Naegleria, Acanthamoeba, and Balamuthia cause rare but life-threatening infections. All three parasites can cause meningoencephalitis. Acanthamoeba can also cause chronic keratitis and both Balamuthia and Acanthamoeba can cause skin and systemic infections. There are minimal drug development pipelines for these pathogens despite a lack of available treatment regimens and high fatality rates. To identify anti-amebic drugs, we screened 159 compounds from a high-value repurposed library against trophozoites of the three amebae. Our efforts identified 38 compounds with activity against at least one ameba. Multiple drugs that bind the ATP-binding pocket of mTOR and PI3K are active, highlighting these compounds as important inhibitors of these parasites. Importantly, 24 active compounds have progressed at least to phase II clinical studies and overall 15 compounds were active against all three amebae. Based on central nervous system (CNS) penetration or exceptional potency against one amebic species, we identified sixteen priority compounds for the treatment of meningoencephalitis caused by these pathogens. The top five compounds are (i) plicamycin, active against all three free-living amebae and previously U.S. Food and Drug Administration (FDA) approved, (ii) TG02, active against all three amebae, (iii and iv) FDA-approved panobinostat and FDA orphan drug lestaurtinib, both highly potent against Naegleria, and (v) GDC-0084, a CNS penetrant mTOR inhibitor, active against at least two of the three amebae. These results set the stage for further investigation of these clinically advanced compounds for treatment of infections caused by the free-living amebae, including treatment of the highly fatal meningoencephalitis.The free-living amebae   •Focused screen of ReFRAME library (159 compounds) on three free-living amebae.•16 priority compounds selected based on potency, advanced clinical studies and CNS penetration.•Naegleria and Balamuthia.Identified compounds with fast killing kinetics against •Balamuthia trophozoite recrudescence.Identified compounds capable of delaying •5 compounds of high interest for treatment of CNS infections with free-living amebae. However, despite their highly pathogenic nature, there are no optimal established clinical therapies. Infection with N. fowleri results in a rapidly progressive Primary Amebic Meningoencephalitis (PAM), which is a severe brain infection and can lead to death in 5–7 days , which is more common in immunosuppressed individuals, although a few cases of Balamuthia infection in immunocompetent patients have been reported  via the olfactory nerves after entering the nostrils through contaminated water. In the United States from 1962 to 2018, 145 cases were documented with only four survivors, a fatality rate of over 97%  or chlorhexidine along with a diamidine (either propamidine or hexamidine)  . HoweverAcanthamoeba or Balamuthia infection), or corneal scrapings (for Acanthamoeba keratitis) are often needed , skin biopsies  is a long and expensive road with the average cost per drug brought to the market of ~$2.6 billion . Screenidonovani , indicatRepurposing, Focused Rescue and Accelerated MEdicinal chemistry (ReFRAME) library, as well as a few additional related compounds, against trophozoites of the three free-living amebae N. fowleri, A. castellanii and B. mandrillaris. This library was previously demonstrated to be composed of high-value compounds for accelerated drug development efforts and has been successfully used to identify new drug options for Cryptosporidium parvum (Entamoeba histolytica and the active compounds identified from this screen (reframedb.org)   were sel22.1Naegleriafowleri:N. fowleri strain KUL (ATCC) were axenically cultured in Nelson's medium supplemented with 10% FBS at 37 °C  were cultured at 28 °C in PYG medium according to a modified technique  were grown in axenic Cerva's medium (2 and sub-cultured every 3–4 days. All the experiments were performed using trophozoites harvested during the logarithmic phase of growth.s medium  at 37 °C2.HFF-1 cells (gift from John Boothroyd lab) were cultured in DMEM with 1  g/L glucose, Sodium Pyruvate and L-glutamine (Gibco) completed with 10% (vol/vol) FBS, and 100 U/mL penicillin/streptomycin at 37 °C with 5% CO2.2Naegleria and Acanthamoeba: The compounds were screened against N. fowleri and A. castellanii using established parameters (N. fowleri (5000 trophozoites) and A. castellanii (2500 trophozoites) were added to 96-well plated compounds to yield final concentrations spanning from 0.078 μM to 10 μM. Negative controls in the screen plates contained 0.5% DMSO and positive controls contained 50 μM of amphotericin B  for N. fowleri and 50 μM of chlorhexidine for A. castellanii. Assay plates were incubated for 48 h at 37 °C for N. fowleri and for 48 h at 28 °C for A. castellanii and at the end of incubation 25 μL of CellTiter-Glo Luminescent Cell Viability Assay (Promega) were added in each well of the 96-well plates to induce cell lysis. The resulting ATP-bioluminescence of the trophozoites was measured at room temperature using an Envision plate reader from PerkinElmer. Compounds that demonstrated >70% inhibition at 10 μM were retested following the protocol mentioned above, but with the following modifications: for the 8 or 16-point dose response curve, compounds were tested in triplicate at concentrations ranging from 0.0015 μM to 50 μM; the assay was performed in 96-well plates in 100 μL volume with 10,000 N. fowleri trophozoites and 5000 A. castellanii trophozoites. All experiments were performed in triplicate in three independent experiments .rameters . For theBalamuthia. For the initial 8-point dose response study, drug testing was performed by seeding 30 μL of trophozoites (3000 parasites/well) into 384-well opaque white plates and incubating with drug or control at 37 °C for 72 h. Viability was assayed using a CellTiter-Glo assay (Promega) as previously described . For the confirmation and follow up assays, we performed dose response experiments in triplicate in trophozoites, with compound concentrations ranging from 3 nM to 50 μM . 0.5% DMSO was used as a negative control and 50 μM of nitroxoline , a reference drug for PAM. Compound selection was based on CNS-permeability, clinical development  and potency. All compounds were tested in biological triplicate at a concentration which is two times the EC50 concentration. Trophozoites of N. fowleri or B. mandrillaris  were treated with the compounds at two times of 48-h EC50 concentration in 96-well plates for 8 h, 24 h and 48 h and viability of N. fowleri trophozoites was measured with CellTiter-Glo Luminescent Cell Viability Assay (Promega) and B. mandrillaris viability was determined after incubation with fluorescein diacetate for 30 min, fixing the trophozoites with 4% PFA and measuring fluorescence using a Tecan Infinite M1000 pro fluorometer following incubation. The percentage viability of trophozoites treated with different compounds at different time points was calculated. 0.5% DMSO was used as a negative control and 50 μM amphotericin B or 15.6 μM nitroxoline  were added, and Luminescence was measured using a Tecan Infinite M1000 PRO. The percentage viability of cysts treated with different compounds at different time points was calculated. 0.5% DMSO was used as a negative control, and nitroxoline and pentamidine were used as positive controls.2.6Balamuthia trophozoites to encyst. To determine if our priority compounds induce encystment, we evaluated the number of cysts present after treatment with the compounds. We plated 50,000 Balamuthia trophozoites in 500 μL media per well in 24 well plates and incubated at 37 °C for 72 h. The compound-treated parasites were fixed by adding 500 μL of 8% PFA and cysts were counted using a hemocytometer. Cysts and trophozoites can be differentiated morphologically under light microscopy. Results are expressed as number of cysts per mL.Nitroxoline was previously shown to stimulate 2.7B. mandrillaris trophozoites (105 cells in 4 mL of Cerva's medium) were exposed to selected compounds at a concentration which is two times the EC50 for 72 h at 37 °C. In parallel, HFF cells were seeded at 5 × 104 per well in four 24-well plates and incubated for 72 h at 37 °C. Compound-treated B. mandrillaris parasites were centrifuged, washed once with PBS and resuspended in 4 mL of complete DMEM media. 0.5 mL of resuspended parasites were transferred in duplicate to each well of a plate containing HFF. Plates were fixed with 4% PFA after 3, 5, 7 and 9 days. The percent of the HFF monolayer lysed was observed using light microscopy and recorded for each timepoint. 0.5% DMSO was used as a negative control and 15.6 μM nitroxoline  needed for each syndrome have unique features  . We previously identified a high overlap between compounds active against the amebae E. histolytica and N. fowleri , 16 hits for A. castellanii (EC50 ≤ 10 μM), and 35 hits for B. mandrillaris (EC50 ≤ 30 μM)  for Balamuthia than Naegleria or Acanthamoeba. Using these criteria, we obtained a list of 87 unique compounds active against at least one of the three free-living amebae. This confirms the results we have previously noted, that compounds active against E. histolytica are often active against Naegleria . Additionally, it has been noted that compounds such as miltefosine or auranofin have activity against multiple parasitic pathogens (The 159 compounds were tested in a single 8-point dose response curve against ≤ 30 μM)  (Supplem≤ 30 μM) . Becauseathogens . Whether3.250s, we selected 41 compounds for further testing. These compounds included 38 hits identified from the ReFRAME library and three compounds that are analogs of hits or which share a common target. The compounds were purchased from vendors and tested in triplicate in an 8 or 16-point dose response curves ranging from 1.5 nM to 50 μM against trophozoites of N. fowleri, A. castellanii and B. mandrillaris  identified as well as having the most potent (low nanomolar EC50) inhibitors (4 compounds). Whether this reflects a biological overlap with susceptibility to Entamoeba , a faster doubling time of Naegleria leading to higher drug susceptibility, inability of Naegleria to easily convert to the resistant cyst form in vitro, or some other cellular factor specific to Naegleria is not known. Among the two remaining free-living amebae, Balamuthia had more drug susceptibility (23 compounds) than Acanthamoeba (18 compounds). The overall positive results, including identification of high-value compounds that have late-stage clinical use data, highlights the benefit of using the ReFRAME compound library as a starting point in our drug discovery efforts. Furthermore, the data reveal that the premise of compounds targeting multiple free-living amebae is valid as 66% of the compounds with confirmed activity were active against multiple amebic species. This is the first identification of a significant number of compounds that have proven efficacy against multiple free-living amebae and is an important advance. Previous efforts have identified miltefosine with activity against Naegleria and Acanthamoeba; the compound is also used against Leishmaniasis (accessdata.fda.gov). Additionally, auranofin has been shown to be active against a wide range of parasites, including Entamoeba, Giardia and Naegleria  are FDA-approved. The anti-Naegleria activity of auranofin and bortezomib has been previously noted; these two compounds were identified as hits against Naegleria in two separate studies  have known CNS penetration. Lestaurtinib and panobinostat displayed nanomolar potency and they are either in late stage of development or FDA approved providing an opportunity of repurposing for the treatment of PAM. Of the compounds active against Naegleria only, three  are in phase III clinical trials and have received orphan drug designation, and other three  have been approved by the FDA. Of special interest is midostaurin, a derivative of staurosporine, which was noted to have high efficacy against Naegleria (EC50 0.6 μM) , drugs highly active against Naegleria can be prioritized for clinical use in cases of presumptive Naegleria PAM as imminent death is otherwise highly likely; compounds that are in late stage of clinical development and CNS penetrant would be priorities in this area. An FDA-approved BBB (blood-brain barrier) permeable compound panobinostat and another CNS permeable phase III compound lestaurtinib were particularly potent against N. fowleri with EC50 in the nanomolar range. Other compounds of similar structure, staurosporine and midostaurin, have also been identified as potent against Naegleria. Lestaurtinib (CEP-701) is an orally bioavailable indolocarbazole alkaloid compound that has activities against tropomyosin receptor kinases, neurotrophin receptors, FLT3, and JAK2  program (in vivo efficacy, the regulatory path forward appears promising.Getting approval for new drugs is a complex and long path. In order to obtain a faster track to the market, a compound that is already approved by the FDA or has significant human use data would be ideal. Moreover, given the overall rarity of the clinical syndromes caused by the free-living amebae, clinical trials are not feasible. With demonstration of efficacy of an FDA-approved drug in an animal model and human safety use data, it is possible to include it in the treatment regimen or approach the FDA for orphan drug designation, as was done for auranofin in 2010 for the treatment of amebiasis . New tre program , 2019b. Overall, our efforts identified many high-value compounds with excellent efficacy against the free-living amebae including compounds that have passed regulatory hurdles and have CNS penetration. The effort and results demonstrate the value of screening established libraries to identify new drug candidates for neglected parasitic diseases.


