because no suitable CT image was available; 68 patients because of stage IV disease; 11 patients because of missing information on stage of disease; and 63 patients because no fatigue data at diagnosis were available   Fig. . PRs witp = 0.02, test for non-linearity p = 0.14), where lower levels of SMR were associated with higher prevalence of fatigue  and at 24 months post-diagnosis .A significant linear association between SMR and fatigue was observed at 6 months post-diagnosis   .Levels of SAT were non-linearly associated with fatigue at diagnosis, where higher levels of SAT were significantly associated with higher prevalence of fatigue


Usnea articulata, Parmotrema tinctorum, and Heterodermia obscurata, found on Mount Cameroon, for potential macrofilaricidal activity. Organic extracts were screened for anti-Onchocerca activity against Onchocerca ochengi isolated from cattle skin using worm motility and MTT formazan assays. Toxicity of highly active extracts was investigated on monkey kidney epithelial (LLCMK2) cells and in BALB/c mice (2000 mg/kg body weight) including effects on liver enzymes. The methanol extract of P. tinctorum (Pammet) was the most active against adult male worms (IC50 = 8.1 μg/mL) with the highest selectivity index (SI = 21.3). U. articulata was the most active against the adult female (IC50 = 36.3 μg/mL) but had a low SI value (3.4). No mortality and no adverse effects were recorded in the acute toxicity test. These two most active extracts had no significant effect on liver enzymes, alanine aminotransferase, and aspartate , but a high AST : ALT ratio (2.59) for Pammet indicates likely reversible adverse hepatic toxicity. The high macrofilaricidal activity and selectivity of P. tinctorum suggest it is a potential source of new macrofilaricides which should be further investigated to identify its bioactive constituents.Onchocerciasis is a parasitic infection affecting a relatively small population globally but has very devastating pathological outcomes. Ivermectin and recently moxidectin are the only drugs approved for clinical management of the disease, both of which have several limitations. In particular, they are efficacious against microfilariae  with no activity against adult worms . Promising anthelmintic activity has been reported in some lichens. This study investigated three lichens,  Onchocerca volvulus, an infectious filarial worm, features among the World Health Organization's neglected tropical diseases [Onchocerciasis (river blindness), caused by diseases . The infdiseases , 3. It tLoa loa coendemicity. Recently, moxidectin, a chemical analogue of ivermectin, was approved for clinical use against onchocerciasis but is also microfilaricidal and has potential serious toxicity [Vector control programmes later complemented by community-directed treatment with ivermectin have made significant achievements in reducing the burden and negative impact of the disease, but onchocerciasis is yet to be eliminated . Chemothtoxicity . Hence, Heterodermia boryi macrolichen against Indian earthworm Pheretima posthuma. Similar results were reported by Kumar et al. [Usnea articulata (L.) Hoffm, Parmotrema tinctorum (Despr. ex Nyl.) Hale, and Heterodermia obscurata (Nyl.) Trevis, Nuovo Giorn, not previously tested for anti-Onchocerca activity, using the in vitro model, Onchocerca ochengi, a known close relative of O. volvulus. The cytotoxicity and acute toxicity of the active lichens were also investigated. In further studies, compounds in the active extracts will be isolated and investigated in view of finding potential new efficacious drugs for treatment of onchocerciasis.Efforts in the discovery of anti-Onchocerca agents especially macrofilaricides have included screening of natural products with promising results . Of inter et al. , againstUsnea articulata (L.) Hoffm, Parmotrema tinctorum (Despr ex Nyl.) Hale, and Heterodermia obscurata (Nyl.) Trevis, Nuovo Giorn, were collected in September 2018, from Mount Cameroon and identified by Dr. Ayuk Elizabeth Orock, a Lichenologist in the Department of Botany, Faculty of Science, University of Buea. The extracts were prepared similarly as described by Bate et al. [The three lichens, e et al. . Brieflye et al. .