This study aimed to elucidate cardiovascular loads (CVL) in construction workers during work and leisure by relative heart rate (RHR) over several days. Furthermore, we sought to evaluate the level of CVL in relation to individual factors, work ability, musculoskeletal pain and subjective general health. From a group of 255 construction workers responding to the baseline questionnaire, the CVL during work and leisure time was determined by recording RHR in 42 workers over 3–4 days. Almost 60% of the workday was spent below 20% RHR. The mean RHR during work for all participants was 16% RHR, with large differences between professions. On average, the 42 workers spent 14% of the workday at a RHR above 33%, and four subjects (10%) had a mean RHR above 33% during work. Eight (19%) of the participants had a mean length of their workday exceeding calculated maximal acceptable work time. Seven persons (17%) experienced on average one or more episode(s) of 5 min or more continuously above 33% RHR. The cardiovascular load at work was significantly associated with age and  Physical demands at work are considered an important risk factor of several musculoskeletal disorders  and measured demands during one single period. Two recent studies have objectively measured cardiovascular load for more than one day within other occupations commonly considered as physically demanding, female hospital cleaners [Work in the construction industry is generally considered physically demanding and construction workers show high prevalence of musculoskeletal pain ,10. Highcleaners  and an ucleaners . Still, It seems a paradox, that physical activity during leisure time is considered health-promoting and essential for maintaining and increasing physical capacity and work ability ,21,22, w2 uptake (2max) or % of maximum HR (HRmax) for the individual. HRmax depends on age and there are several formulas for calculating HRmax [max approach is the fact that resting HR (HRmin) never is zero, hence the percentage of HRmax does not represent load above the resting state. Furthermore, some physically fit individuals exhibit very low HRmin, hence their range of HR-variation (HR reserve) is larger for a given age. The relative HR (RHR or % of HR reserve) takes HRmin into account by subtracting HRmin from the HR measured during work and from HRmax. There is a linear relationship between HR and oxygen consumption during a bout of work or exercise . Hence, ng HRmax . One proIn the present study we aimed to elucidate cardiovascular loads in construction workers during work and leisure by relative HR (RHR) from objective measures over several days. We further evaluated the level of cardiovascular load in relation to individual factors, work ability, MSDs and general health.Subjects for this study were recruited from three large construction enterprises during April–September 2014. A total of 579 employees were invited to fill out a baseline questionnaire and give their consent to participate. Two hundred and fifty-five answered the baseline questionnaire and 161 stated they were also willing to participate in ambulatory technical measurements. From the 161 a sample of 57 was invited to the technical recordings presented in this paper. The 57 were selected to best fit logistics (based on availability and work schedules) and to give a reasonable representation of occupational titles. The construction sector consists of a high proportion of men, hence all participating volunteers investigated by technical measurements in this study are men. Individual characteristics for participants in the technical measurement group and the questionnaire group are shown in All participants were informed of the purpose and content of the study and signed an informed consent prior to participation. The study was conducted in accordance with the 1964 Helsinki declaration and approved by The Regional Committee for Medical and Health Research Ethics in Norway (2014/138/REK south-east D).Participants volunteering for the study answered first the baseline questionnaire before proceeding to a physical examination (including weight and height measurements) carried out by a physician or nurse. If none of the exclusion criteria were present, the participant carried out a physical fitness test determining aerobic fitness and muscular strength. On a succeeding (separate) workday morning, instruments for ambulatory technical measurement were attached. The instruments for recording physical exposure sampled 24-h a day for three to four consecutive days or until deliberately removed by the subject. Measurement were targeted to include at least two working days. On the first day of measurement all participants were given a small diary where they should note time of day they got out of bed in the morning, started work (if workday), ended work (if workday), and went to bed at night for the days they were measured. Additionally, if the instruments at any time were detached subjects were instructed to note time periods when the monitor was not worn. They were also given extra electrodes and instruction on how to attach the measurement equipment if it detached unintentionally. The following self-reported measures from questionnaire were included in this study: seniority, height, weight, smoking status, perceived exertion at work , work abSmoking status was determined by the question: “do you smoke or have you ever smoked?” with the four response alternatives: No, never (0), yes, but not anymore (1), yes, occasionally (2) and yes, every day (3). The responses were dichotomized in to non-smoking (0–1) and smoking (2–3). The question “how physically demanding do you normally find your work?” with a response scale of 13 categories, ranging from “not exhausting at all” to “maximally exhausting” measured self-reported exertion during work . Work ability was measured by a single item taken from the Work Ability Index: “current work ability compared with lifetime best”. The score range for this question is from 0 (“completely unable to work”) to 10 (“work ability at its best”). This single item has previously shown strong predictive value for health outcomes . Self-perceived health was measured using the question: “How is your general health at present?”