The current study had two aims. The primary purpose was to examine the association between serum vitamin D levels and the ergometric evaluation of muscle strength, aerobic capacity, and speed in professional soccer players. The secondary aim was to evaluate the effects of the soccer off-season period on serum vitamin D levels.2max), anthropometry, and blood sampling were performed before (pre) and after (post) the six-week off-season period.Sixty-seven Caucasian male soccer players (age 25.6±6.2 and height 1.81±0.08 m), members of two Greek Superleague Soccer teams and one Football-league championship team participated in this study. Exercise performance testing for the determination of squat jump (SJ), countermovement jump (CMJ), 10 (10 m) and 20 meters (20 m) sprint performance, maximal oxygen consumption  and post  experimental sessions. (b) Vitamin D concentration increased significantly (P<0.001) following the six-week off-season period compared to baseline, while at the same time all measured performance parameters decreased .Analysis of our results showed the following: (a) a significant correlations between serum vitamin D levels and performance parameters in both pre (SJ; P<0.001, CMJ; P<0.001, VO2max in professional soccer players, irrespective the levels of performance. Furthermore, our data reaffirm the importance of UVB on serum vitamin D levels. Moreover, reductions in exercise training stress may also have beneficial effects on vitamin D levels, suggesting a possible association of its levels and the training-induced stress. Our results indicate a possibly bidirectional interaction between soccer performance indices and vitamin D levels.Our findings suggest that vitamin D levels are associated with the ergometric evaluation of muscle strength, as expressed by SJ and CMJ, sprinting capacity, and VO Vitamin D is primarily synthesized endogenously following cutaneous exposure to ultraviolet B radiation (UVB) It should be noted that the majority of the above mentioned studies have been performed in the elderly Paradoxically, a growing number of studies report a high prevalence of vitamin D insufficiency or downright deficiency even in regions with extensive sunlight in both athletic and non-athletic populations 2max Soccer is a sport where aerobic capacity, muscular strength and speed are of vital importance for most of the actions during playing. Indeed, players must perform repeated sprints, stops, jumps, and changes of direction with maximal force development, and in the shortest possible response time. Furthermore, top level players run approximately 10–12 km during a soccer game, and the total distance covered is linearly related to VO2max), 10 and, 20 meters sprint performance in two different occasions, prior to the beginning and at the end of the off-season soccer period. Our second aim was to examine the vitamin D response to the reduced exercise training during the six-week off-season transition period. We hypothesized that in both experimental sessions vitamin D levels would correlate with soccer players’ jumping, sprinting, and aerobic capacity, and that the off-season transition period, of reduced training stress, would favorably affect vitamin D concentration. To the best of our knowledge, no published study has examined the relationship between vitamin D levels and muscle strength, VO2max and speed in professional soccer players, and/or the effects off-season detraining soccer period on its levels in any kind of athletic population.Our study had two aims. The main objective was to examine the potential relationship between vitamin D levels and muscle strength, as expressed by squat jump (SJ) and countermovement jump (CMJ) Initially, seventy seven professional players, members of three soccer teams, were evaluated for possible participation in the study. The exclusion criteria were as follow: a) any medical disorder that would affect vitamin D levels, b) players that their contract was ending before the conclusion of the study, and c) the consumption of supplements containing vitamin D. Ten (10) players failed to fulfill criteria b (8) and c (2) and were subsequently excluded from the study. Sixty seven Caucasian professional male soccer players, members of two Greek Super league teams (n = 45) and one Football league team (n = 22) were consecutively included in the study. The mean values of age (years) ± SD and height (m) ± SD were: age 25,6±6,2 and height 1,81±0,08 respectively.Before testing verbal explanation of the aim of the study and the testing procedures were given to all participants, and written informed consent was obtained. The study was performed in strict accordance with the ethical guidelines of the Helsinki Declaration and was approved by the Ethical Scientific Committee of the University Hospital of Heraklion, Crete, Greece.2max) of 20 to 30 minute total duration  three times per week, divided by one day of rest. This type of activity was selected by the team coaches. All players were tested at two different occasions. Each experimental testing consisted of two days of consecutive measurements. The first experimental testing took place immediately after the end of the competition period in May (pre). The second experimental testing was performed at the beginning of July (post), just prior to the beginning of the preparation period for the forthcoming season. In each experimental periods testing consisted of anthropometric measurements , blood testing for the assessment of vitamin D levels, and ergometry tests for the measurement of SJ (cm), CMJ (cm), VO2max (ml/kg/min), 10 m, and 20 m sprint performance. The first day of each experimental period anthropometric characteristics were measured at 08∶30 am. From 09∶00 to 10∶30 am, venous blood samples were obtained in order to determine the vitamin D concentration. In the afternoon of the same day (17∶00 pm) the players were tested for SJ, CMJ, 10 m, and 20 m sprint performance. The second day of each experimental period, starting at 09∶30 am, our participants were tested for the determination of VO2max. During the study players were instructed to avoid consuming any vitamin D supplements. Before each experimental session, players were informed not to consume any supplement that could promote performance at least 2 days prior to the testing and to avoid any caffeine or alcohol beverages. Moreover, they were instructed to abstain from any exercise training sessions two days prior to each testing in order to avoid any fatigue effects. Detailed nutritional guidelines were given to all players in order to ensure a high (>60%) carbohydrate dietary intake during the study, including a list of a variety of foods, and based on individual resting metabolic rates and the calculated daily energy expenditure as per reported activities (24). Furthermore, players were also asked to maintain their hydration status. All players were familiarized with the testing protocol, as they had been previously tested with the same procedures on several occasions during the last soccer season. The study was performed in Crete, Greece, at latitude of 35,9°N.The duration of the off-season transition period was set to six-weeks, starting at the end of the competition period. During this recuperation period, participants were instructed to avoid any kind of exercise training for the first two weeks. After this two-week period, they were instructed to perform low intensity aerobic running  and weight was obtained using an electronic weight scale . Body fat percentage was assessed by skinfold thickness measurement  using the 4 site formula by Durnin and Womersley Venus blood samples were obtained following a ten-minute period of rest in a lying position, after an overnight fast. Vitamin D levels were assessed using DiaSorin 25 hydroxy vitamin D , repeatability CV = 3–6% and reproducibility CV = 6–11%, according to the laboratory standard operating procedures. Calibrator 1 and 2 (human serum) concentrations are referenced to standard preparations containing highly purified 25(OH) vitamin D. According to the manufacturer the correlation of the immunoenzymatic assay with LC-MS/MS is described by the equation: concentration = 5,6+0,83*LC-MS/MS . The intra assay coefficient of variation ranges between 3–6%. In our study vitamin D levels below 10 ng/ml were considered as severe deficiency, levels between 10–20 ng/ml as deficiency and levels between 20–30 ng/ml as insufficiency.2max) assessment was performed on a motorized treadmill using an automated gas-analysis system , with the use of set procedures of a standard protocol The jumping  and sprinting ability  of the soccer players were assessed with a jumping mat  and infrared photoelectric cells  respectively, according to standard procedures Statistical analysis was performed using the software program SPSS 17.0. Results are presented as means ± SD. The distribution of variables was tested by the Shapiro-Wilk statistical method. Then, Pearson’s  and Spearman’s  correlation coefficients were used to assess the linear relationship between quantative variables. The changes between the experimental periods in the measured parameters within the groups were analyzed by the paired samples t-test for normally-distributed data, and by Mann-Whitney test for non-normally-distributed data. Statistical power analysis was performed  in order to attain 80% power. Analysis was carried out at a confidence level = 95% and confidence interval = 13,6 2max values at the beginning and at the end of the experimental period  and speed at both experimental periods. Our observations are comparable to several studies showing that vitamin D is linearly associated with jumping ability and strength in pre-adolescent girls The pathways via which vitamin D affects muscular strength (as measured by SJ and CMJ) and sprint performance are still hypothetical. However, there are several potential mechanisms conveying these effects. The ergogenic effects of vitamin D may be related to the regulation of muscle protein synthesis which could affect muscle mass, thanks to the presence of vitamin D receptors on skeletal myocytes 2max in both experimental sessions. This finding is supported by an early study which reported increased aerobic capacity as a result of exposure to ultraviolet radiation in collegiate students . Since VO2max is related to soccer performance, as indicated by the well-documented relationship between this parameter and the distance covered during a soccer game 2max could be related to its protective effect on lung function. According to recently published evidence, low vitamin D levels are associated with lower indices of lung function 2max) is depended on optimal lung function 2max by affecting iron metabolism and erythropoietin 2max via its effects on erythropoesis modifying the capacity of the oxygen supply to the exercising muscles and consequently affecting aerobic exercise performance Analysis of our data revealed a linear association between vitamin D and VOInterestingly, analysis of our data showed that although vitamin D increased at the end of the off-season period all measured exercise performance parameters decreased. However, the observed linear correlation at baseline between vitamin D levels and performance was retained at the end of the study, suggesting that vitamin D is related to the ability to perform efficiently during exercise, irrespective the level of performance. These data clearly suggest that although vitamin D does seem to affect neuromuscular and aerobic performance, it does not play the primary role. Indeed, during periods of reduced training stimulus or training cessation (i.e. soccer transition period) there is deterioration in exercise performance In our study the six-week transition period had a boosting effect on vitamin D levels. Indeed, at the first experimental period, although none of our participants was vitamin D deficient (<20 ng/ml) or severe deficient (<10 ng/ml), 55,22% of our players had insufficient vitamin D levels (<30 ng/ml), whereas at the second one only 4,47% were found to be below 30 ng/ml. In regard to the first experimental session our findings are in agreement with studies on soccer players (∼50% <30 ng/ml) Although our data reaffirm the importance of adequate exposure to UVB, recent evidence indicate that the exercise training -induced stress may play a regulatory role on vitamin D levels 2max in professional soccer players. Moreover, our results indicate that, apart from increased exposure to UVB, reductions in exercise training stress may also have beneficial effects on vitamin D levels in elite soccer players. However in order to confirm this hypothesis additional research is needed, also examining indices of inflammation. Furthermore, our findings indicate that vitamin D plays, among other parameters, a secondary supportive role in athletic performance. However, this does not lessen its importance, especially in highly competitive athletes, since in elite athletic level slight changes in performance may determine the outcome of a competition. Our current data may provide an additional tool to sport scientists, coaches, and players to enhance soccer performance.Our study provides evidence of an association between not supplemented vitamin D levels and parameters of aerobic and neuromuscular exercise performance in soccer. In particular, our findings indicated a linear relationship between vitamin D levels and muscle strength as evaluated by SJ and CMJ, sprinting ability , and VOFile S1Supplementary File Data.xlsx which include all performed vitamin D and performance parameters measurements in this study.(XLSX)Click here for additional data file.