γ-irradiated L-glutamine powder, 5% newborn calf serum (NBS), 200 units/mL penicillin, 200 μg/mL streptomycin, and 0.25 μg/mL amphotericin B; pH 7.4. Incomplete culture medium (ICM) did not contain NBS. All reagents used were from Sigma-Aldrich. All experiments were done twice.Complete culture medium (CCM) was prepared using RPMI 1640 media containing sodium bicarbonate. It was supplemented with 25 mM HEPES, 0.3 g 2 in HERACELL-150i incubator (USA), during which time male worms emerged from the masses into the medium and females remained within. Thereafter, the viability of worms and sterility of cultures were evaluated using an inverted microscope  prior to testing and 1 mL of CCM was added in each well containing worm. A primary screen was done to eliminate inactive extracts. Each extract  diluted in CCM to 4x final test concentration was added (1 mL) in triplicate to an adult worm in 3 mL CCM per well giving final concentration of 500 μg/mL. Positive (10 μM auranofin) and negative control (2% DMSO in RPMI) were included. The plate was incubated for 5 days same as above, and male worm viability was assessed using an inverted microscope based on worm motility; inhibition of motility was scored as follows: 100% (no motility), 75% , 50% , 25% (only little change in motility), and 0% (no observable change in motility). Activity against female worms was assessed by visual estimation of percentage inhibition of formazan formation following incubation of the worm masses in 500 μL of 0.5 mg/mL MTT -2,5-diphenyltetrazolium bromide) for 30 min [μg/mL), to determine the IC50 values [This was done as described  with somr 30 min ; activit0 values .μL/well) and incubated as above and grown until fully confluent. Microfilariae (mf) were isolated from cattle skin as described in detail [μL of CCM/well) containing the LLCMK2 cell layer, and their viability and sterility were ascertained for 24 h prior to addition of extracts. Each extract solution was tested in duplicate wells at 500 μg/mL. Positive and negative controls were included followed by incubation as above. Mf viability was scored daily by mean motility as above for 5 days. A secondary screen was done similarly [50 value of one extract only.These were performed similarly as the assays on adult worms described in detail in previous studies , 14 withn detail , 14, susμg/mL. Positive and negative controls were included followed by incubation for 5 days same as above. The medium was discarded, and wells were decolorised by shaking with ICM (IKA Labortechnik KS125 basic shaker) at 600 rpm for 5 minutes twice. MTT (100 μL of 1 mg/mL in ICM) was added, and the plate was incubated as above for 2 hours. Then, the medium was discarded, 100 μL of DMSO was added to dissolve formazan precipitate, and the plate was shaken gently to mix well contents. Well optical densities were read at 595 nm, and percentage inhibition was calculated using the formula:This was also done using monkey kidney epithelial cell . BrieflyU. articulata, and methanol extract of P. tinctorum each in 2% DMSO, as described [O 014/2019). Briefly, fifteen BALB/c mice comprising one control and two test groups of five each  from the animal house of the study laboratory were acclimatised for one week. One animal per test group was fasted overnight with water provided and administered 2000 mg/kg body weight of one extract by oral gavage and further observed [This was done for the most active extracts on the adult worms, the hexane extract of escribed , 16. Guiobserved . With no50 of active extracts. The logarithm of the extract concentration was plotted against activity, to obtain a nonlinear regression curve-fitting and a variable slope sigmoidal dose-response curve. CC50 (concentration at which 50% of cells were dead) was derived from a plot of % inhibition of viability against log extract concentration generated using the same software. Enzyme activities were analyzed using the same software, and an unpaired two-tailed t-test was used to check for any significant difference (P < 0.05) between the control and test groups.Filaricidal activity data were analyzed using GraphPad Prism 5.0 software (USA) to obtain ICP. tinctorum had the highest yield (12.9%) while the methanol extract of H. obscurata had the lowest yield (1.1%), U. articulata (USahex).The methanol extract of μg/mL were active against adult male and female O. ochengi worms with 100% inhibition in both cases in the primary screen. In the secondary screen, the IC50 values ranged from 8.1 to 52.4 μg/mL on adult male worms; the methanol extract of P. tinctorum (Pammet) was the most active with the IC50 value of 8.1 μg/mL. IC50s on adult female worms ranged from 36.3 to 115.2 μg/mL; the hexane extract of U. articulata (USahex) was the most active with an IC50 value of 36.3 μg/mL  were the most active against adult male worms (IC50 = 15.6 μg/mL), and both extracts of U. articulata (USahex and USamet) were the most active against adult female worms (IC50 = 125 μg/mL), showing consistency in activity  giving a value of 101.8 μg/mL.On microfilariae, all the 6 extracts screened at 500 50 values greater than the cut-off point for lack of cytotoxicity (CC50 > 30 μg/mL) [hex) had a CC50 value lower than 30 μg/mL indicating mild cytotoxicity (P. tinctorum (Pammet) which showed a high selectivity index (21.33). Extracts of H. obscurata were more toxic to the mammalian kidney cells with SI values < 1. On O. ochengi microfilariae, USamet recorded an SI value of 1.25.Five extracts had CC0 μg/mL) , and onetoxicity . All extmet and USahex, on O. ochengi adult male and female worms, respectively, showed no sign of acute toxicity at 2000 mg/kg body weight in Balb/c mice 14 days posttreatment. The animals were sluggish within the first 2 hours postdosing, but no mortality was recorded at day 14. When compared with control mice, intake of food and water were similar. Average weights of mice increased in both control and test groups, but the difference between the groups on day 14 was not statistically significant (P = 0.43). No change in physical appearance, behavior, fur, skin, and mucous membranes and physical activity was observed.The most active extracts, PamU. articulata (USahex), which was the most active against female worms, an unpaired two-tailed t-test was done for aspartate aminotransferase (AST) at 95% confidence interval (P < 0.05) because both control and test groups had the same variances. This was also done for alanine aminotransferase (ALT) with Welch's correction because the variances were not the same. There was no significant difference between the control (2% DMSO) and test (USaHex) groups with P values of 0.77860 and 0.5943 for AST and ALT, respectively, as shown in In order to compare the effect on liver enzymes of the hexane extract of met for body weight (P = 0.28), and enzyme activities with P values of 0.59 and 0.52 for AST and ALT, respectively, as shown in Also, no significant difference was found between the control and mice treated with PamP. tinctorum, with high selectivity for the adult male worm. Also, moderate activity was shown by the hexane extract of U. articulata against the adult female with a relatively low selectivity . Overall, the lichen extracts were more active against the adult male worm (lower IC50s) than the female. This difference may be due to the smaller size of the male worm which permits greater penetration and bioavailability in the worm tissue of the bioactive secondary metabolites in the extracts, or action via target(s) uniquely present in the male which needs to be further investigated. The promising properties of P. tinctorum warrant further investigation. This may yield an efficacious macrofilaricide which upon killing the male worm will prevent fertilization of the female and hence abolish production of microfilariae. The resultant effect will be a marked improvement in patients' welfare since microfilariae are largely responsible for the devastating pathology of onchocerciasis. Partial screening of the extracts on microfilariae also revealed activity against this worm stage indicating potential macro- and microfilaricidal activity in the tested lichens. Further studies will be done to fully assess their microfilaricidal activity. A study of P. tinctorum including other Parmotrema species found in Malaysia reported weak antibacterial activity for this lichen, contrary to two others whose extracts and isolated pure natural products were considerably active [All the extracts were active against adult worms with ICy active . Resultsy active .H. obscurata), with CC50 > 30 μg/mL [P. tinctorum) showed high selectivity for the adult male worm  in rats reported high AST : ALT ratios > 3 with no mortality but significant histopathological changes compared to the control animals indicating liver injury [The two most active extracts against male and female worms did not cause death of treated mice with no adverse effects observed. As evidence of lack of adversity, there was no significant difference in activities of liver enzymes between the control and test groups Figures  and 3. The liver . Howeverhe blood . Since nr injury ; this war injury  demonstrr injury ; this maO. ochengi worm, of the P. tinctorum found on Mount Cameroon; hence, it is a potential source of new macrofilaricides. The secondary metabolites in this lichen should be isolated for further chemical and anthelmintic characterization. The considerable activity of U. articulata against the female worm should also be further explored.This study has revealed high activity and selectivity for the adult male


