Left ventricular hypertrophy and diastolic dysfunction are common echocardiographic features of both aortic valve stenosis (AS) and cardiac amyloidosis (CA). These two different entities therefore may mask each other. From recent years, there is a growing body of evidence about the relatively high incidence of wild-type transthyretin (wtTTR) amyloidosis in AS, but there are scarce data on the prevalence of AS in CA, particularly in AL-type amyloidosis. The echocardiographic approach to these patients is not obvious, and not evidence based. We aimed to study the prevalence, severity, and type of AS in patients with CA and also to evaluate the potential of echocardiography in the diagnostic process.Between January 2009 and January 2019, we retrospectively analyzed the clinical and echocardiographic data, and the echocardiographic work up of 55 consecutive CA patients.80% of our CA patients had AL amyloidosis. We identified 5 patients (9%) with moderate to severe AS: two with moderate AS and three with low-flow, low-grade AS (LFLG AS). Further analysis of the latter three patients with dobutamine stress echocardiography revealed pseudo-severe LFLG AS in two, and true-severe AS in one patient.The prevalence of moderate to severe AS is 9% in our population of CA patients, the majority of whom have AL amyloidosis. Dobutamine echocardiography seems to be appropriate for the further characterization of patients with LFLG AS, even with normal ejection fraction. Aortic valve stenosis (AS) is a common valvular heart disease, which significantly reduces life expectancy when it becomes symptomatic . The preAS and CA share common echocardiographic features, such as left ventricular hypertrophy, diastolic dysfunction, and – in many cases – elevated left ventricular filling pressure. Echocardiography may not reveal CA in AS, because the typical signs of CA may be readily explained by the AS. In recent years, a few case studies reported the coexistence of wtTTR cardiac amyloidosis and AS, highlighting the diagnostic, as well as the therapeutic challenges of these cases , 5. The We aimed to study the prevalence, severity, and type of aortic valve stenosis in patients with CA. We also aimed to examine the potential role of dobutamine stress echocardiography in the diagnostic work up of patients with CA and low flow-low grade AS (LFLG AS).We performed a systematic review of CA patients treated in our department, between 2009 January and 2019 January, focusing on the presence of AS. We diagnosed AL-type amyloidosis when all the following criteria were met: the presence of monoclonal light chain in the serum and in the urine; the presence of plasma cell dyscrasia in bone marrow biopsy; and positive tissue biopsy confirming light chain amyloid deposition in any organ. Cardiac involvement was diagnosed when cardiac MRI showed late gadolinium enhancement as a typical sign of CA, echocardiography revealed characteristic abnormalities, or the presence of AL amyloid was detected in cardiac tissue by biopsy, performed either during right heart catheterization or at autopsy. Since June 2016, if no monoclonal light chain was present in the serum, we established the diagnosis of TTR CA after a positive Tc-99-pyrophosphate (PYP) isotope scan (Perugini score 2 or 3), specific for the disease . In prevUnless it was contraindicated, we routinely performed CMR  for differential diagnostic purposes, and as a test to confirm CA. Resting echocardiography was performed in accordance with the topical guidelines  –15. Afte2, but low–flow, low-grade AS (LFLG AS) was diagnosed by echography at rest, we performed dobutamine stress testing to differentiate between pseudo- and true-severe AS, regardless of the left ventricular ejection fraction.During echocardiography, the suspicion of AS was raised by valve morphology in agreement with the criteria set out in the latest European guideline on valvular heart disease . We perfSince most of the variables exhibited skewed distributions, the descriptive statistics are presented as medians with interquartile ranges (IQR), or as percentages. The strength of the associations was calculated with the nonparametric Mann-Whitney test or the chi-square test, as appropriate.Between January 2009 and January 2019 we performed resting transthoracic echocardiography in 55 patients with cardiac amyloidosis: 44 had AL-, 9 TTR (6 with mTTR and 3 with wtTTR), and one patient had AA amyloidosis. Their median age was 65 years.Here we summarize the most important therapeutic and mortality data of this patient population: Specific treatment of the AL patients for the plasma cell dyscrasia was driven by hematologists. In most of the cases, a combination of cyclophosphamide-bortezomib and corticosteroid was introduced first, but the whole spectrum of multiple myeloma medication was used, including lenalidomide, thalidomide, daratumumab and melphalan. Doxycylin was used in the last 4 to 5 years. Two patients had autologous bone marrow transplantation.wtTTR patients did not receive any specific treatment. Four out of the six mTTR patients received 20 mg tafamidis, daily. Generally, our CA patients had no ICD, and none of these 55 patients had heart transplantation.Calculation of mortality rate is not simple, because the follow up period is no more than nine month in the cases of the latest included patients. We think, the most correct way to describe mortality in this case, is to describe the nine month mortality, which was 23/55 (42%) for the whole CA patient population.p-value of the difference was only 0.055.The systematic review identified 5 patients with moderate or severe aortic valve stenosis. Important clinical, biomarker, and echocardiographic characteristics of the patients are shown in Table 2/body surface area (BSA) m2 was calculated in 3 cases, whereas in 2 cases, the calculated AVA was somewhat greater than 0.6 cm2/BSA (0.65 and 0.63 cm2/m2) – indicating moderate AS. In patients with an AVA/BSA less than 0.6 cm2, we performed dobutamine stress echo to distinguish between true-severe AS, and pseudo-severe AS. One patient, with a left ventricular ejection fraction (LVEF) of 48%, had true-severe AS: both echo and EKG suggested amyloidosis, CMR verified CA – and thus, the final diagnosis was AL amyloidosis  did not reach 1.We found a 9% prevalence of moderate-to severe AS among consecutive, unselected CA patients. Of note, the vast majority of our CA patients had AL amyloidosis. This might be explained, in part, that our university hospital specializes also in hematology, in addition to cardiology. Nowadays, the number of patients diagnosed with wild-type TTR cardiac amyloidosis is growing fast . Howeverperry et al. found that the prevalence of moderate to severe AS was 16% in TTR CA [ava et al. included patients with diverse causes of amyloidosis. They examined the results of aortic valve replacement in these patients [In previous years, the prevalence of CA in AS patients was explored using different methods and study designs and was found to be 4.1  to 16% , as welln TTR CA . The retpatients . The autpatients , 3 is, aCardiac amyloidosis was once thought to be a very rare disease , 19. Stu2, the mean pressure gradient is low (< 40 mmHg), and low-flow state is confirmed by the calculated low-stroke volume index (< 35 ml/m2), the guideline recommends performing dobutamine stress echocardiography. This test can differentiate between “pseudo-severe AS”, and true-severe AS. In pseudo-severe AS, AVA will increase together with stroke volume (SV), but the mean transvalvular gradient will not change significantly. In case of true-severe AS, the gradient will increase, but AVA will not change significantly. Of course, the dobutamine test will not aid the correct diagnosis in the absence of left ventricular contractile reserve – in this case, other clinical and imaging parameters should be taken into account. Interestingly, the guideline recommends performing the dobutamine test in LFLGAS only in patients with a reduced LVEF. From the pathophysiological perspective, this may seem reasonable, as one could speculate that only reduced LVEF can be increased by dobutamine. However, there are data on the usefulness and safety of this test in patients with LFLG AS, with reduced SV, but a normal EF. Caval et al [The other novelty of our publication is, that it may draw attention to the use of dobutamine stress echo in LFLG AS patients with preserved ejection fraction in CA. Current guidelines do not recommend to perform dobutamine stress test with echo in LFLG AS patients with preserved ejection fraction (EF) . The latal et al  performeAlthough there are speculations that the pathomechanism of CA and AS may be connected, as amyloid deposition in the aortic valve could induce stenosis, there is no supporting evidence for that. Apparently, true-severe AS and CA coexist in a large number of patients. However, this can rather be explained simply by statistical distribution, as the prevalence of AS is quite high in the elderly, who also have a higher risk for wtTTR. In the above-mentioned studies, 6 to 16% of the AS patients systematically screened for TTR amyloidosis had wtTTR CA , 9, 10. In our study, only one AL patient had significant, true-severe AS. This 66-year old patient with advanced (NYHA Class III) heart failure survived only 2 months after diagnosis. In this case, life expectancy was clearly limited by AS, CA, and the systemic disease of multiple myeloma. In such a case, it is not possible to determine the share of AS and CA in contributing to heart failure. In a UK study , patientperry et al [Sry et al  at CleveBased on our experience, we wish to underline the importance of dobutamine stress echocardiography in our LFLG AS population. This test can identify patients with true-severe AS, who may benefit from AVR or TAVI. We think that further stratification of LFLG AS, even in cases with preserved EF, is necessary in this patient population with CA, to screen for patients, who may benefit from invasive treatment.This is the first publication aimed to determine the prevalence of AS among unselected CA patients. In our patient population, the prevalence of moderate to severe AS was 9% based on resting echocardiography findings. Using dobutamine stress echocardiography, we were able to distinguish patients with pseudo-severe AS from those with true-severe AS, even in case of LFLGAS with preserved LVEF. Our results may draw attention to the coexistence of CA and AS, as well as to the importance of dobutamine echocardiography in determining the nature of AS in CA.The major limitation of our study is its small sample size. Because hematology is a major component of the clinical profile of the 3rd Department of Internal Medicine at Semmelweis University, our study population is not representative of CA patients in Hungary – those with AL amyloidosis are possibly overrepresented. Nevertheless, according to our knowledge, this study is the first to screen consecutive CA patients for AS.


