The aim of this study was to examine the mediation of body mass index (BMI) on the association between per capita sugar consumption and diabetes prevalence using country-related data.In this ecological study, based on 192 countries, data on per capita sugar consumption were obtained from the Food and Agriculture Organization of the United Nations (FAO), on BMI from the World Health Organization and on diabetes prevalence from the International Diabetes Federation. Data on demography and economic factors were obtained from the Central Intelligence Agency, the United Nations and the FAO. Multiple linear regression analysis was performed to investigate the association between per capita sugar consumption and diabetes prevalence, and mediation analysis to detect the mediated percentage of BMI on this association.Each increase of 100 kcal/day per capita sugar consumption was associated with a 1.62% higher diabetes prevalence [adjusted β-estimator (95% CI): 1.62 ]. Mediation analysis using BMI as the mediator demonstrated an adjusted direct association of 0.55  and an adjusted indirect association of 1.07 . Accordingly, the BMI explained 66%  of the association between per capita sugar consumption on diabetes prevalence.These findings indicate that the association between dietary sugar intake and the occurrence of diabetes is mediated by BMI to a large proportion. However, it seems that other mechanisms may explain the association between sugar consumption and development of type 2 diabetes.The online version of this article (10.1007/s00394-020-02401-2) contains supplementary material, which is available to authorized users. There is an ongoing debate whether dietary sugar intake has an impact on the development of type 2 diabetes (T2D). In the last decades, per capita sugar consumption increased, particularly in developing and emerging countries , due to However, findings from observational studies on the relation between total dietary sugar intake and T2D incidence were inconsistent. While an increase of sugar sweetened beverage consumption was consistently associated with T2D , 17, 18,2 − < 30 kg/m2) and obesity (BMI ≥ 30 kg/m2) were collected. All countries with available data on at least half of the considered variables were included in this study. Finally, for the present analysis data from 192 countries were selected .This is a mediation analysis based on an ecological study design, including geographical group-level country data. The study is reported according to the Guidelines for Accurate and Transparent Health Estimates Reporting (GATHER) , and StrData on per capita sugar consumption adjusted for exports and any kind of non-human consumption were obtained for the year 2007 from the Food and Agriculture Organization of the United Nations (FAO) that received the estimated data mainly from national statistical offices . Per capPearson’s correlation coefficients were calculated to describe the correlations between per capita sugar consumption and diabetes prevalence, as well as between per capita sugar consumption and BMI, and between BMI and diabetes prevalence. Multiple linear regression analysis, adjusted for age, per capita GDP, per capita fat intake and total energy supply and rate of rural population, was accomplished to estimate the association between per capita sugar consumption and diabetes prevalence. We performed the mediation analysis by applying the method suggested by VanderWeele . For thin = 22 were missing, for diabetes prevalence n = 4, BMI n = 2, age n = 1, fat intake n = 24, total energy n = 22 and rural population n = 16. All available variables were used for imputation and 1000 data sets were generated. The mediation model was calculated for each imputed data set, and we reported the median value from the 1000 model fits as the estimated parameter together with the 2.5% and 97.5% percentiles as the respective non-parametric 95% confidence interval (CI). If the 95% CI does not include the null-value, the findings are estimated precisely and can be interpreted as “statistically significant”. We chose this simultaneous application of multiple imputation and a non-parametric bootstrap procedure as there is still methodological debate on how confidence intervals for mediation parameters should be calculated. In addition, by combining the two approaches it is guaranteed that the complete statistical variability in the data is properly accounted for, yielding conservative confidence intervals that are rather too long than too short. Statistical analyses were performed in R 3.6.1 and SAS, Version 9.4 by the MI and the CAUSALMED procedure.Standard multiple imputation methods were used to account for missing values, where for per capita sugar consumption 2, with the highest mean BMI in Northern America (28.4 ± 1.0 kg/m2), Central America (27.4 ± 0.6 kg/m2) and Western Asia (27.3 ± 1.5 kg/m2).After performing multiple imputation, data on 192 countries were assessed for analyses. The characteristics for the included countries are shown in Supplement: Tables 1–7 allocated by regions defined by the UN . Mean , p < 0.01). After stratification by region, this positive trend was observed for Asia , p = 0.01) and Africa , p = 0.06), but not for Oceania , p = 0.67) and Europe , p = 0.30) (Supplement: Table 10). Similar findings between per capita sugar consumption and diabetes prevalence were observed after stratification by income classification, showing positive correlations for lower middle and upper middle income countries, and null associations for low and high income countries regarding the association between per capita sugar consumption and diabetes prevalence (Supplement: Table 11).Findings showed a positive correlation between per capita sugar consumption and diabetes prevalence , p < 0.01) and mean BMI and diabetes prevalence , p < 0.01), respectively . Correlation coefficients between exposure or outcome with potential confounding variables were positive for mean age, per capita GDP, per capita fat intake and total energy or inverse correlations for rural population (Supplement: Table 12).There was also positive correlations between per capita sugar consumption and mean BMI (p < 0.01) and BMI (β (95% CI): 1.18 , p < 0.01) , respectively. BMI was positively associated with diabetes prevalence (β (95% CI): 1.16 , p < 0.01) . Adjustment for BMI weakened the association between per capita sugar consumption and diabetes prevalence (β (95% CI): 0.12 , p = 0.81), indicating that BMI is a mediator. In the multiple linear regression model adjusted for age, per capita GDP, per capita fat intake, total energy and rural population, each increase of per capita sugar consumption per 100 kcal/day was associated with 1.62% higher diabetes prevalence (β (95% CI): 1.62 , p < 0.01) : 1.42 , Sensitivity analysis showed similar results when using the most recent data that were available . Performing mediation analysis using the prevalence of overweight or obesity as mediator, the adjusted mediated percentage was 40%  for overweight, and 51%  for obesity, respectively (Supplement Table 9).The findings of our ecological study showed an association between dietary sugar intake and prevalence of diabetes and suggested that the total association can be explained by about two thirds via BMI. In consequence, there might be other mechanisms that explain the association between dietary sugar intake and diabetes.Regarding the impact of dietary sugar intake on the development of T2D, the evidence is inconclusive and findings are controversial , 19. Com13C) and nitrogen (δ15N) as potential sugar biomarker in adults measured in hair, capillary finger stick blood and blood serum [13C might potentially be an objective biomarker to assess dietary sugar intake, since δ13C values were predictive for the intake of added sugar [15N biomarker were inconclusive predicting sugar intake. While results in an Alaska native study population showed a strong association between δ15N and dietary sugar intake [These observations indicate that reporting bias dependent on BMI status cannot be ruled out. Since nutrition data is mainly based on self-reports of participants, it is likely that intake of unhealthy food, including sugar containing products, can be biased due to measurement errors, because especially, overweight participants tend to underreport their true intake –28. As aod serum –54. Finded sugar –54, sugaed sugar –53, as wThe present body of research implies that an increased body weight might be the major explanation for the observed association between dietary sugar intake and diabetes prevalence. A high dietary sugar intake may lead to overweight and obesity which is associated with increased risk of T2D. Since the amount of adipose tissue is enlarged, overweight and obesity increase the secretion of nonesterified fatty acids and different hormones and cytokines, such as leptin, adiponectin and pro-inflammatory cytokines, which are associated with insulin resistance , 56. In Our study had several strengths. This was the first study performing a causal mediation analysis that investigated the impact of BMI as mediator for the association between per capita sugar consumption and diabetes prevalence. Besides this, the used data, including potential economic and health-related confounders, were current and obtained from reliable global organisations. Furthermore, we considered lead time intervals between exposure, mediator and outcome to simulate a prospective sequence between these factors. However, our study had some limitations as well. First, conclusions about causality are hampered due to the ecological study design. Future prospective studies are needed to ascertain more reliable findings regarding the direct association between dietary sugar intake and incidence of T2D based on individual data while using objective biomarkers to assess the true dietary sugar intake. Second, data regarding diabetes prevalence obtained from the IDF also contained individuals with type 1 diabetes or other types of diabetes, which might be approximately around 7–12% and 1–3% in high income countries, respectively . This maIn this ecological study, per capita sugar consumption was positively associated with diabetes prevalence at a global level. This association was mediated by 66% by BMI, indicating, that a large amount of this association can be explained by BMI, but that also other mechanisms exist that could explain the association between dietary sugar intake and the development of T2D. Causality cannot be proved with an ecological study design, and thus further well-conducted studies are recommended that use individual participant data and valid measurements for their sugar consumption.Supplementary file1 (PDF 951 kb)Below is the link to the electronic supplementary material.