Sodium intake in China is extremely high and eating in restaurants is increasingly popular. Little research has explored the sodium level of restaurant dishes. The present study aims to assess the content and sources of sodium in Chinese restaurants.Cross-sectional data were obtained from the baseline survey of the Restaurant-based Intervention Study (RIS) in 2019. A total of 8131 best-selling restaurant dishes with detailed recipes from 192 restaurants in China were included. Sodium content per 100 g and per serving were calculated according to the Chinese Food Composition Table. The proportion of restaurant dishes exceeding the daily sodium reference intake level in a single serving and the major sources of sodium were determined.Median sodium content in restaurant dishes were 487.3 mg per 100 g, 3.4 mg per kcal, and 2543.7 mg per serving. For a single serving, 74.9% of the dishes exceeded the Chinese adults’ daily adequate intake for sodium , and 62.6% of dishes exceeded the proposed intake for preventing non-communicable chronic diseases . Cooking salt was the leading source of sodium in Chinese restaurant dishes (45.8%), followed by monosodium glutamate (17.5%), food ingredients (17.1%), soy sauce (9.4%), and other condiments/seasonings (10.2%). More types of salted condiments/seasonings use were related to higher sodium level.The sodium levels in Chinese restaurant dishes are extremely high and variable. In addition to cooking salt, other salted condiments/seasonings also contribute a large proportion of sodium. Coordinated sodium reduction initiatives targeting the main sources of sodium in restaurant dishes are urgently needed. High sodium intake is a public health concern worldwide, as it is linked to elevated blood pressure, which leads to cardiovascular disease –3. In 20A national target of 20% reduction in salt intake by 2030 has been proposed in China’s health development agenda “Healthy China 2030” . Determi“Action on Salt China” (ASC) is a unit for salt reduction, established in June 2017, in collaboration with Queen Mary University of London in the United Kingdom, The George institute for Global Health in China, the Chinese Center for Disease Control and Prevention, and other key national organizations . The ASCThe RIS baseline assessment survey was carried out in 6 provinces of China  in May 2019. For each province, 2 counties of similar socioeconomic level were selected in the provincial capital city. Then, according to the selection criteria, 16 restaurants mainly offering Chinese cuisine were selected from each county, including 4 large-size, 8 medium-size and 4 small-size restaurants . The det2 and ≤ 3000 m2, or number of seats > 250 and ≤ 1000), medium  and small . Chinese cuisine substantially varied across geography areas, and was categorized into four types of dishes based on the cooking method: cold dish, fried dish, soup, and staples/snacks.To understand how sodium levels vary according to different characteristics of the restaurant dishes, we conducted our analyses by region, restaurant size and dish type. Region was defined as North  and South . Restaurant size was classified into large (surface area > 500 mA mobile-based electronic data collection system (EDC) developed by the Beijing University of Aeronautics and Astronautics was used for data collection. The recipes were collected by in-depth interviews with chefs who were familiar with the preparation and cooking of the dishes. The detailed recipes included the following information: name of the dish, edible percentage, ingredients and condiments/seasonings used (with amounts), dish type and cooking method. To improve the accuracy of the estimated amount of condiments/seasonings used, the investigators showed the weighed amount using a usual spoon or other measuring instruments during the interview. Sodium content of each dish was calculated according to the Chinese Food Composition Table, combining the sodium content of all ingredients and condiments/seasonings used for the dish. Sodium content is reported as sodium density , as well as sodium (mg) per serving.Sodium levels in a single serving of the restaurant dishes were compared to the Chinese DRIs: the daily AI  and PI  for adults aged 18 to 49 years old. Restaurant dishes were compared to the Chinese DRIs because restaurant sodium reduction targets have not been established in China. The proportions of restaurant dishes exceeding the daily sodium AI and PI in a single serving were calculated.Based on the recipes, we classified the sources of sodium in restaurant dishes , soy sauce, and other condiments/seasonings. We then estimated their respective contribution (in percentages) to sodium levels in dishes.P < 0.05 as statistically significant.We reported the median and interquartile range (IQR) for mg of sodium per 100 g, mg of sodium per kcal, and mg of sodium per serving in restaurant dishes, by region , restaurant size , and dish type . Descriptive statistics for restaurant dishes, including %AI (sodium per serving divided by 1500 mg), and % of restaurant dishes exceeding the daily sodium AI and PI were conducted. The respective contributions of the main sources of sodium to total sodium