Most sedimentary ammonium N (NH4+) and nitrate N (NO3−) concentrations were similar; however, NH4+ accumulated on the subsurface of the middle tidal flat. NO3− fluxes (from −0.54 to −0.35 mmol m−2 h−1) were uptake fluxes in the intertidal flats, but NH4+ fluxes (−2.48–3.54 mmol m−2 h−1) changed from uptake to efflux in the seaward direction. Structural equation modeling of the effects of inundation frequency, underground biomass, total carbon (TC), electrical conductivity (EC), and clay proportion on the N processes revealed that these accounted for 67%, 82%, and 17% of the variance of PDNR, minPN, and PNR, respectively. Inundation frequency, underground biomass, TC, EC, and minPN effects on N pools accounted for 53%, 69%, and 98% of the variance of NH4+, NO3−, and TN, respectively. This suggests that future sea level rise may decrease N storage due to increase in coupled nitrification–denitrification and decrease in N mineralization, and the NH4+ flux may change from sink to source in intertidal ecosystems.Knowledge on the distribution of nitrogen (N) pools, processes, and fluxes along hydrological gradients provides a comprehensive perspective to understand the underlying causal mechanisms in intertidal flats, and thus improve predictions and climate adaptation strategies. We used a space-for-time substitution method to quantify N pools, processes, and fluxes along a hydrological gradient. Further, we linked N pools and processes and investigated not only surface but also subsurface sediments. Our results showed a gradual decrease in total N (TN) and mineralization rates ( Intertidal flats constitute major portions of estuaries and are simultaneously some of the most economically important and vulnerable ecosystems on Earth ,2. These3−), rates of nitrification, denitrification, and mineralization increased as inundation increased , while PDNR (nmol g−1 h−1) was calculated as the change in an incubated sample with and without acetylene [Equation (2)]. The minPN (mg kg−1 d−1) was calculated after 14 d sediment incubations as the change in NH4+ and NO3− between the initial and incubated samples [Equation (3)].1N (mmol L−1) is the change in NH4+ concentrations with and without the nitrification inhibitor allylthiourea, Δ2N (mmol L−1) is the change in N2O concentrations with and without acetylene, and Δ3N (mg kg−1) is the change in inorganic N (NH4+ and NO3−) concentrations before and after incubation; T is the incubation time. W (g) is the fresh weight, M (%) is the moisture content, and V (mL) is the air volume of the incubated bottle.The −1 . Each core was pre-incubated for 24 h to establish steady-state exchange conditions. Then each core was cultivated at the time of in situ flooding; the inflow and outflow water samples at time 0 and time of the end of cultivation (40 mL) were collected using a syringe (100 mL), respectively, then filtered through 0.45 μm pore size filters for NH4+ and NO3− analysis. The exchange fluxes of NH4+ and NO3− across the sediment–water interface were calculated according to the following equation (Equation (4)) [F (mmol m−2 h−1) is the NH4+ or NO3− flux, ΔC (mmol L−1) is the change of NH4+ or NO3− concentration, V (L) is the volume of overlying water running above the surface of the core sediment, S (m2) is the surface area of the core sediment, and T (day) is the incubation time.Nine cores (three sampling sites × three replicates) in opaque PVC tubes were installed into a continuous-flow system, placed within a flow-through system. This system was hermetically sealed and opaque and consisted of a square incubation box , a carboy to contain the in situ and unfiltered tidewater, a multichannel peristaltic pump, polyether-ether-ketone transmission tubing, and an acetol plunger with a Viton O-Ring . Tidewater was poured into the incubation box until the water level was 4 cm higher than that of the PVC tube. The pump transferred water from the carboy to continuously displace the water overlying the core at a flow rate of 4.5 L minion (4)) .(4)F= VF-values from the main tests and the t-statistic from the pairwise comparisons were evaluated in terms of the significance of the tested factor among different groups at p < 0.05. Structural equation modeling (SEM) was performed to analyze the causal mechanisms underlying N pools’ establishment, processes, and environmental parameters. The SEMs were implemented using SPSS Amos 21.0 . The best-fit SEM was derived by maximum likelihood and the model fit was determined using P-values, chi-square tests (χ2), root mean square errors of approximation (RMSEA), goodness-of-fit index (GFI), and Akaike information criteria [Differences among the different sites were analyzed using a one-way analysis of variance (ANOVA) and repeated measure analysis of covariance (ANCOVA) in SPSS 22.0  for Windows 10.0 . All data were tested for normality and homogeneity of variance prior to ANOVA using the Shapiro–Wilk test and the Brown–Forsythe test, respectively. If these assumptions were not met, then the raw data were log transformed before any further statistical analysis. The criteria . Plots o−1) decreased with elevated inundation frequencies (p < 0.05), and there were no significant differences for EC (p > 0.05). However, an accumulation of TC and high levels of EC were observed in the subsurface of site B (10–50 cm) (p < 0.05) (p < 0.05)  were much higher than in site C , but similar to that in site B .We found relatively high sedimentary BD in site B at 0–10 and 20–30 cm, but in site A at 10–20 and 30–50 cm . At 0–200–50 cm) . Further0–50 cm) . A predo < 0.05) . In the  < 0.05) . In addi−1) decreased with elevated inundation frequencies and decreased elevation except for the accumulation on the subsurface sediment of site B (4+ (15.83–140.15 mg kg−1) and NO3− (0.78–4.85 mg kg−1) concentrations were similar (p > 0.05) (4+ concentration was highest on the subsurface of site C (p < 0.05) (TN concentration (1.25–3.57 g kgf site B a. Most N > 0.05) b,c, but  < 0.05) b.PNR ranged from 0.03 to 18.38 nmol g−1 h−1 with a crest value in the surface sediment of site C (PNR in the subsurface sediments. The distribution of PDNR (0.51–16.17 nmol g−1 h−1) was relatively consistent, and increased with elevated inundation frequencies and decreased with depth (minPN (1.04–22.34 mg kg−1 d−1) decreased with depth, and relatively high values were found on the surface sediment of site A and subsurface sediment of site B (p < 0.05)  c.4+ and NO3− across the sediment–water interface are shown in 3− was detected at all three zones, varying from −0.54 to −0.35 mmol m−2 h−1 (4+ fluxes (approximately −2.48 to 3.54 mmol m−2 h−1) changed from uptake at site A to efflux at sites B and C  [NR increased by a factor of 1–16 more than was found in the Yangtze estuary, China (0.02–1.12 nmol g−1 h−1) [PDNR and PNR increased in a seaward direction, which might be explained by coupled nitrification–denitrification. Nitrification leads the biological oxidation of NH4+ to NO2− followed by the oxidation of NO2− to NO3−, which fuels denitrification by providing an additional source of NO3− [In the present study, both  studies ,21,32,33 studies ,38. We fg−1 h−1) , but NR  of NO3− . The mag of NO3− ,51,52.PDNR with elevated inundation frequency might be explained by three reasons. First, the increase in inundation may originate an anaerobic environment. Denitrification processes are carried out by denitrifiers, and the predominant heterotrophic microorganisms associated with denitrification are facultative anaerobes under low-oxygen or anaerobic conditions [C. malaccensis from the high tidal flat to the middle tidal flat further negatively affected PDNR (PDNR (Vibrio diabolicus SF16) and isolated from marine sediments can remove 91.82% of NH4+ and 99.71% of NO3− [The increase in nditions . Second,nditions ,54. A deted PDNR a. Root-mNR  [−1) [4+ and NO3− concentrations were similar along the hydrological gradient except for the accumulation of NH4+ concentration on the subsurface sediment of the middle tidal flat. These results suggested that the drought scenario may lead to an increase N storage, whereas N storage would decrease but NH4+ would increase under the sea level rise scenario.In the present study, TN concentrations decreased with elevated inundation frequencies at the surface, corroborating the results of previous studies , althoug−1) ,18,30 an−1) [−1) . At the −1) [−1) , NH4+ anThe SEM results indicated that high TC has contributed to the increase in TN concentration at both surface and subsurface sediment b, consisminPN were positively correlated to both NH4+ and NO3−  [4+ flux changed from uptake to efflux with elevated inundation frequencies  ,28. The quencies . This re instead . NH4+ flalkaline . Indeed,tly high . The alkediments .3− fluxes, however, were similar along the in situ hydrological gradients, and this pattern was similar to the sedimentary NO3− concentration. The sediment sink or source function for dissolved inorganic N greatly changed both spatially and temporally due to the complex transportation and transformation of dissolved inorganic N near the sediment–water interface [3− flux across the sediment–water interface was controlled by NO3− concentration in the water column [4+ diffuses from the overlying water into the sediment under the drought scenario, whereas NH4+ would be released from the sediment into the overlying water under the sea level rise scenario.The NOnterface ,72. The r column . These rPDNR and PNR increased but minPN and TN decreased along an elevated hydrological gradient in the surface sediment. However, in the subsurface sediment, we found elevated minPN and TN concentration in the interaction zone between the high and middle tidal flats, as well as elevated NH4+ concentration in the middle tidal flat. Additionally, our findings show that N storage may increase owing to increasing drought events. Conversely, under conditions of continuous sea level rise, a decrease in N storage could cause a change from NH4+ sink to NH4+ source.Herein, we proposed a conceptual scheme for the fate of N pools, processes, and fluxes on a tidal flat based on our results . This coC. malaccensis. Since plant functional composition is crucial for sediment N pools and processes [Due to the inherent limitations of the study region, we selected sampling sites that were all covered by rocesses ,45, we sPDNR and PNR increased but minPN and TN decreased along an elevated hydrological gradient in the surface sediment. However, in the subsurface sediment, we found elevated minPN and TN concentration in the interaction zone between the high and middle tidal flats, as well as elevated NH4+ concentration in the middle tidal flat. These distribution patterns of N pools and processes may be explained by the decrease in inundation frequency, underground biomass, carbon availability, and the increase in salinity. In addition, NH4+ flux changed from uptake to efflux with elevated inundation frequencies, which coincided with the change in sediment pH from acid to slightly alkaline soil. However, sedimentary NO3− concentrations and NO3− fluxes were similar along the hydrological gradients.Our data indicated that minPN may increase, but PDNR and PNR are likely to decrease, and NH4+ would diffuse from the overlying water into the sediment under a drought scenario. Conversely, the sea level rise scenario may not only lead to an increase in coupled nitrification–denitrification and N removal but also to a decrease in mineralization and immobilization, consequently decreasing N storage and leading to a change from NH4+ sink to NH4+ source.In response to possible future scenarios of climate change, N storage and