We explored the association between excess body fat and academic performance in high school students from Santiago, Chile. In 632 16-year-olds  from low-to-middle socioeconomic status (SES), height, weight, and waist circumference were measured. Body-mass index (BMI) and BMI for age and sex were calculated. Weight status was evaluated with 2007 World Health Organization (WHO) references. Abdominal obesity was diagnosed with International Diabetes Federation (IDF) references. Total fat mass (TFM) was measured with dual-energy X-ray absorptiometry (DXA). TFM values ≥25% in males and ≥35% in females were considered high adiposity. School grades were obtained from administrative records. Analysis of covariance examined the association of fatness measures with academic performance, accounting for the effect of diet and physical activity, and controlling SES background and educational confounders. We found that: (1) having obesity, abdominal obesity, or high adiposity was associated with lower school performance alone or in combination with unhealthy dietary habits or reduced time allocation for exercise; (2) high adiposity and abdominal obesity were more clearly related with lower school grades compared to obesity; (3) the association of increased fatness with lower school grades was more salient in males compared to females.  Excess weight at a young age is associated with cardiometabolic disorders ,2, reducNational reports also indicate that excess weight and risk factors are more prevalent among socially vulnerable groups ,6,8,9. AThe cardiometabolic complications of childhood and adolescent obesity have been widely explored; however, its impact on cognition and educational outcomes has recently received a lot of scientific attention. Certain brain structures continue to develop throughout adolescence; therefore, whether obesity impacts the adolescent brain is highly relevant. Frontal and temporal lobe abnormalities as well as reduced hippocampal volume were found in obese adolescents . Both thBecause paediatric obesity has been related to specific outcomes that are relevant for children’s educational attainment, some authors have examined the association of weight status with academic attainment as measured by school grades and standardised test scores. A negative, significant association was reported for adolescent students in Finland , IcelandMost studies addressing the academic implications of adolescent obesity rely on body-mass index (BMI) as the sole measure. With some exceptions ,26,27, vn = 1791). They were born at term of uncomplicated vaginal births, weighed >3.0 kg, and were free of acute or chronic health problems. At 6 months, infants free of iron deficiency anemia (n = 1657) were randomly assigned to receive iron supplementation or no added iron (ages 6–12 months). They were assessed for developmental outcomes in infancy and at 5, 10, and 15 years [n = 679) were also assessed for obesity risk and the presence of cardiovascular risk factors in a half-day evaluation that included assessment of dietary habits and nutritional content of food intake. Of them, n = 632 (93%) entered high school and were eligible for this study. Ethical approval was obtained by the institutional review boards of the University of Michigan, Institute of Nutrition and Food Technology (INTA), University of Chile, and the University of California, San Diego. Participants and their primary caregiver provided informed and written consent, according to the norms for Human Experimentation, Code of Ethics of the World Medical Association .This was a cross-sectional study within an infancy cohort. The sample was made up of 16–17-year-old adolescents living in Santiago, Chile, from low-to-middle SES. Participants were recruited at 4 months from public healthcare facilities in the southeast area of Santiago (15 years . At 16–1School grades in high school (9th to 12th) and final grade-point average (GPA) were collected from administrative records of the Ministry of Education (Chile). Since schools may have differed in grading policies, grades  were transformed into scores (range of 210–825), following the Ministry of Education criteria. The arithmetic average of each subject taken during each academic year was calculated and the result was compared in the conversion table provided by the Department of Assessment, Measurement and Educational Record, University of Chile, which complies with specifications on behalf of the Ministry of Education. The same procedure was used to convert the GPA into a score. Both school grades and GPA were used as continuous variables. z) were calculated. Weight status was evaluated according to WHO references: normal weight (BMIz from −1 to 1 SD); excess weight (BMIz > 1 SD). Abdominal obesity was defined as WC ≥ 90 cm for males and ≥80 cm for females, according to the International Diabetes Federation (IDF). Total fat mass (TFM) was determined via dual-energy X-ray absorptiometry (DXA) . Because there is no generally accepted definition of obesity based on TFM, we used cutoffs by Taylor et al.: values ≥25% in males and ≥35% in females were considered high adiposity [A physician used standardized procedures to measure the adolescent’s height (cm) to the nearest 0.1 cm, using a Holtain stadiometer, and weight (kg) to the nearest 0.1 kg, using a scale . Waist circumference (WC) was measured with nonelastic flexible tape and recorded to 0.1 cm . Measurements were taken twice, with a third measurement if the difference between the first two exceeded 0.3 kg for weight, 0.5 cm for height, and 1.0 cm for WC. BMI and BMI for age and sex , validated using three 24-h recalls to include weekends  ,34. A liWe approached physical activity (PA) with scheduled, repetitive, and planned PA, accounting for the number of weekly hours devoted to physical education (PE) and extracurricular sports. To measure this, we used a questionnaire that was validated in a previous study using accelerometry-based activity monitors in both elementary and high school children . The queThe present study used family structure and parental education level as proxies of SES. Parental education is an important predictor of children’s educational outcomes . In infaIn Chile, secondary education includes academic high schools, which provide theoretical education in languages, mathematics, history, and sciences; vocational training schools; and adult schools. Data on the type of secondary education attended by participants was retrieved from publicly available records at the Ministry of Education. Aiming to control for potential design biases, we used a categorical variable denoting whether the participant had received iron supplementation or no added iron at 6–12 months.t-test and chi-squared test were used for comparison of continuous and categorical variables, respectively, in male and female participants. Due to sex differences in fatness and academic attainment, we ran separate analyses for male and female participants. To examine the association of excess weight, abdominal obesity, and high adiposity (exposure) with school performance (outcome), we conducted analysis of covariance (ANCOVA). Each fatness measure was tested against school grades (9th to 12th) and overall GPA using two models. Model 1 was unadjusted. Model 2 added parental education, family structure, age at high school completion, type of secondary education , and a variable denoting iron supplementation in infancy (no added iron at 6–12 months). Because diet and physical activity have been found to be associated with academic achievement in studies conducted in Chile [d coefficients. Data were analyzed using Stata for Windows version 15.0 . A p value of 0.05 was used to test for statistical significance.Student’s in Chile ,41,42, an = 679) who participated in a prior obesity/cardiovascular study, 632 (93%) entered high school and were included in the present study. A comparison of the participants included in and excluded from this study is shown in the Of the adolescents (p < 0.001). While the prevalence of obesity was similar in both groups, the prevalence of abdominal obesity (p < 0.001) and high adiposity (p < 0.001) was significantly higher in females. As for SES background, we found that females were more likely to have a father with incomplete higher education (p < 0.001). They were also more likely to be physically inactive compared to males (p < 0.001), although no differences were observed in relation to diet quality.After controlling for sex, we found that female adolescents had better school performance than males (d ranging from 0.51 to 0.67) in males, whereas in females, the effect was moderate (Cohen’s d ranging from 0.33 to 0.45). In males who had a fair-to-healthy diet, but still were obese, obesity was related to lower school attainment across high school, whereas in females who had a fair-to-healthy diet, but still were obese, obesity was associated with lower school performance only in grades 11 and 12. As for the effect of diet quality in nonobese adolescents, we observed that in males, a poor diet quality was related to lower school attainment in grades 11 and 12. A lower GPA was also observed in nonobese males who had unhealthy diets. In nonobese females, unhealthy dietary habits were associated with lower school performance only in grade 11.When we explored the association of fatness with school grades, accounting for the effect of diet quality, we observed that in both males and females, the greatest losses in school attainment were related to the combined effect of increased fatness and poor diet quality. In a first analysis, where fatness was approached with obesity , the comd values ranging from 0.69 to 0.74) and moderate-to-large females   . In partd coefficients indicate that the combined impact of diet and fatness on school grades was moderate in females and moderate to large in males. In males with high adiposity, although having fair-to-healthy dietary habits, we also observed lower school grades compared to males without high adiposity, although having fair-to-healthy diets. The ES for difference ranged from 0.36 to 0.39, denoting a moderate effect of high adiposity on school grades in males who had fair-to-healthy diets. In females with high adiposity, on the other hand, we only found a significant association of fatness with school attainment in grade 12 and GPA and a trend towards a lower school attainment in grade 11. As for the effect of unhealthy dietary habits in participants with normal adiposity, again, the association was more salient in males than in females. Males with normal adiposity and unhealthy diets significantly underperformed compared to males with normal adiposity and fair-to-healthy diets in the 10th, 11th, and 12th grades and in terms of GPA, whereas females with normal adiposity and unhealthy diets significantly underperformed compared to peers with normal adiposity and fair-to-healthy diets only in the 12th grade and in terms of GPA. In a third model, where fatness was measured with total fat mass percentage, we found that the combined effect of high adiposity and unhealthy dietary habits was associated with lower school performance in both sexes . Cohen’sd < 0.30).When we explored the association of fatness with school grades, accounting for the effect of PA on the academic outcome, we observed that in both males and females, the greatest losses in school attainment were related to the combined effect of increased fatness and physical inactivity. When fatness was approached with obesity , the comWhen the exposure was abdominal obesity instead of obesity, we found that the combined effect of fatness and physical inactivity on school grades was moderate in females and moderate to large in males . In maled values ranging from 0.25 to 0.35) and moderate in males . In adolescents with normal adiposity, the effect of physical inactivity on school grades was found to be significant only in the 12th grade  and in terms of GPA . Finally, when fatness was approached with total fat mass percentage, we found that the combined effect of high adiposity and physical inactivity on school grades was large in males and females . In bothWe examined the association of fatness measures with school performance in adolescents of low-to-middle SES in a nonindustrialised country. We found that having obesity, abdominal obesity, or high adiposity was associated with lower school performance alone or in combination with unhealthy dietary habits or reduced time allocation for exercise. Second, the findings show that high adiposity and abdominal obesity are more clearly related with lower school grades compared to obesity. Last, the results also indicate that the association of increased fatness with lower school performance was more salient in males than in females.A number of studies have tested the association of academic outcomes with obesity and their results are similar to ours. In a sample of 6346 adolescents from Iceland, Kristjánsson et al. found that BMI, physical activity, and dietary habits were all independently associated with academic achievement . In thisd value from 0.3 to 0.6 corresponds with a moderate ES, whereas a d value greater than 0.6 corresponds with a large ES. Therefore, the effect of obesity on school performance tended to be