content in restaurant dishes were calculated. As sodium values were not normally distributed, the non-parametric Wilcoxon signed-rank test was used to compare the median of sodium content among restaurant dishes stratified by area, restaurant size and dish type. We used SAS 9.4  for data cleaning and analyses, and considered two-sided The analysis included a total 8131 dishes from 192 restaurants in 6 provinces of China. The sample encompassed 3829 (47.1%) and 4302 (52.9%) foods from the North and the South, and 2285 (28.1%), 4162 (51.2%) and 1684 (20.7%) foods from large, medium and small restaurants, respectively. The main dish type was fried dish (83.9%), followed by cold dish (11.7%), soup (3.0%) and staples/snacks (1.4%).Overall, the restaurant dishes contained on average 487.3 IQR: 291.1, 781.9) mg sodium per 100 g. Sodium levels varied significantly by area, restaurant size, and dish type . The median sodium level (mg) per 100 g in foods with 4 types of condiments/seasonings (644.4 mg per 100 g) was 4.8 times that of foods with 1 type of condiments/seasonings (134.8 mg per 100 g), 1.7 times of those with 2 types (380.7 mg per 100 g), 1.3 times of those with 3 types (506.4 mg per 100 g).Among all dishes included, nearly half (45.1%) contained 3 types of salted condiments/seasonings, followed by those containing 2 types (24.2%) and those containing 4 types (23.3%) condiments/seasonings  and PI (2000 mg), respectively.Our findings of the very high and wide-ranging sodium levels in restaurant dishes are in agreement with those reported in several other studies –28. SuchWidespread use of salted condiments/seasonings is another explanation for the high sodium content in restaurant dishes. In leading Canadian chain restaurants, more than 60% of the dishes contained a salt substitute/enhancer, such as yeast extracts, calcium chloride, monosodium glutamate and potassium chloride . In our Many countries have implemented national or regional initiatives for sodium reduction in restaurants , 31–35, In China, cooking habits and consumers’ preferred taste make sodium reduction difficult in restaurant foods. However, attempts to explore effective sodium reduction strategies for restaurant in China have been made . The RISThe strengths of our study include the large sample of restaurant dishes based on both major types of food served in the restaurants and foods frequently ordered by consumers from 6 provinces in China. The notable differences of restaurant food types between China and other countries , 38, calA number of limitations also exist. Sodium content assessing methods usually include laboratory analysis, menu labelling or online nutrition information provided by restaurant companies, and analysis with a nutrient database . There aIn conclusion, our study shows that the sodium content of the majority of popular restaurant dishes in China are extremely high and variable. Further, the large number of restaurant dishes that exceeded the daily AI and PI in a single serving, along with the widespread use of salted condiments/seasonings, demonstrate the need for a Chinese sodium reduction strategy that addresses all the major sodium sources. Coordinated government-led efforts should be implemented, with the participation of restaurants, food manufacturers, and consumers to reduce sodium level in restaurant foods, raise sodium-reduction awareness and ultimately lower population sodium intake.


The heterogenous cytogenetic and molecular variations were harbored by AML patients, some of which are related with AML pathogenesis and clinical outcomes. We aimed to uncover the intrinsic expression profiles correlating with prognostic genetic abnormalities by WGCNA.2 ≥ 0.4, p < 0.01). ORA detected the enriched pathways for the key co-expression modules. The patients in TCGA cohort were randomly assigned into the training set (50%) and testing set (50%). The LASSO penalized regression analysis was employed to build the prediction model, fitting OS to the expression level of hub genes by ‘glmnet’ package. Then the testing and 2 independent validation sets (GSE12417 and GSE37642) were used to validate the diagnostic utility and accuracy of the model.We downloaded the clinical and expression dataset from BeatAML, TCGA and GEO database. Using R (version 4.0.2) and ‘WGCNA’ package, the co-expression modules correlating with the ELN2017 prognostic markers were identified . ORA revealed that the ‘lightyellow’ module was mainly enriched in DNA-binding transcription factor activity and activation of HOX genes. The ‘saddlebrown’ module was enriched in immune response process. And the ‘lightgreen’ module was predominantly enriched in mitosis cell cycle process. The LASSO- regression analysis identified 6 genes  with non-zero