. Participants had five response alternatives ranging from excellent to poor . Musculoskeletal pain in neck, shoulders, back , elbow, hip, knee and foot/ankle were measured by assessing pain intensity and duration during the previous four weeks. Pain intensity was classified by participants to be no pain (0), mild pain (1), moderate pain (2) or severe pain (3) with the pain duration alternatives 1–5 (1), 6–10 (2), 11–14 (3) and 15–28 (4) days. From these answers a pain score was calculated, ranging from 0 (no pain × no duration) to 12 (severe pain × 15–28 days). A musculoskeletal complaint-severity index (MSI) was computed as a mean of the pain scores for all pain sites . Leisure-time physical activity level was determined by a single item. The participants reported which of the following activities levels that corresponded best to their own level the previous four weeks: (1) Almost completely inactive ; (2) Some physical activity at least four hours per week ; (3) Regular activity ; (4) Regular hard physical training for competition several times per week .2max based on the Åstrand nomogram [Aerobic fitness was established using a standardized cycle ergometer test  . Based onomogram . Handgrip strength was tested according to standardized procedures  by a hanHeart rate recording was carried out with, Actiheart 4 , a small chest-worn monitoring device consisting of two clips attached to standard electrocardiogram electrodes  placed at the apex of the sternum and at the left intercostals at the level of the 6th and 7th costae . Before Based on the diary data, each day was categorized into periods: before work, work, after work, sleep and leisure (off days). In this study the periods analyzed were work time and leisure time on work days (before and after work). Sleep periods and leisure on off days are not reported here. To be eligible in the analysis participants needed to have valid measurement periods (work or leisure) lasting four hours or longer than ≥75% of the length of a normal period. The definition of a normal period was the average of the measured periods. Data were also excluded if beat error exceeded 50% for a measurement period, defined as HR <35 or >230 bpm or >15% difference between two succeeding beats. In addition, all measurement periods were visually checked. Data for the valid time periods within work and leisure categories were then aggregated and averaged for each individual. To evaluate the relative cardiovascular load the RHR during work and leisure respectively, was calculated as follows:max is given by 208 – 0.7 × age [min entered into the equation was a sex- and age-adjusted value obtained from a Norwegian population study (HUNT3) [work and HRleisure in the equation is the mean HR measured for the respective periods. In this equation the HR.7 × age . The HRm (HUNT3)  , the variable with highest predictive value was used in the analysis. For the statistical analyses IBM SPSS Statistics 23  was used. Significance level was set at p = 0.05.Normal distribution of independent variables was tested by the Shapiro-Wilk tests of normality. Differences between questionnaire and technical measurement groups and differences in percentage distribution of RHR ranges between work and leisure were tested with independent samples p < 0.05) differences in smoking status and gender distribution between the questionnaire group and the technical measurement group, with more men and smokers in the latter , too many measurement periods with beat error above 50% or unfulfilled length of measurements criteria. Hence, the sample available for analysis of cardiovascular strain at work was 42. Two subjects did not have any valid measurements outside working hours, thus 40 subjects were available for leisure time HR analysis. A total of 85 days of work were measured with a mean length of measured workday being 8.1 h (±2.2). For leisure time measurements the total was 81 days with a mean length 7.3 h (±2.6). The average RHR during work for all participants was 16.4% (±11.4). The distribution within different ranges of RHR showed that for 58.6% (±28.5) of the work day RHR levels were lower than 20% RHR. Furthermore, for 19.5% (±13.3) and 11.1% (±10.2) of the day RHR level ranged between 20%–29% RHR and 30%–39% RHR, respectively. A small proportion of the day, 5.0 percent (±7.4) of the day in a RHR between 40%–49% and 1.9% (±3.6), the work was accompanied by cardiovascular demands in range of 50%–59% RHR. For the group as a whole, 14.4% (±18.4) of the working day was spent above 33% RHR. Out of 42 subjects, 10% (4) did have a mean RHR above 33% during work, 90% (38) did not. Foremen and project leaders spent fewest minutes above 33% RHR during work (6.5 ± 8.3 min and 22.9 ± 42.4 min), see The average MAWT for this sample was 14.1 (±8.4) hours, while mean length of work shifts was 8.1 (±2.2) hours. Eight (19%) subjects exhibited work lengths exceeding mean calculated MAWT , 34 (81%) did not. Foremen had highest and bricklayers had the lowest MAWT, see Seven persons had on average one or more episode per day of RHR above 33% continuous for 5 min duration or more (exertion periods). Five persons showed one or more episodes of 10 min or more and one person had episodes of RHR above 33% continuously for 15 min or longer. See p < 0.01. Moreover, when compared to work, the distribution of the leisure time periods spent in different RHR ranges showed that a significantly higher proportion was spent below 20% RHR  and significantly lower proportions in RHR ranges of 20%–29% , 30%–39%  and 40%–49% , see The mean load during leisure time was significantly lower compared to work, with mean RHR of 9.2% (±7.8), p < 0.05), indicating decreasing levels of RHR with increasing age. Seniority (years in profession) showed similar tendency, however, did not reach significance criteria . In the adjusted multiple linear regression model age remained significant . Seniority was strongly  correlated to age. Unadjusted linear regression analysis showed RHR during work to be significantly associated with age . Higher levels of 2max was associated to lower RHR during work. The 2max variable remained significant  in the adjusted analysis. Higher self-reported physical activity levels did also seem to lower RHR during work, but was not significant. Further, we were unable to find any association between RHR and hand strength.RHR was dependent on aerobic fitness level, showing a significant association to estimated p = 0.076) and was cancelled out in the adjusted analysis.There was a trend towards lower reported work ability with higher RHR during work. However, this did not reach customary criteria for statistical significance . This relationship was cancelled out in the adjusted analysis. Self-reported perceived exhaustion at work was signficantly associated to levels of RHR. Those reporting high level of perceived exhaustion did also tend to have higher cardiovascular loads during work  had a mean RHR above the recommended threshold of 33% RHR and for the whole group approximately 70 min were in load ranges above this level of cardiovascular load on an average workday. Carpenters, henchmen and bricklayers represented professions with the highest mean RHR. Compared to carpenters and bricklayers, henchmen did have more episodes of both continuous exertion and rest periods, indicating a somewhat different work pattern. Foremen and project leaders seemed to have less physical demands, with lowest levels of RHR, no continuous periods of exertion and highest number of rest periods. This reflects that different professions within construction will have different physical demands, which should be taken into