moderate, yet the effect of abdominal obesity and high adiposity ranged from moderate to large. This suggests that BMI might be underestimating the true effects of obesity on the adolescent brain. Although BMI is the most commonly used method for diagnosing obesity, it has been criticized because it does not always reflect true body fatness, which may be better assessed using body fat percentage and distribution. This also seems to be the case when it comes to understanding how obesity impacts the adolescent brain, specifically the hippocampus and the prefrontal cortex. Adolescence is a period of rapid growth and maturation and is a particularly critical period for hippocampal development and for neural organisation and functional connectivity between the PFC and other brain areas. The hippocampus plays a role in the processing of short- to long-term memory, learning, and emotions, whereas the PFC is involved in executive functions, which allow conscious control, planning, time management, and organisation [A second major finding of our study has to do with the fact that abdominal obesity was the exposure showing the more clear association with school performance, followed by high adiposity and obesity. A nisation . A reducnisation ,13,48. Enisation . Likewisnisation . Furthernisation . Lastly,nisation , which aThe fact that excessive fatness appears to take a special toll on males in terms of school performance is also of interest. Cross-sectional and longitudinal studies conducted in adolescents report results similar to ours. In a sample of Hispanic adolescents in grades 9–11 in Texas (U.S.), obese males had lower scores in both reading and mathematics as measured by the Texas Assessment of Knowledge and Skills (TAKS) in comparison to their nonobese counterparts. However, no relationship was found between obesity and TAKS performance in females . Using dOur findings have implications for public health and education policy and provide schools and parents with potentially significant incentives for encouraging the healthy weight of adolescents. Because academic results are a high priority for schools, educators, and parents, the relationship between students’ health and school success is important to disseminate. Healthy dietary habits, physical activity, and healthy weight in adolescents are important contributors towards good school performance . Thus, tThese results support the connection between weight status and school performance. As most studies have been conducted in the developed world, one strength of the present study is that we provide evidence that is relevant for countries undergoing nutritional and epidemiological transitions. Second, we examined the relationship of adiposity markers and cognition using functional measures of cognition that were aimed at providing translational evidence that can be used for health promotion and brain protection in young populations. Furthermore, very few studies have examined the relation between weight status and school performance using adiposity markers other than BMI. Also, we used DXA to measure TFM. Yet, several limitations should be acknowledged when interpreting these results. Because our sample consisted of adolescents from low-to-middle SES, we are not able to extrapolate these results to the overall population of Chilean adolescents. Although a limitation, this SES bias makes our findings particularly relevant and thus could be taken as a strength for several reasons. The prevalence of overweightness/obesity is higher in adolescents of low-to-middle SES according to nationally conducted surveys ,6. Also,In Chilean adolescents of low-to-middle socioeconomic background, we found a negative and significant association of several fatness measures with school performance, independent of other influences, including diet quality and time allocation for PA. Having obesity, abdominal obesity, or high adiposity might be a potential risk factor for lower academic performance in males, but not always in females. When increased fatness is combined with unhealthy eating and physical inactivity, it becomes a risk factor for lower school performance in both males and females.


Background: The association between decreased heart rate variability (HRV) and increased internalizing symptoms is well documented. Adolescence is a critical period for the development of mental health problems, in particular internalizing symptoms. Previous research has illustrated sex differences in adolescent HRV, such that females have reduced short-term resting state HRV compared to males. Studies on long-term ecological recordings of HRV in adolescents are scarce. The aims of the present study were, (a) to test if adolescent females show decreased long-term HRV and cardiac complexity (CC) compared to males, and (b) to explore whether sex and HRV and CC measures, as well as their interaction, would predict internalizing symptoms.Materials and Methods: HRV was recorded in n = 166 adolescents (86 girls), on a normal school day. HRV and CC measures were calculated on the interbeat interval time series.Results: Females showed lower HRV and CC in most of the assessed indices. Internalizing symptoms were mainly predicted by HRV whereas sex only predicted symptoms of social anxiety. The interaction between sex and HRV did not predict internalizing symptoms.Conclusions: Results suggest that reduced HRV should be considered as a potential contributor to exacerbating internalizing symptoms in adolescence. Girls with reduced HRV and CC might be prone to the development of internalizing disorders. HRV is a promising tool for the early identification of vulnerability. Studies have shown that prevalence rates for internalizing disorders  are higher among females than males  refers to the variations in the length of successive interbeat intervals (IBIs), though it is not a unitary concept. By measuring the length of successive intervals, HRV is calculated, and it can be quantified using time domain measures . However, these measures do not give information about the sources of variability; instead, frequency domain measures of the HRV are used. Thus, use of the high frequency  band power as an index of vagally mediated HRV is widely accepted, i.e., the variability that depends on the inhibitory action of the autonomic nervous system's parasympathetic branch. The low frequency  band power has traditionally been interpreted as an index of sympathetic cardiac control. However, recent studies challenge this interpretation and suggest that the LF seems to be a mix of sympathetic, parasympathetic, and other factors Billman, . Apart fInterest in HRV and cardiac complexity (CC) has grown in the last 30 years. Goldberger  providedn = 43, mean age = 15), and found lower SDNN values for females, but no differences in HF power. Silvetti et al.  could explain the incongruence of results, although most of them point out that cardiac regulation is less flexible in adolescent girls compared to boys. Importantly, HRV and CC in regular school settings  have rarely been studied, though this ecological assessment could provide helpful knowledge about heart regulation in everyday conditions  to test if adolescent females show decreased HRV and CC compared to males and (b) to explore whether sex and measures of HRV and CC, as well as their interaction, predict levels of internalizing symptoms.The Bioethics Committee at the university approved of all procedures, and all participants and their parents or legal guardians provided written consent.n = 82 per group is required to achieve a statistical power of 90% to detect effect sizes in HRV case-control studies. In order to achieve this sample size, and considering the average number of students per school enrolled in the TRANS project, five of the 12 schools were randomly selected and invited to participate in the present study. All of them agreed, and therefore data from 169 adolescents were collected. The exclusion criteria were: suffering from severe mental retardation; a neurological, developmental, or psychiatric disorder . The final sample (N = 166) comprised n = 86 girls and n = 80 boys.Data for the present analyses were taken from a sizable longitudinal project  to all participants. In the following month we conducted cardiac recordings on a regular school day within the academic context but only when participants were not going to be doing any physical or out-of-the-ordinary activities during the time of the recording. Participants left the classroom in the early morning (around 8:00 a.m.) and went to a private room to have their BMIs calculated via measurements of height and weight. Subsequently, the researcher put two electrodes on each participant's chest and attached a portable device to the electrodes (see the Cardiac Assessment section). Participants then returned to their classrooms with the devices activated, and cardiovascular function was continuously measured for the first 120 min of that day. Finally, each participant was scheduled for a structured interview with one of the researchers. Adolescents who had consumed alcohol, drugs, and/or caffeinated beverages during the previous 4 h, adolescents with an acute illness, or those who were menstruating were assessed another day.We used the Revised Child Anxiety and Depression Scale . The time series was filtered with the Physionet  a low-pass band filter at 1,100 ms, a high-pass band filter at 400 ms, and a central interval filter were applied. Using a window of 11 intervals  but excluding the central interval, the average over the window was calculated. If the central interval laid outside 20% (0.20) of the window average, this interval was excluded and the window was advanced to the next interval, resulting in the mean of excluded IBIs .The reporting of cardiac measures follows GRAPH guidelines  intervals. We used the AVNN , a measure of HR, and RMSSD.Frequency domain measures provided the total spectral power of all NN intervals in each frequency band by transforming the time series into the frequency domain through the fast Fourier transform. LF ranges from 0.04 to 0.15 Hz and HF from 0.15 to 0.40 Hz.All HRV measures were calculated using the Physionet  is a method designed by Peng et al. , which aN  are calculated. Secondly, each pair of adjacent points is aggregated  to get a time series of length N/2, and the mean and standard deviation of that time series are calculated. We repeate this aggregation process for 3, 4, 5, and usually up to N/10 adjacent data points. Since our IBI time series included around 9000 values, we repeated the aggregation process for 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 adjacent data points and then plotted the mean and standard deviation of each level of aggregation on a logarithmic graph. When the number of aggregated values increases, the relationship between mean and standard deviation remains the same (invariant) and can therefore be described as a linear relationship, with the slope of the line being the scaling exponent h. Notice that the scaling exponent for random fluctuations is 0.50 and for deterministically regular processes it is 1 . If we take the variance instead of the standard deviation, the slope of the line is 2h. A MathWorks Matlab code (available upon request) was developed to calculate the exponent. The fractal dimension is then calculated according to the formula FD = 2-h.For the fractal dimension (FD), the allometric aggregation method examines the invariance of the relationship between the mean and the standard deviation of a data series as the data points are iteratively aggregated, thus decreasing the resolution  of the series. First, the mean and standard deviation of the time series of length m points remain similar at the next point, is calculated for each of the coarse-grained time series. Less entropy implies higher regularity and predictability. In the current study, entropy for scale factors 1, 5, 10, 15, and 20 was calculated using the software available from https://physionet.org/physiotools/mse/.The multiscale entropy analysis  and set to 0.007. Since HF power values were not normally distributed , we took the natural logarithm of these values (lnHF) before conducting statistical analyses that require data to be normally distributed. After this transformation, the lnHF distribution was normal . Cardiac measures were adjusted by the average HR to control for HR-derived mathematical bias. The cardiac measures that correlated negatively with HR were divided by the average HR. Those which correlated positively (only DFA 1) were multiplied by the average HR, in line with Gasior et al. , frequency-domain measures in the second set (p < 0.025), DFA and FD in the third set (p < 0.017), and entropy measures in the fourth set (p < 0.01). Multiple linear regressions analyses were also performed with each internalizing symptoms subscale as the dependent variable, and both sex and cardiac measures as predictors. In step 1, sex  and each cardiac measure were entered as predictors. In step 2, the interaction between sex and each cardiac measure was added. To prevent for multicollinearity, values of cardiac measures were standardized. All analyses were conducted using the IBM SPSS Statistics v.20.0.0 package.We analyzed differences in cardiac measures between