Sleep dysfunction is a core symptom in bipolar disorder (BD), especially during major mood episodes. This study investigated the possible link between subjective and objective sleep disturbances in inter-episode BD, changes in melatonin and cortisol levels, and circadian melatonin alignment. The study included 21 euthymic BD patients and 24 healthy controls. Participants had to wear an actigraphy device, keep a weekly sleep diary and take salivary samples: five samples on the last evening to determine the dim light melatonin onset (DLMO) and one the following morning to measure rising cortisol. Sleep quality was assessed by the Pittsburgh Sleep Quality Index (PSQI) and Regensburg Insomnia Scale (RIS), and circadian alignment by the phase angle difference (PAD).p = 0.022) (significant after controlling for age and gender), and higher RIS scores ; (2) subjective a longer mean TST (p = 0.024) and TIB (p = 0.002) (both significant after controlling for age and gender), longer WASO (p = 0.019), and worse SE (p = 0.036) (significant after controlling for gender); (3) actigraphically validated earlier sleep onset (p = 0.002), less variation in sleep onset time (p = 0.005) and no longer TST (p = 0.176); (4) no differing melatonin levels (4.06 ± 2.77 vs. 3.35 ± 2.23 p = 0.352), an 1.65 h earlier DLMO  (significant after controlling for gender), and a phase advance of melatonin  (significant after controlling for gender); and (5) no differing cortisol awakening response (16.97 ± 10.22 vs 17.06 ± 5.37 p = 0.969).In comparison to healthy controls, BD patients had: (1) higher PSQI (5.52 ± 3.14 vs. 3.63 ± 2.18; Patients with BD, even in euthymic phase, have a significantly worse perception of their sleep. Advanced sleep phases in BD might be worth further investigation and could help to explain the therapeutic effects of mood stabilizers such as lithium and valproate. Bipolar disorder (BD) is a chronic, severe, and recurring mood disorder that may also affect cognitive performance, and circadian rhythm , ratings measuring subjective sleep quality and actigraphy were employed as proposed by the ISBD Chronobiology Task Force , 21 of them suffering from BD. All patients were in clinical remission and underwent out-patient treatment in the LWL-University Hospital Bochum. The control subjects were recruited among students and employees of the Ruhr-University. Structured Clinical Interviews for DSM-V  ≥ 13 indicates distinct and ≥ 24 striking psychophysiological insomnia symptoms. The internal consistency is α = 0.89  with a lower detection level of 1 pg/ml. Inter- and intra-assay coefficients of variation were 7.6–13.0% and 6.1–10.8%, respectively. Morning cortisol levels were measured in morning salivary samples using an electrochemiluminescence immunoassay (ECLIA) with a lower detection level of 0.054 µg/dl and inter- and intra-assay coefficients of variation of 2.5–14.8% and 1.7–9.3%, respectively.Participants were instructed to take five salivary samples on the last night and a sixth sample the subsequent morning. The evening samples were used to calculate the DLMO and had to be taken four hours before the median sleep onset time in dim light . The sixth sample was taken 30–60 min after awakening (Cortisol awakening response). Participants stored the samples in their fridge; samples were centrifuged at 1250To measure the circadian phase of melatonin we used the DLMO, which is defined as the time at which the melatonin level began to rise above a certain threshold while kept under dim light conditions. The melatonin level had to either exceed 3 pg/ml. If this threshold was not reached, we did conduct a visual inspection of the concerning sample curve and did determine a DLMO if a clear rise of concentration was visible. Salivary melatonin sampling and estimation of DLMO have been recognized as a valid measurement of DLMO in BD by the Chronobiology Task Force of ISBD . To analyse the study cohort, descriptive statistical methods were used. Quantitative data are presented as mean and standard deviation. For comparison of categorical and continuous variables, chi square tests, unpaired t-tests, and Mann–Whitney U test were used where appropriate. A p-value of less than 0.05 was interpreted as significant. Univariate analysis of covariance (ANCOVA) was conducted for age and gender, given the significant group differences to control for the influence of age and gender. For all analyses, p < 0.05 was required for significance.Statistical analyses were performed with SPSSp < 0.001) and gender (p = 0.005) (Table p = 0.001) and had more close relatives with SD (38.1% vs. 16.7%) compared to healthy controls. All healthy controls, but only 38.1% of the BD group were employed at time of inclusion, none of them working shifts. The mean age of onset of the first mood symptoms was 24.10 ± 9.75 years, but mean age of first diagnosis was 33.66 ± 11.03 years in BD patients. 95.2% of the BD patients had experienced inpatient care before (5.14 ± 3.72 times). Almost all patients (95.2%) received psychotropic pharmacotherapy; every other (53.4%) reported a history of suicidal behaviour; and every third (33.3%) had attempted suicide before. Their average HAMD-17 score was 4.38 and the average YMRS score was 1.9, thus excluding a current major episode.BD  and control group  differed significantly in age (p = 0.022) and in the RIS . Despite these differences, none of the groups reached a mean score high enough to indicate overall bad sleep. On an individual level, six BD patients and two healthy controls reached the PSQI cut- for bad sleep and five BD patients as well as one healthy control reported distinct psychophysiological symptoms of insomnia according to RIS. According to the sleep diaries, patients had a longer subjective TST (p = 0.024), and TIB (p = 0.002),BD group and healthy controls differed significantly in the PSQI  had a worse SE , fell asleep earlier  and sleep onset time varied less , which could indicate a more stable sleep schedule. There were no significant differences for actigraphically validated TST.As in Table p = 0.352). The DLMO was on average 1.65 h earlier in BD patients in comparison to healthy controls . When computing the PAD to quantify possible circadian misalignment, we found a significant phase advance of melatonin in BD patients compared to healthy controls . Two examples of melatonin curves are displayed in Fig. There was no significant difference in average melatonin levels between BD patients and healthy controls (4.06 ± 2.77 vs. 3.35 ± 2.23 There was no significant difference between the mean cortisol levels 30 min after rising time in both groups. The average sampling time was at 8:08 am (SD = 1:24) for controls and at 8:17 am (SD = 2:05) for BD. Three individuals were excluded from analysis: two had strikingly low cortisol levels that was attributed to sampling error and one failed to deliver enough saliva. All of them were part of the BD-group.A larger PAD was highly correlated with longer WASO, a worse SE and a longer duration of illness . Concurrently we found a correlation between an earlier DLMO and a higher WASO as well as a longer duration of illness . Longer time passed since the last episode experienced was highly correlated to higher cortisol levels  and lower TST . Longer time since the last manic episode was associated with a worse SE and a lower TST both in actigraphy and sleep diary : r = − 0.559, p = 0.01; TST (sleep diary): r = − 0.486, p = 0.03). We found age at first diagnosis to be negatively correlated with SE . We also found a correlation of between a higher score in HAMD-17 and a higher score in PSQI as well as in RIS . There were no correlations between any sleep outcome variable and YMRS scores, age at first phase and number of depressive episodes.p = 0.052).Group differences in PSQI , subjective TST  and subjective TIB  remained significant after controlling for age and gender, while group differences in RIS , actigraphically measured sleep onset , SD of said sleep onset , WASO and SE , DLMO  and PAD  did not. DLMO, PAD and actigraphically measured WASO and SE remained significant, when we only controlled for the covariate gender, while there developed a trend towards a longer TST in our BD group when we controlled for the covariate age . This trend was further strengthened by also controlling for gender  higher PSQI (significant after controlling for age and gender), and higher RIS scores; (2) subjective a longer mean TST and TIB (both significant after controlling for age and gender), longer WASO, and worse SE (significant after controlling for gender); (3) actigraphically validated earlier sleep onset, less variation in sleep onset time, and no longer TST; (4) no differing melatonin levels, an 1.65 h earlier DLMO, and a phase advance of melatonin (both significant after controlling for gender); and (5) no differing cortisol awakening response.Individuals suffering from BD had significantly higher PSQI and RIS score. Those findings of subjectively worse sleep are also a common feature of interepisode BD  episode might not be a measurement to test for disease stability and could even be associated with a higher risk of relapse, thus higher cortisol levels and shorter TST are both features of mania in BD  and circadian variables , since group differences in these variables remained significant when only controlling for gender. The finding of a worse sleep quality with more WASO and a subsequently worse SE in aging populations is consistent with previous meta-analysis (Ohayon et al. Interestingly TST also shortens during aging (Ohayon et al. Although there is evidence for an more WASO (Ohayon et al. There are several limitations to our study. Firstly, our study had a naturalistic design and several drop outs due to unsufiicient actigraphic data. In consequence there were group differences in age and gender and a smaller then expected group size, which might have influenced our results. Secondly, it is important to recognize that our sampling window for DLMO was rather short due to the high cost of melatonin analysis. Although we could identify most individuals’ DLMO in our sampling window, there were some cases where an individual’s first sample was already above our DLMO threshold, which forced us to assume this person’s DLMO. A higher sampling frequency and a larger sampling window would be worthwhile. Thirdly, although subjects received detailed, easy-to-understand information, they had to retrieve saliva samples on their own at home, which might have hampered the results due to non-compliance or misunderstanding. Lastly, our sample size was rather small, so our findings would need to be confirmed in a larger setting and—until then—must be interpreted with great caution.We found that sleep in interepisode BD is perceived as significantly worse. Although objective sleep variables did not remain significant throughout ANCOVA, there was a trend towards a significantly longer TST and patients showed a lower SE and higher WASO. Our results hint at a circadian misalignment with an earlier DLMO and a larger PAD in interepisode BD, which might further support Kripke et al.  ’s theor