consideration when evaluating the construction sector. The physical demands of construction supervisors  have previously been very scarcely investigated .et al. work in progress). This finding indicates that work behavior may be altered the on first day of measurement. Still, compared to the general working life [Previous studies investigating masons, bricklayers and plasterboard work (carpenter task) during a full workday have found mean RHR ranging from 21 to as high as 39% RHR, with factors as brick and plasterboard sizes as important load varying factors ,16,17. Fing life , construThe mean MAWT for this sample was 14 hours, and a mismatch between length of workday and MAWT were found in approximately 1 in 5 individuals. A mean RHR of 24.4% would represent MAWT equal to the average workday of 8.1 h. Thus, all professions were within these limits. With this said, the distribution within RHR ranges and exertion/rest periods does imply that construction work is not a physiologically steady-state situation, but is rather fluctuating between levels of cardiovascular load. Therefore, this kind of work may not fulfil the assumptions behind the MAWT-equation, which is set pace ergometer cycling . Hence, Age was significantly associated with RHR during work. Increasing age was associated with reduced cardiovascular load, indicating that younger workers had higher cardiovascular loads during work, compared to older workers. Similarly, Gupta and colleagues found seniority to be significantly more prevalent in workers with low RHR during work, compared to those with high RHR [2max decreased significantly with age in our sample (results not shown), and there were no significant age difference between professions measured.high RHR . Possiblhigh RHR . It is p2max was significantly associated with RHR during work. An increase in aerobic fitness will result in work being less physically demanding, with lower RHR as long as the level of physical demands remains unchanged. Even though there are differences in physical demands between professions, the relative demands for each person will be individually determined by level of fitness. We recorded large individual differences in the relative physical demands within the same profession. There is an ongoing discussion concerning the paradoxical effect of physical exercise: seemingly negative effects of high physical activity at work and the health-improving effects of physical activity in leisure time ,45. Our et al. found that women needing to exceed their physical demands regularly during work had reduced general health and increased level of musculoskeletal complaints, however, men had no such problems [Construction work has been associated to development of MSDs and reduction in work ability, and heavy physical work commonly is considered a major risk factor ,2. Our dproblems . Thus, tThe methods and design used in this study were chosen to provide a thorough objective description of the cardiovascular demands in construction work. The continuous measurement over several days provide a more solid foundation when describing general demands compared to studies with task or short period measurements, which may serve other purposes.max presented by Tanaka and coworkers [min is difficult at the workplace. Therefore, we based HRmin on the sex- and age-adjusted population means. The study participants were drawn from a variety of occupational titles within construction and will thereby give a good overall description of this work sector. However, they were male employees at Norwegian large-scale enterprises and data may not be generalized small enterprises and builders of private homes. In addition, there is possibility of selection bias of participants. From the 579 invited, 255 answered the questionnaire, 161 volunteered for technical measurement, and a sample of 57 were selected. Still, the participants monitored in the present study did not differ from the questionnaire group in any variable investigated, except smoking and gender. Concerning gender, there were only 18 females in total answering the questionnaire, hence females were weakly represented. However, at present this is the normal gender distribution in the construction sector. Long-term follow-ups are needed to determine the long term health effects of cardiovascular load during work. The formula for HRoworkers  producedCardiovascular demands in construction are characterized by mainly work in ranges of relative heart rate below 39%, with few continuous periods above one-third of capacity. Few minutes are spent in high load intensities needed to achieve training effect. Cardiovascular load differs between professions within construction and both age and aerobic fitness are individual factors influencing cardiovascular load at work.


Stem rust (SR) is one of the most economically devastating barley diseases in Kazakhstan, and in some years it causes up to 50 % of yield losses. Routine conventional breeding for resistance to stem rust is almost always in progress in all Kazakhstan breeding stations. However, molecular marker based approach towards new SR resistance genes identification and relevant marker-assisted selection had never been employed in Kazakhstan yet. In this study, as a preliminary step the GWAS (genome-wide association study) mapping was applied in attempt to identify reliable SNP markers and quantitative trait loci (QTL) associated with resistance to SR.Barley collection of 92 commercial cultivars and promising lines was genotyped using a high-throughput single nucleotide polymorphism  Illumina iSelect array. 6,970 SNPs out of 9,000 total were polymorphic and scorable. 5,050 SNPs out of 6,970 passed filtering threshold and were used for association mapping (AM). All 92 accessions were phenotyped for resistance to SR by observing adult plants in artificially infected plots at the Research Institute for Biological Safety Problems in Dzhambul region of Kazakhstan. GLM analysis allowed the identification of 15 SNPs associated with the resistance at the heading time (HA) phase, and 2 SNPs at the seed’s milky-waxy maturity (SM) phase. However, after application of 5 % Bonferroni multiple test correction, only 2 SNPs at the HA stage on the same position of chromosome 6H can be claimed as reliable markers for SR resistance. The MLM analysis after the Bonferroni correction did not reveal any associations in this study, although distribution lines in the quantile-quantile (QQ) plot indicates that overcorrection in the test due to both Q and K matrices usage.Obtained data suggest that genome wide genotyping of 92 spring barley accessions from Kazakhstan with 9 K Illumina SNP array was highly efficient. Linkage disequilibrium based mapping approach allowed the identification of highly significant QTL for the SR resistance at the HA phase of growth on chromosome 6H. On the other hand, no significant QTL was detected at the SM phase, assuming that for a successful