males and females using one-way MANOVAs. Then, Cohen's Quintana . The resM = 14.96, SD = 0.46, range = 13.78–16.40 years old; mean body mass index [BMI] = 17.68, SD = 2.74) and 80 boys .The sample in the present study was comprised of two groups: 86 girls . Females also showed lower CC than males . The effect sizes can be considered medium as most of them were close to 0.50. When we analyzed adjusted cardiac indices, similar results were obtained but the differences between males and females increased. Furthermore, differences also became significant for the α2 exponent  and entropy at scale factors 5  and 10 .No significant differences in age or BMI were found between males and females. Sex differences in cardiac measures are shown in Table p > 0.007).Means, standard deviations, and ranges of total internalizing symptoms are shown in Table Hierachical multiple regression analyses predicting internalizing symptoms from sex and unadjusted cardiac measures are shown in Table Scatter plots of HR and lnHF (both unadjusted by HR) with social anxiety, depressive and total anxiety symptoms are shown in Figure p < 0.05). HR predicted social anxiety , panic , obsessive-compulsive symptoms , depression , and total anxiety . LnHF predicted social anxiety , panic , obsessive-compulsive symptoms , depression , and total anxiety . The α1 exponent was a significant predictor for social anxiety , panic , obsessive-compulsive symptoms , depression , and total anxiety . The α2 exponent predicted panic , obsessive-compulsive symptoms , and total anxiety . The FD was a significant predictor for panic , obsessive-compulsive symptoms , depression , and total anxiety . Entropy at all scale factors predicted obsessive-compulsive  and total anxiety  symptoms. Social anxiety symptoms were predicted by entropy at scale factors from 5 to 20 . Separation anxiety symptoms were predicted by entropy at scale factor 5 . Panic symptoms were predicted by entropy at scale factors 1 and 5 . Finally, depression symptoms were predicted by entropy at scale factors 1, 5, and 20 . No interaction effects of sex and cardiac measures were found.When we adjusted cardiac indices by HR, sex was a significant predictor of social anxiety symptoms  in females. Low vagally-mediated HRV has been repeatedly reported to be associated with anxiety ; Silvetti et al. .Considering the complex regulation of HR, we found the DFA α2 exponent to be higher (closer to 1) in males than in females, but only when adjusted by HR. DFA quantifies the fractal-like properties of IBI time series data. The α2 exponent gives a measure of these properties for long-range (>11 beats) correlations, and Peng et al.  showed td = 0.46 for adjusted and Cohen's d = 0.53 for unadjusted data) in that the FD of males' HR was higher than the FD of females . Again, the physiological meaning of the small differences in the absolute mean values prevents a clear interpretation of this finding, but both the α2 exponents and the FD values point to poorer HR regulation in females. Furthermore, sex differences in HR entropy were clear at scale factors 15 and 20 with unadjusted data, and at scale factors from 5 to 20 with adjusted data, with females showing more regular and predictable HRs. By increasing the scale factor, the differences also increased . Together, these findings clearly underline the more complex regulation of HR in males than in females. We should say that HR scaling properties underlie the role of vagal activity (indexed in this study by the HF band power), as pointed out by Balle et al. .We might be able to understand the lack of sex differences in self-reported anxiety and depression from a diathesis-stress model at the onset of anxiety and depressive disorders. According to Christiansen  and HydeTo sum up, our results provide some evidence supporting the role of HRV and CC as relevant factors for the early identification of physiological vulnerability of adolescent girls. From a clinical point of view, the fact that girls and boys do not significantly differ in self-reported levels of anxiety or depression may mask the girls' vulnerability, and consequently, it might hinder the development of strategies to protect girls against anxiety.Although we consider reduced HRV and CC as vulnerability factors for IDs, we are aware that a more global, neurovisceral view . What is different, depending on sex, is HR regulation, which is significantly less variable and complex in females, and might be making them more prone to developing IDs. From physiologically based interventions  would be equivalent, though we cannot be sure. Lastly, we did not register the different phases of the menstrual cycle. Although we assessed menstruating participants on another day, it has been suggested that all menstrual stages have an effect on the brain areas related to HR regulation (Bäckström et al., Despite these shortcomings, the present study uses a large sample of adolescents that is homogeneous with regards to the age range studied. Furthermore, the fact that adolescents were monitored under ecological conditions improves the external validity of the findings. Altogether, it makes the results of our study robust and helpful in comprehending how sex differences in HR regulation could contribute to the onset of internalizing disorders.AF-V has contributed in the study design, collection, analysis and interpretation of data, and in the writing of the manuscript. AD has contributed to the collection and analysis of data and in writing the manuscript. MB has contributed to the study design and editing the manuscript. XB is the main researcher of the more in-depth project where this study took place. He has contributed to the study design, analysis and interpretation of data, as well as in writing and editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The studies included in this review and meta-analysis revealed that dietary fats and fatty acids had no effects on the risk of CRC.Dietary fat intake is associated with the risk of colorectal cancer (CRC); however, the results of epidemiological studies on this are controversial. Therefore, this study aimed to summarize the available scientific evidence regarding the association between dietary fat and the risk of CRC. We conducted a systematic search of PubMed, Web of Science, and the Cochrane library for articles related to dietary fat and the risk of CRC. The summary relative risks with 95% confidence intervals (CI) were calculated via a random effect model. Begg’s test was used to detect publication bias. A total of 18 articles were identified. The pooled relative risk with 95% CI for the risk of CRC were 1.00 (95% CI: 0.90–1.12), 0.97 (95% CI: 0.86–1.10), 1.08 (95% CI: 0.92–1.26), and 0.99 (95% CI: 0.93–1.04) for total fat, saturated fatty acid, monounsaturated fatty acid, and polyunsaturated fatty acid, respectively. No significant associations were found in subgroup analyses. Begg’s test for all exposures revealed no publication bias (total fat,  Colorectal cancer (CRC) is one of the most common cancers in both men and women worldwide ,2,3. TheAmong factors associated with the development of CRC, modifiable risk factors, such as physical activity, smoking, alcohol drinking, and diet, have drawn attention ,11. In pAccordingly, the need for meta-analyses that qualitatively assess individual studies to integrate recent literature on the association between total fat and individual fatty acid intake and the risk of CRC is evident. Therefore, in this study, we reviewed the available literature and summarized the results by conducting a meta-analysis of prospective cohort studies.A systematic literature review and meta-analysis of studies on the association between dietary fat intake and the risk of CRC were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline . PubMed,Duplicates were removed using the ENDNOTE program. Titles and abstracts were reviewed to check whether the selected articles satisfied the inclusion criteria. If the eligibility for inclusion could not be determined based on the title and abstract only, the full text was reviewed for the final inclusion. Two investigators performed the selection process independently, and differences in opinion between investigators regarding study selection were resolved by discussion.The inclusion criteria were as follows: (1) Original research, (2) prospective cohort study, (3) dietary fat as the major exposure factor ), (4) CRC as the major outcome , and (5) relative risk (RR) or hazard ratio (HR) and the 95% confidence interval (CI) were reported. The exclusion criteria were as follows: (1) Papers other than an original research  and (2) not conducted in humans . For studies overlapping in studied populations, the most recently published article was included. The following information was extracted by reviewing the full texts of the selected articles: Name of authors, year of publication, country, study population, sex, age at baseline, total number of participants, follow-up period, method of diet assessment, diagnostic criteria of CRC, intake levels of fat or fatty acids, RR/HR and 95% CI, and confounders. Quality evaluation was performed according to the Newcastle-Ottawa quality assessment scale-cohort studies , which c2 tests were used to evaluate the heterogeneity of studies included in this analysis. Studies were deemed heterogeneous if I2 was >50% or the p value in the Cochrane’s Q test was <0.1 [p <0.05.To combine the effect size between dietary fat intake and the risk of CRC from each study, the RR or HR and 95% CI of the group with the highest fat intake relative to those of the group with the lowest fat intake were used. The weighted values needed to combine the effect sizes were calculated by identifying the standard deviation of a log-transformed RR, and the summary RR was determined using a random effect model. Cochrane’s Q and Higgin’s Iwas <0.1 . Subgroun = 8156), articles lacking information (n = 44), articles which were not original studies (n = 4065), articles not involving human participants (n = 8787), and articles unrelated to the topic of this study (n = 7288) were excluded. Among the 66 remaining studies, those with overlapping datasets (n = 6), those without RR or HR and 95% CI (n = 3), and those that were not prospective cohort studies (n = 39) were excluded. Overall, 18 articles were included in this meta-analysis , and no significant association was also observed . Similarly, the pooled results of the seven articles reporting the MUFA-CRC association were not significant . The results of the 14 articles on PUFA, including omega-3 and omega-6 fatty acids, were pooled, and no significant association between PUFA intake and risk of CRC was found .In the subgroup analysis of the association between total fat intake and CRC according to the continent in which the study was conducted, heterogeneity was reduced in the United States and Europe, but the association was not significant . SimilarTo verify the reliability of these results, we conducted the leave-one-out analysis to assess the influence of each study on the summary RR. When the study by Chyou et al., (including men) was excluded , the assp = 0.3). Moreover, SFA showed an asymmetrical trend at the lower right side of the funnel plot; however, Begg’s test also showed no publication bias (p = 0.1). Similarly, no publication bias was observed on the funnel plots, and Begg’s test results for MUFA and PUFA also showed no publication bias . Publication bias was visually analyzed using the funnel plot, and the test statistics were determined using Begg’s and Mazumdar’s rank correlation test. The funnel plot for total fat intake showed an asymmetrical trend, but Begg’s test showed no publication bias  and risk of CRC showed a positive association ,43. The In our study, we found that PUFA intake was not associated with CRC. Some studies have reported a positive association between levels of PUFA intake and CRC ,50, wherAlthough multivariable-adjusted RRs were reported in individual studies, the range of adjustment varied across studies, with the possibility of residual confounding. However, the possibility of reverse causality or information bias was minimized by integrating results of the prospective cohort study. Moreover, unlike previous studies, the current study assessed the quality of articles included in the meta-analysis to prevent the distortion of research results by poorly designed studies or from small study effects. Finally, because no meta-analyses integrated the research results on this topic since 2011, this study included studies published after 2011 to enhance the evidence level on the unclear association between dietary fat intake and CRC. This study systematically reviewed research articles published since the 1990s and integrated the results to assess the influence of dietary fat or fatty acids on the risk of CRC. No significant association was observed between total fat, SFA, MUFA, and PUFA intakes and the risk of CRC. Moreover, subgroup analyses by sex, continent, and follow-up period revealed no significant differences. Because numerous studies have been published in the United States and Europe, large-scale prospective studies and clinical trials involving a greater diversity of countries and races should be conducted.