No changes were observed over time for subjective measures of physical function and QOL. Results showed RT could be an effective means to improve objective physical function in BCS. Further research is needed to clarify the effects of RT on subjective physical function and QOL.Breast cancer survivors (BCS) exhibit decreased physical function and quality of life (QOL) following cancer treatments. Resistance training (RT) may elicit positive changes in physical and mental well-being. This study assessed 27 BCS, pre-and post-intervention (six months) on the following variables: muscular strength (via one repetition maximum (1RM) of chest press and leg extension), physical function  and QOL (via the Short Form-36 survey). RT consisted of two days/week of ten exercises including two sets of 8–12 repetitions at 52%–69% of their 1RM. A repeated measures analysis of variance revealed BCS significantly ( Approximately 1.6 million new cases of cancer were expected to be diagnosed in the United States in 2014. Of these cancer diagnoses, it was estimated that nearly 300,000 were breast cancer . Studieset al. (2004) reported that BCS experience significant decrements in their perceived physical function at the cessation of their primary treatment for breast cancer [et al. (2011) found that 17 months after the completion of primary treatment for breast cancer, BCS exhibited 21% lower strength for chest press and 23% lower strength for leg extension compared to age-matched healthy physically inactive women. Similarly, in this same study the authors found that BCS reported an 11% lower subjective physical function as measured via the Short Form-36 Health Survey (SF-36) and demonstrated lower objective physical function scores that were approaching significance compared to age-matched healthy physically inactive controls [The detrimental effects of cancer and cancer related treatment on the physical function and QOL of BCS vary in magnitude and longevity . Ganz ett cancer . Unfortut cancer . Specificontrols . The faci.e., Short Form Health Survey and Functional Assessment of Cancer Therapy—General). To date there has only been one study examining the efficacy of resistance training exercise to increase objective physical function in cancer survivors. Jankowski et al. (2008) implemented the Continuous Scale Physical Functional Performance (CS-PFP) test in a group of older cancer survivors and found that components of the CS-PFP were significantly higher compared to the control group after a resistance training exercise intervention [Over the past several years, researchers have investigated the effects of various exercise modalities and intensities on QOL in cancer patients and survivors. According to a recent meta-analysis, there are numerous studies that have indicated resistance training (RT) interventions can successfully improve QOL among cancer patients and survivors . Despitervention . The voirvention . ObtainiStudies reporting the negative physical and psychological changes that BCS encounter, and the lack of studies investigating non-pharmacological approaches to combat these negative changes, warrant an investigation of interventions to improve the conditions of this population. Thus, the purpose of the present investigation was to determine the efficacy of resistance exercise training on improving physical function, measured objectively, and subjective physical function and QOL in BCS. It was hypothesized that BCS would demonstrate improvements in both objective and subjective physical function as well as QOL in response to a six-month resistance training program.Female BCS (stages 0-III), having completed treatments at least six months prior to beginning the study were recruited. Participants were recruited via advertisements posted at various local establishments and announcements made at local breast cancer support groups. Participants currently taking or who had completed hormone suppressant therapies were eligible to participate in this study. Participants still receiving hormone suppressant therapies were eligible for the study because after initial treatments are completed, hormone suppressant therapies are typically prescribed for an additional period of three to five years. Excluding women still taking hormone suppressant therapies would have significantly decreased the number of women eligible for the study. Participants already participating in vigorous exercise programs at baseline of the study and/or those with uncontrolled hypertension (≥160/≥100 mmHg), uncontrolled diabetes, uncontrolled heart disease, or who were still going through cancer treatment were not allowed to participate in the study.Prior to baseline assessments, participants were given a physician consent form to take to their primary health care providers to be evaluated for participation in the study. Upon clearance by their physician, participants were provided an opportunity to ask any questions about the research study requirements or expectations. After all questions and concerns had been addressed, participants completed a written informed consent document. Participants then completed a demographic and medical history questionnaire and were given an appointment for baseline assessment. This study was approved by the Institutional Review Board at The Florida State University.At their scheduled appointment, participants arrived to the testing laboratory for their first baseline appointment, at which point participants completed the SF-36 for a baseline QOL assessment of physical function, and both physical and mental well-being. Following the questionnaire, the participants had their resting blood pressure, resting heart rate taken and their upper and lower body strength measured. Upper and lower body strength were assessed using the chest press and leg extension exercises, respectively . After a warm-up, participants were progressed towards the maximum weight that they could lift one time through a full range of motion to achieve a one repetition maximum (1RM). The 1RM tests for both upper and lower body were obtained within a 10–15 min time frame after the initial warm-up set and were performed according the guidelines for strength testing as outlined by the American College of Sports Medicine .r = 0.97) and is sensitive to change, induced by exercise, with an effect size of 0.8 [On the second baseline visit, which occurred one week later, participants had their physical function measured objectively via the CS-PFP test. The CS-PFP test was developed using data collected on older adults with a broad range of physical abilities . This tee of 0.8 ,21. The e of 0.8 .The CS-PFP is based on routine tasks, performed at maximal effort within the bounds of safety and comfort. A total of 10 tasks are administered, and a combination of time, distance, and weight is used to quantify performance. Tasks quantified using both weight and time include: (1) carrying of weight and (2) carrying groceries. Tasks quantified by time alone include: (1) transferring laundry from a washer to a dryer, (2) putting on and removing a jacket, (3) floor sweeping, (4) climbing stairs, (5) getting down and up from the floor, and (6) picking up four scarves from the floor. Tasks that are quantified by distance alone include: (1) a six-minute walk and (2) highest reach. Each task is scored 0–100, based on an empirically derived range from data gathered on older adults with a broad range of individual functional abilities .Time was used to calculate speed (1/t), so that higher numbers reflected higher function for each unit of measure . Each task is scaled 1–100 according to the following formula:The total physical functional performance score  is the average corrected score of all tasks. The CS-PFP total can also be broken down into five domains representing upper body strength, upper body flexibility, lower body strength, balance and coordination, and endurance.The laboratory for the administration of the CS-PFP test was set up to adhere to the published dimensions  and was TM chest press, leg press, leg extension, biceps curl, triceps press down, overhead press, seated row, leg curl, abdominal crunch, and lower back hyperextensions. During each resistance exercise session, participants performed a warm-up by walking for approximately five minutes, and concluded their session by performing stretches that targeted all the major muscle groups. Total exercise session time was approximately one hour. Exercise intensities for the chest press and leg extension were calculated as a representation of upper body and lower body intensity, respectively. When assessing the intensity (percentage of 1RM) for a particular four-week period, the 1RM test just prior to the four-week period was used for calculating percentage of 1RM lifted. A complete account of the resistance training intervention is published elsewhere [After the completion of baseline testing, each participant was provided an appointment for their supervised twice weekly resistance training sessions. For each exercise session, participants were prescribed a target goal of two sets of 8–12 repetitions at 60%–80% of their 1RM. Exercise machines included the MedXlsewhere .p ≤ 0.05. All data are presented as means ± standard deviations.A time effect, with an effect size of 0.85, maintaining α = 0.05 and 1-β = 0.80, indicated that a sample size of at least 10 participants was required to detect changes in total CS-PFP scores over time based on a previous finding by Ades and colleagues . Sample A total of 51 BCS potential participants inquired about the study; 10 declined participation after initial screening; and nine did not meet qualifying criteria. Thirty two participants completed baseline testing; however five women did not return after initial testing. Two women were unable to complete the RT invention due to medical complications that surfaced during the course of the study and stopped all participation in the research project. One participant developed uncontrolled hypertension during the 18th week of the study and her physician would not grant clearance to remain in the study. The second participant, during the 8th week of the study was diagnosed with a reoccurrence of cancer that metastasized into her bones and was not permitted to continue with the study. Since an intention to treat analysis was implemented a total of 27 participants were utilized to carry out the research project.n = 20) of the participants were Caucasian, while the remaining 13% (n = 3) were African American. Analysis of the participants’ baseline characteristics revealed