Background and Objectives: A deficiency exists in the criterion-referenced cut-points for field-based cardiorespiratory fitness (CRF) in Latin American youths. The aims of the present study were two-fold: (1) To identify the ability of CRF estimated by the 20-m shuttle-run test (20mSRT) to differentiate between “healthy” and “unhealthy” phenotypes (by adiposity) in adolescents; (2) to assess the association between obesity and relative peak oxygen uptake (VO2peak) in a large and diverse sample of Latin American youths. In total, 72,505 adolescents aged between 13 and 15 years were recruited from Chile and Colombia (47.5% girls). Materials and Methods: The waist circumference (WC) and waist-to-height ratio (WHtR) were used to identify body adiposity markers. CRF was measured using the 20mSRT (VO2peak). Receiver operating characteristic curves and logistic regression were used to determine the discriminatory ability of CRF to predict body adiposity parameters. Results: For boys and girls, VO2peak showed a significant predictive capacity to detect body fat (area under the curve [AUC] > 0.62). The sensitivity of VO2peak was medium (>63%) for all age- and sex-specific cut-points, with optimal cut-points in 13- to 15-year olds for obesity identified as 43.77 mL·kg−1·min−1 and 38.53 mL·kg−1·min−1 in boys and girls, respectively. Conclusions: According to these cut-points, adolescents with low CRF were more likely to be obese either by WC or WHtR. The CRF cut-points can be used as quantitative markers for a healthier body in Latin American adolescents. Physical inactivity and low levels of CRF are major threats to public health. However, their prevalence is rapidly increasing in developing countries, such as among Latin American children and adolescents ,2. MoreoCRF is an equally powerful predictor of mortality risk, similar to traditional risk factors, such as smoking, obesity, hypertension, dyslipidemia, and type 2 diabetes mellitus, in both adolescent and adult populations . An impo2peak, laps, or speed), children with values below the recommendations were more likely to be obese, either by BMI, WC, or both, regardless of factors such as age, screen time, and level of physical activity levels. This result corroborates other studies that reported CRF to be an independent risk factor for obesity and reinforces the need for the development of CRF health-related standards to discriminate cardio-metabolic health in youths. Thus, comparing the differences in the criterion-referenced standards for the different predictive equations used to estimate aerobic capacity will help to identify different phenotype risks  from the CRF levels estimated by 20mSRT performance and will be of significant public health value. Moreover, while there are criterion-referenced cut-points for CRF in many high-income countries, only one study has been performed in Latin American countries [Although physical fitness and body adiposity may be correlated, they are not synonymous and indicate differing disease risks. In youth studies, several authors have shown that those with low levels of adiposity and low CRF are at a greater cardiovascular risk than high-adiposity adolescents with adequate CRF, independent of muscular fitness and body composition ,10. Receountries .2peak) in a large and diverse sample of Latin American youths.Youth fitness assessment guidelines have called for a better understanding of the inter-relationship between physical fitness and body composition . TherefoThis study was based on a secondary data analysis of two separate and independent samples drawn from two different countries: Chile  and Colombia . Participants were considered eligible for this study if they were aged 13.0–15.0 years (grades 8–9), and maximal effort exercise was not contraindicated. The complete methodology of this study is described in detail [http://informacionestadistica.agenciaeducacion.cl/#/bases). The study authors (A.G.-H.) entered a written data use agreement with the Chilean Ministry of Education.Briefly, SIMCE, which has been administered annually by the Chilean Ministry of Education in November since 2011 , uses a https://www.educacionbogota.edu.co/archivos/Temas%20estrategicos/Documentos/Resultados_PruebasSER-Bienestar_Fisico_Ciudadania_y_Convivencia.pdf). The survey was approved by the Review Committee for Bogotá’s District Secretary of Education .The Prueba SER survey was performed by Bogotá’s District Secretary of Education in November 2014 and 2015. These were cross-sectional surveys of ninth grade students recruited from public and private schools from all 20 “localidades”  within the District Capital of Bogota . Further details can be obtained from the website (available from: https://www.urosario.edu.co/CEMA/Inicio/#cema), to standardize the assessment process and to minimize inter-observer variability. Body mass was measured using digital weigh scales to the nearest 0.1 kg; height was measured using a stadiometer to the nearest 0.1 cm. Body mass index  was subsequently derived and BMI z-scores calculated using age- and sex-specific reference data from the World Health Organization, with obesity defined as ≥2SD above the mean [The measurements of weight, height, and WC were carried out at school applying standardized procedures; all the instruments were verified before measuring each subject. Before data collection, examiners were trained prior to testing by the Chilean Ministry of Education and Universidad del Rosario-Centro de Estudios en Medición de la Actividad Fisica “CEMA”  small, medium, and large, respectively [®  and SPSS 24.0® software  were used for all analyses, and a p value < 0.05 was considered to be statistically significant.Descriptive statistics were calculated for all variables . All variables were checked for normality of distribution before analysis using Kolmogorov–Smirnov test and Q-Q plots. None required transformation due to normally distributed. Cohen ng tests   cm; WHtR, 0.44 (0.44−0.44); and CRF, 42.47 (42.42−42.52) mL·kg−1·min−1 using the Léger et al. equation [The final sample comprised 72,505 adolescents aged 14.02 (95% CI 14.01−14.03) years. Means (95% CIs) for the sample were: BMI, 22.65 (21.63−21.68) kg/mequation . The pre2peak showed significant predictive capacity for obesity . In boys, when considering the full sample (13–15 years), the best cut-point for VO2peak estimated by the equation of Léger et al. to detect body fat by WC and WHtR were 43.77 mL·kg−1·min−1 (12.51 METs). For girls, when considering the full sample (13–15 years), the best cut-point to detect body fat by WC and WHtR were 38.53 to mL·kg−1·min−1 .For boys and girls  presented discriminatory ability in identifying a poor health profile (estimated by body fat parameters) in both girls and boys aged 13–15 years. Additionally, the suggested cut-points for low CRF were associated with obesity in all ages and sexes.−1·min−1 and 41 mL·kg−1·min−1 for girls and boys, respectively. In another similar study including 16,619 British children’s from Liverpool aged 11–13.9 years [−1·min−1 and 46.6 mL·kg−1·min−1 for girls and boys, respectively. In the present study, the optimal cut-points, when we considered all samples by WC or WHtR, were 43.77 mL·kg−1·min−1 and 38.53 mL·kg−1·min−1 for boys and girls, respectively. Another study published by our team including Colombian children and adolescents proposed cut-points of 47.9 mL·kg−1·min−1 in boys and 34.4 mL·kg−1·min−1 in girls age 9–12 years and 48.0 mL·kg−1·min−1 in boys and 33.8 mL·kg−1·min−1 in girls aged 13–17 years [2peak was low for girls. In this sense, it was observed that the positive predictive value was low for both sexes aged 13–15 years , suggesting an important number of false positives, especially in the girls group, and reinforcing the need for sex-specific targeted interventions in the Latin-American setting. This positive predictive value is directly related to the low prevalence of obesity in the overall sample (range: 6.9% to 10.8%). However, a major limitation is that cut-points can only be applied to populations in which the condition has a similar prevalence to the population tested or to individuals with a similar risk of a positive result. Furthermore, low CRF in adolescence is largely a reversible condition. Fitness-enhancing interventions in youths have been shown, on average, to improve CRF by approximately 10% [Our study extended the results of a recent study in 8740 Canadian children aged 8.0–12.9 years that showed a negative association between the 20mSRT and cardiometabolic risk (as assessed by adiposity measures) and that the CRF cut-points from ROC analyses had a good discriminatory power for obesity [.9 years , the valtely 10% , includitely 10% .−1·min−1 and 35 mL·kg−1·min−1 for boys and girls aged 8–18 years, respectively. The cut-points proposed in our study are higher than those suggested by Lang et al. [Two reviews ,30 regarg et al.  and RuizCollectively, these findings suggest the existence of an important relationship between CRF and fat that is detectable as early as adolescence and that could be used to guide prevention efforts and, consequently, reduce cardiometabolic risk factors. The deleterious consequences ascribed to obesity could be counteracted, to some extent, by maintaining appropriate levels of CRF ,31. In oSuch interventions—for example, in the school environment—should involve promoting physical activity to increase CRF levels  and were2peak from a single 20mSRT, and standard testing methods with directly measured oxygen consumption are required to confirm our cut-points.Our study has several limitations that should be considered in the interpretation of the findings. First, the cross-sectional design of our study does not allow us to explain causality. Second, the assessment of other cardiometabolic risk factors  could have shown additional information on prognostic assessment. Finally, we predicted VOThe major strength of this study is the potential usefulness of the cut-points in field settings by health professionals in Latin American countries. Because the 20mSRT is a simple, low-cost method of CRF assessment, its use may provide a feasible way to study the link between physical activity and health among adolescents at the population level . A final2peak cut-points could be used by health professionals and in the school environment as a low-cost assessment in the field setting to help identify youths at risk for poor health.Despite the cross-sectional design of the present study and that the AUC showed only moderated values, our results highlight the ability and usefulness of CRF (as estimated by the 20mSRT) in detecting fat in adolescents. Our findings also suggest that the proposed cut-points for low CRF are associated with obesity, in both sexes. Therefore, these VO