Dietary Guidelines for Americans 2015–20 recommend replacing sugar sweetened beverages (SSBs) with plain water in order to promote adequate hydration while reducing added sugar intake. This study explored how water intakes from water, beverages, and foods are distributed across the day. The dietary intake data for 7453 children (4–18 y) and 15,263 adults (>19 y) came from the National Health and Nutrition Examination Survey (NHANES 2011–2016). Water was categorized as tap or bottled. Beverages were assigned to 15 categories. Water intakes (in mL/d) from water, beverages, and food moisture showed significant differences by age group, meal occasion, and time of day. Plain water was consumed in the morning, mostly in the course of a morning snack and between 06:00 and 12:00. Milk and juices were consumed at breakfast whereas SSBs were mostly consumed at lunch, dinner, and in the afternoon. Children consumed milk and juices, mostly in the morning. Adults consumed coffee and tea in the morning, SSBs in the afternoon, and alcohol in the evening. Relatively little drinking water was consumed with lunch or after 21:00. Dietary strategies to replace caloric beverages with plain water need to build on existing drinking habits by age group and meal type. Dietary Guidelines for Americans 2015–2020 have recommended replacing sugar-sweetened beverages (SSBs) with plain drinking water . The maiThe 2015 Dietary Guidelines for Americans recommended a shift to reduce added sugar consumption to less than 10 percent of calories per day [For example, past analyses of NHANES data have shown a significant effect of age. Teenagers and young adults consumed the most fruit juices, SSBs, and water. Adults and older adults consumed much less SSBs but drank more coffee, tea, and alcohol . EducatiA socio-economic gradient was recently observed for the consumption of tap water . AnalyseAligning daily beverage choices with healthy eating patterns is a key component of many dietary intervention programs ,9,10. HoThe timing of beverage consumption in the course of the day may have additional implications for adequate hydration. There is an emerging mythology about the correct time to drink water in the course of the day. One strategy is to drink water 30 minutes before a meal, during a meal. and after a meal, but no more . AnotherOne recent suggestion was that mild dehydration may occur in a transient manner when water and fluids are not consumed, either because of poor access to water or beverages or because of poor drinking and eating habits . The preConsumption data for drinking water, beverages, and foods came from 3 cycles of the nationally representative National Health and Nutrition Examination Surveys (NHANES), corresponding to years 2011–2012, 2013–2014, and 2015–2016 . The thrThe NHANES 24-hour recall uses a multi-pass method, conducted by a trained interviewer using a computerized interface. Respondents report the types and amounts of all food and beverages consumed in the preceding 24 hours, from midnight to midnight ,19. RespFor children 4–5 y, dietary recall is completed entirely by a proxy respondent  . ChildreNHANES participants were stratified by sex and age. The age group cut-points were: 4–8, 9–13, 14–18, 19–30, 31–50, 51–70, and >70 y. These age groups generally correspond to the age groups used by the IOM. Race/ethnicity was defined as non-Hispanic white, non-Hispanic black, Mexican American, Other Hispanic, and other/mixed race. Family income-to-poverty ratio (IPR) is an index of socioeconomic status; the cut-points for the IPR were <1, 1–1.99, 2–3.49, and ≥3.5.Plain drinking water was split into tap and bottled. Beverages were classified into 15 categories: milk and milk beverages, milk substitutes (soy milk), citrus juices, non-citrus juices, diet soda, regular soda, ready-to-drink tea, ready-to-drink (RTD) coffee, fruit drinks, sports drinks, energy drinks, hot tea/coffee, alcoholic beverages, flavored, carbonated or enhanced water, and supplemental beverages. The present analyses of water intakes from beverages were for beverages only; for example, milk consumed with cereal  was counted in the food category. The USDA Food and Nutrient Database for Dietary Studies (FNDDS), used to establish energy and nutrient content of individual diets, has been revised in parallel to each NHANES cycle .The NHANES 24-hour recall for each participant provides information on the amount in grams of each food and beverage consumed. The present results were for mL of water content from selected beverages, and not for the volume of the beverages themselves (which may not be 100% water). Moisture from foods was calculated as well. Eating occasions were classified as follows: breakfast, morning snack, lunch, afternoon snack, dinner, and evening snacks. That information was obtained by self-reporting. The distribution of water and beverage intakes was also captured by time of day. The 24 h temporal profile of water and beverage consumption was framed in 3 h intervals, starting at 06:00 (6 a.m.) until midnight. The time intervals were 06:00–09:00; 09:00–12:00; 12:00–15:00, 15:00–18:00, 18:00–21:00, 21:00–00:00, and 00:00–06:00.The necessary IRB approval for NHANES was obtained by the National Center for Health Statistics (NCHS) . Adult pThe survey-weighted mean intakes of total water were evaluated overall and by age group, sex, race/ethnicity, and family income-to-poverty ratio. All analyses accounted for the complex survey design of NHANES and reflected the dietary behaviors of the US adult population from 2011 to 2016. The consumption of water and beverages was evaluated for the entire population and for population sub-groups. Survey-weighted means and corresponding standard errors were obtained. All analyses were conducted using SAS software, version 9.4  by using SURVEYREG, SURVEYMEANS, and SURVEYFREQ procedures.Men consumed more total water and more beverages than did women; there was no sex effect for water consumption. Non-Hispanic Whites consumed the most water, the most beverages and the most total water; lowest water consumers were Non-Hispanic Blacks. Mexican Americans drank the most bottled water. Water and beverage consumption also increased with the income to poverty ratio (IPR). Income effects were observed for water and beverages, the effect for tap water was particularly strong (496 vs. 821 mL/d). The present results are among the first to document the timing of water and beverage intakes around the clock in a large and representative NHANES 2011–2016 sample of US children and adults. The present results have important implications for the promotion of healthy beverage choices, notably the ongoing attempts to replace SSBs with plain drinking water.There is very little science on population water consumption patterns during the day. One recent study, conducted in Greece, explored the fluctuation in water intakes and hydration indices during the day, looking for signs of transient dehydration in a sample of healthy adults . While wThe present data add to past work on the impact of caloric beverages consumed separately or with a meal on total energy intakes. In experimental studies, when caloric beverages were presented shortly before or with a pizza meal, no energy compensation was observed. Caloric beverages consumed freely at meal times added calories to the meal ,24. By cExcessive SSB consumption is thought to contribute to childhood obesity . DietaryThe present analyses support the CDC initiative but for a different reason. The CDC report notes that more than 95% of children are enrolled in schools and typically spend 6 h at school each day. We note that those times are in the morning , which are the peak times for water consumption. There seems to be less competition during the morning snack from other beverages. Another productive strategy would be to promote water consumption with the school lunch meal.We were surprised to see that water did not figure prominently in the afternoon snack. Though the total amount of water consumed was comparable to that during the morning snack, those beverages were SSBs, tea, and alcohol (for adults). Promoting water consumption by children in the afternoon may be a potential intervention strategy. By contrast, promoting more water consumption in the morning might be a viable strategy for adults. There are opportunities to increase water consumption at lunch. By contrast, water is unlikely to displace morning coffee, especially with older adults or the afternoon tea .The social gradient in water consumption has been addressed before . Plain dThis study had limitations. First, the NHANES 2011–2016 data are based on dietary self-reports, still the default practice in large population-based studies. Second, the within-day variations by meal and time interval were close but not quite the same. For example, the consumption of water was high between 06:00 and 12:00, but it was associated not with breakfast but with a morning snack. People who reported consuming a morning snack did not necessarily have breakfast. Finally, the NHANES 2011–2016 data are cross sectional, not allowing for causal inferences to be made. The potential impact on water consumption patterns on health outcomes of interest cannot be determined.Present analyses of diurnal fluctuations in water intakes can inform dietary strategies for maintaining adequate hydration while reducing the consumption of added sugars. The most effective strategies for behavioral change are those that build on existing habits and consumption patterns .