the women were 64 ± 7 years of age, weighed 73.5 ± 14.8 kg, and had a height of 163.1 ± 6.1 cm. F = 32.913, p ≤ 0.01, ES = 0.568) and leg extension strength  = 51.073, p ≤ 0.01, ES = 0.671). There was a significant 17% increase in upper body strength from baseline to three months and a further significant increase of 7% from three months to six months, for an overall 25% improvement from baseline to six months. Similarly, there was a significant 15% increase in upper body strength from baseline to three months and a further significant increase of 9% from three months to six months, for an overall 26% improvement from baseline to six months. The participants displayed a steady progression of weight lifted for upper and lower body exercises throughout the six-month intervention. For weeks 1–4, participants exercised at an intensity less than the target intensity (60%–80% 1RM); with the participants achieving an intensity of 52% ± 9% 1RM for upper body and 52% ± 5% 1RM for lower body. Beginning in weeks 5–8, participants achieved an exercise intensity of >60% 1RM for both upper and lower body and continued to maintain compliance to the study design of >60% 1RM for the remaining weeks of the intervention. At no point in the study did the participants exercise over 69% of their previous four-week 1RM for upper or lower body. Throughout the intervention, the participants were able to significantly increase their upper and lower body strength. There were significant time effects observed for chest press strength  = 12.457, p ≤ 0.05, ES = 0.324), upper body flexibility  = 3.452, p ≤ 0.05, ES = 0.117), lower body strength component  = 25.817, p ≤ 0.05, ES = 0.498), balance and coordination component  = 18.613, p ≤ 0.05, ES = 0.417), endurance component  = 19.569, p ≤ 0.05, ES = 0.429), and total function  = 23.896, p ≤ 0.05, ES = 0.479). From baseline to six months, the participants experienced significant improvements in their upper body strength (+12%), lower body strength (+17%), balance and coordination component (+12%), endurance component (+12%), and total function (+12%). Scores at baseline for total function ranged from 36 to 82 units; with six participants achieving a score that fell below the threshold score of 57 units, which is needed for independent living. At the six-month mark, the scores for total function ranged from 50 to 93 units, with two participants remaining at a score that fell below the threshold score of 57 units. Despite the significant ANOVA for upper body flexibility, examination of the main effects failed to show any significant changes among any of the three time points. There were significant time effects observed for the subcomponents of total function of upper body strength component . Similarly, there were several significant correlations between percent change in lower body strength and percent change in several components of the CS-PFP test. Specifically, percent change of lower body strength was significantly related to percent change in total CS-PFP function . There were no significant correlations found between percent change in CS-PFP and percent change in SF-36. A complete list of correlations between percent change in CS-PFP and percent changes in upper body and lower body strength and SF-36 test can be found in There were several significant correlations between percent change in upper body strength and percent change in several components of the CS-PFP test. Specifically, percent change of upper body strength was significantly related to percent change in total CS-PFP function . The women demonstrated significant improvements in total function and all subcomponents of the CS-PFP, thus the hypothesis that a resistance training intervention would improve objection function in BCS was supported. No changes were reported for subjective levels of physical or mental QOL, measured via the SF-36 Health Survey; thus the hypothesis that a resistance training intervention would improve QOL and subjection function in BCS was rejected.et al. (2008) implemented the CS-PFP in a group of older cancer survivors (cancer type not specified) and found that after four months of RT, both upper body and lower body strength components of the CS-PFP were significantly higher as compared to the control group [et al. (2008). The baseline values for the women from the present study were seemingly higher for the upper body strength component (63.5 ± 16.3 units) as compared to the baseline values from Jankowski and colleagues (59 ± 29 units). Similarly, for the lower body strength component, the women from the present study had higher baseline values (58.5 ± 14.9 units) compared to Jankowski and colleagues (45 ± 16 units). These discrepancies are likely due to the older population (71 ± 5 years) with which Jankowski and colleagues studied, as compared to the present study where the sample population was younger (64 ± 7 years). Although time effects were not reported by Jankowski et al. (2008), pre-to-post differences were calculated to be +20% for the upper body strength component and +11% for the lower body strength component [Women participating in the study showed excellent capabilities to improve objective physical function; however, the same cannot be said for the subjective levels of physical function or for QOL. To date the present study was the first to implement the CS-PFP test among BCS for an assessment of objective physical function. Results showed that the participants increased total function by 12%. While there are no studies examining BCS to which the results of the present study can be compared, Jankowski ol group . Total pomponent . These iomponent . The sixet al. (2002) reported an even larger improvement (+24%) in total function in a group of older, disabled women with coronary heart disease after six months of resistance training [Although, there is a lack of literature in which to compare the CS-PFP results of the present study with cancer survivor populations, the results found align with published data in other female chronic diseased populations. Kingsley and colleagues (2005) reported that 12 weeks of resistance training in women with fibromyalgia, resulted in a 14% increase in total CS-PFP function . Whereastraining . These rp = 0.08) lower physical function scores as compared to healthy age-matched women who were physically inactive [The ability for BCS to increase physical function is especially important given the fact that after the completion of cancer treatments, BCS have significantly  found that lower body strength was significantly related to lower body function as measured with chair stands in a group of elderly individuals [et al. (1998) who found that lower extremity strength was significantly related to chair stand performance and gait speed in older adults [et al. (2000) found a significant relationship between upper body strength and subjective self-reported function in elderly women [et al. (2012) state that 10%–64% of BCS report upper body morbidities from six months to three years after diagnosis [The significant correlations that were found between both upper and lower body strength and total CS-PFP function are similar to that found in previous studies investigating strength and function. While the present study is unique in that CS-PFP was used to assess objective function in BCS, previous studies have assessed function using other modalities in older adult populations. Fukagawa ividuals . These rr adults . While tly women . These fiagnosis . These miagnosis . The resThe lack of QOL improvement for the BCS in the present study was inconsistent with most previous literature investigating QOL changes in cancer survivors after an exercise intervention. Many studies have reported that following resistance training interventions of various durations, intensities, and volumes have produced significant improvements in QOL in BCS ,34,35. TThe present study had several limitations that may have hindered the ability to accurately interpret the results. The present study had a seemingly smaller sample size than other studies of similar design, which may have hindered the obtainment of statistical significance for some of the variables assessed. Another limitation of the current study was a lack of a true control group, which may have lessened the magnitude of the results reported. Lastly, the usage of the SF-36 QOL instrument may not have been sensitive enough to accurately represent the physical and mental changes that BCS experienced over the course of the intervention. In fact, previous literature suggests that self-reported questionnaires, such as the SF-36, are not sensitive enough to allow individuals to accurately report subtle changes in physical function that may be clinically relevant .In conclusion, our findings indicate that a resistance training intervention of moderate intensity was well tolerated among BCS. All women displayed high levels of adherence to the attendance of exercise sessions and reported no adverse physical incidences as a result of the intervention. With the exception of upper body flexibility, the women improved all other subcomponents, as well as total function, measured via the CS-PFP. It is also noteworthy that at the end of the six-month intervention, upper and lower body strength as well as objective physical function measures were increased to levels that mimicked those achieved by healthy inactive post-menopausal women. This implies that a resistance training intervention is capable of helping BCS achieve similar levels of strength and function that they may have had prior to their diagnosis and treatment of breast cancer. Also noteworthy is the fact that the participants of the present study were able to make the aforementioned gains from a light to moderate intensity resistance training intervention (52%–69% 1RM). The efficacy of a light to moderate intensity resistance training program should be emphasized among health care practitioners and can possibly motivate cancer survivors to begin a more conservative resistance training program who are hesitant to begin strenuous activity. The present study was unable to detect any changes in QOL among the participants; however, from the literature reviewed, the QOL assessment tool may have not been the best choice for the BCS population. Furthermore, the significant gains in objective levels of physical function that the participants achieved should be more heavily considered as opposed to the subjective assessment of QOL, as it provides a more accurate depiction of their true physical capacities and physical well-being.