GWASmapping experiment a larger size population with more diverse genetic background should be tested. Obtained results provide additional information towards better understanding of SR resistance in barley.The online version of this article (doi:10.1186/s12870-015-0686-z) contains supplementary material, which is available to authorized users. Puccinia graminis f. sp. Tritici, Pgt, is one of the most powerful factors affecting barley production in Kazakhstan. Sometimes annual yield losses exceed 50 % [Pgt in the US, including well characterized Rpg1 [rpg4 [Rpg5 [Rpg2, Rpg3, and rpg6 [Pgt pathogen races MCCF and QCCJ by using conventional bi-parental genetic mapping approach. In addition, in response to serious threat of the new and highly infectious race of Pgt (TTKSK) in Africa and Near East, also known as Ug99, Steffenson with coauthors were able to identify qualitative Pgt-TTKSK locus on the long arm of chromosome 5H [Barley is the second after wheat most important for Kazakhstan cereal crop with total annual grain yield of more than 2 mln tons . SR causzed Rpg1 , 4, rpg4g4 [Rpg5 , 7, and and rpg6 . Most ofosome 5H  showed tosome 5H .The other approach aimed for both known and novel agronomically important loci identification in cereals is based on AM, and high-throughput platforms with GWA scans . In barlIn Kazakhstan the genetic aspects of stem rust resistance were not studied well. Spring two-rowed barley represents over 90 % of all barleys in this country. Currently, despite regular use of conventional breeding procedures, there is no data regarding pathogen races classification in the region , and so http://bioinf.hutton.ac.uk/iselect/app/index.pl of the James Hutton Institute, Scotland). The site provides information of chromosomal positions for 3,129 markers while remaining 1,921 SNPs were markers with unknown (U) positions. Additional information for each individual U marker can be retrieved from the Triticeae toolbox website (https://triticeaetoolbox.org). The total genetic length of the whole genome based on the genetic distances between mapped markers was 990.9 cM. The number of SNPs per chromosome with known positions ranged from 306 on chromosomes 1H and 4H to 608 on chromosome 5H, and suggested that average coverage for each marker was 0.33 cM.The 92 spring barley accessions from Kazakhstan  between resistance at the HA and the SM phases, this result was in large discontent with allele status of Rpg1 gene  showed high resistance to the Pgt at the HA, and 33 out of tested 92 accessions showed complete resistance to the pathogen population at the SM stage of growth  suggesting that there are no significant associations for Pgt resistance in the MLM experiment. On the other hand, it is interesting to note that QQ plots in the GLM test showed a higher correction efficiency of the population structure in comparison with MLM-QQ plot , the GWAS mapping scans showed different results  shown that SNP SCRI_RS_216088 on 1H is part of the NB-ARC domain which is shared by a number of R genes in plants [In total, the GWAS allowed the identification of 17 candidate SNPs associated with SR resistance at two growth phases Table . The useIn this study GLM and MLM tests predictably showed different outcome, as the MLM is a statistically more strict approach, and no significant QTL was detected in former model after application of the Bonferroni correction. On the other hand, the results of MLM-QQ plots clearly showed overcorrection in the test due to the use of K and Q matrices. Meanwhile GLM-QQ plot shows nearly perfect distribution of scores with slight deviation from the reference line in the region of 0.75-1.50 Fig. . This rePgt resistance at the HA stage, but failed detecting any associations at the SM stage. Presumably, despite large set of SNP data, the studied 92 accessions is a too low number of samples for searching of a robust QTL of SR resistance in the particular environment. The genetic position of identified SNPs of the QTL at the HA stage did not coincide with chromosomal positions of known genes, such as Rpg1, rpg4/Rpg5, Pgt-TTKSK, and, therefore, they can be potentially new genetic factors related to SR resistance. Assuming that barley resistant mechanism to Pgt is triggered by pathogen races common in the Central Asia, particularly in Kazakhstan, the results add new insights in complex problem associated with resistance to SR.The resistance to SR is among most serious problems preventing barley grain yield increase in Kazakhstan. In this study we applied GWASmapping approach using 9 K SNP Illumina array for 92 local barley accessions and phenotypic scores for SR resistance at the different growth stages. The result shows that genome wide genotyping of spring barley accessions from Kazakhstan using the 9 K Illumina SNP array was highly efficient. The accessions were separated to three distinct subgroups and the level of the genetic diversity of total population was comparable to the results reported by Alqudah with co-authors . The AM The collection of 92 two-rowed spring barley accessions was selected from six different breeding stations of Kazakhstan, and it is part of larger collection, which earlier studied in field trials in seven regions of the country . The lisEvaluation of resistance of barley genotypes was done in the Dzhambul region of Kazakhstan  at the Research Institute for Biological Safety Problems. Plants were grown in two randomized replication rows, with 30 cm distance between the rows. Plots were inoculated in the spring at the tillering stage with a mixture of isolates representing the most prevailing races of the pathogen in Central Asia. To analyze the accumulation of infection and high disease pressure, every five experimental plots were sown with universally susceptible varieties. Assessment of stem rust disease conducted at heading time and seed milky-wax maturity phases was done using modified Cobb Scale , and meaRpg1 gene according to [DNA samples were extracted and purified from single seeds of individual cultivars using commercial kits . The DNA concentration for each sample was adjusted to 50 ng/ml. All samples were genotyped by the rding to , 4. Alsording to  at the Trding to , 28.k. The output from STRUCTURE was analyzed for delta K value (ΔK) in STRUCTURE HARVESTER [k values, Q-matrix for three identified clusters was developed. GWASmapping of QTL governing stem rust response in the set of 92 accessions was performed using 5,050 informative SNPs, GLM and MLM tests [The SNP dataset was filtered using a 10 % cutoff for missing data and markers with minor allele frequency ≥ 0.10 were considered for GWAS. Numbers of hypothetical groups ranging from k = 1 to 10 were assessed using 50,000 burn-in iterations followed by 100,000 recorded Markov-Chain iterations. To estimate the sampling variance of population structure inference, five independent runs were carried out for each ARVESTER . On the LM tests , 30, andLM tests .