The aim of the investigation was to compare the occurrence of post-activation performance enhancement (PAPE) after drop jumps, or heavy sled towing, and the subsequent effect on repeated sprint ability (RSA).Ten young basketball players (17 ± 1 yrs) performed, in randomized order, RSA test with changes of direction after a standardized warm up followed by drop jumps, heavy sled towing, or no exercise (control condition). Neuromuscular assessments composed of two maximal voluntary contractions of the knee extensors, peripheral nerve stimulation, and surface electromyography (EMG), responses were recorded before and immediately after the RSA. The EMG signal of leg muscles during sprinting were also recorded as well as the blood lactate concentration.P = 0.033), total time (P = 0.031), and slowest time (P = 0.029) compared to control condition, while heavy sled towing did not change RSA outcomes (P > 0.05). All conditions exhibited a decrease of doublet high frequency stimulation force (pre-post measurement) (P = 0.023) and voluntary activation (P = 0.041), evidencing the occurrence from peripheral and central components of fatigue after RSA, respectively, but no difference was evident between-conditions. There was a significantly greater EMG activity during sprints for the biceps femoris after drop jumps, only when compared to control condition (P = 0.013).The drop jumps improved the RSA mean time (Repeated drop jumps were effective to induce PAPE in the form of RSA, while heavy sled towing had no effect on RSA performance in young basketball players. Furthermore, both conditioning activities exhibited similar levels of fatigue following the RSA protocol. Thus, drop jumps may be used as an alternative to induce PAPE and thus improve performance during sprints in young male basketball players.The online version contains supplementary material available at 10.1186/s13102-021-00395-w. Basketball is a team sport which consists of brief high-intensity efforts with frequent changes of direction, interspersed with short recovery intervals –4. ThesePost-activation potentiation (PAP) is a potential strategy to improve performance during high-intensity activities in basketball. PAP is defined as an acute involuntary improvement in muscle force generating capacity  , after aAccordingly, the purpose of this study was to investigate and compare the use of drop jumps and heavy sled tows as CAs to improve performance during repeated sprint efforts, with directional changes. Additionally, we examined the effects of these protocols on selected markers of fatigue. Our hypotheses were that both, drop jumps and heavy sled towing, would induce PAPE, however, the drops jumps would be more effective due to lower level of residual fatigue.F test family were alpha = 0.05 and power = 0.90. Using the time until exhaustion during cycling at a supramaximal power output after control (157 ± 42 s) and the one preceded by drop jumps protocol , the minimal required sample size of 9 participants was estimated. A basketball team was contacted and fifteen young male basketball players (the players were from the under 17 (n = 7) and 19 (n = 8) categories and had experience of competing at a national level) were recruited to participate in the current study after meeting the following inclusion criteria: (1) basketball training experience ≥ 4 years; (2) absence of recent (< 3 months) musculoskeletal and joint injuries; and (3) absence of recent (< 3 months) regular use of any ergogenic substance . The exclusion criteria were: (1) suffering an injury during the period of investigation (n = 2); (2) not tolerate the assessment procedures (n = 1); and (3) withdraw from the team (n = 1). Finally, 10 participants  were selected. Prior to the commencement of the study, the athletes were informed about benefits and risks of experimental procedures and signed an informed consent form. For the underage participants, their parents were informed and signed the consent form. All experimental procedures of the present study were approved by the Research Ethics Committee of the Sao Paulo State University (#2.540.512/2018) according to the Declaration of Helsinki.The minimal required sample size was determined using G*power software . The inpA randomized cross-over design was adopted and all sessions were completed at the end of the pre-season and at the beginning of the competitive season, over a 3-week period. All assessments were performed on the same official basketball court, at the same time of day, with a consistent temperature of 29.3 ± 2.8 °C and a relative humidity of 52.2 ± 8.5%. All exercise sessions were separated with a minimum of 48 h and a maximum of 72 h.Firstly, participants completed a familiarization session to the RSA test, to the CAs , and to the neuromuscular assessments. In the following two sessions, participants initially performed a standardized warm-up . After 5 min of passive rest, a protocol for the evaluation of neuromuscular function  was conducted. Following neuromuscular evaluation, participants performed one of the CAs  or remained at rest for 5 min . After interventions , the RSA test was conducted with simultaneous recording of surface electromyography (EMG) of lower limb muscles. The reason for different intervals after CAs is reported in “The RSA test, which included a change of direction (“L” format), used in this study consisted of 10 × 30 m maximal sprints with each sprint involving 5 rapid directional changes (6 runs of 5 m), interspersed with 30 s of passive recovery. This test was selected because it has been previously shown to mimic real game-play demands, with good reliability , 22. All2 > 0.99). The peak force of MVCs was defined as the mean force recorded during 100 ms of the force plateau ) were calculated and used as indices of sarcolemmal excitability  for the RSA outcomes and the delta changes between conditions for parameters of neuromuscular assessment was also calculated.The results are presented as means ± standard deviations and 95% confidence intervals (CI95%). A two-way repeated measures analysis of variance (ANOVA) was used to identify the effect of time and condition on neuromuscular assessment parameters. A repeated measures of ANOVA was used to compare RSA test performance outcomes and the percent changes of neuromuscular assessments between conditions. In all cases, the Mauchly´s test of sphericity was applied, and the Greenhouse–Geisser Epsilon correction was used when the sphericity criteria were not met. When necessary, the analyses were completed with SIDAK post hoc test. A significance level of p-value = 0.033), slowest time , total time , while best time  did not change. RSA outcomes were different between the drop jump and control condition with improvements shown following drop jumps in mean time , total time and slowest time . However, heavy sled towing did not present any differences compared to the drop jumps (ESs = 0.02 (CI95%: − 0.25 to 0.30) for best time, 0.12 (CI95%: − 0.18 to 0.41) for mean time, 0.12 (CI95%: − 0.17 to 0.41)for total time, and 0.23 (CI95%: − 0.08 to 0.55)for slowest time and control conditions (ESs(CI95%) = − 0.25 (CI95%: − 0.57 to 0.07), − 0.23 (CI95%: − 0.51 to 0.05), − 0.23 (CI95%: − 0.51 to 0.05), and − 0.17 (CI95%: − 0.49 to 0.15), respectively (P ≥ 0.110).The results of RSA performance during control, drop jumps and heavy sled towing conditions are presented in Fig. P = 0.821), RMS (P = 0.684) and MdF (P = 0.904). An effect for time was found for MVC force . In addition, a main effect for MdF during MVCs revealed a higher value after RSA for all conditions . There was no interaction between conditions for force at Db100 (P = 0.591), the ratio Db10/Db100 (P = 0.126), as well as for M-waveamp (P = 0.509). A main effect for time existed with a decrease after RSA for force at Db100  and the ratio Db10/Db100 . Finally, there was no interaction between conditions for VA (P = 0.953), however, a main effect of time revealed the existence of central fatigue in all experimental conditions . The delta changes between conditions for all parameters of neuromuscular assessment did not differ (P > 0.05) . There was also a difference between conditions for RMS in the biceps femoris, showing higher RMS values in the drop jumps condition, compared to control .The EMG data during RSA testing is presented in Fig. −1; heavy sled towing: 0.83 ± 0.29 mmol.L−1; drop jumps: 0.93 ± 0.57 mmol L−1; P = 0.655) and after the RSA . Besides, the drop-jump condition presented a lower significant difference for lactate concentrations prior to RSA testing when compared with other conditions .No difference between conditions was observed for lactate concentrations at baseline  in the PAPE response. Specifically, the drop-jump condition exhibited a higher RMS in the The improvement in RSA performance after drop jumps may be important for basketball performance with effect size varying among small to moderate. A previous study has reported a correlation between RSA mean time, and distance covered during high intensity actions throughout matches . In a reThe improvement in RSA performance induced by drop jumps may be attributed to a potential PAP effect; however, for methodological reasons, we did not measure this occurrence with the twitch verification in the present study. Previously, de Poli et al.  revealedrectus femoris from the fourth, eighth, and tenth sprints in the drop-jump condition, when compared to the first sprint, and a higher level of activity in the drop-jump condition compared to the control. The RMS of the EMG is considered as a global index of motor unit recruitment for the specific exercise performed. One of the proposed mechanisms for PAP is the enhanced recruitment of higher order motor units following a CA [rectus femoris after a taekwondo kick movement with an elastic band. More recently, de Poli et al. [rectus femoris, vastus lateralis, vastus medialis and gluteus maximus). These recent results are in agreement with another recent study by Zagatto et al. [biceps femoris in the drop-jump condition, may partially explain the PAPE observed as it would be related to a lower fatigue of this muscle when compared to the other conditions [The main results for RMS during the RSA test showed a significant reduction in RMS of ing a CA . Thus, ting a CA  found ani et al.  investigo et al. , who meanditions . This, inditions . Howeveramp) induced by repeated sprinting, corroborating previous work [It is interesting to note that despite sprinting performance was greatest following use of drop jumps protocol, the assessment of neuromuscular function revealed similar fatigue levels post-exercise in all trials. The results showed a combination of a decrement in peripheral (Db100 and ratio Db100/Db10) and central parameters  to improve the linear and multilinear sprint performance of young basketball players. As this exercise is very easy to prescribe and implement, it may be regularly included at the end of warm-up and/or re-warm up sessions during basketball games.Drop jumps were effective to induce PAPE via improvements in repeated sprinting ability, while heavy sled towing elicited no effect in young basketball players. Furthermore, both activities induced similar levels of fatigue after the repeated sprinting protocol.Additional file 1.Supplementary Material: Supplementary Table 1. Outcomes of optimal recovery interval related to conditioning activity drop jumps. Supplementary Table 2. Outcomes of optimal recovery interval related to conditioning activity heavy sled towing.