The demand for bioenergy caused an increased use of logging residues, branches and treetops that were previously left on the ground after harvesting. Residues are stored outdoors in piles and it is unclear to what extent fungi transform this material. Our objective was to quantify the amount of wood degrading fungi during storage using quantitative real-time PCR (qPCR) to detect basidiomycetous DNA in logging residues, a novel approach in this field. We found that the qPCR method was accurate in quantifying the fungal DNA during storage. As the moisture content of the piled logging residues decreased during the storage period, the fungal DNA content also decreased. Scots pine residues contained more fungal DNA than residues from Norway spruce. Loose piles had generally more fungal DNA than bundled ones. Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) residues correspond to about 55 and 20% of the stem volume, respectively  may be correlated with the degree of material degradation. Hence, we make the following assumption: the more basidiomycetous DNA measured, the higher the basidiomycetous colonization with degradation potential in the logging residue. By understanding the pattern of basidiomycetous colonization in stored logging residues better, this knowledge can be used to (1) better understand the potential effect of storage on basidiomycetous colonization and (2) optimize storage methods further.This paper is based on the same samples as described in Picea abies (L.) Karst.) and two were dominated by Scots pine (Pinus sylvestris L.), all 70–100 years old. The experimental setup is illustrated in The detailed description of the experimental setup and sampling is given in ®-Werke Gmbh & Co., Staufen, Germany). The same sample batch was used for analyses by From each chipped sample, about 0.4 kg was randomly taken, dried at 50 °C for 24 h, and grinded first coarsely  and then finely , and as negative controls non-target DNA , all fungi obtained from the Norwegian Forest and Landscape Institute’s fungal culture collection (http://www.skogforsk.no/skogpatologi/database/searchform.cfm) were grown in petri dishes on cellophane over malt agar medium and incubated at 25 °C. To verify the specificity and sensitivity of the primers also on degraded wood samples, known dual mixtures of basidiomycetous DNA and host tree (Serpula-degraded and Trametes-degraded pine wood) together with internal control pGEM plasmid DNA were used. The presence of amplified qPCR product in the expected size range was verified by agarose gel runs. Negative controls consisted of both water and purified Scots pine DNA.To test the primer specificity for basidiomycetes, pure cultures of organisms representing the target DNA (basidiomycetes) were used as positive controls (bas) was performed using SYBR Green PCR Mastermix  and the reference pGEM was quantified with TaqMan Universal PCR Master Mix .The qPCR detection of basidiomycetous DNA .The internal standard pGEM for calibrating DNA extractability was quantified as described by bas a standard curve was prepared from DNA isolated from four basidiomycetous fungi , using serial diluted samples of 0.03–0.0003 ng of DNA giving a standard curve of y = 3.0922 − 0.176x .For quantification of DNAbas the primer concentrations of 60 nM and 3 µl of extracted DNA solution from each sample were used for each reaction. The qPCR cycling was carried out using cycle parameters of 95 °C for 10 min to activate the polymerase, followed by 40 amplification cycles of 95 °C for 15 s and 60 °C for 1 min with signal thresholds set automatically.For qPCR detection and quantification of DNAThe qPCR was performed with ABI PRISM 7700 . After amplification the data were analyzed and plotted (fluorescence vs. cycle number) using the Sequence Detection System, version 1.7a, Software Package . The extent of amplification was calculated as a mean Cq value of 2 technical replicates for each sample. All PCR reactions were performed in singleplex conditions under standard PCR cycling parameters. Undiluted, 10×, 100×, 1,000× and 10,000× diluted experimental sample concentrations were tested to investigate the presence of compounds inhibitory (impurities) to PCR amplification and to ensure that the amount of template fell in the linear range of the standard curve. The 100× dilution showed no indications of inhibition and will thus be presented. Five biological and two technical replicates were used for each sample.bas in logging residues from five forest stands as dependent parameter against the following independent categorical parameters: tree species, harvesting times, storage, within bundle placement, storage time. Precipitation, moisture, bundling time, mean temperature, dry matter loss and calorific value were continuous parameters in the model  for all categorical parameters and linear regression for all continuous parameters. In addition, we tested interaction among wood moisture and storage method. First we tested whether pre-storage (leaving the logging residues on the harvesting site during the winter) had a significant effect on the DNAbas between Stands 1 and 2. Because it did not, we included all five stands in the same model. Means in categorical parameters were compared by Tukey–Kramer HSD test at P < 0.05). In all cases, a null hypothesis was rejected at the 5% level of significance. All statistics were done using JMP .We analyzed the amounts of DNAhe model  and 2. Tbas, while the non-target DNA used as negative controls  was not amplified under the conditions used. Likewise, the primers used for the calibration of DNA extractability between samples amplified only the pGEM plasmid used as an internal control in these studies. The qPCR can detect down to one average size fungal genome  corresponding to the lowest point on our standard curve (∼0.0003 ng).The qPCR primers used in our assay were specific for tested DNAbas in logging residues. It detected DNAbas in the range of 0.01–0.69 ng DNAbas/mg in chipped logging residue  is an indicator that the sampling was representative and that the selected primers were efficient. This same general pattern was found in all stands: the lowest DNAbas value was detected at the start of the storage period, followed by increase in the wetter period in spring 2009, followed by a decrease during the dryer period at the end of the storage, suggesting that our detected values may reflect the environmental conditions impact on basidiomycetous growth. Our DNAbas values were considerably higher than those found by bas in samples tested in our study.Our qPCR assay was able to quantify DNA residue . That thAlthough the qPCR assays are highly sensitive with high resolution and correlation to mass loss at the early stages of decay, they may lack accuracy in advanced decay stages due to substrate depletion . We did The accuracy of the qPCR based quantification of fungal biomass depends on extraction efficiency and purity of extracted DNA. The mean and modus concentrations of extracted DNA in our samples were even 95 and 83 ng/µl, respectively. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA. The mean and modus value for 260/280 ratio in our extracted DNA measured by NanoDrop  was 1.8, which is accepted as “good quality” DNA. The absorbance ratio 260/230 is used as a secondary measure of nucleic acid purity and expected values are commonly in range 2.0–2.2. Mean and modus values in our samples were 1.4, suggesting that contaminants absorbing at 230 nm, such as carbohydrates or phenols, may have been present. Impact of contaminants was avoided by diluting samples, and dilution 100X ensured optimal accuracy.bas. When In spite of the increasing use of qPCR for fungal biomass estimation, it is known that there is variation in ITS copy numbers (per ng DNA) among fungal species , which wn = 133, R2 = 0, 68). Of all the parameters we tested, only harvesting time, storage time, precipitation, temperature, wood moisture content, tree species and storage method significantly affected the amount of DNAbas detected in the logging residues . Generally, the amount of DNAbas decreased at the end of the storage , since basidiomycetous fungi have restricted growth at low temperatures.Material from Stands 4 and 5, with the lowest DNAbas , to the amount of DNAbas . We deteing 2008 . The moiing 2008 . Althougp = 0.0398), precipitation (p < 0.001) and the wood moisture*storage method (p = 0.0397) contributed significantly in explaining DNAbas content  in spring 2009, after 270 days of storage, was associated with higher DNAs values  and 2F. material  further p = 0.0091) to the amount of DNAbas in logging residues  than in spruce-dominated ones , and there was larger variation in values in pine than in spruce residues  and correspondingly low values at spruce stand 4 (mean 0.02 ng DNAbas), probably contributed to the significant differences among the tree species.The high variability among the DNAp < 0.0001) and also in combination with moisture (p = 0.0397) contributed significantly in explaining the amount of DNAbas in logging residues . Compared to start, the DNAbas was significantly higher in bundles  and highest in loose residues .The storage methods alone  and microbial activity will result in dry matter loss in logging residues. In bas content. Hence, our results indicate that that storage of logging residues for maximum 460 days under these conditions give low levels of decay. Because we found higher values of DNAbas in loose residues compared to bundles, the practice of bundling does not seem to promote more basidiomycetous growth than leaving the residues in loose piles. Neither did pre-storing of residues during winter seem to make a difference to DNAbas content.We show that moisture is a key factor for basidiomycetous growth in piles. During storage time of maximum 460 days the moisture content in logging residues gradually decreased together with DNAOur data suggest that basidiomycetous growth may be temporarily stimulated when the material has the optimal water content, such as in periods of increased precipitation, This supports the practice of covering the piles during storage.•bas is a method, capable of detecting the basidiomycetous biomass in logging residue material.We show that qPCR assay to quantify the DNA•bas, indicating the presence of potentially decomposing basidiomycetes, decreases at the end of the storage period together with moisture content, there seems to be a small danger of fungi significantly transforming logging residues stored for less than 460 days at given conditions.Because the DNA•bas than Norway spruce residues.Scots pine residues had more DNA•bas than bundled ones.Loose piles had generally more DNA10.7717/peerj.887/supp-1Supplemental Information 1Click here for additional data file.