Crassicauda boopis is known to infect the kidneys and vascular system of mysticetes included Balaenoptera physalus and has been recently reported in Mediterranean waters. Identification at the species level relies on the observation of morphological features of the adult parasites, but field conditions during necropsy and the massive reaction of the host's immune system often prevent optimal conservation of the extremities. Moreover, larval stages of Crassicauda have never been described and no sequences are available in public databases to help such identification. Adult and larvae of Crassicauda were isolated from four specimens of B. physalus and studied with morphological and molecular techniques. Specimens of C. anthonyi, C. grampicola and Crassicauda sp. isolated from Ziphius cavirostris, Grampus griseus, Stenella coeruleoalba and Tursiops truncatus respectively were studied as well. Sequences of nuclear markers 18S and ITS-2 and of mitochondrial gene cox1 were obtained and phylogenetic relationships within the genus Crassicauda were analysed. Analysis of the ITS2 grouped the different species in accordance with morphological identification, as already evidenced in literature for other Spirurida. A higher intra-specific variability was observed for the cox1 gene, for which two species (C. grampicola and C. anthonyi) did not appear as monophyletic in the tree. Well-developed non-attached larval specimens in the intestinal lumen of a whale calf were molecularly identified as C. boopis, allowing new insights on the life cycle of this species. This work broadens the genetic database on cetaceans parasites, allowing species identification even in challenging field conditions or in poor conservation of the samples; moreover, the first morphological description of C. boopis larvae is provided.  •Crassicauda boopis, C. anthonyi, C. grampicola and a Crassicauda sp. are molecularly studied here.•Crassicauda are provided.First morphological descriptions of larval stages of •cox1 for identification.Phylogenetic analyses show ITS-2 as the more efficient marker compared to •Crassicauda boopis are described.New insights on life cycle of  Different organ tropism and consequent pathogenic impact are described among the members of this genus, whose localization spans over subcutaneous tissues, cranial sinuses and the urogenital system , humpback  and blue whales   . Reportss, 1758)  and the s, 1758) . Recentlnean Sea  and nemanean Sea . Thrombonean Sea .Crassicauda spp. have been formulated (Meganyctiphanes norvegica (Crustacea: Euphasiacea) or other crustaceans included in rorquals diet could play a role in a hypothetical indirect life cycle of C. boopis, in which infective larvae would get into the host during filter-feeding. Nevertheless, a direct transmission whale-to-calf has been hypothesized during lactation, because of the presence of adult nematodes in the kidneys of waning calves  acquire information about migration and localization of immature stages of C. boopis inside the final host.The aim of this work is to: i) morphologically and molecularly characterize adult and larval stages of nematodes isolated from Mediterranean fin whales, ii) contribute to the genetic database of Crassicauda species to test the presence of molecular markers useful for species discrimination.In order to reach molecular identification, analysisof nuclear and mitochondrial markers was performed on different 22.1Portions of adult nematodes  were isolated from the vena cava, ureters and kidneys of three fin whales stranded along Italian Tyrrhenian coasts in the period 2008–2013 . Three cB. physalus), stranded in the same geographical area. In particular, seven larvae were collected from nodules embedded in the intestinal wall (INL) and four included in the mesenteric artery walls (MAL) of one adult fin whale infected also by adult nematodes in the kidneys ; nine free larvae were collected from the intestinal lumen (FIL) of a whale calf (#270-13) in absence of any adult nematode , and from the pterygoid sinuses of a Risso's dolphin , were morphologically identified as C. anthonyi  and C. grampicola, Johnston and Mawson, 1941 , respectively. One cephalic portion, one male tail and one female tail of both species were deposited at the Natural History Museum of London (NHMUK) . Finally, fragments of Crassicauda sp. isolated from subcutaneous tissues of two bottlenose dolphins  and of one striped dolphin  were included in the study. Due to the lack of the cephalic and caudal extremities, these nematodes could not be identified to species level. Data of the hosts and nematodes analyzed in this study are listed in Specimens of adult nematodes isolated from the kidneys of a Cuvier's beaked whale (All the collected nematodes (larvae and adults) were preserved in 70% ethanol. Adult parasites  and the whole body of larvae, selected for morphological identification, were clarified in Amman's lactophenol solution and in 10% glycerol and 70% alcohol solution respectively. All samples were subsequently observed and measured with a light microscope  by NIS-Elements D software (Nikon).2.2C. anthonyi; 3 specimens of C. grampicola; 6 fragments of Crassicauda sp. , according to the manufacturer's instructions.DNA was isolated from the following specimens: 7 adult nematodes from fin whales; 4 larvae from mesenteric arteries and intestinal nodules and 3 larvae from the intestinal lumen of fin whales; 2 specimens of auda sp. . Extract2, 0.25 mM dNTPs , 1X PCR buffer, 0.5 μM of each forward and reverse primer, 1U Platinum Taq DNA Polymerase (Invitrogen). Molecular biology grade water was added up to the final volume. Cycling conditions comprised an initial activation step at 94 °C for 2 min, followed by 35 cycles of 94° for 30 s, 58.7° for 30 s, 72 °C for 80 s, with a final extension step of 72° for 7 min. The PCR products were resolved through electrophoresis runs in 2% agarose gel with SYBR® Safe DNA gel stain . The amplicons of PCR (fragments of expected size 1700 bp) were directly sequenced at Macrogen  using PCR primers. Additionally, a couple of internal primers were designed specifically with software Primer3 to facilitate sequencing the 18S fragment, i.e. forward primer 437F (5′-AACTAAGAACGGCCATGCAC-3′) and reverse 1279R (CTCTCGGCATGAGGAGGTAG-3′) .The small subunit  was amplified by polymerase chain reaction, using the primers G18S4–F (5′-GCTTGTCTCAAAGATTAAGCC-3′) and reverse 136-R (5′- TGATCCTTCTGCAGGTTCACCTAC-3′) . The PCR® database using the software BLAST . The consensus sequences were assembled with the program SeqMan available in the DNAstar package. The consensus sequences were compared with the non-redundant data base available in the GeneBankre BLAST .cox 1 gene region. Primers D (5′-GAGTCGATGAAGAACGCAG-3′) and reverse B1 (5′-GAATCCTGGTTAGTTTCTTTTCCT-3′) were used for the ITS-2 region , 1X PCR buffer, 0.5 μM of each forward and reverse primer, 1U Platinum Taq DNA Polymerase (Invitrogen). Molecular biology grade water was added up to the final volume. Cycling conditions comprised an initial activation step at 94 °C for 2 min, followed by 35 cycles of 94° for 30 s, 58° for 30 s, 72 °C for 30 s, with a final extension step of 72° for 5 min.The PCR for ITS-2 region was performed in a 30 μl reaction, comprising 3 μl DNA, 2.5 mM MgClcox 1 region was performed in a 30 μl reaction volume, comprising 3 μl DNA, 2,5 mM MgCl2, 0.5 mM dNTPs , 1X PCR buffer, 1.25 μM of each forward and reverse primer, 1U Platinum Taq DNA Polymerase (Invitrogen). Molecular biology grade water was added up to the final volume. Cycling conditions comprised an initial activation step at 95 °C for 2 min, followed by 35 cycles of 94° for 40 s, 50° for 30 s, 72 °C for 30 s, with a final extension step of 72° for 5 min.The PCR for ® Safe. The PCR products (fragments of expected size 510 bp and 710 bp for ITS-2 and cox 1 respectively) were sequenced at Macrogen  using the same PCR primer pairs and chromatograms processed as previously described.The PCR products were resolved through electrophoresis runs in 2% agarose gel with SYBR2.3cox1 and ITS2 sequences obtained in the present study as well as orthologous counterparts belonging to Crassicauda genus available in GenBank , were created in accordance to in accordance to (see results). When available, sequences obtained from members of the same superfamily were included as outgroup. The multiple alignments were performed with the program Muscle (cox1), the alignment was performed at codon level. To guarantee phylogenetic trees reliability, distantly related outgroup sequences were maintained only when alignment quality was high and no evidences of substitution saturation were detected. Phylogenetic trees were computed with the MEGA7 software . To assess the statistical support to the tree topologies, 10.000 bootstrap replicates were calculated.Independent data set including the 18S, m Muscle  implemenm Muscle . When cosoftware  accordin33.13.1.1C. boopis  were deposited at the NHMUK (Crassicauda boopis 2015.10.11.1–4).Specimens of C. boopis in literature  lying free in the lumen of vena cava, narrowing towards the head, with an evident constriction at 53.5 ± 2.12 μm (range 52–55 μm) from the buccal opening, with a diameter of 90.5 ± 10.5 μm (83–98 μm). The buccal cavity appeared laterally compressed (21.5 × 108 μm); two lips in lateral position, triangular in shape (74 μm wide at the base and 30 μm high), each with a small apical labial papilla. Two lateral and four submedial cephalic papillae were visible on the anterior end (rior end i. The esrior end a (esophaThe posterior end (measured specimens n = 3), found in the ureters, had a genital constriction at 5600 ± 503 μm (range 5000–6000 μm) from the tip of the tail, where the vulva opened ventrally b. The piMale. A cephalic extremity (measured specimens: n = 1) was strongly fixed in the renal parenchyma and the fragment was 8.5 cm long. In the anterior part of the parasite a constriction was observed at about 50 μm from the top, giving the extremity a triangular shape. The mouth was characterized by a slit with two evident triangular lips (88 μm wide at the base and 33 μm high) with two small labial papillae and two well visible lateral cephalic papillae. The buccal cavity appeared laterally compressed (20 × 110 μm).The posterior end (measured specimens n = 3), found in ureters, was narrowing with a coiled tail (2 turns); the cloaca opened at 1040.6 ± 57.2 μm (999-1106 μm)-from the tip of the tail, with no spicules. Eighteen caudal papillae were visible c.3.1.2All measures in this section are average values; please refer to Intestinal nodules. Larvae (n = 7) contained in the intestinal mucosa of one of the adult whales showed the following morphometric features: total length of the body 8356.0 μm, width 81.5 μm. Anterior end was sharp; two lips with terminal rounded tooth, 2 lateral and 4 submedial prominent papillae were observed . Morphological features were generally