coefficients. The risk score generated from the 6-gene model, was associated with ELN2017 risk stratification, relapsed disease, and prior MDS history. The 5-year AUC for the model was 0.822 and 0.824 in the training and testing sets, respectively. Moreover, the diagnostic utility of the model was robust when it was employed in 2 validation sets (5-year AUC 0.743–0.79).We established the co-expression network signature correlated with the ELN2017 recommended prognostic genetic abnormalities in AML. The 6-gene prediction model for AML survival was developed and validated by multiple datasets.The online version contains supplementary material available at 10.1186/s12967-021-02914-2. The prognosis of AML is characterized by clonal cytogenetic and molecular variations harbored by leukemic cells, the prognostic significance of which is validated by previous studies and integrated into the ELN 2017 risk-stratification system .Over the recent years, the increasing genomic and expression data has emerged by next generation sequencing, which provided us the chance to identify genetic and transcriptomic markers relating to clinical outcomes. To improve the power of test for correlation analysis, we utilized the BeatAML database, which was the largest RNAseq dataset for AML patients by now . BeatAMLIn this study, we found significantly correlated gene modules to NPM1, RUNX1 and TP53 mutation, the enriched cell signaling pathways of which were identified. The integrated hub genes across modules were input into LASSO analysis and established prediction model of OS, which was validated by 2 external datasets. Our work offered the landscape of expression signatures relating to ELN2017 prognostic markers and revealed the key hub genes and pathways.http://www.vizome.org/aml/). 14 available markers were selected for further analysis, including ELN2017 risk stratification, complex karyotype, del(7), RUNX1-RUNX1T1, CBFB-MYH11, biallelic CEBPA mutation, MLL3-KMT2A, DEK-NUP214, GATA2-MECOM, FLT3-ITD, NPM1 mutation, ASXL1 mutation, RUNX1 mutation and TP53 mutation. Then the expression dataset was download in the form of RPKM. After the data was integrated, 421 non-APL AML patients with prognostic markers and RNAseq data were selected for WGCNA. Since the patients in BeatAML accepted various experimental target therapy and non-standard treatment, which lead to unignorable bias for survival analysis. So, we included other expression datasets with survival data to establish and validate the prediction model. We downloaded the OS and expression data from TCGA database  . The microarray data and survival information was obtained from GEO database (https://www.ncbi.nlm.nih.gov/geo/), for GSE12417 [The clinical, genetic and transcription matrix was downloaded from BeatAML database  . After the minimal module size was set as 30 genes, the average linkage hierarchical clustering was performed to divide the whole gene set into modules based on TOM-based dissimilarity. Then module membership  and gene significance (the correlation coefficients between gene and prognostic markers) were calculated by Pearson’s method. Modules eigengenes (MEs), the first principal component of expression matrix, were correlated to the target prognostic markers. Correlation coefficient R2 ≥ 0.4 and p value < 0.01 were set as the criteria for significant correlation between MEs and prognostic markers. Then the hub genes were identified by gene significance ≥ 0.2, module membership ≥ 0.8 and q. Weighted < 0.01 .The whole gene set of RNAseq data were used to construct the co-expression network, by R software (version 4.0.2) and ‘WGCNA’ package . The hiehttps://string-db.org/) was used to predict PPI (protein–protein interaction) network information based on the previous evidence and experiments. After mapping the gene symbols into STRING database, minimal criteria for extracting PPI pairs was 0.4. The nodes were calculated and ranked by connectivity degree method, 10 top nodes were screened by cytoscape software (version 3.7.2) and cytohubba plugin.STRING  database (https://david.ncifcrf.gov/) was used for gene enrichment analysis based on the Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The package ‘ReactomePA’ was implemented for analysis based on Reactome database (https://reactome.org/) [To demonstrate the biological function and implication on cell signaling of significantly correlated MEs, DAVID  (Databasme.org/) . The enrThe univariate Cox proportional hazard regression analysis was employed to investigate the association of hub genes and OS of AML patients in TCGA AML cohort. Then to minimize the overfitting, we performed the iterative regression analysis (LASSO) to reduce dimensionality of inputted variables, and establish the