Obesity is increasing in developing countries. This study aimed to identify the association between rapid infancy weight gain and obesity risk among early school-age children.A total of 581 singletons  whose mothers participated in an antenatal multiple micronutrient supplement trial in rural western China were followed from birth to between 7 and 9 years of age. Height and weight were measured at birth, 1.5 years, and between 7 and 9 years. At the 7- to 9-year time point, body composition was determined using bioelectrical impedance analysis. Multilevel mixed analysis was used to test the associations between rapid weight gain in infancy (from birth to age 1.5 years) and body size and composition or overweight/obesity among early school-age children.P < 0.001), mid-upper arm circumferences (P < 0.001), percentage body fat (P < 0.001), and fat mass index (P < 0.001) at 7 to 9 years of age after adjusting for biological and social economic factors, genetic factors, and perinatal and postnatal factors. These associations appeared to be independent of gender, economic status at early school age, and maternal nutritional status at enrollment. Rapid growers may have approximately 3 times the risk of being overweight/obese during the early school-age years .Overall, 31.2% (181 of 581) of the infants showed a weight-for-age Z score gain greater than 0.67 between birth and 1.5 years, indicating rapid weight gain. Approximately 5.7% (33 of 579) of the subjects were overweight (BMI-for-age Z scores [BAZ] >1 and ≤2) or obese (BAZ >2). Rapid infancy weight gain was associated with a higher BAZ (Rapid infancy weight gain is a risk factor for being overweight/obesity among early school-age children in rural western China. We propose that social and biological determinants, such as economic status, physical activity, and feeding practice, should be targeted to prevent obesity. Systematic reviews11 have consistently shown that rapid infancy weight gain is associated with an increased risk of obesity in later life. However, most studies included subjects born before the 21st century.13 It is unclear whether the association between infancy weight gain and later obesity is still valid today. The objective of our study was to evaluate the association between rapid infancy weight gain and later body size and composition or overweight/obesity in a 2004 Chinese birth cohort study.To prevent obesity, both the risk factors and the critical periods for the development of obesity must be clearly identified. In a large birth cohort study,14 The trial was conducted in 2 poor rural counties in Shaanxi Province in northwest China from August, 2002 to January, 2006. The 2 counties have experienced rapid economic growth over the last decade because of a thriving coal mine industry.This study is a prospective study of growth in the offspring of mothers who had participated in a cluster-randomized, double-blinded, controlled trial (ISRCTN08850194).14 Briefly, pregnant women in the same village received the same daily antenatal micronutrient supplementation from study enrollment until delivery. Postnatal growth was measured at birth, every 3 to 12 months of age, every 6 months between 12 and 30 months of age, and at 7 to 9 years. In total, 5828 mothers were invited to participate in the study. They were followed during and after pregnancies; 1388 live singletons born in 2004  completed the follow-up study from birth to 30 months. A total of 562 children were excluded: 5 died, 407 moved out of the study area (and were therefore excluded for having a different socioeconomic profile), and 150 were missing birth weight data or weight data at 1.5 years. Among the 826 remaining children, 41 refused to participate, and 204 were unknown locations. Therefore, the final sample consisted of 581 children (349 boys and 232 girls), representing 70.3% of the original 826 eligible participants. Ethical approval was obtained from the Science and Research Ethics Committee of Xi’an Jiaotong University. In each case, a parent provided signed consent, and verbal assent was obtained from each child.The details of the trial have been described elsewhere.Birth weight and length were measured by hospital nursing staff within 1 hour of delivery. Birth weight was measured with an electronic scale  with precision to the nearest 10 g. Birth length was measured to the nearest 0.1 cm using a portable measuring board with fixed head piece. Weight and length at around 1.5 years were available from routinely infant home visit performed by trained research fieldworkers and nurses and were documented in the infant's personal health record. Weight was measured to the nearest 10 g with the electronic scale, excluding the weight of children's clothes. Recumbent length was measured to the nearest 0.1 cm on the length board. At early school-age, weight, standing height, mid-upper arm circumference (MUAC), and body composition were assessed by trained investigators. The subjects were dressed in underwear only and were barefoot. Weight and body composition was evaluated using bioelectrical impedance . Standing height was measured to the nearest 0.1 cm using a steel strip stadiometer . In our cohort, the coefficient of variation for weight at birth, 1.5 years, and early school-age were 13.2%, 10.2%, and 15.7%, respectively, and for length/height were 5.2%, 3.8%, and 4.4%, respectively. MUAC was measured on the right arm at the level of the upper arm midpoint mark to the nearest 1 mm using a nonelastic tape .15 and computed using the birth size Z scores R script.16 Sex- and age-adjusted Z scores for weight, height, and BMI at 1.5 years and early school-age were calculated using the World Health Organization (WHO) reference-data growth charts.18 Rapid infancy weight gain was defined as a gain in Z score greater than 0.67 for weight between birth and 1.5 years.8 BMI-for-age Z scores (BAZ), MUAC, percentage body fat (PBF), and fat mass index (FMI) were used as markers of overweight/obesity. Children with a BAZ between +1 and +2 were classified as overweight, and those with a BAZ above +2 were classified as obese.19 PBF was FM divided by total body mass times 100. FMI was FM in kilograms divided by the height in meters squared, which was calculated to adjust the body composition for height.BMI was calculated as the body weight in kilograms divided by the height in meters squared. Sex- and gestational-age adjusted birth weight and length Z scores were calculated according to global standards27 Parental height and weight were measured at early school-age according to the standard procedures. Information on other variables were collected by face-to-face interview using self-designed structured encoded questionnaire. All of these data were recorded during the original trial and the follow-up study. We adopted the hierarchical approach proposed by Victora et al28 to develop a conceptual framework, which grouped all covariates into 3 domains . Biological and socioeconomic factors included: gender , age, birth weight, gestational age, family economic status at early school age , parental educational level , and parental occupation (farmers or not). Genetic factors included: parental height (below 10th percentiles among study objectives or not) and parental BMI (above 25 kg/m2 or not). Perinatal and postnatal factors included: parity (primipara or not), maternal malnutrition at enrollment (yes or no), antenatal micronutrient supplementation , numbers of supplementation consumed , infant feeding method (appropriate or not), activity level (appropriate or not), and medical history (serious diseases during infant or not). Gestational age was computed as the number of completed weeks of gestation from the date of the mother's last menstrual period to delivery. Family economic status was based on an assets index divided into dichotomies to indicate the poorer and wealthier households. The mothers were considered as primipara if they reported “no previous pregnancy” or “no children living in the household” at enrollment, depending on which question was asked at that time. Maternal MUAC at enrollment was used as an indicator of short-term nutritional status, and maternal malnutrition was defined as women with MUAC below 23.5 cm.29 Appropriate feeding was defined as exclusive breastfeeding until 6 months and the appropriate introduction of complementary food . Providers chose one of 6 categories  in response to a question “How many minutes did the infant in your care spend on outdoor activity per day.” We dichotomized their responses to determine whether appropriate (>60 minutes/day) or not.For all analyses, we included all corresponding covariates in the models based on their associations with rapid infancy weight gain or with childhood body size and composition based on previous studies.t tests  or Mann–Whitney U test , and categorical data were analyzed with χ2 tests. Because of the hierarchical structure of the data, we adopted 3-level mixed analysis to estimate the effects of rapid infancy weight gain on markers of overweight/obesity at early school age with township to level 3, village to level 2, and individual to level 1; the outcomes included the BAZ, MUAC, PBF, and FMI (continuous variables). Following the conceptual hierarchical framework, sequential models were constructed and tested as follows: with no adjustment (model 0); with adjustments for biological and socioeconomic factors (model 1); with adjustments for all variables in model 1 plus genetic factors (model 2); and with adjustments for all variables in model 2 plus perinatal and postnatal factors (model 3). Another 3-level mixed analysis was used to test the effects of rapid infancy weight gain on the risk of overweight/obesity (dichotomous variable) at early school age, with the same adjustments that were used in models 0 to 3. In all these models, the presence or absence of rapid infancy weight gain and potential confounders were treated as fixed effects, whereas township, village, and individual were treated as random effects. To test the impact of those lost to follow-up, an additional analysis was conducted after imputation of missing values using expectation-maximization. The main results were consistent between analysis on those follow-up and imputed analysis. Furthermore, we performed sensitivity analysis excluding preterm  born children. We also performed sensitivity analysis to examine the potential modifying effects of gender, family economic status at early school age, and maternal nutritional status at enrollment on the associations between rapid infancy weight gain and BAZ, MUAC, PBF, and FMI. All reported P values were 2-tailed, and the level of significance was set at 0.05. The analyses were performed using SPSS version 20.0 .The data were checked manually for completeness and were double-entered into EpiData version 3.1 . We performed range and logic checks of data to ensure the accuracy. Initially, mean ± standard deviation (SD) or median with interquartile range were used to describe normally or abnormally distributed continuous variables, respectively. Count and proportions were used to describe categorical variables. We compared baseline characteristics between the follow-up group and the lost-to-follow-up group, and also we compared the characteristics between subjects experiencing rapid infant weight gain or not. Continuous data were analyzed with Figure P = 0.009), PBF (P < 0.001), and FMI (P < 0.001) than the boys; however, the total prevalence of overweight/obesity was significantly higher among the boys than that of the girls  in childhood among children born in the 21st century. Approximately one-third of the children in this cohort grew rapidly between birth and 1.5 years of age. Our results showed that rapid infancy weight gain was significantly associated with an increase in BAZ, MUAC, PBF, and FMI of the order of 0.5 SD at early school age. Importantly, rapid infancy weight gain increased a child's risk of being overweight/obesity at early school age by almost 3 times after adjustments for other confounders.11 Previous studies conducted in developed countries have shown that children with rapid infancy weight gain tend to have a higher BMI and are at increased risk of being overweight during childhood,30 adolescence,31 and adulthood.32 A prospective cohort study8 of 848 full-term children suggested that children with accelerated growth between birth and 2 years of age (an increase in weight-for-age Z scores > 0.67) had a greater BMI at 5 years of age. In addition, another study33 of 136,971 regularly followed children in south China found that rapid growers had a higher risk of being overweight (BAZ >  +2) at 4 to 5 years of age than the others. Note that BMI is a marker of general change in body size, while body composition can reflect a detailed change in body components. In our study, children with rapid infancy weight gain had a higher PBF and a higher FMI at early school age. These findings align with those of other studies showing that rapid early postnatal growth may lead to higher levels of total body fat in childhood.35 A prospective study of 233 white adults suggested that rapid infancy weight gain was associated with an elevated risk of obesity, higher total body fat, PBF, and visceral adipose tissue mass.32 However, in our study, we could not explore the effect of rapid infancy weight gain on total body fat deposition , which exerts distinct effects on metabolic syndrome, diabetes risk, and circulating adipokine levels.36The effect of early rapid growth on later obesity risk that was observed in this cohort expands the findings of some previous reviews.37 Our study was conducted in the backward, rural region, which had low levels of education. We could see a great influence of economic status on the associations between rapid infancy weight gain and body size, body composition. Not only social inequalities but also children themselves should be responsible for their nutritional status. A study among an international sample of 9 to 11-year-old