Recommendations for cardiovascular disease prevention advocate lowering both cholesterol and low-density lipoprotein cholesterol systemic levels, notably by statin intake. However, statins are the subject of questions concerning their impact on male fertility. This study aimed to evaluate, by a prospective pilot assay, the efficacy and the toxicity of a decrease of cholesterol blood levels, induced by atorvastatin on semen quality and sexual hormone levels of healthy, normocholesterolaemic and normozoospermic men.Atorvastatin (10 mg daily) was administrated orally during 5 months to 17 men with normal plasma lipid and standard semen parameters. Spermatozoa parameters, accessory gland markers, semen lipid levels and blood levels of gonadal hormones were assayed before statin intake, during the treatment, and 3 months after its withdrawal.Atorvastatin treatment significantly decreased circulating low-density lipoprotein cholesterol (LDL-C) and total cholesterol concentrations by 42% and 24% (p < 0.0001) respectively, and reached the efficacy objective of the protocol. During atorvastatin therapy and/or 3 months after its withdrawal numerous semen parameters were significantly modified, such as total number of spermatozoa , vitality , total motility , morphology , and the kinetics of acrosome reaction (p < 0.05). Seminal concentrations of acid phosphatases (p < 0.01), α-glucosidase (p < 0.05) and L-carnitine (p < 0.05) were also decreased during the therapy, indicating an alteration of prostatic and epididymal functions. Moreover, we measured at least one altered semen parameter in 35% of the subjects during atorvastatin treatment, and in 65% of the subjects after withdrawal, which led us to consider that atorvastatin is unsafe in the context of our study.Our results show for the first time that atorvastatin significantly affects the sperm parameters and the seminal fluid composition of healthy men.NCT02094313.ClinicalTrials.gov:  In the current context of prevention of cardiovascular risks, a decrease of total cholesterol and low-density lipoprotein cholesterol (LDL-C) plasma levels is an important goal for public health ,2. StatiSterols are essential in male reproductive physiology, notably for steroidogenesis, spermatogenesis and fertilization -12. DuriSince lipid metabolism disorders are associated with impaired fertility  or alterThis study is a prospective pilot assay that evaluates the efficacy and the toxicity of a decrease of cholesterol blood levels induced by atorvastatin on the fertility of healthy men without confounding factors. For the first time the effects of atorvastatin were analyzed on human sperm parameters, accessory gland secretions, semen lipid composition and testosterone and gonadotropins systemic concentrations in normocholesterolaemic and normozoospermic subjects.Volunteers were recruited in accordance with the 1975 Helsinki declaration on human experimentation, under a protocol approved for research by a Regional Ethical Committee and the French Agency for the Safety of Health Products (AFSSAPS) and was registered in Clinical Trials Register with the identifier number: NCT 02094313. Written informed consent was obtained from subjects before inclusion in the study.The main objective of this pilot study was to estimate the toxicity and the efficacy of atorvastatin on fertility of normocholesterolaemic and normozoospermic men by analyzing its effects on sperm parameters. The secondary objective was to assess the evolution of gonadotropins and total testosterone plasma levels, lipid composition of sperm cells and seminal fluid, spermatozoa capacitation ability and accessory glands markers.Inclusion criteria were healthy men, 18 to 65 years old, with normal conventional semen parameters and negative semen culture according to the 1999 WHO standards  and to DConsidering this protocol as a pilot study to evaluate toxicity and efficacy, sample size estimation was fixed according to a one-/multi-stage Fleming design. This design with one group and multi-stages (between 1 and 5) can be seen as filtering steps leading to the “go/no go” decision type. They are among those most used in phase II trials in oncology but remain far more rarely implemented in other areas .With typThe safety/toxicity was evaluated by measuring the effects of atorvastatin on sperm parameters according to the 1999 WHO standards   for a power greater than 90% (1-β = 97%).Thirty-nine subjects were assessed for eligibility , unless otherwise indicated.Blood was kept on ice in heparin-coated tubes and then centrifuged 15 min at 1500 g at 4°C. Assays were performed on an automated clinical chemistry analyzer  based on enzymatic colorimetric  or electrochemiluminescence assays  according to the manufacturer’s instructions.The ejaculates were collected by masturbation after 3 to 5 days of abstinence. Immediately after liquefaction semen parameters were evaluated according to the WHO guidelines, 1999 . Sperm mFor semen lipid determination, samples were centrifuged at 500 g for 5 min. The resulting pellet containing the sperm cells was washed twice in Phosphate-buffered Saline (PBS) by centrifugation . The supernatant corresponding to seminal fluid was centrifuged  to remove cell debris. Lipids present in spermatozoa and in seminal fluid were extracted following a modified Folch procedure . The pho2). P-Tyr and acrosome integrity were measured after 5 min and 3 h of incubation. P-Tyr of P80 and P110 proteins, was monitored by western-blot using a monoclonal anti-phosphotyrosine antibody  and a mouse monoclonal anti α-tubulin antibody as indicated in a prior study [Pisum sativum agglutinin conjugated to fluorescein isothiocyanate  in PBS. Sperm cells were observed blindly using epifluorescence microscopy (×400). Sperm cells with an intact acrosome membrane show a marked fluorescence in the acrosome region  using parametric paired tests. The different parameters measured during and 3 months after the end of atorvastatin treatment were compared with initial values , by one-way paired ANOVA with Holm-Sidak multiple comparison post-test when the distribution was normal; if not we applied a nonparametric post-test (Dunn’s Multiple Comparison Test). For each comparison, the corresponding effectiveness was checked to verify the robustness of the analysis.Baseline clinical and biochemical characteristics of studied subjects are summarized in Tables The reduction of cholesterol level in serum did not significantly affect semen cholesterol and phospholipid levels, whether in spermatozoon or in seminal fluid Table . Howevervs. 