UQCRH, which encodes a structural complex III (CIII) subunit. We generated a mouse model with the equivalent homozygous Uqcrh deletion (Uqcrh−/−), which also presented with lactic acidosis and hyperammonaemia, but had a more severe, non‐episodic phenotype, resulting in failure to thrive and early death. The biochemical phenotypes observed in patient and Uqcrh−/− mouse tissues were remarkably similar, displaying impaired CIII activity, decreased molecular weight of fully assembled holoenzyme and an increase of an unexpected large supercomplex (SXL), comprising mostly of one complex I (CI) dimer and one CIII dimer. This phenotypic similarity along with lentiviral rescue experiments in patient fibroblasts verifies the pathogenicity of the shared genetic defect, demonstrating that the Uqcrh−/− mouse is a valuable model for future studies of human CIII deficiency.Mitochondrial disorders are clinically and genetically diverse, with isolated complex III (CIII) deficiency being relatively rare. Here, we describe two affected cousins, presenting with recurrent episodes of severe lactic acidosis, hyperammonaemia, hypoglycaemia and encephalopathy. Genetic investigations in both cases identified a homozygous deletion of exons 2 and 3 of  UQCRH in two children presenting with recurrent episodes of metabolic crisis. A mouse model harbouring the equivalent variant in Uqcrh shared similar biochemical phenotypes of impaired CIII activity and abnormal OXPHOS supercomplex structures, but with more severe manifestation.This work describes the first confirmed pathogenic variant in  UQCRH variants in mitochondrial disease cases.Mitochondrial diseases are a group of disorders and are varied in terms of both their clinical manifestations and their genetic causes. They are characterised by mitochondrial dysfunction, predominantly resulting in decreased energy production in the form of ATP made by the oxidative phosphorylation (OXPHOS) system. Mitochondrial diseases can be caused by changes within the mitochondrial genome itself (mtDNA), or in nuclear genes that encode mitochondrial proteins . More than 300 of these genes have been associated with mitochondrial disease, and this diversity often makes obtaining genetic diagnoses in affected individuals difficult. UQCRH is a mitochondrial protein encoded by the nuclear genome and is a subunit of complex III, one of 5 multi‐subunit complexes that make up the OXPHOS system. There have not been any previous reports of UQCRH so we opted to generate a mouse model with the equivalent Uqcrh deletion. The mice showed similar symptoms of lactic acidosis and high ammonia levels, though the presentation was more severe and was constant rather than episodic, leading to early death. Biochemically, skin fibroblasts from the affected human subjects and tissue from the mutated mice were strikingly similar, displaying impaired activity of complex III, no expression of the UQCRH/Uqcrh protein, a smaller size of assembled complex III, an increase in larger OXPHOS supercomplexes and decreased expression of other complex III subunits. This strongly suggested that the shared UQCRH deletion was the cause of these similar biochemical defects. Crucially, we also showed an amelioration of the complex III deficiency when introducing a wild‐type  copy of UQCRH via a lentiviral delivery system, further demonstrating that the UQCRH deletion was causative of the disease.Here, we describe two affected cousins who presented clinically with episodes of metabolic crisis, manifesting with severe lactic acidosis, high ammonia levels, low glucose levels and brain abnormalities. Between these episodes , the affected individuals were otherwise healthy. Whole exome sequencing identified a 2.2 kb dropout of sequencing reads predicting a homozygous deletion of 2 out of the 4 coding regions of the gene, confirmed by Sanger sequencing (c.55‐528_243+473del). We could not identify any other individuals with variants in UQCRH. Confirmation of this variant as causative enables pre‐symptomatic accurate diagnosis of at‐risk individuals in the family, demonstrating the clinical importance of providing a genetic diagnosis in mitochondrial disease cases. Furthermore, the viable mouse model described here represents a valuable organism for the study of complex III biology and mitochondrial impairment more generally. Indeed, this mouse holds great potential to be used for new therapeutic strategies targeting CIII defects or related mitochondrial disorders.This study documents the first cases of mitochondrial disease due to variants in et al, et al, et al, et al, et al, et al, et al, et al, et al, et al, Mitochondrial diseases are a group of complex genetic disorders characterised by mitochondrial dysfunction that result in clinically heterogeneous presentations, mostly affecting tissues with high energy requirements  is one of the five complexes of the mitochondrial oxidative phosphorylation (OXPHOS) system. CIII transports electrons from ubiquinol to cytochrome c and translocates protons across the inner mitochondrial membrane, contributing to the electrochemical gradient that is then used by complex V to drive ATP synthesis. In vertebrates, CIII comprises 11 subunits assembled with the support of at least four additional factors , CYC1 (encoding cytochrome c1) and UQCRFS1 (encoding the Rieske protein containing an iron‐sulphur cluster centre), while the exact function of the other eight  remains to be fully elucidated  and UQCRH is thought to be a structural component linking the cytochromes c and c1 . The clinical phenotypes associated with CIII deficiency are heterogeneous, but the most common clinical presentations are early‐onset recurrent metabolic crises characterised by lactic acidosis and hypoglycaemia , observed in only one patient, was associated with episodic metabolic decompensation  have been associated with episodic severe metabolic acidosis and hyperammonaemia, hypoglycaemia and lactic acidosis in four patients , a male infant, is one of three children of healthy, consanguineous, British/Pakistani parents Fig . At the The second affected individual (II‐4) is the only child of consanguineous parents and is the first cousin of the proband (II‐1). He was well until the age of 3 years and 4 months when he presented to the hospital with vomiting and diarrhoea, high lactate and compensated metabolic acidosis. Over the next three years, he presented an average number of eight times per year to the hospital, with vomiting, fever or abdominal pain, and was treated with intravenous fluids. He experienced high lactate levels on four occasions (maximum 10 mM). On one occasion, at the age of 4 years and 8 months, he presented with a pH of 7.046, lactate of 10 mM, ammonia of 306 μM and blood glucose of 0.5 mM. Apart from a single episode of pancreatitis, the patient developed normally. At his last follow‐up visit, he was 8 years old, since then he showed no episodes of metabolic crisis for 2 years. A comprehensive clinical history of both patients is provided in Appendix Table HIVEP3 (NM_024503) c.7043C>T; p. Pro2348Leu and TCTEX1D4 (NM_001013632) c.140C>T; p. Pro47Leu. Neither of these variants were likely pathogenic due to presence in control populations  and were predicted benign using in silico tools. However, WES coverage data  in humans, which is absent in mice  showed a more severe phenotype. Uqcrh−/− mice were viable, but born at a lower Mendelian ratio and presented with failure to thrive after birth. Indeed, crossings of heterozygous mice resulted in 161 pups, of which 13.66% were homozygous mutants , and below the expected 25%, indicating an important function of Uqcrh during embryonic development. After weaning, Uqcrh−/− mice were smaller  and 13.0 g (64% of wild‐type), respectively  and slightly decreased blood glucose levels   , similar to the patients during episodes of metabolic crisis  Fig . In cont mM) Fig . Uqcrh−isis Fig .Uqcrh−/− mice presented with tremor and 11 out of 15 Uqcrh−/− mice showed abnormal gait indicating motor dysfunctions (at 9 weeks of age) , hyperphosphatasaemia (increased ALP), decreased calculated total iron‐binding capacity (TIBC) and strong hyperglycaemia  Fig . The glo age Fig  indicatimice Fig , but ele age Fig . FinallyUQCRH deletion, we investigated the function of a primary dermal fibroblast cell line from the proband (II‐1) and multiple tissues from Uqcrh−/− mice. A sample of fibroblasts from the second individual II‐4 was not available. Biochemical analysis confirmed a specific decrease of CIII activity to approximately 60% compared to control cell lines . Western blot analysis showed that doxycycline induction of wild‐type UQCRH increased the steady‐state protein levels of UQCRC2 in patient fibroblasts in a dose‐dependent manner  in patients . We confirmed the appearance of assembled CIII with a loss of UQCRH in both the human patients and Uqcrh−/− mice although they were of lower abundance compared with the respective controls  and mouse heart tissue . The affected individuals are cousins and presented with recurrent episodes of metabolic decompensation, including severe lactic acidosis, hyperammonaemia, hypoglycaemia and encephalopathy. Notably, the patients were asymptomatic between these episodes. The identification of the UQCRH variant using WES and autozygosity mapping prompted functional studies on patient‐derived fibroblasts, which demonstrated a functional consequence of the identified UQCRH variant   compared with expected Mendelian ratios, thus suggesting that it is important but not fundamental to proper embryonic development.We were unable to identify any additional patients with crh Figs . HomozygUqcrh−/− mice displayed a metabolic phenotype with lactic acidosis and hypoglycaemia  highlighting the phenotypic heterogeneity in humans  had occasional episodes of hypoglycaemia and lactic acidosis, rapidly corrected by glucose infusions  levels, which are usually observed in respiratory chain defects, impacting upon glucose homeostasis in several ways. We observed that, when not treated, the hypoglycaemic burden on the mice developed into persistent hyperglycaemia. Whether this is an adaptation mechanism of the mouse is yet unknown.The contrast between episodes of hypoglycaemia observed in patients versus the hypoglycaemia developing gradually to constant hyperglycaemia in the Uqcrh−/− mice, there was a striking similarity in the biochemical analysis of each. Firstly, both exhibit a decrease in CIII activity but retain significant residual activity. The decrease in the activity of CIII observed in Uqcrh−/− mice was approximately 50% . Complexome analysis confirmed that neither the patient nor the Uqcrh−/− mouse expressed the UQCRH (Uqcrh) protein  supplemented with heat‐inactivated foetal bovine serum (Gibco), 200 µM uridine (Sigma‐Aldrich) and penicillin/streptomycin amphotericin B solution (Lonza). Cells were regularly tested for mycoplasma contamination; all cells used in the experiments were tested mycoplasma negative.The parents of the recruited children provided informed written consent for their participation in a study approved by the South Manchester Ethics committee  and the University of Manchester to determine the genetic cause of the condition affecting their children. Patient and normal human dermal fibroblasts (NHDF) (Lonza #CC‐2509) were cultured at 37°C and 5% COet al, Autozygosity mapping was undertaken on DNA samples from the proband (II:1), his two