children indicated that greater physical activity was associated with lower odds of obesity.38 Outdoor activity during infant was within the range of controlled confounding in our study. Recent evidence suggested that fathers had a unique and key role in shaping their children's physical activity behaviors.40 More researches were needed to explore the efficient behavioral interventions that could improve the living style and health of children.An evidence from 70 countries found that obesity was positively associated with GDP among individuals with lower levels of education.42 The evidence for a lower growth rate in breastfed children compared to formula fed children has been rather consistent.43 Formula has higher protein and energy density than human milk, which stimulates the secretion of insulin-like growth factor 1.44 Insulin-like growth factor 1 improves protein synthesis and cell proliferation, and then accelerates growth and increases muscle mass and adipose tissue.45 In our study, whether being exclusively breastfed until 6 months was associated with rapid growth, which may suggest that rapid infancy weight gain could be associated with a diet-induced altered metabolic state in infancy. Rapid infancy weight gain has also been associated with changes in appetite regulating hormones ,46 which result in increased appetite.47 A cohort of term appropriate-for-gestational-age infants46 showed that an increase in sex- and age-independent SD scores for weight during infancy was associated with lower concentrations of adiponectin, which may predict future insulin resistance.48 Moreover, a birth cohort study found an association between higher androgen metabolite excretion and faster postnatal weight gain among girls.49 It could be hypothesized that rapid infancy weight gain programs overactivity of the hypothalamic-pituitary-adrenal axis, which result in central obesity and insulin resistance.49 A prospective observational study reported that maternal prepregnant BMI was positively associated with infant weight gain from birth to 1 year.50 Therefore, the mechanisms linking rapid infancy weight gain and childhood obesity may also include transgenerational factors, such as genetic or epigenetic factors, or familial behaviors. Prospective studies that carefully characterize childhood growth, body composition, and genetic, endocrine, metabolic, nutritional, and social factors would advance our understanding of these complex associations.Our results support etiologic hypotheses concerning critical periods or pathways for obesity development. Identifying the mechanisms that underlie the association between rapid infancy weight gain and later obesity is likely to be important for understanding how to prevent obesity throughout life. Some hypotheses about possible mechanisms have already been suggested based on previous observational studies and experimental models.The main strength of this analysis is the long-term follow-up. Prospective data for potential confounders and growth were available in this study, which decreased the risk of recall bias or unreliable measurements. As with any observational study, caution should be exercised when inferring causality based on the findings. However, our findings were consistent before and after adjustments for potential confounding by most known variables that are plausibly associated with infancy and childhood growth. The associations were still consistent in the sensitivity analysis.P < 0.05). It is difficult to speculate whether this difference significantly affected the observed associations, but we consider this possibility unlikely. We assumed that the sample attrition followed a missing-at-random pattern; therefore, the results of the present study can be generalized to the original sample. Although our study was conducted within the context of a clinical trial for antenatal micronutrient supplementation, we did not find any evidence of modified associations within the trial intervention arm. The relatively small sample size reduced the power to detect differences in the risk of being overweight/obesity between rapid growers and nonrapid growers. We need to be prudent in making conclusions about the association between rapid growth and risk for overweight/obesity. However, we had a more than 99% power to detect differences in markers of overweight/obesity of the order of 0.5 SD between groups. Other study limitations included a lack of information about body composition or other biochemical markers of chronic disease risk during infancy. It is possible that changes in infant adiposity, body composition, and even biochemical markers might predict future obesity risk more clearly than simple weight gain does. We are not aware of any long-term studies with such detailed characterization during infancy. However, birth weight and infant weight are easily measured, and such information is widely available; therefore, such information could be easily used to screen large populations. We used the international standards developed by the WHO to calculate the Z scores and to identify rapid weight gain and overweight/obesity, thereby enabling more meaningful comparisons across different cultures and settings. Besides, the most frequent definition for rapid growth was a Z-score change > 0.67 in weight-for-age,51 which may be readily clinically interpreted as crossing upward by at least 1 percentile band on standard growth chart. Finally, physical activity during school age was not within the range of controlled confounding in our study. As physical activity was assessed by a self-reported questionnaire, which may be confounded by recall bias. Moreover, we only assessed the frequency of physical activity during school age, but not included the information about intensity and volume. Although we performed an extensive adjustment for a large number of potential confounders, unobserved confounding factors might still occur among the observed associations, as is possible in any observational study.Our study also has some limitations, such as the high sample attrition rate. Among the children with available weight information at birth and 1.5 years of age who were still living in the study areas, 70.3% participated in the follow-up measurements at early school age, 99.7% of these children also participated in the body composition assessments. Mothers who were farmers were less likely to be excluded from the current analyses than included (91.4% vs 86.4%, This study concluded that rapid infancy weight gain is a risk factor for obesity at early school age among children born in the 21st century in rural western China. Rapid infancy weight gain is a risk factor for childhood obesity. Using growth charts to identify at-risk infants could reduce levels of child overweight and obesity by enabling health care professionals to target prevention more effectively. Further research needs to evaluate the clinical validity and feasibility of this risk factor. Further exploration of genetic and postnatal environmental factors that might influence rapid postnatal growth could uncover modifiable mechanisms that underlie the association between growth in infancy and risks for later obesity. Longer follow-up studies of this study population and other populations are needed to assess whether these associations persist and other associations develop in adolescence and adulthood.


Lung protective ventilation, LPV, is recommended for ever wider patient populations, even those without primary lung disease. Low tidal volume ventilation is a first-hand remedy for LPV that is facilitated by dead space reduction. Inspiratory flow pattern affects gas exchange. In ARDS patients, it was recently shown that a long period during which inspired tidal gas is in contact with resident alveolar gas, mean distribution time, MDT, as well as a high end inspiratory flow, EIF, immediately before interruption of inspiration augment exchange of C02 by reduction of airway and alveolar dead space.The hypothesis was tested that MDT and EIF affect C02 exchange in man without lung disease in principle as in ARDS. The study should provide guide lines in support of LPV at health as well as in lung disease.I, post inspiratory pause time, TP, and shape in form of ramps with increasing or decreasing flow rate. Signals for flow rate and C02 were analysed in the format of the single breath test C02 from which the volume of C02 eliminated during each ordinary or modified tidal breath, VTC02, was derived. Changes of VTC02 were explained by changes in airway dead space and by effects on intra-alveolar gas mixing inversely reflecting alveolar dead space.During preparation for cerebral surgery in 8 patients without lung disease, breaths with 20 inspiratory wave forms were delivered with different inspiration time, TI 33%, TP 10% and square inspiratory flow, the change of C02 volume exchanged per breath, DVtC02%, varied with MDT and EIF: DVtC02% = 17 · lnMDT + 13 · EIF +1.4In relation to ordinary breaths with TIn each subject the influence of lnMDT and EIF was highly significant. Positive effects on C02 exchange by longer MDT and higher EIF reflected both a reduced airway dead space and improved intra-alveolar gas mixing indicating a reduced alveolar dead space.TC02 by 17% at constant inspiratory flow and 27% at increasing inspiratory flow. The most efficient way to modify inspiration was to prolong TP at the expense of a shorter TI and eventually a shorter expiratory time. The more efficient C02 exchange allows a reduction of tidal volume by about 10%.Modification of inspiratory flow pattern could increase VP, is a tool for more efficient C02 exchange that can easily be applied for lung protective ventilation at reduced tidal volume in patients without or with lung disease.Modification of inspiratory flow pattern, particularly by a longer T


There is growing interest in using wild animals to monitor the real-life cocktail effect of environmental chemicals on male reproduction. However, practical difficulties, such as long distances to the laboratory, generally prolong the time between euthanisation and specimen handling. For instance, tissue fixation is often performed on frozen material or on material where deterioration has started, which may affect tissue morphology. This study examined the effect of pre-fixation delay and freezing on mink testicular endpoints in order to determine robust endpoints in suboptimally handled specimens. Sexually mature farmed mink (n=30) selected at culling were divided into six groups and subjected to different time intervals between euthanisation and fixation or freezing: 0 hours (fixed immediately post mortem), 6 hours, 18 hours, 30 hours, 42 hours, or frozen 6 hours post mortem and thawed overnight. Unaffected endpoints when pre-fixation storage was extended to 30 hours included: area and diameter of the seminiferous tubules, length and weight of the testes, and acrosomes marked with Gata-4. Epithelial height, Sertoli cells marked with Gata-4 and cell morphology were affected endpoints after 6 hours of storage. Freezing the tissue prior to fixation severely altered cell morphology and reduced testicular weight, tubular diameter and area. Morphological changes seen after 6 hours included shredded germ cells and excess cytoplasm in seminiferous tubular lumen, chromatin rearrangements and increased germ cell death. Extended delay before fixation and freezing affected many endpoints in the mink testicular tissue. Some of these endpoints may mimic chemically induced effects, which is important to consider when evaluating specimens from wild animals for environmental toxicity. There is growing concern that male reproduction is affected by chemicals in the environment. Chemical cocktails are more likely to cause this damage than single compounds. One way to determine such changes is to use wild mink as a monitor species and evaluate testicular toxicity using the most sensitive method, histopathology ,2. SinceA number of studies have linked adverse male reproduction in humans ,5, mammaIn laboratory studies, the effect of a single chemical on male reproduction is commonly investigated. However, during recent years there has been increasing interest in the effect of interactions between chemicals found in the environment, which has been named the “cocktail effect”. New chemicals are continually being produced and the effect of the resulting chemical cocktail is unclear and will vary depending on the environment. The recent report from the World Health Organisation on endocrine disruption underlined this issue and urged future investigations to take it into consideration when studying reproductive disturbances in humans and wildlife . One wayMink has been suggested as a suitable sentinel species in environmental monitoring ,17. MinkNeovison vison) from a commercial fur farm  in Sweden were collected at culling. One mink at a time was euthanised at the fur farm using carbon dioxide (95 vol.