60.9 ± 1.8% before, p < 0.05), with no effect either on progressive motility or on sperm motion parameters. When the effects were analyzed per subject, 2 out of 17 men were necro- and asthenozoospermic according to the WHO 1999 standards, notably after withdrawal of treatment  induced a significant decrease in serum levels of total cholesterol and LDL-C achieving the efficacy goal of the protocol. Atorvastatin treatment affected semen parameters significantly. It also decreased the total number of spermatozoa in ejaculate and sperm vitality, increased morphological abnormalities and total motility, and altered acrosome reaction kinetics. Seminal concentrations of acid phosphatases, α-glucosidase and L-carnitine were also decreased. All these results hint at deleterious effects of atorvastatin on testicular, prostatic and epididymal functions.At the end of the study, 9 subjects (53%) had an alteration of at least one semen marker; two men were necro-asthenozoospermic and had a low value of neutral α-glucosidase. We had specified that if one subject or more presented toxicity on sperm or accessory gland markers, the treatment was considered unsafe. The results obtained led to the decision to stop the protocol and to conclude in the efficacy but also the toxicity of atorvastatin for semen parameters. To further evaluate the fertilizing ability of spermatozoa, capacitation and acrosome reaction were measured in capacitating conditions. To our knowledge, fertilizing ability has never been studied in the context of a cholesterol-lowering therapy in humans. Atorvastatin had no significant effect on early (cholesterol depletion from plasma membrane) or terminal markers (P-Tyr levels of proteins P80 and P110) ,47 of caOur study showed that atorvastatin therapy does not alter cholesterol and phospholipid composition of human spermatozoa and seminal fluid. This is consistent with our previous study showing that normo- and hypercholesterolaemic men had similar amounts of cholesterol and phospholipids in semen . The samSimilarly, modifications of systemic cholesterol metabolism do not significantly modify cholesterol and phospholipid levels in semen. These data could explain the absence of effects of atorvastatin on systemic levels of total testosterone and gonadotropins. Nevertheless, sperm production and quality were decreased and prostatic and epididymal markers were altered. Such data indicate clearly that atorvastatin therapy affects testicular, epididymal and prostatic functions. Testis, epididymis ,52 and pWe could also consider that atorvastatin might induce local inflammation or oxidative stress with persistent effects on prostatic and epididymal secretion. A recent study has characterized prostatic acid phosphatases regulation in androgen-dependent and -independent manner , suggestvia these different products leading to alterations of intracellular signaling pathways in genital tract tissue or sperm cells, associated or not with an oxidative stress. Once again, animal models could be an interesting perspective to characterize atorvastatin effects on the genital tract.This persistent effect could indicate that atorvastatin effects on the genital tract could be also mediated by a messenger or an intermediary that persists after the end of therapy. In 2005, Niederberger raised the question of the adverse effect of atorvastatin on human fertility . One hypet al., in 1992  and changed their seminal fluid composition. These consequences occurred after only 5 months of treatment. Considering the long duration of statin treatment, for which the clinical benefit with respect to cardiovascular diseases is beyond question, potential negative consequences on reproductive function should be taken into account when deciding to initiate such a treatment, notably for young adults.The authors declare that they have no competing interests.HPR participated in the design of the study and its coordination, carried out semen assays and drafted the manuscript. FB participated in the design of the study, its coordination, in the inclusion of the patients and helped to draft the manuscript. BS participated in the design of the study, carried out the semen lipid and filipin assays and helped to draft the manuscript. SM carried out the carnitine assay. GG participated in the design of the study and helped to draft the manuscript. BP performed the statistical analysis and helped to draft the manuscript. GM performed blood biochemical analyses. ASG performed the physical examination of the patients, including testis evaluation. JRD helped to draft the manuscript. GG conceived the study and participated in its design and coordination and helped to draft the manuscript. LJ conceived the study and participated in its design and in the inclusion of the patients, and helped to draft the manuscript. IT conceived the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.Flow diagram. After a screening visit (visit 0) allowing inclusion and exclusion of subjects by the measurement of the clinical, blood and semen baseline parameters, serum and semen parameters of subjects were assayed in the laboratory during 4 visits corresponding to the beginning of atorvastatin treatment (10mg daily) (visit 1); a control visit to ensure good tolerance to treatment and its efficiency after 2 months of therapy (visit 2) and the measurement of the effects of a 5-month atorvastatin intake (visit 3) and the residual effects after 3 months of treatment withdrawal (visit 4).Click here for fileProspective studies analyzing statin effects on human gonadal steroidogenesis and semen quality. *after 6 months of therapy, ref.: reference, treat.: treatment, normochol.: normocholesterolæmic; hyperchol.: hypercholesteroleamic. Test: testosterone, Num: sperm number, Motil: sperm motility; Morph: sperm morphology; Acc. glands: Accessory glands; Cap: capacitation; AR: Acrosome reaction.Click here for file