unaffected siblings (II:2 and II:3) and affected cousin (II‐4) by using the Genome‐Wide Human SNP Array 6.0  as described previously . Reads were aligned with the human reference genome version GRCh37/Hg19. Single‐nucleotide substitutions and small insertion and/or deletion variants were identified with a modified GATK variant‐calling pipeline. Filtering was undertaken to remove variants with a minor allele frequency of > 1% in the Exome Variant Server, Exome Aggregation Consortium , 1000 Genome databases or those seen previously within an in‐house dataset of over 600 individuals. Analysis was confined to the > 2 Mb regions of homozygosity defined by the autozygosity mapping.Uqcrh knockout(−/−) mice (C57BL/6NCrl‐Uqcrhtm1b(EUCOMM)Wtsi/Ieg) were generated by allele conversion of C57BL/6NCrl‐Uqcrhtm1a(EUCOMM)Wtsi/Ieg mouse line originating from EUCOMM ES clone EPD0378_3_C07. For further details on the construction of this clone, see the page at the IMPC portal Wtsi).Uqcrh and the neomycin cassette using a cell‐permeable Cre recombinase . Heterozygous mice were intercrossed to generate homozygous mutants.The resulting mice were genotyped to verify mutation and the two‐exon deletion according to genotyping protocol, available on the Infrafrontier web page (www.mouseclinic.de). Uqcrh−/− homozygous mice and their wild‐type controls were fed ad libitum with moist food provided fresh twice a day in addition to the normal chow food. Food intake was monitored during daily health checks, and no issues were observed in food intake in the Uqcrh−/− mice.Mice were maintained in IVC cages with water and standard mouse chow according to the directive 2010/63/EU, German laws and GMC housing conditions  and approved by the responsible authority of the district government of Upper Bavaria.If not stated otherwise, all tests were performed following standard procedures as described before ; during the post‐weaning period from 5 to 6 weeks of age ; at 6–7 weeks of age ; 7–8 weeks of age ; 8–9 weeks of age ; 9–10 weeks of age , and 10–11 weeks of age . Animal number may contain more than one cohort and is always age and sex‐matched.Bodyweight was measured for the different cohorts at different time points: at 1–2 weeks of age . The open‐field test evaluates the motor function and spontaneous exploration and was performed as previously described  the animals crossed in the first 30 s after transfer into the viewing arena were counted. During observation in the arena, a trained observer categorised gait  and recorded the occurrence of tremor during observation.Data generated by the Open Field test and SHIRPA were obtained at 8 and 9 weeks of age .To evaluate sensorimotor gating via assessment of the acoustic startle reflex (ASR) and its prepulse inhibition (PPI), an ASR/PPI test was applied. It consists of a large sound‐attenuated chamber that isolates the animal in the presence of background noise (65 dB). A loudspeaker is located in the upper part of this chamber and the animal is enclosed in a transparent cylinder placed upon a piezoelectric motion sensor platform that transduces movements of the animal into electrical signals that are recorded and analysed. A session begins with an initial stimulus‐free acclimation period of 5 min (except for background noise), followed by 5 startle stimuli alone (110 dB) trials to measure ASR response. PPI was assessed for a startle stimulus level of 110 dB with prepulse levels of 67, 69, 73 and 81 dB preceding the startle pulse at an inter‐stimulus interval of 50 ms . High‐throughput electrocardiogram (ECG) and echocardiogram (ECHO) recordings in conscious mice were performed to assess the morphology and functionality of the heart  and electrocardiography (ECG) were performed at 6‐week‐old mice  and mice aged 8‐9 weeks . Animal number may contain more than one cohort and always age and sex‐matched.Final blood samples were either collected from the retrobulbar vein plexus under isoflurane anaesthesia, by heart puncture after cervical dislocation, or collected from the trunk after decapitation (1–2‐week‐old pups) in Li‐heparin‐coated tubes . Collected blood samples were centrifuged at 5000xg for 10 min at 8°C and plasma separated within one hour of blood collection. Clinical chemistry parameters were measured immediately using an AU480 analyser (Beckman‐Coulter) and adapted reagent kits from Beckman‐Coulter according to the manufacturer’s instructions, as described previously  and OXPHOS enzyme activity measurements were performed as previously described  and OXPHOS complexes were assessed using DDM‐solubilised enriched mitochondria samples as previously described (Oláhová ® Mini Protease Inhibitor Cocktail (Roche) and PhosSTOP™ (Roche). Protein concentrations were obtained using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. 20 µg of protein sample was loaded onto a 10% SDS–polyacrylamide gel (Bio‐Rad), and electrophoresis was performed. The lysate was subsequently transferred to a nitrocellulose membrane (Thermo Fisher Scientific). The membrane was blocked with Odyssey® Blocking Buffer (TBS) (LI‐COR), thereafter incubated overnight at 4°C in primary antibody and subsequently in secondary antibody for 45 min at room temperature in the same buffer. As primary antibodies, rabbit polyclonal UQCRH  and mouse monoclonal VCP  for loading control were used. As secondary antibodies, IRDye 800CW anti‐rabbit (Gt)  and IRDye 680CW anti‐mouse (Gt)  were utilised. For fluorescent detection of proteins, Odyssey Infrared Imaging System and Odyssey® software (LI‐COR) was utilised.Proteins from mouse livers were extracted by tissue disruption in ice‐cold RIPA lysis and extraction buffers (Thermo Fisher Scientific) supplemented with 1x cOmpleteet al, et al, et al, Sample preparation and blue‐native electrophoresis (BNE) of mouse tissue and human cells were carried out as described previously  using the In‐Fusion HD Cloning Kit (Takara Bio/Clontech). The UQCRH containing vector was packaged into infectious lentiviral particles using Lenti‐X Packaging Single Shots (Takara Bio/Clontech) to transfect HEK293T according to the manufacturer’s guidelines. Media containing infectious lentivirus was harvested after 48 h and centrifuged at 500 g for 10 min to remove cellular debris and the supernatant was used to transduce patient fibroblasts at a ratio of 1:3 with fresh media and 4 mg/ml polybrene overnight. The media was replaced and puromycin (2 µg/ml) added to select for successfully transduced cells. Transduced cells were cultured in puromycin for 7 days before being maintained in standard high glucose. The expression of wild‐type UQCRH was induced by adding doxycycline  to the media and incubating for 72 h.Wild‐type UQCRH was cloned into pLenti6.3/V5‐TOPO® TA Cloning® Kit (Invitrogen) for the expression of the gene in human control dermal fibroblast (Lonza #CC‐2509) and patient fibroblasts. Generation of transgenic fibroblast cell lines was performed following a previously described transfection protocol  at 9 weeks of age at the time of necropsy. The tissues were fixed in formalin for 24 h and embedded in paraffin for histological and immunohistochemical analysis. Immunohistochemistry was performed on 2 µm‐tick sections in an automated immunostainer . Briefly, sections were incubated with primary antibodies against UQCRC2  and VDAC1/porin  with a streptavidin–peroxidase detection reagent. As a negative control, the primary antibody was omitted. Digital images were captured with the NanoZoomer® 2.0HT  digital slide scanner. A scoring system was used to quantify the expression levels of UQCRC2 and VDAC1/Porin: 0 = no staining; 1 = weak staining; 2 = moderate staining; 3 = strong staining. The score for each section was calculated as the mean of 4 high‐power fields . Microscopy was carried out with a Zeiss LSM 880 confocal microscope. Fluorescence intensity was analysed with the help of ImageJ . The intensity was measured in 10‐14 cells for each microscopic field for a total of three macroscopic fields for each type of cell. Both immunohistochemical and immunofluorescence analyses were performed in blinded conditions.Immunohistochemical staining was performed as previously described . Ultrathin sections were cut using an ultramicrotome  and examined in a transmission electron microscope (Zeiss EM 902). Digital images were captured with a slow‐scan 2K CCD camera .Anaesthetised animals were perfused with 1.5% glutaraldehyde and 1.5% formaldehyde in 0.15 M HEPES (pH 8.0), and dissected hearts were stored in the same fixative at 4°C for at least 24 h. For epoxy resin embedding, heart samples were post‐fixed in 1% osmium tetroxide, stained t‐test, Wilcoxon rank‐sum test, linear models, or two‐way ANOVA and with Tukey HSD post hoc tests, or Fisher’s exact test depending on the assumed distribution of the parameter and the questions addressed to the data .The selected sample size per sex and genotype was sufficient to find a medium difference of one standard deviation with a power of 0.8 and an alpha of 0.05. If not stated otherwise, data that were generated by the German Mouse Clinic were analysed using R (version 3.4). For each parameter, we calculate measures like mean, SD, effect size and, respectively, median and IQR in our standardised automatic R‐scripts. In general, no data point is excluded from the analysis unless there is clear evidence of, for example, technical failure of the experimental machine. For many of the parameters, there exist pre‐established threshold values as indicators for invalid measurements, these data points are replaced by status codes. Tests for genotype effects were made by using ta Table . Wild‐tyFor immunohistochemistry and enzymatic activity, statistical analyses were performed using Prism 6 and 9 . Results are given as mean ± SEM. Student’s t‐test (unpaired samples) was applied to compare two groups, one‐way ANOVA , to compare more than one group and correct for multiple comparisons.HP, VG‐D, KT, RWT, WGN, MHA conceived and initiated the study; HP, VG‐D, HF, KT, RWT, WGN coordinated the study; JEU, KT, JAA‐P, OVA, LB, PS‐B, JC‐W, Y‐LC, LG, RG, KPH SMH, TK‐R, PM‐K, BR, JR, NS, IT, NFC, SV, RGF, JM, IW planned and performed the experiments; JAA‐P, OVA, LB, PS‐B, JC‐W, Y‐LC, LG, RG, SMH, TK‐R, PM‐K, BR, JR, NS, IT, NFC, MAO, GM, SV, RGF, JAM, JEU, KT, WGN, IW analysed the data; UG performed electron microscopy imaging; SM coordinated mouse line generation; CB, EJ, JHW, CSa clinically assessed the affected individuals and obtained patient samples; KG, SL, CSt, KP were involved in project management; RG, VG‐D, JAA‐P, BR, SL, KT, RWT, WGN, MS, SV drafted and worked at the manuscript; MHA, HF, VG‐D, WW provided oversight and resources; all authors read and approved the manuscript.The authors declare that they have no conflict of interest.https://www.omim.org/entry/613844?search=UQCRH&highlight=uqcrh#titleOMIM: https://gnomad.broadinstitute.orggnomAD: https://www.newcastle‐mitochondria.com; https://www.mouseclinic.de/Authors’ websites: https://rarediseases.info.nih.gov/diseases/8295/mitochondrial‐complex‐iii‐deficiency; https://www.infrafrontier.eu/infrafrontier‐and‐rare‐diseasesRare diseases resource links: https://www.mousephenotype.org/blog/2021/02/27/rare‐disease‐day‐2021/International Mouse Phenotyping Consortium (IMPC): http://marrvel.org/Human&Mouse Genetic Resource: AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Source Data for Figure 3Click here for additional data file.