%) in a specially constructed euthanisation box until cardiac arrest, followed by pelt ablation. This euthanisation process conforms with EU legislation and Swedish Board of Agriculture standards [Thirty healthy, 10-month-old, sexually mature mink (tandards . The farTreatment group 0 h (n = 5): Directly after the pelt was removed, body weight, left testis weight and length were measured, one animal at a time, followed by fixation of left testis tissue (within 10 minutes post mortem).Treatment groups 6, 18, 30, 42 h and 6 h + frozen (n = 5 per group): After euthanisation and pelt ablation, the animals were transported intact to the laboratory and left at room temperature (21°C) for 6, 18, 30 or 42 h. Measurements of body weight, left testis weight and length and fixation of left testis tissue were performed after 6, 18, 30 and 42 h. The five animals in the last group (6 h + frozen) were frozen (-20°C) 6 h post mortem and then thawed at room temperature (21°C) for 30 h prior to measurements of body weight, left testis weight and length and fixation of left testis tissue.Absolute testicular weight was used instead of relative weight, because the testes are less influenced by body weight than most other tissues ,21. No eTesticular tissue is known to be difficult to fix and traditional formalin is discouraged due to its poor preservation of morphological details ,2. BouinTwo transverse tissue slices (approximately 2 mm thick) were cut in serial fashion from the left testis and fixed in modified Davidson’s fluid for 24 h at 4°C. They were then rinsed and stored in phosphate buffer until all 30 mink had been fixed. The testis slices were then trimmed to appropriate size with a razor blade to fit in the embedding cassettes, followed by dehydration in increasing concentrations of ethanol. Testis slices fixed in modified Davidson’s fluid were used for both resin and paraffin embedding.For morphological evaluations, one testis slice from each mink, was embedded in a water-soluble resin  and sectioned with glass knives into 2 μm thin sections using a microtome . All sections were stained with haematoxylin, eosin (HE) and toluidine.For immunohistochemistry and semi-automatic image analysis, one testis slice from each animal was embedded in paraffin wax and cut into 4 μm thin sections using a sledge microtome . One testis section from each mink was mounted on Superfrost Plus Gold slides  for immunohistochemistry and one section was placed on Superfrost slides  for image analysis. For the image analysis, the sections were stained with a variant of periodic acid-Shiff staining to obtain maximum colour differences between the seminiferous epithelium and the interstitial tissue.The sections were deparaffinised in xylene (twice for 5 minutes each) and rehydrated (100% (twice), 95% and 70% ethanol for 5 minutes each, followed by distilled water), incubated for 5 minutes at room temperature (21°C) in periodic acid (0.5%), rinsed in distilled water several times, incubated in Shiff’s reagent (40 minutes) and washed in running water for 25 minutes. To counterstain, the slides were submerged in Weigert for 10 seconds, followed by 7 seconds in a mix of 95% ethanol and 1% HCl, and then rinsed in running water for 30 minutes. Sections were dehydrated through rising ethanol concentrations , transferred into xylene  and mounted with Pertex.2HPO4 and 0.02% KH2PO4. The endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide diluted in methanol. Immunolocalisation of GATA-4 antibody was identified using the ImmunoCruz goat ABC Staining System . In brief, tissue sections were treated with blocking serum goat  for 30 minutes and excess serum was blotted from the slides. GATA-4 (C20)  antibody was diluted in PBS 1:50 and the sections were incubated in the dark for 20 h at 4°C. The sections were rinsed with PBS between each of the subsequent steps. Secondary antibody (donkey anti-goat)  was applied to each section and incubated for 30 minutes, followed by AB enzyme reagent for 30 minutes. Immunoreactivity was visualised using 3.3´-diaminobenzine tetrahydrochloride , to which H2O2 was added to visualise the bound enzyme activity as brown colour. Sections were finally rinsed in H2O, dehydrated and mounted with Pertex. Negative controls were run by excluding the primary antibody on one slide and by replacing the primary antibody with non-immune serum from goat  on one slide. The slides were counterstained with haematoxylin for 10 seconds.Immunohistochemical localisation of Gata-4 antibody was evaluated as a marker to distinguish between different cell types in the seminiferous epithelium of the testis. The slides were deparaffinised in xylene  and rehydrated (100% (twice), 95% and 70% ethanol for 2 minutes at each concentration, followed by distilled water (twice) for 5 min each). Antigen were retrieved by submerging the slides in 0.01 M sodium citric buffer (pH 6.0), followed by pressure-heating for 20 minutes in a pressure boiler at 120°C . After cooling, the slides were rinsed in phosphate-buffered saline (PBS). The PBS used for the entire immunoprotocol had pH 7.4 and contained 0.8% NaCl, 0.02% KCl, 0.106% NaOne person (E. Sporndly-Nees) performed the morphological evaluation of the cells in the seminiferous tubules. The evaluation was based on both visual comparison of unblinded slides under the microscope and comparison of digital images . A total number of 649 images were taken . Factors compared between the treatment groups were overall organisation of seminiferous epithelium and tubular lumen at 10x and 20x magnification. To compare cellular details , 40x and 60x magnifications were used. The specific cell types studied in the seminiferous epithelium were: Sertoli cells, spermatogonia, primary spermatocytes , round spermatocytes, elongated spermatids and Leydig cells. One cell type at a time was studied within each group  and described. When all cell types within each group had been described, a comparison was made between the groups to describe how the cell morphology changed over time and by freezing. Staging of the seminiferous tubules were carried out, when possible, in all sections.One person (E. Sporndly-Nees) performed the morphological evaluation of the immunohistochemical sections. The evaluation was based on visual comparison of unblinded slides under the microscope using 10x, 20x, 40x and 60x objective lenses. Gata-4 stained Sertoli cells and acrosomes were identified when possible.Digital images of paraffin sections were taken with a Nikon Microphot-FXA microscope using a 10x objective lens. A series of images from each animal were taken in a grid-like pattern to make sure that no tubular overlap occurred. A total of 1022 round-shaped, straight-cut seminiferous tubules were evaluated. Mean (range) number of seminiferous tubules measured in each group was: 0 h: 61.4(37–93); 6 h: 31.6(20–46); 18 h: 30.4(21–38); 30 h: 29.4(14–50); 42 h: 29.8(13–38); and 6 h + frozen: 21.8(13–26). Morphometric measurements of area, diameter and epithelial height of seminiferous tubules were analysed by semi-automatic image analysis using a method designed specifically for testicular tissue (below). The measurements were performed by one investigator (A. Fakhrzadeh) and all images were coded.A semi-automatic image analysis technique was developed to measure area, epithelial height and diameter of the seminiferous tubules. A semi-automated programme that includes user input to produce a correct delineation was used to find the boundaries of the seminiferous tubules. The programme determines various measures for each pixel belonging to an edge. The gradient image has information about edges and was computed using the vector gradient approach . From thThis cost function was plugged into the livewire algorithm ,27 impleAfter delineating a tubule, its area was given by the number of the pixels inside the drawn boundary, and its radius was computed as the average distance from all pixels on the drawn boundary to the tubule’s centre of mass. For the tubules with visible lumen, the lumen was segmented using the same procedure as described above. The epithelium height was then computed as the average distance between the border of the lumen and the border of the tubule.2), diameter (μm) and epithelial height (μm) of seminiferous tubules) were based on several measurements per mink and a hierarchical ANOVA was therefore used. Two models were used, one including the fixed effect of time  and the other the effect of freezing . The analyses were carried out with SAS software  using the MIXED procedure. Residuals were analysed and were considered normally distributed with equal variances. Significance levels for pair-wise comparisons were corrected for multiple testing using Tukey’s method. All values presented in diagrams represent mean±S.E. and results were considered significant at p<0.05.The experimental unit was mink. The gross morphology (body weight (kg), left testis weight (g) and length (mm) was based on one measurement per mink and therefore analysed by one-way ANOVA. The morphometric measurements  or by freezing prior to measurements (6 h + frozen group) Tables .The area of seminiferous tubules decreased over time and after freezing Tables  and 2. TAt 0 h post mortem, the tissue was well-organised, with an intact epithelium and a well-defined lumen in the seminiferous tubules. Germ cells were well connected and attached to the basement membrane . Germ ceAt 6 h post mortem, many of the seminiferous tubular lumen were occupied, mostly by excess cytoplasm but also by round and elongated spermatids, and only a few empty lumen were seen. The epithelium was well-organised and germ cells were attached to the basement membrane . OverallAt 18 h post mortem, the seminiferous tubular epithelium showed increased disorganisation, but the peritubular cells were still attached to the seminiferous epithelium. All lumen of the seminiferous tubules was occupied by cytoplasm as a blurred haze, sometimes mixed with round and elongated spermatids . SpermatAt 30 h post mortem, the seminiferous epithelium was further disorganised, with germ cells located at abnormal positions . The perAt 42 h post mortem, the longest interval studied, it was difficult to distinguish between the different cell types in the seminiferous epithelium. There was a loss of intercellular connections, which contributes to distance and disorganisation between the germ cells. The peritubular cells were detached from the seminiferous epithelium in many tubules . Only a Testicular tissue frozen 6 h post mortem and thawed before fixation showed a complete loss of organisation in the seminiferous tubular epithelium. The seminiferous tubules were still identifiable, but resembled a dense mass in which peritubular cells were visible but detached from the epithelium. All cell nuclei had condensed chromatin and appeared pyknotic . The excThe tissue fixed directly post mortem (0 h) showed distinct staining for Gata-4 of Sertoli cell nuclei in the seminiferous epithelium . In addiIn all treatments , there was no staining of Sertoli cells. The acrosomes remained marked all through the different time periods and freezing Fig –3d. TherTesticular histopathology is the most sensitive and reliable method for detecting testicular toxicity ,2. In thOne of the first signs of autolysis observed in histological examination of testicular tissue in the present study was shredded cytoplasm and germ cells occupying the seminiferous tubular lumen, which was apparent after only 6 h of storage between euthanisation and fixation. Shredding of epithelial content to the lumen of the seminiferous tubules can be a sign of tissue damage. A review by Creasy  noted thIn a review paper, Mathur and D'Cruz  state thToxic damage can lead to germ cell death, but the dead germ cells are phagocytised by Sertoli cell within 24 h, resulting in generalised germ cell depletion . No specIt is recommended that numbers of vacuoles within the tubular epithelium be examined . No vacuThis study showed that testicular weight was not affected by storage of mink carcasses for up to 42 h, but that it was significantly reduced by freezing the animals. Testicular weight is a known measure of reproductive health, with a decrease generally indicating germ cell loss or impaired spermatogenesis associated with decreased fluid production by the Sertoli cell ,2. TestiTesticular length and body weight are both commonly used endpoints in toxicological studies. In the present study these endpoints remained constant even though the tissue had been exposed to prolonged storage for up to 42 h prior to measurements or to freezing, which confirms their applicability as endpoints. According to Sonne et al. , there iEpithelial height and diameter of the seminiferous tubules are frequently used parameters to evaluate male reproduction, as known environmental chemicals, such as cadmium, nickel and di (n-butyl) phthalate, affect these parameters –33. In tA recent editorial published in Nature Methods  notes thIn the present study, the polyclonar antibody Gata-4 stained Sertoli cell nuclei in mink testicular tissue fixed immediately post mortem. Gata-4 also stains Sertoli cells in bulls, mice, rats, dogs and boars –45, makiBefore generalising the present findings to other animal species, the size of the animal, type of fur/ feathers and where the testes are situated should be considered, as these may affect autolysis. As an example, autolysis of human liver is slower than autolysis of rat liver at 18°C . The appIn the present study the group 6 h + frozen was chosen to show how freezing effected endpoints. Why we chose to leave the animals 6 h post mortem and prior freezing was to mimic the situation in the field where it can take the hunter several hours to get back with the animals before it can be frozen. Prolonged time prior to fixation affected epithelial height severely between 0 h and 6 h resulting in difficulties to separate the freeze and time effect, and a 0 h + frozen would have been desirable. To adjust for this limitation the 6 h + frozen was compared with 6 h and thereby the freeze effect was isolated.Knowledge of the interval between death of the animal and fixation of testicular tissue is important when interpreting the results of morphometric and histopathological analyses. Even when fixation is delayed for up to 30 h post mortem, certain endpoints are not affected, such as area and diameter of the seminiferous tubules, length and weight of the testis and acrosomes marked with Gata-4. Endpoints that proved to be affected already with a 6 h delay in post mortem storage were epithelial height, Sertoli cells marked with Gata-4 and morphology of the cells. In addition, some morphological changes due to delayed fixation may be misinterpreted as toxic damage. Freezing tissues prior to fixation severely affected most endpoints evaluated.Due to the finding that the interval between death of the animal and fixation may be crucial for obtaining valid results, future research should investigate other testicular endpoints and methods to validate their use if the pre-fixation time is delayed.