similar to those of the larvae included in the intestinal nodules. Total length of the body was 10,613.8 μm, width 107.3 μm. A triangular shaped head, bulging at the base, was observed. Excretory pore at 171.7 μm from anterior extremity  had a length of 30,417.4 μm and width of 151.1 μm. The anterior end was sharp, with two lips and six cephalic papillae . The buccal cavity was compressed and measured 63.9 μm × 15.2 μm. Excretory pore was located at 269.3 μm from anterior extremity  and C. boopis (Acc. number KY263809) already deposited. Thus these sequences proved useless to discriminate among different Crassicauda species.Amplification and sequencing of 18S gene resulted in 23 good quality sequences and their alignment (1600 bp long) proved them to be all identical. The Blast research in GeneBank revealed them to be highly similar (99.88%) also to the sequence of C. boopis were split in two clusters. All specimens belonging to C. grampicola exhibited identical sequences. The same evidence was reported for specimens of C. anthonyi. Specimens, represented by fragments of adult parasites, assigned to Crassicauda sp. exhibited a single sequence. Two ITS-2 sequences of the larvae found free in the intestinal lumen (FIL) appeared to be identical to that of C. boopis. On the other side, larval specimens included in the intestinal nodules (INL) and in the mesenteric artery's wall (MAL) were part of a genetically distant cluster (p-distance ranging between 9 and 9.2%) compared to C. boopis cluster. The phylogenetic analyses of all sequenced individuals is provided in Amplification of ITS-2 fragment gave 22 sequences  of good quality (Acc. Numbers MK631888 - MK631909). The specimens, represented by fragments of adult parasites belonging to the species cox1 gene gave 19 sequences  of good quality (Acc. Numbers MK621821 - MK621839). The phylogenetic tree based on the cox1 sequence provided a much complex pattern. The samples of adult C. boopis and free intestinal larvae were classified in a single cluster. On the other hand, specimens of C. anthonyi and C. grampicola were intermingled in two independent clusters comprising substantially identical sequences despite the species difference (Crassicauda sp. formed a single monophyletic cluster. Two sequences from mesenteric (MALm) and intestinal larvae (INL7) respectively formed, similarly to what observed with the ITS2 gene, an independent group, loosely related (p-distance ranging between 8.2 and 8.5%) to the C. boopis one. The analysis of the topology of the obtained cox1 sequences reveals that, with the exception of adults Crassicauda sp. and C. boopis, other species of Crassicauda do not form monophyletic groups.Amplification of fference . SamplesDetails of all the obtained sequences are reported in 4Crassicauda, i.e. C. crassicauda , C. giliakiana Skrjabin and Andreeva, 1934, C. anthonyi, C. bennetti Spaul, 1926, C. grampicola, C. boopis (syn. C. pacifica), C. magna Johnston and Mawson, 1939 (syn. C. duguyi), C. tortilisC. delamureana Skrjabin, 1966, C. fuelleborni Baylis, 1932, C. costataC. carbonelli Raga and Balbuena, 1990. These species occur in the kidneys , reproductive system , pterygoid sinuses (C. grampicola) and subcutaneous tissues and “gill slit” gland (C. magna) of the host   . The disCrassicauda species. is incomplete, but hypotheses on transmission, migration and development of larvae have been formulated for C. boopis, by observing pathological findings in infected fin whales , as reported for other nematode families  (and cox1 gene as well - p-distance to C. boopis = 8.2%). If this variability can be explained by a remarkable within-species variability or if it is due to the detection and sequencing of still unclassified Crassicauda species remains to be elucidated. Unfortunately, the absence of additional data on ITS-2 sequences from other Crassicauda species prevents a further specimen characterization. We speculate that at least two other species should be molecularly characterized to perform comparison with the larvae recovered, i.e. C. crassicauda and C. tortilis. The first one is the only other species of Crassicauda described in fin whales. In the study by C. crassicauda were simultaneously present with adult C. boopis and with larvae migrans in the mesenteric arteries; no hypothesis on the life cycle of this species have been formulated. On the other side, C. tortilis is the only other species described in the kidneys of a mysticete . Assuming that similar final localization could correspond to similar intra-somatic migration route - as observed for Crassicauda sp. in Cuvier's beaked whale's kidneys (Crassicauda is characterized by a relatively low host specificity inside the suborders of the odontocetes and mysticetes, a wider molecular assessment inside the genus would be essential to characterize related species and investigate the life cycle with confidence.In this study, larval nematodes with morphological features of progressively more advanced developmental stages were isolated from mesenteric arteries, intestinal walls and intestinal lumen of fin whales, respectively. The phylogenetic analysis performed on 18S sequences of these larvae and of all adult nematodes from the different hosts allowed to assign all of them to the genus families  includedfamilies . Interesood flow . In our  kidneys  and conscox1 gene revealed a less interpretable pattern, since samples morphologically classified in the same species demonstrated a heterogeneous mitochondrial genetic background and were not monophyletic in the tree. The actual causes of the observed phenomenon remain elusive and further analysis based on more extended dataset should be performed to investigate this issue. Therefore the need for collaborative efforts among research groups and the definition of shared reference genes and regions to be sequenced appears pivotal to understand the evolutionary relationship among Crassicauda genus members. Nevertheless, although preliminary, the obtained evidences and especially the comparison between morphological and genetic features, suggests ITS2 to be a more reliable marker for taxa identification within the Crassicauda genus.Sequencing and phylogenetic analysis of the mitochondrial Crassicauda, in order to establish an updated, shared and handier classification scheme.Overall, the results of the present study emphasize the limits of current classification criteria and the need of a much more extensive and systematic analysis of the morphometric and genetic features of the genus Crassicauda spp.This work contributes both to enrich the genetic sequence data bank and the molecular taxonomy of this genus; it represents an important starting point for identification of ruined fragments or immature stages of


Hospital acquired infection (HAI) and multiple organ dysfunctions (MODS) remain a leading cause of death in pediatric intensive care unit (PICU) despite the great efforts to control it.Our objective was to assess the mRNA of TNFα and BCL2 for prediction of HAI and/or MODS in our community.Fifty children, admitted to PICU, were included in the study after exclusion of cases of end-stage renal failure, end-stage liver failure and congenital immune deficiency. Serial Blood samples were collected for complete blood count (CBC) and other routine investigations. Gene expression of (TNFα and BCL2) was quantified using quantitative real time PCR (qRT-PCR). Centers of disease control (CDC) criteria were used to detect HAI, and organ failure index (OFI). Pediatric logistic organ dysfunction (PELOD) and pediatric risk of mortality (PRISM) scores were used for follow up. The results were compared between the group who acquired HAI and who didn't. Gene expression was tested with a ROC curve to detect its ability to predict HAI.The overall complication (HAI and/or MODS) rate was 52%, Complicated cases had a significantly longer duration of stay in PICU (0.002) and in overall hospital stay (p = 0.013) and a higher death rate (p = 0.000). On day1; TNFα, BCL2 and lymphocytic count were lower in patients who developed complications , all had the ability to predict the complications with AUC . On day 4: TNFα and BCL2 returned to normal levels while the lymphocytic count still lower in complicated cases, p = 0.001 and AUC = 0.73.TNFα and BCL2 on admission can predict HAI and MODS (AUC = 0.7 and AUC = 0.8), but were of no use in the follow-up, however, the lymphocytic count is a rapid, easy and cheap test to assess the immune state with a good predictive and follow up values. •st to show mRNA biomarkers in acquired immunosuppressed critically ill Egyptian children.To our knowledge, our work is the 1•The current study provides a new evidence that immune suppression can be measured and opens the way for further studies.•We mainly found that TNFα and BCL2 can predict HAI and MODS on admission but they were of no use in the follow-up.•Lymphocytic count, a rapid, easy and cheap test, can assess the immune state with a good predictive and follow up values. Early dThe immune system is massively activated with any critical insult, which is called severe inflammatory response syndrome (SIRS) and can lead to early mortality, then the patient tries to restore the immune balance by the compensatory anti-inflammatory syndrome (CARS), however uncontrolled CARS lets the patient be susceptible to infection and can lead to MODS and late mortality . The CARCritically ill neonates, children, and adults who die from nosocomial sepsis and MODS had prolonged lymphopenia (absolute lymphocyte count ‹ 1000 cells for more than 7 days) .Apoptosis is not only a marker, but it has been shown to have a direct role in immune dysfunction, and that was obvious in murine studies, where prevention of apoptosis improved survival in sepsis . QuantitTumor necrosis factor alpha (TNFα), a proinflammatory cytokine, is a potent activator of many cell types such as macrophages/monocytes and NK cells and can induce cell survival or cell death by activation of the extrinsic pathway of apoptosis . TNFα ha2This study aimed to assess the ability of mRNA biomarkers (BCL2 and TNFα) to predict patients at risk of HAI, sepsis and MODS, in our community.3This is a cohort study, carried out in the Pediatric Intensive Care Unit (PICU) in Benha University Hospital in cooperation with the Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Benha University, during the period from April 2017 to October 2017.Inclusion criteria: all patients admitted to PICU between 2 and 12 years old.Exclusion criteria:•End-stage renal disease requiring chronic dialysis therapy.•End-stage liver disease: cirrhosis with evidence of portal hypertension,•Congenital immunodeficiency.•Readmitted cases.•Referred patients.