Picea jezoensis is completely dependent on coarse woody debris for seedling establishment. To understand the fine-scale spatial genetic structure (FSGS) of this species, a 5-ha plot was established in central Hokkaido, and 531 individual trees were categorized into four life-stages  on the basis of age and size. The FSGS of the established seedlings and later growth stages was investigated using 11 nuclear simple sequence repeat loci. A STRUCTURE analysis of seedlings and saplings established on fallen logs revealed that genetically related individuals were spatially localized between adjacent logs. We also found a significant FSGS in early life-stages based on a decline in the kinship coefficient calculated between individuals over shorter to longer spatial distances. Furthermore, the estimation of dispersal kernels indicated the frequent occurrence of short-distance seed dispersal. These results indicated that genetically related seedlings and saplings regenerated on the same or nearby fallen logs. In contrast to the results for the early stages, mature-stage trees showed no significant FSGS. We ran a simulation to examine the hypothesis that the FSGS could be eliminated by demographic thinning during life history processes. We calculated values for simulated offspring generated under three sets of conditions; i.e., by removing (i) inbred individuals, (ii) randomly chosen individuals, and (iii) all individuals on the specific fallen logs. However, the results for the FSGS were significant for all simulated data sets. This indicated that inbreeding depression, stochastic loss, or eradication of establishment sites by local disturbances alone could not explain the lack of FSGS among mature-stage trees. Therefore, it is possible that the colonization history of mature trees present on the study site might differ from that of the current offspring.Conifers in northern forests, such as fir and spruce, preferably regenerate on coarse woody debris, including fallen logs, stumps, and snags. In northern Japan, the sub-boreal conifer species  Picea, Abies, and Tsuga. These coniferous species successfully regenerate at safe sites, which are most often decaying logs and areas of coarse woody debris (CWD)  , and somis (CWD) , the Pacis (CWD) , and Easis (CWD) . CWD is is (CWD)  and feweis (CWD) . Previouis (CWD) . HoweverAbies sachalinensis, a CWD-dependent regeneration species, A. sachalinensis. In contrast to A. sachalinensis, which mainly regenerates on CWD but can also regenerate directly on forest soil  within wild populations; i.e., the non-random spatial distribution of genotypes , is a coest soil , the esteedlings . Furtherall size . Consequall size . Okada ee-stages . HoweverSp is the reciprocal of the neighborhood size under certain conditions  strong restriction of safe-sites decreases the number of mother trees contributing to offspring and results in a strong FSGS at the early demographic stages and (2) accordingly, the FSGS at the early demographic stage should be weaker at the later demographic stages. Finally, we examined the factors affecting the FSGS, such as inbreeding depression, stochastic loss of individuals, and the eradication of establishment sites by local disturbances using simulation studies.In the present study, we estimated the FSGS in Fraxinus mandshurica var. japonica  was established at the fluvial terrace of the Iwanazawa Forest Reserve, University of Tokyo Hokkaido Forest (UTHF) in central Hokkaido , Japan. The FSGS of co-occurring tree species; namely, japonica , Cercidiaponicum , and Betwicziana  were invP. jezoensis plants and identified four growth stages in the study plot: mature, juvenile, sapling, and seedling. We observed all 123 mature P. jezoensis individuals with a diameter at breast height (dbh) ≥20 cm, and all 39 juveniles with a dbh ≥5 cm but <20 cm. Individual trees with a dbh <5 cm were differentiated into two categories by age as determined by the number of bud scale vestiges. For saplings and seedlings, we selected half of the individuals established on fallen logs in this study, so that 190 saplings aged ≥3 years and 179 seedlings aged <3 years were included in this study. The locations of the areas of CWD and individual trees were identified using a digital compass , and their needle leaf tissues were collected for DNA analysis.In this study site, there were 112 fallen logs the diameters of which were larger than 20 cm at the base. Fallen logs were regarded as a major component of CWD in this forest. We measured individual GC1, GD1, GG3 . The following 11 microsatellite loci, GD1, GG3 , PaGB3, 3, PgGB5 , EATC1E0EATC2G05 , Pj4, Pjand Pj24  were anaFIS was also calculated considering the null alleles using INEst , ranging from 1 to 10. To determine the most likely value for K, we first used the methods suggested in the original description of STRUCTURE, which involves comparing the log probability to the data (ln Pr [X|K]) for each run to evaluate the genetic structure. Then, we calculated ΔK   for all lated ΔK  to deterpriate K  for the Fij (coancestry) /(1–Qm), where Qij is the probability of identity in state between two randomly chosen genes from i and j, and Qm is the average probability of identity by state between two randomly chosen individuals from the reference population. Assuming that the reference population is the parental generation, Fij is expected to have values derived from the pedigree information  , in which spatial distances were permuted randomly among pairs of individuals. Furthermore, to compare the intensity of the FSGS among the four stages, we calculated Sp, a parameter indicating FSGS intensity, using the formula Sp = bF/(F1–1). In this formula, bF is the slope of Fij against the logarithm of the distance between individuals, and F1 is the average kinship coefficient among individuals in the first distance class  in this study. The value for bF was estimated based on the regression of all spatial distances between individuals, and the significance level was calculated using 1000 permutation tests. All the FSGS-related calculations were performed using SPAGEDI ver. 1.4  . In the ll-sibs) . For thever. 1.4 .Fis) as a kinship coefficient between genes within each individual  To examine the deaths of random offspring, we randomly extracted 123 individuals from the offspring data without replacement and repeated this procedure 10 times to generate 10 different datasets. This randomization procedure was conducted using Visual Basic for Applications. In the first and second simulations, the number (123) of surviving offspring was the same as that of mature trees. (3) To determine the effects of local disturbances such as flooding, which may eradicate all offspring on specific CWD, we generated six datasets by excluding all offspring on CWD with (i) the largest number of offspring; (ii) the largest and second largest number of offspring; (iii) the largest, second, and third largest number of offspring; (iv) the largest, second, third, and fourth largest number of offspring; (v) the largest, second, third, fourth, and fifth largest number of offspring; and (vi) more than eight offspring, resulting in 250, 232, 216, 200, 186, and 122 surviving individual data sets, respectively. The number of datasets from (vi) was approximately the same as that of the mature trees. We calculated test statistics for the FSGS of the simulated offspring datasets , to examine whether the FSGS was significant in each dataset. In these simulation-based analyses, all the statistical calculations for the FSGS of actual mature trees, and actual and simulated offspring were determined using the same methods as previously mentioned for the examination of FSGS at each growth stage, but without the reference population.Finally, to examine whether demographic thinning by inbreeding depression, stochastic loss, or both during the transition from adolescent to mature stage affected the FSGS among mature trees, we simulated seedling and sapling deaths under three sets of conditions. We first combined all 179 seedlings and 190 saplings into a dataset of 369 offspring. We then generated the following datasets. (1) To examine the deaths of inbred offspring; i.e., death by inbreeding depression, we generated a set consisting of 123 offspring with low HS) of each growth stage ranged from 0.743 to 0.751, and the allelic richness  ranged from 14.01 to 15.19 (Table FIS) was the highest and lowest in the seedling and mature stages (0.241 and 0.113), respectively. This was also observed for the FIS values  estimated using the Bayesian approach taking into account the null alleles . The 11 loci were polymorphic in mature trees, with 3 to 46 alleles per locus and mean HE values of 0.743 , which all had high null allele frequencies, ranging from 0.090 to 0.185. Therefore, these loci were excluded from the calculations of Fij and Fis. There was no significant linkage disequilibrium between any pair of loci analyzed.The genetic diversity  between successive K values , saplings (-0.013), and seedlings (-0.011) were all significant , but were not significant for mature trees  were much lower than those for juveniles (0.009), saplings (0.014), and seedlings (0.012). This indicated that the FSGS was stronger in the adolescents than in the mature trees.Correlograms of the average Fis) at all four growth stages was significantly high  was the highest, and the values then decreased with life history for saplings , juveniles , and mature trees .The average value of the individual inbreeding coefficient (a), shape parameter (b), average dispersal distance (d), and immigration rate (m) were calculated to be 81.816, 3.097, and 59.9 m and 86.6%, respectively, for pollen dispersal in the seedling stage. Furthermore, the corresponding values for seed dispersal were 3.823, 0.571, and 40.6 m and 74.4%, respectively, for the seedling stage , the F1 and bF values remained significantly high . For the sets of offspring simulated under the two sets of conditions related to random death and eradication of all the offspring on specific CWD, the average Fis, F1, and bF values for all the data sets were significantly high . Furthermore, the Sp values (0.004–0.010) for all the sets of simulated offspring were higher than those of mature trees (0.001).Fine-scale spatial genetic structure values were significant for seedlings and saplings observed on the study site, and for all the sets of simulated offspring under the three conditions, except for mature trees , indicating that the genetic clustering of adolescents was stronger than that of mature trees  values (0.046–0.061) for adolescents were much lower than those expected for half-sibs (0.125). Moreover, the average Fij values for seedlings and saplings for Cluster 1 in the STRUCTURE analysis  were also much smaller than the kinship coefficients expected for half-sibs. The significant FSGS and low F1 values for adolescents indicated that spatially limited seed dispersal should generate a half-sib family structure around seed parents, but this structure may be weakened by the overlapping of seed shadows.The STRUCTURE analysis indicated that adjacent areas of CWD accommodated genetically related progeny at the center of the study plot Figures . This mies Table . AlthougA. sachalinensis, at the same study site, for which the average distance of seed dispersal was 23.7 m  for A. sachalinensis in the same plot . The stronger FSGS for P. jezoensis may be due to its stronger dependency on CWD for establishment than that of A. sachalinensis.Even in the case of anemochory, the seed shadow of each mother tree would create a spatial genetic structure during the initial regeneration process . The pros 23.7 m . It is pame plot , the disFis value was high at the seedling stage and decreased for the later growth stages  in this study was much lower than that of other tree species that relay on wind-pollination and wind-dispersal of seeds (0.002–0.011)   , and tha (0.003) .P. jezoensis. In conclusion, we cannot attribute the decline in the FSGS throughout the life history of P. jezoensis to demographic thinning alone. It is probable that the regeneration processes responsible for each generation have changed significantly over long periods. Thus, not only demographic thinning but also other factors, such as differences in colonization histories between the mature and offspring populations, and seeds derived from multiple sources, should be considered in explaining the low Sp values and lack of FSGS in mature P. jezoensis populations.However, according to the results of the simulations under the conditions of death by inbreeding depression, stochastic loss, and local disturbance, the FSGS values for all simulated offspring datasets were significant  also suggests that the substantial number of areas of CWD accumulated after the initial establishment and regeneration of the younger mature trees. In addition, an artificial disturbance appears to have occurred at the study site in the past. The distribution of juveniles reflected the track of an abandoned forest railway and the present-day forest road. As there are large broadleaf trees growing at the center of the study plot, it seems that few areas of CWD are available for the regeneration of the present juveniles. The limited area available for regeneration may lead to a weak but significant FSGS in the juvenile stage in spite of its small sample size (Figure P. jezoensis (Figures e Figure . Recentlezoensis . In factP. jezoensis. Moreover, our FSGS simulations suggested that present mature individuals may have been established by different disturbance regimes compared to the other demographic stages. It might be one of the characteristics of CWD-dependent tree species that different genetic variations could be created through spatiotemporal differences in the production of areas of CWD.In conclusion, the number and distribution of establishment sites, such as CWD, may be attributable to the FSGS in each generation of KK, CL, and SG designed the study and interpreted the results. KK was responsible for the laboratory work. KK and AN analyzed the data. All authors contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Firmicutes and its class Clostridiales were elevated, meanwhile the abundance of Lachnospiraceae, Alistipes, and Odoribacter showed significant differences among the three groups. Based on LC-MS results, bile acid, secondary bile acid, serotonin and taurine level had varying degrees of changes in alcohol model. From paraffin sections, tissue damage was observed in liver and colon. These findings provide direct evidence that alcohol intake affects the composition of GM, enable a better understanding of the function of GM in the microbiota-gut-brain (MGB) axis, and give a new thought for alcohol addiction treatment.Alcohol abuse is a major public health crisis. Relative evidences supported that the gut microbiota (GM) played an important role in central nervous system (CNS) function, and the composition of them had changed after alcohol drinking. We sought to explore the changes of GM in alcohol dependence. In our study, the GM of mice with alcohol administration was detected through analyzed 16S rRNA gene sequencing and the fecal metabolites were analyzed by LC-MS. The microbial diversity was significantly higher in the alcohol administration group, the abundance of phylum  Alcohol abuse lead to a series of healthy problems, like alcoholic liver disease, cardiovascular diseases and depression, some works reported that gut microbiota (GM) played an important role in treating these diseases . AlcoholWith the interest in GM in recently years, many evidences indicated that GM were closely related to the immune system, nervous system, obesity and diabetes, and damaging GM would lead to severe pathologies, metabolic disease, cancer and irritable bowel syndrome . GeneralMany studies showed that GM composition in alcohol abusers was different from healthy people , and GM Thirty female BALB/c mice  were purchased from Jiesijie . All mice were housed at ambient temperature (21°C) in a room maintained on a reversed 12L:12D cycle . Purchased mice were taken 1 week to adapt the environment before starting the experiments. All animal studies have been approved by China Ethics Committee and performed in accordance with the ethical standards.Alcohol was purchased from Maotai , executive standard GB/T26760. ELISA kits were purchased from Fankewei , chemicals of LC-MS were purchased from Thermo Fisher Scientific .Thirty female mice were randomly equally divided into three groups, two of them are experimental groups, another group is viewed as control group (CT). One of the experimental group named active drinking group (ADG) was treated in two-bottle drinking mode. One bottle with increasing alcohol concentration  was given for each 2 days to train mice adapting alcohol, and another bottle filled with water. The placement of two bottles was exchanged everyday to avoid side preferences . AnotherAfter 8 weeks feeding, open field exploration and light-dark transition test were taken to evaluate the anxiety levels by measuring the general locomotor activity with CT mice. ADG and FDG mice were extracted alcohol solution for 24 h, and then tasting anxiety levels in the withdrawn mice by open field exploration and light-dark transition test.Spontaneous exploration of sports were analyzed using automated activity chambers with camera  . Before The light-dark apparatus was made up of an automated activity monitor with a dark box and insert to create an equally spaced light and dark compartment . The entire apparatus was positioned in a sound-attenuating chamber. The light side was illuminated to a degree of 60 Lx, and 5 Lx in the dark side. Each mouse was placed into the light side and allowed to freely explore the chamber for 5 min. The light-dark apparatus was cleaned with 75% ethanol solution and dried before the next mouse was tested. A photo beam-based tracking system was used to track the movement and calculate the time spent in each area. Anxiety-like effects were detected by increasing time spent in the dark compartment .Fecal sample was collected in metabolism cages. After the behavioral test, mice were placed in the metabolism cages sterilized by absolute alcohol before collection. Fecal samples were stored immediately at −80°C and extracted using OMEGA soil DNA kit . DNA quantity and quality was assessed using a NanoDrop 2000 .2O making up to 20 μL. The following cycling parameters were used: maintain at 95°C for 3 min, 25 cycles , and a final extension at 72°C for 10 min . The V3 and V4 regions of the 16S rDNA gene were selected for PCR. The primers were barcoded 338F (50-ACTCCTACGGGAGGCAGCA-30) and 806R , where the barcode was an eight-base sequence unique to each sample. The 20 μL PCR reaction mixture was composed of 4 μL of 5× FastPfu buffer, 2 μL of 2.5 μM dNTPs, 5 μM each of forward and reverse primers, 0.4 μL FastPfu Polymerase, 10 ng Template DNA, and ddHr 10 min .Serotonin levels were detected in sera by ELISA method according to the manufacturer instructions .The livers and colons were soaked in 10% formalin for 24 h , dehydraThe extraction procedure was performed as previously described . Brieflyt-test. Linear discriminant analysis effect size (LEfSe) was used for identifying differences in population abundance, and assessing the magnitude of the effect of each species abundance on the differences  which is a unity mark set artificially for a taxonomic unit , and the statistical analysis of biological information is usually carried out at the similar level of 97% of OTU. Similarity or difference in composition of sample communities was analyzed using principal component analysis (PCA). Statistical analysis between multiple samples was performed using One-way ANOVA. Significant differences between the two microbial communities were analyzed using Student’s ferences . DiffereFigure 1). Staining of ADG showed the slight damage of colon mucosa, staining of FDG showed the shedding of colon villus, partial necrosis and colon chronic inflammatory cell infiltration. Moreover, staining of ADG and FDG showed slight congestion and loosening of liver cytoplasm.The liver is the main site for alcohol metabolism and the colon is a habitat for the GM. To investigate functional and pathologic outcomes in recipient mice after alcohol administration, colon and liver sections were examined after 8 weeks feeding  and alcohol  intake of ADG had increased and tended to stability. The results suggested that mice of ADG adapted to alcohol and took the initiative to drink, mice of FDG gradually adapted to the lifestyle of only drinking alcohol. Weight of FDG mice showed significant decrease compared with CT group .Mice of ADG and FDG had less feed intake than CT group. After 8 weeks feeding, food . After 8 weeks feeding, the mice of two groups were withdrawn from alcohol for 24 h. Both of them were performed open field exploration test and light-dark transition test to confirm whether the mice of ADG or FDG were addicted in alcohol. Mice of ADG  and FDG  spent more time in the dark side, the distances traveled and time in the center of the open field exploration box significantly reduced  compared to the CT group. The result indicated that the alcohol groups performed anxiety and depression compared with no alcohol group and alcohol-withdrawn mice shown more anxiety and depression than before withdrawal. Compared with the CT group , the serotonin levels of the experimental groups were increased, and FDG had a significant differences .We used open field exploration test and light-dark transition test to investigate the differences of the physiological state in the three groups . ACE and Chao1 were estimated to be 472.9335/477.2634 in ADG, 444.8102/449.9084 in FGD and 449.4612/456.777 in CT group. Respectively, there were little differences between ADG and CT group. However, the observed richness (Sobs) was different from 409.9 in ADG and 433.9 in FDG . The Shannon and Simpson indices were 4.441124/0.027667 in ADG, 4.37675/0.026892 in FDG and 4.411108/0.027046 in the CT group, the higher Shannon and lower Simpson index with the same change in the three groups indicated higher community diversity. The results suggested that there were no significant change in community diversity in the three groups, but FDG had an increasing tendency compared with the other two groups.Rarefaction curves indicated that the bacterial community was well represented because the OTU level had no changes as randomly selected number of sequencing analyzed increased. The curves characterize species abundance and species uniformity, in the horizontal direction, abundance of species is reflected by the width of the curve. The higher the abundance of species, the greater the range of the curve on the horizontal axis, and the shape of the curve(smoothness) reflects the homogeneity of the species in the sample, the smoother the curve, as well as the more uniform the species distribution. Community richness is the number of bacterial species assigned by OTUs detected in the samples. Richness estimates were obtained from the observed number of species by extrapolation using estimators such as the ACE and Chao1 indices  . Circos , which suggested that Bacteroidetes, Firmicutes, Proteobacteria and Deferribacteres had a high abundance in the three groups and each group had different abundance in the four dominant species, and also indicated changes in Clostridiales and Bacteroidales, the two orders with the highest abundance. Ratio of Firmicutes to Bacteroidetes was increased in the experimental group, abundance of Clostridiales was also observed higher in FDG than that in CT group.The analysis of species composition demonstrated that bacteria from 30 samples had different richness and diversity. Hundred and twenty one genus in 30 samples were detected, and