Clausena dunniana in a subtropical broad‐leaved forest.Understanding the joint effects of plant development and environment on shifts of intraspecific leaf traits will advance the understandings of the causes of intraspecific trait variation. We address this question by focusing on a widespread species C. dunniana at two major topographic habitat types, the slope and hilltop, within the karst forests in Maolan Nature Reserve in southwestern China. We measured individual plant level leaf traits , leaf area, leaf dry‐matter content (LDMC), and leaf thickness) that are associated with plant resource‐use strategies. We adopted a linear mixed‐effects model in which the plant size  and environmental factors  were used as independent variables, to estimate their influences on the shifts of leaf traits.We sampled 262 individuals of C. dunniana, of which plant size explained less variances than environmental factors. (2) With increasing plant size, C. dunniana individuals had increasingly smaller SLA but larger sized leaves. (3) The most influential environmental factor was topographic habitat; it drove the shifts of all the four traits examined. Clausena dunniana individuals on hilltops had leaf traits representing more conservative resource‐use strategies  than individuals on slopes. On top of that, local‐scale environmental factors further modified leaf trait shifts.We found that (1) plant size and the environmental factors independently drove the intraspecific leaf trait shifts of C. dunniana in the subtropical karst forest of Maolan. Compared with plant size, the environment played a more critical role in shaping intraspecific leaf trait variations, and potentially also the underlying individual‐level plant resource‐use strategies.Plant size and environment independently shaped the variations in intraspecific leaf traits of  Clausena dunniana in the subtropical karst forest of Maolan. Compared with plant size, the environment played a more critical role in shaping intraspecific leaf trait variations, and potentially also the underlying individual‐level plant resource‐use strategies.Plant size and environment independently shaped the variations in intraspecific leaf traits of  We predict that leaf traits of C. dunniana would shift toward more conservative resource‐use strategies  with increasing plant size, and this trend be more pronounced in high light, low moisture, and infertile environments , compared to dark, moist, and fertile environments .Here, we explore how development and environment jointly (including their interactions and relative importance) drive the shifts of intraspecific leaf traits of 22.1Clausena dunniana (Rutaceae) is a deciduous compound leaved plant, with an adult height of 2–5 m. Its compound leaf is composed of 5–15 leaflets. The leaflet blade is ovate to lanceolate in shape, with a length of 4–10 cm and a width of 2–5 cm. Clausena dunniana is mainly distributed at 300–1,500 m a.s.l. in montane forests in southwestern China  is located in Libo County of Guizhou Province in southwestern China. Local annual mean temperature is 15.3°C, with January mean of 5.2°C and July mean of 23.5°C. Mean annual precipitation is 1,752.5 mm. The bedrock of the reserve consists of Carboniferous and Permian limestones and dolomites, with elevation varying from 430 to 1,078 m a.s.l. Zhou, . In the 2.2C. dunniana at two major topographic habitat types, the slope  and the hilltop , within the forests of six peak‐clusters  evenly distributed in two regions  about 10 km apart within the reserve  method  of its stem/trunk using an electronic digital vernier caliper  for individuals with basal diameter <5 cm, or using a diameter tape for larger individuals . We measured the height of the sampled individual using a pole with a rod level. With respect to leaf sampling, we randomly sampled 2–10 fully expanded intact compound leaves located at the outer layer of the upper portion of the crown of the plant using a tree pruner . The sampled leaves were placed in black plastic bags, stored in portable refrigerating box at above 0°C temperature and transported back to the laboratory in the same day. Then, the leaves were placed in fresh water for rehydration overnight. In the next day, the sampled compound leaves were scanned using a flatbed scanner, and leaf area was measured as the sum of one‐sided projected fresh lamina surface area of all the leaflets without petiole and rachis, using ImageJ software . A principal component analysis (PCA) was conducted on basal diameter and plant height, and the first principal component of the PCA, which explained 98.4% of the total variance, was used to represent plant size, larger value of the first principal component represents larger sized plant. Correlations between the predictors  were estimated, and no high correlation (Pearson' r ≥ .5 or ≤ −.5) was detected.The plant size‐dependent and environmental factors  mediated shifts in leaf traits  were estimated by linear mixed‐effects model using the “lmer” function in the Lth (ijkt) of individual plant i at site j of region k. Variation in ijkt was modeled as a function of plant size (PS), topographic habitat (TH), and local environmental conditions , and rock‐bareness rate (RB)), plus the two‐way interactions between PS and the three environmental factors, as fixed effects. The two‐way interactions were included to examine if size‐dependent leaf trait shifts were modified by the environment. A random intercept (jkμ) was specified for site j nested within region k, as random effect. The model structure was:β1 through β4 are fixed‐effect coefficients representing variation in SLA, LA, LDMC, or Lth due to variation in plant size, topographic habitat, canopy height, and rock‐bareness rate, respectively; γ represents fixed‐effect coefficient for second‐order interaction of these factors; and α represents the mean trait value of the individual plant averaged over all sites and regions. The random term jkμ was assumed to be normally distributed as μjk∼N0,σμ. Before analysis, all continuous predictor variables were standardized by subtracting the mean and dividing by the standard deviation  variable of a linear mixed‐effects model was the SLA, LA, LDMC, or  Helmus,  in R; we Helmus, , suggestMuMIn package  and conditional (Rc2) pseudo‐R2 values  ranged from 0.054 to 0.211, the total proportion of the variance explained by the models (Rc2) including the random part ranged from 0.146 to 0.491 ; formal analysis ; investigation ; methodology ; writing – original draft . Ya Jiang: Data curation ; formal analysis ; investigation ; methodology ; writing – original draft . Hong Qian: Writing – original draft (supporting); writing – review and editing (supporting). Yanjiao Mao: Data curation (supporting); investigation (supporting); writing – original draft (supporting). Chao Zhang: Investigation (supporting); writing – review and editing (supporting). Xiao‐Xin Tang: Funding acquisition (supporting); writing – review and editing (supporting). Yi Jin: Conceptualization (lead); formal analysis (supporting); funding acquisition (lead); investigation (supporting); methodology (supporting); project administration (supporting); writing – original draft (lead). Yin Yi: Funding acquisition (supporting); writing – review and editing (supporting).


No study has assessed the feasibility and safety of cervical pedicle screw implantation in patients with vertebral artery dominance (VAD), a common vertebral artery (VA) variation which can increase VA injury (VAI) risk. This study was to assess morphological characteristics of the subaxial cervical pedicles and surrounding critical structures, and identify their correlations in patients with VAD.Computed tomography arteriography scans of 152 patients were used for retrospectively measuring parameters including pedicle outer width (POW), the distance from the lateral pedicle border to the closest part of VA (DPVA), diameter of VA (DVA), area of VA (AVA), area of transverse foramen (ATF) and occupational ratio of transverse foramen (TF). Moreover, correlations among some critical parameters were assessed.One hundred eight males and 44 females, with a mean age of 55.9 years were included. POW was smaller on the dominant side than on the non-dominant side, whereas DPVA, DVA, AVA, ATF, and TF were larger on the dominant side than those on the non-dominant side. On both sides, POW < 4 mm and POW + DPVA < 5 mm were observed most frequently at C3 and C4. On both sides, POW was correlated to ATF, and ATF was correlated to DVA and AVA. DPVA was correlated to ATF on the dominant side.Patients with VAD exhibited smaller POW on the dominant side, most frequently at C3 and C4. Dominant VA may indirectly affect POW. TF may be a key determinant of DPVA and POW. Cervical pedicle screw (CPS) fixation, first introduced by Abumi , provideVertebral artery injury (VAI) is one of the major concerns during CPS fixation. Although VAI is uncommon in cervical spine surgeries –9, the cGiven the frequency of VAD, it is critical to assess methods for safer and more efficacious CPS placement in patients with VAD. To our knowledge, however, no study has focused on the morphological characteristics of the subaxial cervical pedicle and surrounding structures just in patients with VAD. Therefore, we conducted a morphological analysis of these characteristics based on computed tomographic arteriography (CTA) and analyzed correlations among these various structures.Our hospital ethics committee approved this retrospective image-based study. We reviewed 697 patients who underwent CTA at our hospital from January 2012 to March 2019. Based on the findings of previous studies , 17, we Patient inclusion criteria were 1) VAD, 2) computed tomography (CT) scan range C1–C7, 3) CT scanning using 256-slice CT, 4) age ≥ 18 and ≤ 60 years, and 5) complete clinical and imaging data. Patient exclusion criteria were 1) severe cervical fracture and dislocation , 2) severe cervical deformity such as kyphosis > 20 degrees, scoliosis > 10 degrees, basilar invagination, or Chiari malformation, 3) ankylosing spondylitis, 4) diffuse idiopathic skeletal hyperostosis, 5) cervical spine surgery history, 6) VA embolization, VA atherosclerosis, or a single VA, and 7) other diseases such as tumor or severe osteoporosis.All measurements were performed on a CT advanced workstation 4.4 . Scan parameter settings were as follows: layer thickness 0.625 mm, layer spacing 0.625 mm, window width 1300 HU, and window level 400 HU. Measurement accuracy was ±0.1 mm. Measurements were performed on the axial CT image showing the maximum pedicle outer width; due to the maximum pedicle outer width shown by this image, it was considered that this image presented the center of the pedicle or reflected the content at the center of the pedicle. Furthermore, the adjacent upper axial CT image of that one and lower one were also measured for all parameters and the data measured from the three images were averaged for analysis. Multiple-planar reconstruction was used to conduct multiple-point observation and identification for accurate measurement.2; 5) ATF, area of transverse foramen (TF), was calculated by the method similar to AVA; 6) ORTF, occupation ratio of TF (ORTF = AVA/ATF)  POW, the distance between the lateral cortical border and medial border at the most stenotic area of the pedicle; 2) DPVA, the distance from the lateral pedicle border to the closest part of VA; 3) DVA, diameter of the VA, for oval artery, the diameter was defined as the average of the longest axis distance and the shortest one of the VA; 4) AVA, area of VA, was calculated according to DVA (AVA = π × (DVA /2)Statistical analysis was conducted using SPSS version 24.0 . The reliability of measurement data was tested using the intraclass correlation coefficient (ICC); ICC ≥ 0.75 was considered to show good reliability. If the data were found to have good reliability, the average value of data was subsequently adopted for statistical analysis.2 test or Fisher’s exact test. Pearson’s correlation coefficient was used to evaluate the quantitative associations among parameters. A P <  0.05 (two-tailed) was considered significant for all tests.The Shapiro-Wilk test was used to test the normality of all datasets. Continuous data are presented as mean and standard deviation (SD) when normally distributed or as median and interquartile range (IQR) when not normally distributed. Continuous parametric data were compared using Student’s t test and categorical data by χBased on our inclusion and exclusion criteria, 152 patients with VAD  were enrolled, including 108 males (71.1%) and 44 females (28.9%) of mean age 55.9  years, mean body weight of 57.2  kg, and median height of 167  cm. Most cases with VAD (69.1%) were on the left side. Twenty cases (13.2%) exhibited VA pathway variation, with VA entry into the TF at C5, and 85% of these cases had a nearly closed TF. Therefore, DPVA and ATF were not measured at C6. Ten cases (6.6%) did not have an intact TF, including two cases with incomplete TF at C3, one at C4, five at C5, and ten at C6. Therefore, ATF was also not measured at these levels. All data of these 30 cases were excluded for correlational analyzes. Detailed demographic information is summarized in Table P <  0.001) . However, there were no significant differences between two sides in POW and DPVA at C5 (P > 0.05). The DVA was also larger on the dominant side than the non-dominant side , and the bilateral difference was significant at every level (P <  0.001). The AVA calculated based on DVA exhibited a similar trend. The ATF was significantly larger on the dominant side than the non-dominant side . Similarly, ORTF was significantly greater on the dominant side than the non-dominant side . More detailed information is presented in Table The average intra-reader and inter-reader reliabilities according to the ICC were > 0.75 (good) for all parameters. The mean POW of all cases was significantly smaller on the dominant side than the non-dominant side  (Table P = 0.006)  or AVA (P = 0.054) on the dominant side; however, POW was correlated with DVA  and AVA  on the non-dominant side and with ATF on both sides. POW was also correlated with DPVA on both sides.POW was not correlated with DVA (P = 0.001) on the dominant side but not the non-dominant side . ATF was correlated with DVA and AVA on both sides  was lower than in previous studies (from 38.5 to 73%) –15, thisP <  0.001). Furthermore, POW was more frequently < 4 mm on the dominant side compared to the non-dominant side (P <  0.001), indicating that difficult or unfeasible CPS insertion is more common on the dominant side in VAD patients [P <  0.001), the frequency of POW + DPVA < 5 mm was also higher on the dominant side (P = 0.006), suggesting that the bilateral difference in DPVA is insufficient as a metric to guide the choice of CPS insertion. Nonetheless, both POW and POW + DPVA measures suggest a higher risk of pedicle border breach and ensuing VAI on the dominant side than the non-dominant side.POW is the most important factor influencing the safety and feasibility of CPS insertion. According to several previous studies, the lateral wall is the thinnest of the cervical pedicle walls , 19. Thepatients , 20. Morpatients , 8, 21. P <  0.001). Similarly, POW + DPVA < 5 mm was most common at C3 and C4, so surgeons should be more careful in choosing CPS treatment at these levels for patients with VAD. With the development and application of new surgical navigation methods, the ability to accurately identify the best CPS insertion position and angle has significantly improved [POW < 4 mm was found more frequently at C3 and C4 on both sides but was most frequent on the dominant side  at lower levels (Fig. The major limitation of this study is the retrospective design, which may introduce selection bias. Second, the study did not include severe cervical deformities, and so may not be applicable to VAD patients with bony malformations. The last one is that we cannot avoid potential measurement bias.This is the first study to report the morphological characteristics and correlations of the pedicles and surrounding structures from C3 to C6 in patients with VAD. Smaller cervical pedicles were found from C3–C6 on the side with the dominant VA. POW < 4 mm and POW + DPVA < 5 mm were found most frequently at C3 and C4 in VAD patients, and CPS insertion is not recommended at pedicles with these characteristics. Dominant VA may affect indirectly POW, and TF may be a key determinant of DPVA and POW.