Magnetic resonance spectroscopy (MRS) studies have previously described metabolite changes associated with aging of the healthy brain and provided insights into normal brain aging that can assist us in differentiating age-related changes from those associated with neurological disease. The present study investigates whether age-related changes in metabolite concentrations occur in the healthy cervical spinal cord. 25 healthy volunteers, aged 23–65 years, underwent conventional imaging and single-voxel MRS of the upper cervical cord using an optimised point resolved spectroscopy sequence on a 3T Achieva system. Metabolite concentrations normalised to unsuppressed water were quantified using LCModel and associations between age and spinal cord metabolite concentrations were examined using multiple regressions. A linear decline in total N-Acetyl-aspartate concentration  and Glutamate-Glutamine concentration  was seen within our sample age range, starting in the early twenties. The findings suggest that neuroaxonal loss and/or metabolic neuronal dysfunction, and decline in glutamate-glutamine neurotransmitter pool progress with aging. Human senescence is associated with deterioration in physical performance which, in part, can be attributed to age-related neurodegeneration of the spinal cord. Decline in motor agility and gait are ubiquitous features of aging which can begin from the fourth decade of life; typically, walking becomes slower, with shortening of stride length and a tendency to stoop in vivoConventional MRI of the spinal cord lacks the necessary sensitivity to detect these microstructural changes. T2 hyperintensities which are commonly seen in the aging brain are only rarely seen in the spinal cord 1H magnetic resonance spectroscopy (MRS), which allows the quantification of metabolites in human tissue, has been widely used to study healthy aging of the brain in humans in-vivoTo date no studies of the spinal cord have addressed metabolic changes associated with normal aging. In this study, which was carried out in healthy volunteers, we therefore aimed to (i) investigate whether age was associated with changes in concentrations of commonly quantified metabolites and (ii) explore the effect of gender on metabolite concentrations.All subjects provided written, informed consent prior to taking part in the research, which was approved by the NRES Committee London Bloomsbury .Healthy volunteers were prospectively recruited from amongst university staff and respondents to adverts within the university and neurology outpatient clinic. A minimum of two subjects per decade of life (between the ages of 20–65) were recruited in order to achieve a good spread of ages. The age of subjects, from youngest to oldest were; 23, 24, 25, 28, 30, 30, 31, 31, 33, 33, 36, 40, 43, 44, 46, 48, 52, 54, 55, 56, 65 and 65 years old. Participants found to have severe spondylitic changes 2; voxel size = 0.6×0.6×3.0 mm3;number of excitations (NEX) = 2; 13 contiguous coronal slices, and (b) Dual-echo PD/T2-weighted sequence with TR = 4000 ms; TE = 15/80 ms; FOV = 256×160 mm2; voxel size = 1.0×1.0×3.0 mm3; NEX = 2; 12 contiguous sagittal slices.All scans were performed using a 3T Achieva system , with the manufacturer’s 16-channel neurovascular coil. An MR compatible cervical collar was worn by all volunteers as this has recently been shown to considerably reduce motion artefacts during scanning 3 (2.3 ml) were prescribed and centred on the C2/3 intervertebral disc  package P values of <0.05 were taken to be statistically significant.All statistical analyses were performed using IBM SPSS statistical package version 22.0 . Associations between metabolites and age were examined using linear regression of the metabolite as response variable on age, with gender, linewidth (FWHM) and voxel volume covariates; a quadratic term in age was also entered to examine evidence of non-linearity and removed if p>0.1. Gender differences reported from these models are adjusted for age, linewidth (FWHM) and voxel volume. Twenty-five healthy participants were prospectively recruited and scanned. Three participants were excluded from the analysis due to poor spectral quality. Therefore, twenty-two healthy participants  with a mean age of 40.5 years, standard deviation (SD) 13.1, range 23–65 were included in the final analysis.There was no statistical evidence of a non-linear relationship between metabolite concentrations and age. Older age predicted lower spinal tNAA concentration  and lower Glx concentration  . Age wasGlx concentration was significantly higher in men, mean (SD) 7.27 (0.97) mmol/L, than females, mean (SD) 5.73 (1.05) mmol/L , but no gender differences were seen with other metabolites .0 convergence We used a single voxel MRS protocol optimised for improved SNR to permit quantification of Glx from the spinal cord We aimed to evaluate whether age is associated with changes in metabolite concentrations of the upper cervical cord, as is seen in the brain. Using a recently optimised MRS protocol NAA is a non-essential amino acid which is synthesised by neuronal mitochondria and found exclusively in neurones and their processes Interestingly, in healthy brain aging, reductions in tNAA have been widely reported in grey matter regions Glutamate (Glu), the major excitatory neurotransmitter in mammals plays a major role in the coordination of basic propulsive movement synergy for locomotion at the spinal level The observed association between age and Glx in the spinal cord is in keeping with previous MRS investigations in the brain which have consistently shown declining concentrations of Glu with older age in multiple brain regions including the frontal white matter, parietal grey matter, motor cortex, anterior cingulate cortex, hippocampus, basal ganglia and striatum et al. however found no differences in Glu and Gln concentrations between men and women in the corona radiata and mesial motor cortex et al, Glu levels were significantly higher in men than women at any stage of the menstrual cycle. Although the gender differences in spinal Glx concentrations observed in this study are in keeping with some earlier reports from the brain, further studies, with larger sample sizes will be needed to confirm the validity of this finding and to investigate whether spinal Glx levels vary with female hormone levels.An interesting observation in the current study was that of higher concentrations of Glx in men than women. MRS studies in the brain measuring Glx and Glu concentrations have differed on whether gender differences exist. Higher levels have been reported in men compared to women in the parietal grey matter and dorsolateral prefrontal cortex In the present study no evidence of association was seen between tCho, tCr and Ins concentrations in the spinal cord and age. Although changes have been reported in these metabolites in the brain with aging and have been interpreted as reflecting changes in glial proliferation, those changes are not thought to occur uniformly in the brain, with regional variation commonly reported. tCho concentrations have been reported to be higher in the corpus callosum, parietal lobe, frontal grey matter and pons in older people To the best of our knowledge, this is the first report of associations between metabolite levels and age within the spinal cord. We have shown a possible effect of aging on tNAA and Glx levels, which should be taken into consideration when planning serial MRS imaging of the spinal cord in clinical and research settings. However, it will take further longitudinal studies to determine the rate of change in metabolites over time in healthy aging and whether metabolite concentrations decline at differing rates in spinal grey matter and white matter. Due to exploratory nature of the study, our sample size was relatively small and absolute metabolite concentrations observed within of our cohort should therefore be interpreted with some caution until future work, using larger sample sizes, further characterises absolute metabolite concentrations by age group. Future experiments should also allow additional scanning time for the inclusion of an experimentally measured macromolecular spectrum, as this has been shown to improve the accuracy of spectral quantification Table S1Details of RF pulses (default voxel dimensions). *Maximum B1 of the coil used was 13 µT.(DOCX)Click here for additional data file.Table S2Details of RF pulses (default voxel dimensions).(DOCX)Click here for additional data file.