We report here the first complete transcriptome analysis of the dorsal (dDG) and ventral dentate gyrus (vDG) of a rat epilepsy model presenting a hippocampal lesion with a strict resemblance to classical hippocampal sclerosis (HS). We collected the dDG and vDG by laser microdissection 15 days after electrical stimulation and performed high-throughput RNA-sequencing. There were many differentially regulated genes, some of which were specific to either of the two sub-regions in stimulated animals. Gene ontology analysis indicated an enrichment of inflammation-related processes in both sub-regions and of axonal guidance and calcium signaling processes exclusively in the vDG. There was also a differential regulation of genes encoding molecules involved in synaptic function, neural electrical activity and neuropeptides in stimulated rats. The data presented here suggests, in the time point analyzed, a remarkable interaction among several molecular components which takes place in the damaged hippocampi. Furthermore, even though similar mechanisms may function in different regions of the DG, the molecular components involved seem to be region specific. Therefore, the time point chosen was sufficiently late in relation to the initial stimulation, but before the onset of spontaneous seizures; therefore reflecting molecular processes that most likely are involved in epilptogenesis.The stimulation of the PP during electrode placement surgery evoked depolarization of DG granular cells, and electrode positioning was adjusted for maximal amplitude of recorded depolarization. Furthermore, stimulation induced the occurrence of populational spikes . After aExtensive pyramidal neuronal loss was observed in CA1, CA2 and CA3 in tissue sections from rats that were stimulated when compared to Sham controls . The dor® 2500 platform. The sequencing run produced a total of 1,355,974,797 100-bp reads, with 94% of bases over Q30. An average of 84.7 million sequences were produced for each sample. Sequences were aligned with TopHat2 to Rattus norvegicus Ensembl Rnor 5.0 assemble. The average sequence alignment rate was 91.2%. HTSeqCount and DESeq2 packages were used for the estimation of gene expression and statistical analysis. After data processing, a list of differentially expressed genes with p < 0.05 (after correction for multiple tests) was generated. The EdgeR package was used to calculate the Biological coefficient of variance of the present data (BV = 0161), and the RNASeqPowerTotal RNA was extracted from each sample, converted into cDNA libraries using Illumina TruSeq Stranded mRNA LT and sequenced in a HISeqfor a complete list refer toWe found a total of 2,621 differentially expressed genes (p < 0.05) when comparing control dDG with stimulated dDG: 1,425 genes were up-regulated and 1,196 were down-regulated in stimulated samples. For the vDG we found 2,053 genes differentially expressed when comparing control vDG with stimulated vDG: 1,157 genes were up-regulated and 896 were down-regulated in the stimulated condition  Adgre1 JAK3/STAT5 pathway, and the morphological characteristics of the tissue observed by Nissl staining  were differentially regulated in different portions of the DG, indicating distinct axon guidance mechanisms functioning throughout the dorsal ventral hippocampal axis. Eph receptors and their respective ligands, the Ephrins, are also an important class of cell navigation and axon guidance molecules in the nervous systemThe presence of various differentially regulated genes involved in axonal guidance such as class 3 Semaphorins, Ephrins and Integrins is noteworthy in the present study. Moreover, different members of these families of molecules are uniquely differentially regulated in the dorsal or the ventral portions of the DG. Semaphorins consist of a family of more than 20 secreted and membrane-bound proteins that may function as guidance cues during development and in the adult brain2324Dcx (doublecortin) in both the dDG and vDG indicates the presence of migrating newly generated neuroblastsMag (Myelin associated glycoprotein), Mbp (Myelin basic protein) and Plp1, present only in the vDG. This may indicate a greater extent of sprouting and rewiring taking place in the vDG, leading to an increase in the myelination process.An increase in neurogenesis of DG granule cells is a well-known phenomenon associated with epilepsyalpha 3 and beta 2) and a reduction of other subunits (such as the delta and rho-3) in both regions of the DG, as well as a down-regulation of the Gabra5  in the dDG. Furthermore, changes in voltage-gated ion channels may also have an important role in the increase of excitability in the context of epileptogenesis. We observed an increase in the expression of Scn4a (Nav1.4), in both the dDG and vDG, a specific increase in Scn9a (Nav1.7) in the dDG and an increase in Scn7a (Nav2.1) in the vDG. The sodium channel isoform Scn4a is typically found in skeletal muscleScn9a is associated with dorsal root gangliaScn8a (Nav1.6) had an average of 9,285 counts; whereas, Scn4a had an average of 285 counts and Scn9a an average of 154 counts. Nevertheless, these channels were significantly up-regulated in stimulated animals, suggesting an aberrant expression of sodium channels. Furthermore, their differential regulation may indicate some participation in the epileptogenic process. Voltage-gated potassium channels are a diverse family of genes that also have a key role in the regulation of neuronal excitabilityKcnc2 (Kv3.2) was reduced in the dDG. It is worth noting that changes in the expression of such channels may have significant effects on neuronal firing, thus, rendering neurons more susceptible to seizuresA common hypothesis for the induction of epileptogenesis is a change in the balance between excitation and inhibition in neural networks. It has been proposed that one possible mechanism for this change in balance would be a preferential loss of GABAergic neurons3133Pdyn  a member of the opioid familySst (somatostatin) exclusively in the vDGPdyn is also associated with seizure suppression activity in the perforant pathway stimulation modelNeuropeptides are an important class of molecules involved in the regulation of neural tissue excitabilityApoENervous tissue contains high levels of cholesterol, and because cholesterol carrier proteins do not cross the intact blood brain barrier it is synthesized locally in the brainThe transcriptome data produced and analyzed in the present