Lcn2, Defb1, Defb3, S100a8, S100a9), keratinocyte differentiation markers  and inflammatory cytokines (eg Il1b and Il17) in healthy murine skin. This effect was paralleled by inflammation and disturbed skin barrier, as well as an injury response resulting in epidermal hyperproliferation, as observed in murine and xenografted adult human skin. This contact response and disturbed barrier induced by dithranol might lead via a vicious loop between AMPs such as S100a8/a9  and cytokines such as IL‐1β to an immune suppressive environment in the skin. A better understanding of the skin's physiologic response to dithranol may open up new avenues for the establishment of novel therapeutics (including AMP‐related/interfering molecules) for certain skin conditions, such as alopecia areata.Topical dithranol is effective in autoimmune conditions like alopecia areata, inducing hair regrowth in a high percentage of cases. Exact mechanisms of dithranol in alopecia areata, with seemingly healthy epidermis besides altered hair follicles, are not well understood. To better understand dithranol's mechanisms on healthy skin, we analysed its effect on normal murine as well as xenografted human skin. We found a strong increase in mRNA expression of anti‐microbial peptides (AMPs) (eg  Gene expression of keratinocyte differentiation markers, anti‐microbial peptides (AMPs) and inflammatory markers was analysed in dithranol‐treated murine skin and ears. In addition, we investigated the role of AMPs in induction of inflammation on healthy skin by analysing effects of topical application of S100a8/a9 proteins on murine skin. To investigate a potential role of the aryl‐hydrocarbon receptor (AhR) in dithranol's mechanisms, we treated AhR‐deficient mice with dithranol and monitored inflammatory response.3Dithranol was applied topically on shaved dorsal skin and ears of BALB/c mice and concentrations were increased every other day  Figure . Histolo4Lcn2, Defb1, Defb3, S100a8, S100a9), keratinocyte differentiation markers  and inflammatory cytokines (Il1b and Il17) in both healthy murine dorsal . The effect of dithranol on the expression of keratinocyte differentiation regulators, cytokines and AMPs was paralleled by inflammation and disturbed skin barrier, as well as an injury response resulting in epidermal hyperproliferation of the skin. We observed a significant increase in macroscopic thickness of dorsal and ear skin after a single application of dithranol and thickness steadily increased with every following application  and previous work indicated that the former AMP can serve as a very sensitive biomarker to detect an inflammatory response even at subclinical level and independent from the pathomechanism.Real‐time qPCR analysis of dithranol‐treated murine skin and ears revealed a strong increase in expression of AMPs  (a) and erythema index (b) in dorsal skin of healthy BALB/c mice compared to vehicle‐treated controls. Unpaired t test was used for statistics. Bars represent mean±SD (n=10); *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.Click here for additional data file.Figure S2. Effect of dithranol on AhR‐deficient mice and AhR bearing C57BL/6J controls. a) and c) Dithranol strongly increased macroscopic skin thickness of dorsal skin (a) and ears (c) in AhR‐deficient mice and control mice. No significant difference was observed between the groups. Arrows indicate concentration of dithranol. b) and d) Increased epidermal thickness and cellular infiltrate of dithranol‐treated dorsal skin and ear skin was observed in all mice. Representative H&E images are depicted , scale bar =50µm. Multiple t test was used for statistics (n=7). AhR, aryl‐hydrocarbon receptor; n.s., not significant.Click here for additional data file.Table S1. RT‐PCR Primer sequences and corresponding annealing temperatures.Click here for additional data file.


Trichoderma strain was isolated, and its potential to alleviate salt stress and diminish cucumber root rot caused by Fusarium oxysporum was evaluated.Salt stress threaten the growth of plants, and even aggravate plant disease. In this article, salt-tolerant Trichoderma isolates were isolated from samples of sea muds and algae collected from the South Sea of China. Among these, the isolate HN082102.1 showed the most excellent salt tolerance and antagonistic activity against F. oxysporum causing root rot in cucumber and was identified as T. atroviride. Its antagonism ability may be due to mycoparasitism and inhibition effect of volatile substances. The application of Trichoderma mitigated the adverse effects of salt stress and promoted the growth of cucumber under 100 mM and 200 mM NaCl, especially for the root. When T. atroviride HN082102.1 was applied, root fresh weights increased by 92.55 and 84.86%, respectively, and root dry weights increased by 75.71 and 53.31%, respectively. Meanwhile, the application of HN082102.1 reduced the disease index of cucumber root rot by 63.64 and 71.01% under 100- and 0-mM saline conditions, respectively, indicating that this isolate could inhibit cucumber root rot under salt stress.Twenty-seven T. atroviride isolated from marine habitat showing antagonistic activity to F. oxysporum, and the results provide evidence for the novel strain T. atroviride HN082102.1 in alleviating salt stress and diminishing cucumber root rot, indicating that T. atroviride strain HN082102.1 can be used as biological control agent in saline alkali land.This is the first report of salt-tolerant  Also, rcapacity . What’s capacity . Therefo capsica , Fusariuxysporum , Alternaa solani , and so ali land . The salF. oxysporum is an important soil-borne disease in cucumber. Various control methods are used to avoid losses in cucumber yield due to root rot. Physical methods such as sunlight or ultraviolet radiation [Root rot caused by adiation  and chemadiation  are commadiation . So, resadiation .Trichoderma species are widely distributed in nature worldwide, and have been successfully used as biofertilizers, biopesticides and bioremediation agents [Trichoderma played an important role in reducing the adverse effects of salt stress in the desert shrub Ochradenus baccatus [Trichoderma can be used to control plant disease mainly due to their antagonistic and mycoparasitic activity [Trichoderma could alleviate the negative effects of salt stress and diminish root rot in cucumber.n agents , 15. It baccatus . Trichodactivity –19. Alsoactivity  and promactivity –23. So, F. oxysporum [ITS) regions of rDNA.Diseased cucumber plants with typical symptoms of root rot or damping-off  were collected from cucumber fields in Hainan province, immediately placed into plastic bags and stored in iceboxes. The pathogenic fungus was isolated by the tissue separation method, and the pathogenicity of the purified fungus was determined according to Koch’s postulates . The isoxysporum  and moleTrichoderma spp. were isolated from the sea mud samples using a dilution method with a modified potato dextrose agar medium (mPDA)  [Trichoderma isolates were selected, and purified by single spore isolation [Samples of forty-six sea muds and three algae were collected from marine habitats in three districts of Hainan Province, China: Dongfang City , Sanya City , and Lingao County . Samples were placed in sterile polyethylene bags, stored in an icebox and transported to the laboratory as soon as possible.  bengal) . The algsolation , then prTrichoderma isolates isolated above were cultured on PDA amended with various concentrations of NaCl , and incubated at 28 °C. The diameters of colonies on NaCl-amended media were recorded on the 3rd, 5th, and 7th day.To assess salt tolerance, all Trichoderma isolates isolated above which could grow on PDA medium containing 9% NaCl and F. oxysporum HGL.1 were dual cultured on PDA in the presence or absence of 100 mM NaCl salt stress [To evaluate the antagonistic activity against the pathogen of cucumber root rot, all t stress . The plaTrichoderma strain showing salt tolerance and antagonistic activity were carried out. For morphological identification, hyphal morphology and colony growth patterns of salt-tolerant Trichoderma isolate were observed at 72, 96, 120, 144, and 168 h post inoculation on PDA, synthetic low nutrient agar , and corn meal dextrose agar . After inoculation on SNA and CMD media for 72 and 168 h, characteristics of the conidia, conidiophores, chlamydospores, and sori were examined and photographed under optical microscope  and stereoscopic microscopes , then compared with a published description [The morphological and molecular identification of the cription .Trichoderma isolate were extracted using the Cetyl Trimethyl Ammonium Bromide (CTAB) method as described by Stewart and Via [tef1) was amplified using the EF1–728 F/TEF1LLErev primer pair [tef1 PCR was performed in a 96-well PCR instrument . The PCR products were purified using a ZTOPO-TA Fast Cloning Kit , and sequenced by Guangzhou Tianyi Huiyuan Biotechnology Co., Ltd. . The tef1 sequences were deposited in GenBank (NCBI) to get accession number. The phylogenetic tree based on tef1 sequences were construct in MEGA-X using the Neighbour-Joining method.For molecular identification, the genomic DNAs of  and Via  and the mer pair . The PCRTrichoderma isolate against the F. oxysporum isolate HGL.1 was evaluated by the dual culture method as described by Morton and Stroube [Trichoderma and F. oxysporum grow onto them, and the plates were incubated at 28 °C. Seven days later, the radial growth radii of HGL.1 were recorded, and the interaction of hyphae on the cover glasses was observed under an optical microscope. Then mycelia in the dual culture plates were dyed using trypan blue staining method to determine whether the pathogen was killed by Trichoderma isolate [The antagonistic activity of the salt-tolerant  Stroube  under sa isolate , and theTrichoderma isolate against the F. oxysporum