•Patients stayed less than 48 h in PICU and 4 days in hospital .rd day of hospital admission.HAI was defined according to CDC criteria , as a loPediatric logistic organ dysfunction (PELOD), organ failure index (OFI) scores were used to estimate the severity of cases of MODS in PICUs and to describe correctly the clinical course of illnesses observed in critically ill children . RoutineApproval of the study was obtained from the Ethical Committee of Scientific Research, Faculty of Medicine, Benha University. Written informed consent was taken from the parents of each child.The work has been reported in line with the STROCSS criteria .3.1Two venous blood samples (1 mL each) were taken from each subject at the first and the fourth days of their hospital admission. Venous blood samples were taken on (EDTA), mixed well, stored at −80 °C for further assessment of TNFα and BCL2 gene expression as follow:3.2quantitative Real-Time PCR assay (qRT-PCR):1.Total RNA Extraction: Total RNA Extraction was performed using 100 μl EDTA whole blood specimen of each subject via purelink RNA Mini kit (Life technologies) according to the manufacturer instructions.2..Ultraviolet Spectrophotometric Quantification of RNA by nanodrop 2000 Spectrophotometer  To ensure significance, A260 readings should be > 0.15. An absorbance of 1 unit at 260 nm corresponds to 40 μg RNA/ml. The ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity; pure RNA has an absorbance ratio (260/280) of 1.9–2.3 [ 1.9–2.3 .3.Relative quantitation of mRNA of the respective genes by real-time PCR using syber green reagents on 2 steps:Gene expression was assessed by 3.2.1The first step qRT-PCR was for conversion of RNA into complementary DNA (cDNA) in a Veriti™ Thermal Cycler (Applied Biosystems), using High-Capacity cDNA Reverse Transcription kit . The RT master mix for reverse transcription of each subject contained 2 μl RT buffer (10X), 0.8 μl dNTPs mix (25X), 2 μl RT random primers (10X), 1 μl MultiScribe™ Reverse Transcriptase, 1 μl RNase inhibitor, 4.2 μl nuclease-free water. Then the PCR mix for reverse transcription of RNA into cDNA included 10 μl RTmaster mix (2X) and 10 μl Extracted RNA. The Thermal cycling conditions for RNA reverse transcription were primer annealing at 25 °C for 10 min, reverse transcription for at 42 °C for 15 min and inactivation at 85 °C for 5 min.3.2.2The second step qRT-PCR was for quantitation of CD203c and ST2L gene expression in a Stepone real time PCR system . Singleplex reactions were done. Non-templete controls were included in each run. This step was performed using SensiFAST™ Sybr Hi-Rox Kit . Human β-actin was the endogenous control housekeeping gene. Melting curve analysis was done in each run to confirm the specificity of real-time PCR assay. The primers used were as follow: BCL2 (271bp); FP: 5′-GCCAGCTGCACCTGACGCCCTTC-3′, RP:5′-CCGCATGCTGGGGCCGTACAGTT-3' , TNFα , 0.05 μl ROX solution, 1  μl FP, 1  μl RP, 2  μl cDNA and up to 20  μl nuclease-free water. The Real time thermal cycler conditions were as follow; initial denaturation for 10  m at 95 °C, 45 cycle; denaturation at 95 °C for 15s, annealing at 52 °C for 30s and extension at 72 °C for 30s.3.3−ΔΔCT [According to the RQ manager program, the data were produced as sigmoid shaped amplification plots in which the cycle number was plotted against fluorescence . The samples of the control group were used as calibrators so the expression levels were set to 1. The relative quantities of human BCL2 and TNFα genes were normalized against the relative quantities of the endogenous control (human β-actin) so gene fold expression changes were calculated using the equation 2−ΔΔCT . As show3.4The data were coded, entered and processed on the computer using SPSS (version 24). The results were represented in tabular and diagrammatic forms then interpreted. Cases were grouped and compared as follows: HAI vs. no HAI. Mean, standard deviation, frequency, and percentage were used as descriptive statistics.2) for categorical data. Values of p < 0.05 were considered statistically significant.Comparisons between groups were made using student-t test for continuous variables and the Chi-Square test (ΧReceiver operating characteristics (ROC) curve and areas under the curve (AUC) were calculated for each marker, as well as p-values that test the null hypothesis that the area under the curve equals 0.50. Correlations were performed using Pearson coefficient.4st and 4th days of admission, patients were divided into two groups according to their outcome; the first group (complicated), included children who acquired HAI and/or MODS and second group (Non-complicated) includes children who passed the critical insult without any complications.This cohort study was conducted on 50 children admitted to PICU, who survived the early death (72 hr), samples were collected in the 1Clinical data of those patients are described in 4.1st and 4th days of admission are presented in Fig. 4Comparison between gene expression in both groups in the 1The Area under the receiver operating characteristic (ROC) curve for predicting complications, st day: lymphocytic count, TNFα and BCL2 were significant, while in the 4th day: only lymphocytic count was significant. The results of statistically significant AUC were as following:BCL2 day 1: AUC = 0.8, At cut off value of (1.56), sens.= (76.9) specif. =(70.8).TNFα day 1: AUC = 0.7, At cut off value of (1.53), sens.= (66.7) specif. =(84.6).Lymph. day 1: AUC = 0.67, At cut off value of (2.55), sens.= (58.3) specif. =(76.9).Lymph. day 4: AUC = 0.734, AT cut off value of (2.1), sens.=(66.7) specif.=(76.1).4.2Pearson correlations were performed between genes expression and indicators of disease severity as OFI, PELOD, length of stay in PICU and length of stay in the hospital and described in We observed no statistical difference in gene expression of BCL2 or TNFα in patients regarding their sex, age or body mass index.5The immune response activation (or deactivation) can significantly affect the outcome in sepsis, trauma or any medical condition, and may vary between individuals. In patients with a similar infection, there is great variability reported in the clinical profiles and outcomes. The risk of HAI, MODS and their outcomes are influenced by host predisposition, this may be explained by genetic variability between patients .Genetic studies might allow for earlier differentiation between patients with immune-inflammatory response to either infection or trauma, allowing for more focused and timely treatment. Molecular profiles can identify a good versus a poor immune response, and enable us to detect patients at risk of HAI, sepsis and MODS.In this pilot study, we aimed to evaluate mRNA biomarkers of BCL2 and TNFα as tools for monitoring the immune response in critically ill children in our community.Previous studies were conducted to assess the risks of HAI or MODS in critically ill patients, In our cohort, After exclusion of patients died in the first 3 days of admission, we found 2 patients categories: 1- Patients who passed the critical illness and fully recovered. 2- Patients had HAI once or more or who even progressed to MODS and died.As immune dysregulation is the most important pathogenic mechanism underlying both HAI and MODS ,21,22 anst HAI attacks, blood culture was positive in 64.3% of them and the most common organisms were Gram negative bacilli, Klebsiella and staphylococci. Twenty-four percent of patients developed septic shock, MODS and died. In previous studies, HAI rate ranged from 30% to 55% and pneumonia was the most common HAI. Blood culture positive results ranged from 35% to 55%, the most commonly isolated species were: Klebsiella pneumoniae, Pseudomonas, Acinetobacter, and Staphylococcus aureus [In our study, 28% of patients fulfilled the CDC criteria for the diagnosis of HAI, pneumonia was the most common cause of the 1s aureus .We observed that surgical patients were more liable to HAI than medical and septic patients, but in previous studies, the risk was equal, with a minimal increase in complications in septic patients .We observed that the history of previous PICU admission, delayed developmental milestones and chromosomal abnormality were associated with higher rates of complications. Also, Perronet et al., observed that children who developed HAI were more likely to have genetic or chromosomal abnormalities . These rst day of admission and the difference increased by the 4th day. In previous studies, persistent lymphopenia was highly predictive of mortality in medical [In our study, lymphocytic count was significantly lower in the patients complicated with HAI and/or MODS than the other patients in the 1 medical ,27, sept medical , and tra medical . The lymth day sample. This difference can be explained as TNF-alpha is released in the circulation in the first few hours and rapidly disappears after that [TNFα, the proinflammatory cytokine, is tightly related to regulation of host innate immunity, inflammation, and apoptosis. In a previous study, TNFα response <200 pg/mL throughout 7 days after positive culture was associated with persistent nosocomial infection, while recovery above 200 pg/mL was associated with resolution of infection (p < 0.05) . Low TNFter that .th day.Lymphocytic apoptosis is recognized as the core feature of the critical illness induced immunoparalysis. Apoptosis is regulated by a wide group of pro-apoptotic and anti-apoptotic genes, BCL2 (the anti-apoptotic gene) was used to detect the enhanced lymphocyte apoptosis and identify patients at higher risk of HAI in previous studies in children and adults . In our We observed that up-regulation of apoptosis and down-regulation of proinflammatory markers, which are major elements of CARS, were associated with HAI and MODS.To our knowledge, this is the first study of mRNA biomarkers to detect the acquired immune suppression in critically ill children in our community. This study provides a new evidence that immune suppression can be measured. This finding requires confirmations in larger studies and by assessing other mRNA biomarkers which affect the immune response.6Nosocomial sepsis and MODS remain a major cause of morbidity and mortality in PICU. Early detection of patients at risk would be of great help to provide them with the suitable immunomodulators. Lymphocytic apoptosis is one of the causes of immunosuppression in critically ill patients. Our results showed that patients who developed HAI and/or MODS had a lower lymphocytic count, lower levels of BCL2 (the anti-apoptotic gene) and lower level of TNFα (the proinflammatory cytokine). TNFα and BCL2 on admission can predict HAI and MODS (AUC = 0.7 and AUC = 0.8), but were of no use in the follow-up, however, the lymphocytic count is a rapid, easy and cheap test to assess the immune state with a good predictive and follow up values.Small sample size, heterogeneous population not classified by etiological causes.Further studies should classify patients according to their outcomes in grades and their causes of admission as surgical, medical and sepsis, with larger numbers of patients for a sufficiently powered study.Not commissioned, externally peer reviewed.Approval of the study was obtained from the Ethical Committee of Scientific Research, Faculty of Medicine, Benha University. Written informed consent was taken from the parents of each child.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Authors contributed equally in the study.No conflicts of interest.ChiCTR1800018506.The corresponding author.