each relative abundance of genus were shown in . Circos  is a visOdoribacter and Ruminococcaceae_UCG-014 abundance had a significant change in ADG  . The abundance of Lachnospiraceae, Alistipes, and Odoribacter had a significant difference between FDG and the CT group . LEfSe was used to determine the taxa that best characterized each population. LEfSe scores measure the consistency of differences in the relative abundance between taxa in the groups analyzed with a higher score, thus indicating higher consistency. LDA showed distinct taxa in the microbiome of the CT group versus the alcohol administration group .Compared with the CT group, Figures 9A,B). As shown in Figure 10, the content of taurine, butyric acid, bile acid and secondary bile acids was increased in ADG. Higher concentrations of serotonin than the CT group were found in FDG. Moreover, taurine was found increased in ADG while decreased in FDG.The result of LC-MS showed that the principal component analysis (PCA) nicely clustered FDG and the pooled QCs in alkaline condition both positive and negative ion mode. There was clearly a difference between FDG and the CT group, and ADG showed no significant difference with CT group  patients , 90% 5-HT synthesized in the gut by enterochromaffin cells (ECs), myenteric neurons and mucosal mast cells , was a bTaurine (2-aminoethane-sulfonic acid), an organic osmolyte, regulated cell volume and maintained cellular integrity in the heart, muscle, retina, and throughout the CNS . Again, Clostridium and Eubacterium, which belonged to the Firmicutes phylum, had capability to produce secondary bile acids (Clostridium species were reported to possess high 7a-dehydroxylation activity to produce deoxycholate based on cholate (Clostridia and increasing deoxycholate levels. Moreover, levels of bile acids and secondary bile acids were increased in ADG and FDG, which apparently indicated the changes of GM composition might contribute to body metabolism under alcohol damage. In summary, based on genomics and metabolomics, the gut microbial community and relevant metabolomics in mice model showed significant difference in alcohol addiction mice. Overall, our associative findings pave the way to understanding the changes of microbiota composition.Bile acids, produced in liver from cholesterol, modulated gut microbial composition through activation of innate immune genes, and also metabolized by the GM . Moreovele acids . Deoxychle acids . But deole acids . Notablyle acids . Particu cholate , which wQL and GW contributed to the conception and design of the work and specific experiments. GW, ZF, QZ, and JP performed the experiments. GW, JQ, DX, WZ, and XW contributed to analysis and interpretation of the data. GW, HZ, CD, QD, and LG wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Humulus lupulus L. (HOP extract) was evaluated for its anti-influenza activity. The ability of the extract to interfere with different phases of viral replication was assessed, as well as its effect on the intracellular redox state, being unbalanced versus the oxidative state in infected cells. The radical scavenging power, inhibition of lipoperoxidation, and ferric reducing activity were assayed as antioxidant mechanisms. A phytochemical characterization of the extract was also performed. We found that HOP extract significantly inhibited replication of various viral strains, at different time from infection. Viral replication was partly inhibited when virus was incubated with extract before infection, suggesting a direct effect on the virions. Since HOP extract was able to restore the reducing conditions of infected cells, by increasing glutathione content, its antiviral activity might be also due to an interference with redox-sensitive pathways required for viral replication. Accordingly, the extract exerted radical scavenging and reducing effects and inhibited lipoperoxidation and the tBOOH-induced cytotoxicity. At phytochemical analysis, different phenolics were identified, which altogether might contribute to HOP antiviral effect. In conclusion, our results highlighted anti-influenza and antioxidant properties of HOP extract, which encourage further in vivo studies to evaluate its possible application.A hydroalcoholic extract from female inflorescences of  Nowadays, influenza remains one of the main causes of morbidity worldwide, with seasonal epidemics and periodic pandemics. The disease is caused by an enveloped, RNA virus, which infects mainly the upper airways, but complications at lower respiratory tract can occur, especially in children and elderly .Although different strategies have been approached to prevent the disease and/or manage its complications, the only anti-influenza drugs approved by FDA belong to two classes of inhibitors, which target the viral matrix protein 2 and neuraminidase . ResearcSeveral intracellular pathways that influenza virus activates are redox-sensitive , 8, and Humulus lupulus L. (Fam. Cannabaceae) are widely used in the brewing industry as preservative and flavouring additives; in fact, H. lupulus is considered as an essential ingredient in beer, contributing to the bitter flavour and the characteristic hoppy aroma and providing preservative and antimicrobial effects  × 100, where Acontrol was the absorbance of the vehicle while Asample is that of the tested sample.The assay was carried out by the ferric thiocyanate method according to Di Sotto et al. . Briefly3 × 6H2O  and the samples were mixed for 2 minutes, and then a ferrozine solution  was added to the mixture. The ferrous ion-ferrozine complex, corresponding to the reducing activity of the sample, was measured at 562 nm and the percentage of activity was calculated as follows: 100 × (Acontrol − Asample)/Acontrol, where Acontrol is the absorbance of the vehicle, while Asample is that of the tested sample.The activity was evaluated by the ferrozine assay, according to previous published methods . Brieflyn represents the number of experiences.For the antioxidant studies, all values are expressed as mean ± SE and n represents the number of replicates for each treatment.For the antiviral studies, all values are expressed as mean ± SD and Statistical analysis was performed by GraphPad Prism™ software .t-test was applied to determine the statistical significance between two different experimental conditions. The values of P < 0.05 were considered significant.The one-way analysis of variance (one-way ANOVA), followed by Dunnett's multiple comparison posttest, was used to analyze the difference among different treatments, while the Student's E = Emax/[1 + (1050LogEC/A)HillSlope], where E is the effect expressed as increase in the ferrous chelation at a given concentration of agonist, Emax is the maximum ferrous chelating activity, IC50 is the concentration that produces a 50% of the inhibitory response, A is the agonist concentration in molar, and HillSlope is the slope of the agonist curve.The concentration-response curves were constructed using the “Hill equation”: The HPTLC analysis showed the presence in the chromatogram of different polyphenols, evidenced as fluorescent spots, and better visualized by derivatization with NPR and anisaldehyde . Among tColorimetric determinations highlighted a polyphenols/tannins ratio of about 4, while the flavonoid content agreed with that declared by the supplier . On the H. lupulus plant, with lower levels of quercetin, naringenin, kaempferol, and xanthohumol. Ferulic acid was also found in a methanolic extract from cones of H. lupulus [H. lupulus cones and young shoots [According to our results, Inui et al.  highligh lupulus . Further lupulus . Quercetg shoots , 37.v/v), which allows a poor hydrolysis of the glycoside. In fact, the rutin hydrolysis has been found to occur under acidified conditions with high concentrated hydrochloric acid and extended heating times [H. lupulus extracts was reported: in spite of a very low phenolic amount in the aqueous extracts, the hydroalcoholic samples were found richer in epicatechin, chlorogenic acid, gallic acid, catechin, benzoic acid, ferulic acid, and o-coumaric acid [Our sample of HOP extract did not contain quercetin and naringenin, in spite of a high amount of rutin. This could be a consequence of the mild extraction condition  of HOP on kidney epithelial MDCK cells, well known to be highly permissive to influenza virus. As shown in ∗P < 0.05 starting from 50 μg/ml) and concentration-dependent manner, with an IC50 value of 99  μg/ml. Western blot analysis of viral proteins similarly showed a dose-dependent reduction of hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and matrix protein1 (M1) in infected and HOP-treated cells compared to the infected ones  and treated with HOP at different times from the infection: before (b.i.), during (d.i.), and after (p.i.) or before, during, and after the infection (b.d.p.i). As shown in the charts of On the basis of this preliminary evidence, the highest concentration of 140 In the attempt of explaining the molecular mechanism exerted by the HOP extract on influenza virus-infected cells, we decided to evaluate different steps of virus life cycle. First, we analyzed the effect of HOP extract during the 1 h infection. For this step, we could hypothesize a virucidal effect of the extract or an effect on an early phase of the viral life cycle. HOP extract was incubated directly with the virus and then the mixture used to infect the cells, as described in Materials and Methods. PR8 production, measured by the hemagglutinating assay, was significantly inhibited, both when high and low m.o.i. of virus were used, but with a higher percentage of inhibition when low m.o.i. was used  which disappeared in the presence of HOP . These din vitro antioxidant assays were performed. The radical scavenging properties of the sample, based on hydrogen and electron transfer, were evaluated against the synthetic chromogenic DPPH and ABTS radicals. Particularly, the HOP extract (0.001–2 mg/ml) inhibited, in a significant and concentration-dependent manner, both DPPH and ABTS radicals, as confirmed by the IC50 values  is the main source of ROS production . Indeed,0 values . The pos0 values , althoug extract . A weakl lupulus .50 value about 44-fold higher than that of Trolox  than the positive control Trolox . As estihibition . ROS spehibition .Taken together, present results suggest that the HOP extract is able to interfere both directly and indirectly with the ROS-mediated cell injury. It is also plausible that HOP extract might interfere with NOX4 enzyme or with downstream pathways that are activated by oxidative stress.H. lupulus have been widely ascribed to its phenolic constituents. The most significant antioxidant activities are displayed by prenylated chalcones  mainly due to their prenyl group [Antioxidant properties of yl group . Prenylfyl group , 52. TheH. lupulus, focusing on its ability to both directly counteract the viral replication and viral protein synthesis and indirectly increase the hosting cell defense by antioxidant mechanisms, likely due to its phenolic content.In the present study, we described the antiviral properties of a hydroalcoholic extract from the female inflorescences of H. lupulus and its characteristic constituents is available in literature. Buckwold et al. [Very limited evidence on the antiviral properties of d et al.  found noH. lupulus cones represent an important source of phenolic compounds, some of which are reported to produce inhibitory effects against several viral infections [fections –58. Partfections –61 and sfections , 63. ThiIn conclusion, our results suggest that the HOP extract is able to exert dual antiviral and cytoprotective effects, which can be useful in the prevention and treatment of influenza. Further investigations are encouraged in order to define the possible application of the HOP extract as an anti-influenza remedy or in combination with conventional antiviral drugs.