isolate HGL.1 were evaluated by the method described by Dennis and Webster [Trichoderma and F. oxysporum were inoculated, respectively. For non-volatile metabolites, Trichoderma was cultured on cellophane covering on the medium for 2 days and then F. oxysporum was inoculated on the medium after the cellophane was uncovered. The cultures were incubated at 28 °C in darkness for 7 days, and the colony radius of the pathogen was measured to calculate the inhibition ratio of mycelial growth.The inhibitory effects of volatile and non-volatile metabolites produced by the salt-tolerant  Webster , 34 in tCucumis sativus L.) of the cultivar Chuanlv 21 were surface-sterilized with 8% NaClO solution for 1 min, washed three times with distilled water, placed into distilled water for germinating, and then sown in plastic pots  with 0.5 kg composite soil . After 10 days, seedlings of uniform size were selected and the following treatments were compared: (1) 100 mL water + 100 mL spore suspension of Trichoderma (1 × 107 cfu/mL) (marked as “T”); (2) 100 mL water + 100 mL water (marked as “control”); (3) 100 mL 100 mM NaCl solution+ 100 mL spore suspension of Trichoderma (1 × 107 cfu/mL) (marked as “100S + T”); (4) 100 mL 100 mM NaCl solution+ 100 mL water (marked as “100S”); (5) 100 mL 200 mM NaCl solution+ 100 mL spore suspension of Trichoderma (1 × 107 cfu/mL) (marked as “200S + T”); (6) 100 mL 200 mM NaCl solution+ 100 mL water (marked as “200S”). All treatments were arranged in completely randomized design (CRD), and at least 30 cucumber seedlings were used for each treatment. After 14 days, all the plants were harvested to measure their growth parameters and physiological-biochemical indexes.Cucumber seeds .Nine cucumber seedlings from each treatment were harvested randomly, and the fresh leaves were collected for measuring the content of chlorophyll (Chl), soluble protein and malondialdehyde (MDA), and the activity of catalase (CAT) and peroxidase (POD).The quantitative analysis of chlorophyll was done according to the method of Lichtenthaler and Wellburn . The chlChl content (mg/g) = (Chl. concentration × Extraction volume × dilution factor)/fresh weightLipid peroxidation was recorded in terms of concentration of malondialdehyde (MDA) by the method of Heath and Packer . AbsorbaThe content of soluble proteins was measured according to the method described by Bradford . Two hunTo analyse the activities of POD and CAT, 1.0 g fresh leaves were ground in 25 mL sodium phosphate buffer (50 mM pH 7.8) and centrifuged at 12000 rpm (4 °C) for 20 mins. The supernatant was collected to measure the activities of POD and CAT referred to the methods described by Samantary  and Kar Trichoderma (1 × 107 cfu/mL) + 30 mL spore suspension of F. oxysporum (1 × 107 cfu/mL) (marked as “T-Fo”); (2) 100 mL water + 30 mL spore suspension of F. oxysporum (1 × 107 cfu/mL) (marked as “Fo”); (3) 100 mL water + 30 mL water (marked as “Control”). Before inoculated by pathogen, the cucumber roots were injured by inserting transplanting shovel into soil near the plant to damage the roots. All treatments were arranged in completely randomized design, and there were at least 40 cucumber seedlings for each treatment. The disease index and control effect of all 240 cucumber seedlings were assessed on 10 days post inoculation (dpi) using the grading standard of Li et al. [Cucumber seeds (Chuanlv 21) were sown separately in plastic pots  containing 2 kg composite soil . When the third leaves emerged, one half of the seedlings were irrigated with 100 mL of 100 mM NaCl solution, and the other half were treated with water. The following treatments for the seedlings irrigated with salt solution or without were compared: (1) 100 mL spore suspension of P < 0.05. Different superscript letters within the same column indicate statistically significant differences. All experiments were repeated at least three times.Statistical analyses of quantitative data were performed using IBM SPSS Statistics 21 software  and included one-way or two-way analysis of variance (ANOVA) followed by Tukey’s test and Dunnett’s t-test at Fusarium-like morphological characteristics were obtained from diseased cucumber plants exhibited typical symptoms of root rot. Three isolates  were randomly selected and cultured on PDA medium. The colonies of the three isolates initially showed white cotton flocculent on PDA medium and later became light red   showed that isolate HN082102.1 was clustered with T. atroviride   Fig. K. TherefT. atroviride HN082102.1 exhibited mycoparasitism to F. oxysporum HGL.1 causing root rot in cucumber in the presence or absence of 100 mM NaCl stress , the total chlorophyll content of cucumber leaves decreased from 1.29 mg gTrichoderma was used under 100 mM and 200 mM NaCl (Fig. Trichoderma was used under 100 mM and 200 mM NaCl (Fig. Trichoderma was applied, which were 20.94 and 33.23% under 100 mM NaCl and 50.78 and 21.83% under 200 mM NaCl (Fig. The content of soluble protein of leaves decreased significantly by 21.92 and 38.26% under 100 mM and 200 mM NaCl compared with control, and increased by 29.48 and 51.23% when aCl Fig. G. The MDaCl Fig. H. The acT. atroviride was applied under 100 mM NaCl. So, 100 mM of NaCl was used in the following experiment.What’s more, depending on the results above, the indexes above were not significantly different from or better than the control when T. atroviride isolate HN082102.1 could control root rot of cucumber under salt stress. In Greenhouse, when seedlings were treated with T. atroviride HN082102.1, cucumber root rot caused by F. oxysporum was significantly inhibited (Fig. The Trichoderma spp. were isolated from samples of 46 sea muds and 3 algae collected from marine habitats, and the salinity resistance and antagonistic activity of these isolates were examined. HN082102.1 isolated from algae exhibited the strongest salt tolerance and better antagonistic activity and therefore was selected to be used in the further research.In this study, 27 T. atroviride HN082102.1 under NaCl stress, the growth parameters increased, suggesting that T. atroviride may alleviate the adverse effect of salt stress to the cucumber growth. The findings were consistent with the results of Rawat et al. [Trichoderma isolates alleviated the negative impacts of salt stress in rice. The application of Trichoderma increased root length of plants, thereby promoting the plant to absorb nutrients and water from the soil and enhancing its ability to counteract salt stress [The growth parameters of cucumber, that are the length, fresh weight, and dry weight of shoots and roots, decreased significantly under NaCl stress, which might be attributed to increased osmotic stress, nutrient deficiencies, and disturbance of various physiological and biochemical mechanisms –43. Whent et al. , which rt stress , 46.T. atroviride HN082102.1 significantly increased the content of chlorophyll and soluble protein and reduced the MDA content in cucumber under salt stress. Previous studies also reported the similar results [Trichoderma to plants under salt stress increased IAA and GA levels and decreased ABA levels. Martínez-Medina et al. [Trichoderma application under salt stress may be related to its effect on plant hormones. The increased soluble protein content associated with Trichoderma application was probably beneficial in reducing the osmotic potential of plant cells [Trichoderma reduced salt stress in cucumber, which may be attributed to reduce ROS production in treated plants.The application of  results , 47. Zör results  conclude results  demonstr results  reporteda et al.  and Resea et al.  also fount cells , therebynt cells  and chicnt cells . An incrnt cells . ExogenoTrichoderma their activity increased more. To prevent the membrane damage caused by O2− and H2O2, POD reduces H2O2 to water [2O2 to water and molecular oxygen [Trichoderma increased the activity of antioxidant enzymes and was therefore conducive to scavenging more ROS produced under salt stress, thus reducing stress-related damage.The activity of the antioxidant enzymes POD and CAT increased under salt stress, and after the application of to water , and CATr oxygen . IncreasT. atroviride had a significant inhibition effect on cucumber root rot, which was consistent to the reports described by Rawat et al. [Trichoderma obtained tolerance toward root rot by upregulating the activities of POD, polyphenol oxidase, phenylalanine ammonia lyase, and by increasing the content of total phenols. Likewise, Bae’s [Trichoderma in cucumber. These results suggest that metabolites released by Trichoderma isolates not only act directly on the pathogen but also trigger the plant to release defense-related compounds. Here, it was found that the effect of isolate HN082102.1 on cucumber root rot was somewhat decreased in saline soil, which may be caused by a decline in Trichoderma metabolites. Anyway, this is the first report of salt-tolerant T. atroviride isolated from marine habitat can alleviate salt stress and diminish cucumber root rot caused by F. oxysporum. Taken together, T. atroviride HN082102.1 can be used as biological control agent in saline alkali land.With or without salt stress, the application of t et al.  and Kasht et al. . Singh et et al.  demonstre, Bae’s  study deT. atroviride isolated from marine habitat showing antagonistic activity to F. oxysporum. The application of T. atroviride HN082102.1 markedly increased tolerance to salt stress and reduced the severity of root rot caused by F. oxysporum under saline conditions in cucumber. The increasing of ROS scavenging and maintaining the osmotic balance are key observations of the ability to tolerate salt. Therefore, T. atroviride strain HN082102.1 has the potential to improve plant growth and disease resistance serving as biological control agent in saline alkali land.This is the first report of salt-tolerant