Background: Viral influenza infection causes serious health issues especially when an outbreak occurs. Although influenza virus vaccines are available and each year manufactures modify the vaccine depending on the expected mutated strain, it is still far from satisfactory, mainly in young children and older adults. Therefore, a product that can support and shape the immune system to protect against viral flu infections is highly essential. Methods: A functional food water-soluble mixture of pomegranate, red grape, dates, olive fruit, figs, and ginger extracts, termed herein “Protector”, was prepared and tested in stimulating/modulating the production of specific cytokines, and hemagglutinin inhibition (HAI) antibodies following viral flu vaccination in mice. Results: A single intraperitoneal or multiple oral administration for 1–7 days of “Protector” significantly increased the production of interferon (IFN)-γ and interleukin (IL)-12 in blood, spleen, and lungs of mice. When “Protector” was orally administered for one week following a single vaccine injection (primary immunization) or for two weeks (one week apart) following double vaccine injections (secondary immunization), mice significantly produced higher titers of HAI antibodies. This increase in HAI antibodies was associated with Pillow-inducing significant and different changes in vaccine-induced IFN-γ, IL-12, IL-6 and IL-22 following primary and secondary immunizations. Conclusions: “Protector” administration reinforces the protective immune parameters against viral flu infection. Therefore, after performing preclinical toxicology studies and ensuring its safety, “Protector” should be considered a potential product to be tested in clinical trials to conclude its efficacy in reducing the devastating effects of flu infection in humans and its outbreaks. The immune system components including cells, antibodies, cytokines and many other chemical mediators act in orchestric and systemic ways to overcome intruders. Therefore, keeping the immune system vigilant and robust is the factor to eliminate diseases as well as fight infections. Following an inflammatory condition or a viral infection, immune and other tissues produce cytokines to activate and regulate the immune response. During viral infection, cytokines such as type I interferon (IFN), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, are induced following the innate immune response, initiate inflammation via stimulating the production of more cytokines, limit virus replication, and provide signals to activate adaptive immunity . ActivatAlthough IL-6 is an important inflammatory cytokine in the initial phase of influenza viral eradication , it has One of the epidemic viral infections that occur each year; killing hundreds of thousands and hospitalizing millions of people is the flu virus . InfluenOne of the essential ways to keep the body’s immune system alert is by eating the appropriate type of food that contains the necessary nutrients and eliminating/reducing the “food” that culminates the immune system. To assist in this, we have developed a water-soluble mixture of edible extracts composed of pomegranate, red grapes, dates, olive fruit, figs, and ginger, termed herein “Protector”. Each extract of this mixture has been shown to modulate the immune system in favor of either stimulating an event or cascade of events in the immune system ,27,28,29All “Protector” fruit extracts were purchased from Naturex, France. Each extract is standardized and quantified based on a specific group of compounds in each extract with complete certificate of analysis. “Protector” consists of pomegranate extract whole dried fruit including peel (400 mg) with drug extract ratio (DER) 5–6:1, dried red grape extract including peel and seeds (200 mg) with DER 10:1, dried dates fruit extract (100 mg) with DER is 2:1, olive fruit dried extract (100 mg) with DER 3–4:1, figs dried extract (150 mg) with DER 2:1, and ginger dried extract (50 mg) with DER 50:1. So in 1 g of “Protector”, the mixture is 4:2:1:1:1.5:0.5.The protocols applied were according to the World Health Organization (WHO) for “Evaluating Efficacy and Toxicity of Herbal medicines” and the Federation of Laboratory Animal Sciences Association (FELASA), for Biological and Toxicological studies and were approved by the Ethics Committee at University of Petra. The present animal studies were performed on female Balb/c mice weighing 25 ± 3 g. Animals were housed in a pathogen-free environment at 22 °C with 12 h light/dark cycle at the University of Petra animal care unit. Before each experiment, animals were divided, placed in appropriate cages and were acclimatized for five days to laboratory conditions.n = 4). One negative, one positive control and three treated groups. Animals received a single intraperitoneal (i.p.) injection of 1 mL sterile PBS containing 0.2 µm-filtered amounts  of “Protector” or Eriobotrya japonica water extract (EJ), as a positive control and a stimulator of IL-12 and IFN-γ –like); and (3) B/Hubei-Wujiagang/158/2009 NYMC BX-39 (B/Wisconsin/1/2010-like). Each 0.5 mL pre-filled syringe contains 15 µg haemagglutinin of each of the three strains  in a buffered saline solution. A buffered saline solution consists of the following excipients–sodium chloride, potassium chloride, sodium phosphate–dibasic dihydrate, potassium phosphate–monobasic and water for injection. The vaccine is prepared from virus grown in the allantoic cavity of embryonated eggs, concentrated, purified by zonal centrifugation in a sucrose gradient, split by octoxinol 9 (Triton X-100), inactivated by formaldehyde and then diluted in phosphate buffered saline solution to the required concentration. No adjuvant or preservative is added. The vaccine may contain traces of formaldehyde (≤30 µg), octoxinol 9 (≤200 µg) and neomycin (<20 pg). Vaxigrip does not contain more than 0.05 µg ovalbumin per dose.For preparation of the vaccine, 0.4 mL of sterile PBS was added to each Vaxigrip (45 µg/0.5 mL) vial. This yielded (50 µg/mL) and then a volume of 100 µL/mouse  was injected subcutaneously (s.c.) in the right thigh of each mouse ,43.Primary and secondary immunization protocols were performed using six groups of mice (5 mice/group). The primary immunization protocol was as follows: on day 1, groups 2 and 3 were immunized s.c. with a single injection (5 µg/mouse) whereas group 1 was injected with sterile PBS . Also stAs for the secondary immunization protocol, mice (groups 5 and 6) were immunized s.c. with two injections (5 µg each injection/mouse), whereas group 4 with sterile PBS, on days 1 and 14. Also starting on day 1, mice were orally administered three times a day with 400 µL of sterile distilled water containing 375 µg of “Protector” (group 6) or just water (groups 4 and 5) until day 7 and then from day 14 to day 21. Mice were kept under observation for another week  and then were sacrificed and tissues were collected as described above.The influenza virus binds to red blood cells (RBCs) and forms agglutination. Thus, having antibodies against the influenza viruses prevents the virus from binding to RBCs and thus no agglutination occurs . FirstlyMeasurements of mouse tissue-extracted cytokines were accomplished by sandwich ELISA developed following the manufacturer’s recommendations . Absorbance values of each well of 96-well plate  were read at 450 nm by GloMax-Multi Detection system , transformed to cytokines concentrations (pg/mL) and then normalized to (pg/g of protein/sample).p value of <0.05 was considered significant.All data in the figures are presented as the mean ± standard error and assessed by using one-way ANOVA analysis followed by a Tukey’s test for multiple comparisons (SPSS version 17). After 24 h of intraperitoneal administration, “Protector” significantly increased the production of IFN-γ in peripheral blood and spleen  in a dosTo evaluate the time profile of this increase in IFN-γ and IL-12 production, “Protector” at 10 µg dose was administered i.p. in mice. “Protector” significantly increased the production of IL-12 and IFN-γ in blood, spleen, and lungs over time . In compBefore performing the following studies, safety oral dose experiments on “Protector” were completed and found that mice did not show any sign of toxicity for 14 days at the maximum dose tested of 2000 mg/kg (data not shown). Since “Protector” was found to induce cytokines following 24–48 h of intraperitoneal administration and we still need to find if “Protector” gets absorbed in vivo and induces a similar response to intraperitoneal administration, we evaluated “Protector” following multiple oral dosing in inducing cytokines. The target organs tested were peripheral blood, spleen as a secondary lymphoid organ and lungs, as a target of respiratory infections. Oral administration of “Protector” (375 µg × 3/day) significantly increased the production of IL-12 and IFN-γ in blood, spleen, and lungs in comparison to 0-time negative control (group 7) . In compSince “Protector” was found to enhance cytokines production in tissues following systemic as well as multiple oral administration, we further evaluated if “Protector” can enhance HAI antibodies production following immunization with an influenza vaccine. These antibodies are measured as best protective degree against influenza infection in humans ,43. VaxiWe also investigated if this increase in anti-influenza hemagglutination antibody is associated with cytokines modulation. The cytokine modulation pattern in blood, spleen, and lungs following primary (14-day period) and secondary (28-day period) immunizations with the Vaxigrip vaccine were similar. The vaccine: (a) significantly increased IFN-γ, IL-12, IL-6 levels in blood; (b) significantly increased IFN-γ and IL-12 in spleen and lungs; but (c) did not