prediction model of variables with non-zero coefficients. We conducted a bootstrap aggregation approach, and the tenfold cross-validation by ‘glmnet’ package. The TCGA AML cohort was randomly assigned into training (50%) and testing (50%) sets. The risk scores of individual patients were calculated based on the expression level of gene variables with non-zero coefficients. The cutoff value was determined by function ‘surv_cutpoint’ of package ‘survminer’. Kaplan–Meier analysis and time-dependent ROC were performed by ‘survival’ and ‘survivalROC’ packages. Furthermore, the testing set and independent validation cohorts, GSE12417 and GSE37642, were employed to validate the robustness of diagnostic accuracy on overall survival by the prediction model. Then the risk scores of AML patients were compared by unpaired t test, between ELN2017 risk groups, with or without MDS history, newly diagnosed or relapsed patients. Furthermore, the risk scores and other possible prognostic factors, like age, blast percentage, FAB subtypes, type of induction treatment, race, sex, risk stratification of ELN2017 and transplantation, were inputted into multivariate Cox analysis to validate whether the risk score was an independent risk factor for AML survival.2 > 0.85 , ‘saddlebrown’ module  and ‘lightgreen’ module . The ‘violet’ module was initially identified as significantly correlated with NPM1 mutation (R2 = 0.46/p = 3e-23), while the following detail analysis on NPM1/FLT3-ITD combinations indicated that this module was not significantly related to any combination. So, the ‘violet’ module was excluded from further analysis. According to the criteria of hub genes, 973 hub genes were identified for further analysis  and NPM1 mutation  respectively. The further analysis on combinations of NPM1 mutation and FLT-ITD, the ‘lightyellow’ module was only found to be related to NPM1 mutation, regardless of the presence of FLT3-ITD. Despite the correlation coefficients didn’t meet the criteria, the potential negative correlations were detected for the ‘lightyellow’ module with ELN2017 risk stratification, complex karyotype, RUNX1-RUNX1T1, CBFB-MYH11, CEBPA biallelic mutation, ASXL1, RUNX1 and TP53 by p value less than 0.01.The ‘lightyellow’ module included 143 genes and was positively correlated with FLT3-ITD . The results of ORA for genes in the ‘saddlebrown’ module were shown in Fig. This module included 60 genes and was significantly correlated with RUNX1 mutation . The results of ORA for genes in the ‘lightgreen’ module were shown in Fig. This module included 352 genes and was significantly correlated with TP53 mutation , and result in better validation . TherefoThe risk scores were calculated for 6-gene expression signature for the individual patients in TCGA AML cohort. The risk scores of patients in the adverse ELN2017 risk group, were significantly higher than that of favorable group , 45 out 109 genes . The results of ORA indicated the genes of ‘saddlebrown’ were mainly enriched in pathways involving in immune response, cytokine signaling and antigen presentation. These genes are also implicated in the enriched KEGG pathways, including antigen processing and presentation, hematopoietic cell linage, phagosome, and cell adhesion , RUNX1-RUNX1T1, CBFB-MYH11, CEBPA biallelic mutation, MLLT3-KMT2A, DEK-NUP214, GATA2-MECOM and ASXL1 mutation. This may partly be attributed to insufficient samples. A quantity of gene variations for one specific abnormality , ASXL1) may also abolish the accuracy of our analysis.The 6-gene signature with non-zero coefficients was identified by LASSO penalized regression analysis. AML patients with traditional risk factors  have higher risk scores of 6-gene signature Fig. A–C. The We identified the significantly correlated gene co-expression modules with prognostic cytogenetic or molecular markers of ELN2017. The sequential ORA illustrated the involved pathways for the key modules. Additionally, the novel prediction model was established with robust and superior diagnostic utility based on hub genes obtained from WGCNA.Additional file 1:Figure S1. The hierarchical cluster dendrogram of BeatAML samples. The tips of dendrogram referred to sample ID in BeatAML database. No obvious outliers were found in this figure.Additional file 2: Figure S2. The whole PPI network for the ‘lightyellow’ module.Additional file 3: Figure S3. The whole PPI network for the ‘saddlebrown’ module.Additional file 4: Figure S4. The results of module-trait relationship by WGCNA using TCGA database.Additional file 5: Table S1. The hub genes obtained from WGCNA for AML.Additional file 6: Table S2. The overlapped genes of modules correlating with NPM1 significantly, by analysis based on TCGA and BeatAML database, respectively.