modulate IL-6 in spleen or lungs . HoweverThe present study has shown that “Protector” administration in mice enhanced the production of IL-12 and IFN-γ in peripheral blood, secondary lymphoid organ and lungs following intraperitoneal as well as oral dosing. The latter implies that the constituents of “Protector” gets absorbed following oral administration and stimulates immune cells. Such immune cells, for instance, dendritic cells, macrophages, and NK cells are known to produce IL-12 and IFN-γ, respectively ,4,5. AlsThe kinetic patterns of the IFN-γ and IL-12 productions in blood, spleen, and lungs following a single i.p. injection revealed that these tested cytokines persist in the peripheral blood and the organs for at least 48 h . In compIt has been shown that some of the “Protector” constituents, non-water extracts, or isolated compounds exert anti-inflammatory activity in inflammatory models ,35,36,37In this study, we used Vaxigrip, a known commercial human viral flu vaccine, to induce the production of HAI in mice. However, it was used to test if “Protector” has an enhancing effect on the Vaxigrip-induced HAI response . The preViral flu infects the epithelial cells of the lungs, and within hours it induces proteins that block the innate immune system. Such blocking pathway prevents viral RNA from being recognized by PRPs, and this lowers IFN-related genes activation . RecentlIn conclusion, “Protector” administration reinforces the protective immune parameters against viral flu infection. Therefore, after performing preclinical toxicology studies and ensuring its safety, “Protector” should be considered a potential product to be tested in clinical trials to conclude its efficacy in reducing the devastating effects of flu infection in humans and its outbreaks.


White matter hyperintensities (WMHs) are frequently detected in migraine patients. However, their significance and correlation to migraine disease burden remain unclear. This study aims to examine the correlation of WMHs with migraine features and explore the relationship between WMHs and migraine prognosis.A total of 69 migraineurs underwent MRI scans to evaluate WMHs. Migraine features were compared between patients with and without WMHs. After an average follow-up period of 3 years, these patients were divided into two groups, according to the reduction of headache frequency: improved and non-improved groups. The percentage and degree of WMHs were compared between these two groups.P < 0.001) and had a longer disease duration . Furthermore, 33 patients completed the follow up period (15 patients improved and 18 patients did not improve). Patients in the non-improved group had a higher frequency of WMHs  and median WMHs score .A total of 24 patients (34.8%) had WMHs. Patients with WMHs were significantly older (39.0 ± 7.9 vs. 30.6 ± 10.4 years, WMHs can predict unfavorable migraine prognosis. Furthermore, WMHs may have a closer association with age than migraine features. Migraine is a chronic debilitating headache characterized by recurrent moderate-to-severe headache attacks and autonomic nervous system related-symptoms . GloballLarger or confluent WMHs are usually observed in cerebrovascular diseases and cognitive decline cases , 11. HowA total of 69 migraine patients  were consecutively recruited from the Headache Clinic of the Department of Neurology, The First Affiliated Hospital of Xi’an Jiaotong University from February 2012 to November 2016. Inclusion criteria included: (1) patients with migraine who fulfilled the International Classification of Headache disorders (ICHD)-3 (β) criteria [All enrolled patients were required to complete a standard questionnaire to collect basic clinical information at the Headache Clinic. This questionnaire assessed the demographic characteristics, past history, family history and features of migraine, and any accompanying symptoms and self-rating depression scale (SDS). Migraine features include disease duration, attack frequency, attack duration and the visual analogue scale (VAS) score. In addition, the questionnaire also investigated the history of smoking, hypertension, diabetes, oral contraceptive use, past vascular events, heart diseases, tumors, intracranial organic diseases and other medical conditions.All participants underwent MRI scans at the Department of Radiology, The First Affiliated Hospital of Xi’an Jiaotong University, using a 3.0 T GE Discovery MR scanner and a standard 8-channel phase array head coil. High-resolution structural images were acquired using a three-dimensional T1-weighted sequence with the following parameters: TR, 10.276 ms; TE, 4.9 ms; matrix, 256 × 256; section-thickness, 1 mm; FOV, 256 mm. T2-weighted images were acquired with the following parameters: TR, 6000 ms; TE, 104.4 ms; matrix, 385 × 384; section-thickness, 5 mm; FOV, 240 mm. FLAIR images were acquired with the following parameters: TR, 9102 ms; TE, 168.7 ms; matrix, 288 × 224; section-thickness, 5 mm; FOV, 240 mm.n ≤ 5), 2 (hyperintensity < 3 mm and n ≥ 6), 3 (hyperintensity 4–10 mm and n ≤ 5), 4 (hyperintensity 4–10 mm and n ≥ 6), 5 (hyperintensity ≥11 mm and n ≥ 1), and 6 (confluent). The sum of scores from each location was considered as the final score [WMHs were visible as hyperintense lesions on FLAIR images, and as isointense or slightly hypointense lesions on T1-weighted images. MRI scans were assessed for the number and features of WMHs, including the appearance, number, size and anatomical location. All MRI scans were reviewed by an experienced neurologist and neuroradiologist. The degree of WMHs was assessed using the Scheltens visual rating scale . Brieflyal score , 16. Accn = 45) were recruited for a follow up visit. A total of five patients were excluded due to percutaneous closure of the patent foramen ovale, and another seven patients dropped out from the follow up study. The study flow chart is illustrated in Fig. In December 2016, patients who were enrolled in the study for a mean period of > 24 months  or non-steroidal anti-inflammatory drugs, according to the recommendation of physicians.t-test was used to compare the means of normally distributed variables. Non-parametric tests were used for non-normally distributed data. A correlation analysis between WMHs and patient prognosis was performed using Spearman correlation. Further, logistic regression was conducted to evaluate contributing factors to migraine prognosis. Statistical analyses were performed using the SPSS 23.0 software. A P-value < 0.05 was considered statistically significant.Demographic and clinical characteristics were tabulated using descriptive statistics, including percentages, quartiles  and means . Chi-square test and Fisher’s exact probability test were used to test for differences in categorical data. Student’s n = 16), visual and sensory aura (n = 2), and brainstem aura (n = 1). Five patients were presented with chronic migraine, while the remaining patients were episodic. Moreover, a total of eight patients (11.6%) were smokers, and another five patients (7.2%) were hypertensive, but none of the enrolled patients were diabetic.A total of 69 patients with an average age 33.6 years old (range: 14–54 years old) were enrolled in the present study. Among these patients, 52 patients (75.4%) were females. The demographic characteristics and clinical features of migraine are summarized in Table P = 0.000). Among the disease burden related variables, disease duration was significantly higher in the WMH group than in the non-WMH group . Furthermore, a moderate positive correlation was observed between age and disease duration , which indicate a possible confounding effect of age in the association between disease duration and WMHs. A scatter plot was preformed to show the changing trend of disease duration with age. The trend was similar between the improved group and non-improved group  and WMH group (the presence of WMHs or a WMH score of ≥1) (Table n = 24), most lesions were punctuate (22 patients), and two patients presented with confluent lesions   Table .Table 2CP = 0.027, Table P = 0.030).Among the 18 patients in the non-improved group, 10 patients had one or more WMHs, while WMHs were detected in only two of 15 patients in the improved group, and the difference was statistically significant  and aura  were the independent risk factors associated with the non-improved outcome.P < 0.001) and had a significantly longer disease duration  and attack duration . Patients without aura also had a higher incidence of WMHs than those with aura . The difference was obvious although it did not achieve the statistical significance.In the follow up group, patients without aura were significantly older than those with aura  and long-term follow up should be conducted to confirm these results. Future studies should also employ more stable parameters that assess disease burden, in order to further confirm the clinical significance of WMHs. It is worth mentioning that in the present study, we could not definitively investigate other WMH-associated risk factors, including hypertension, diabetes, hyperlipidemia, hyperhomocysteinemia, hyperuricemia, hypercoagulability, heart diseases, kidney diseases, inflammation and autoimmune diseases. These conditions may impact the strength of our conclusions regarding the nature of WMHs and their effect on migraine. Future studies should be controlled for the confounding effects of the above mentioned conditions.This study suggests that WMHs can predict short-term unfavorable migraine prognosis, thereby providing a new insight into the clinical significance of WMHs in migraine. Meanwhile, it demonstrates that WMHs in migraine patients are generally mild, mostly located in the frontal and parietal lobes, and may have a closer association with age than headache characteristics.