study reveal the functional profiles and possible components of the molecular mechanisms that most likely are involved in epileptogenesis in an animal model that displays HS. The gene ontologies enriched in the present dataset implicate several biological processes such as immune-response, the interplay between neural and immune systems, cell death, neural circuitry re-wiring, changes in cell excitability, neurotransmission and metabolic changes. All of these processes are likely to play a role, probably simultaneously, in the induction of epileptogenesis. Of particular interest is the possibility of interplay between the immune system and the granule neurons. This may involve signaling pathways, trophic molecules, cell migration and cell projection guidance molecules, such as those that attract and guide the migration of immune-system cells. These mechanisms may change the migration and projection pattern of neural cells, contributing to the generation of ictogenic tissue.Many individual processes mentioned above have been previously associated to some degree with epileptogenesis. However, the most important contribution of the present study is that by generating a large and comprehensive dataset, one can more precisely identify specific molecular components of the many biological systems likely contributing to the structural and functional changes that take place in the damaged hipppocampus. Furthermore, the present data also indicate that even though similar mechanisms may be found in different regions of the DG, the components involved seem to be region specific. Finally, our results identified a large number of novel potential targets that may ultimately help inhibit the epileptogenic process.ad libitum access to standard rodent chow and water. All procedures were performed in accordance with the ethical standards for animal experimentation at the University of Campinas-UNICAMP. The experimental protocol was approved by the UNICAMP animal research ethics committee, which evaluates experimental protocols according to current accepted ethical practices and legislation regarding animal research in Brazil .In the present study, three-month-old male Wistar rats (n = 8) were used. Rats were housed under a 12 h/12 h light cycle on a ventilated rack with Rats were anaesthetized with an isoflurane/oxygen mixture (2%/98%) at 2 L/min using an acrylic induction box. We maintained anesthesia throughout the entire surgical procedure with the aforementioned mixture and administration rate by using a mask adapted to a stereotaxic apparatus. After a deep anesthesia state was verified by the lack of pedal and corneal reflex, we positioned the animal on a stereotaxic apparatus . The skull was exposed by an incision on the scalp, the Bregma was localized and bone perforations were performed in the following coordinates: +4.5 mm lateral, −7.6 mm posterior; −4.5 mm lateral, −7.6 mm posterior; +2 mm lateral, −3 mm posterior and −2 mm lateral, −3 mm posterior. We performed three perforations in the spaces between the aforementioned coordinates for the fixation of stainless steel screws. One additional perforation was performed 2 mm posterior to the lambdoid suture, in the midline for the placement of a reference electrode , the reference electrode was lowered just over the surface of the dura mater.®, 2 channel, 10× gain, dc coupled, 0.06 Hz high-pass filter, 10 kHz low-pass filter) connected to a main amplifier , and the signal was digitalized at 10 kHz (NIDaq). After electrode and screw positioning, dental acrylic cement was applied in order to hold and stabilize all elements.Bipolar stimulation electrodes  with a 1-mm separation were positioned into the perforant pathway . Monopolar recording electrodes  were positioned into the DG . We optimized the final positioning of the recording and stimulation electrodes based on polarity, occurrence of population spike and maximum amplitude of evoked potentials in the DG 49. We pe® Astro-Med S88 stimulator. We recorded DG field potentials throughout the stimulation with the same configuration described for the electrode placement surgery . They were quickly decapitated and the brain was immediately removed and frozen at −60 °C using dry ice and n-hexane.®). These, were immediately stained with Cresyl Violet, dehydrated with an ethanol series and stored at −80 °C. The DG was divided into dorsal and ventral regions according to previously described morphological criteria®) system, and tissue was mechanically collected in separate tubes using a surgical microscope and micro-forceps  according to the manufacturer instructions. Each sample had different identifier barcodes, which allowed many samples to be sequenced in the same lane. We sequenced cDNA libraries in a HiSeq® 2500 (Illumina®) in High Output mode, producing 100-bp, single-read sequences. The sequencing run produced a total of 1,355,974,797 100-bp reads, with 94% of bases over Q30. An average of 84.7 million of sequences was produced for each sample. We aligned sequences with TopHat2 (http:// http://ccb.jhu.edu/software/tophat/index.shtml) to Rattus norvegicus Ensembl Rnor 5.0 assemble. The average sequence alignment rate was 91.2%. The HTSeqCount and DESeq2 packages  were used for the estimation of gene expression and statistical analysis. HTSeqCount performs the counting of aligned sequences to genome elements, such as exons and genes. DESeq2 employs a negative-binomial distribution for data normalization, corrects for outliers and the contribution of low expression genes for the analysis of variance, and it uses differential expression statistical Wald tests and corrects for multiple tests employing the Benjamini and Hochberg procedures. After data processing, a list of differentially expressed genes with a statistical significance set at a p < 0.05 (after correction for multiple tests) was generated. We used the differentially expressed genes list for gene ontology analysis with the Metacore® software (Thomas-Reuter).We extracted RNA from microdissected tissue samples with Trizol®) following the manufacturer instructions. Relative gene expression was analysed using the 2−ΔctΔct method, for this analysis, primers pairs for the genes: c3, cd18, socs3, mbp, npy, snc4a, sema3a, sema3c, dcx, stat5, adger1, apoe, cdk1, cyp51a1, fdft1, pdyn, sst and y1, were designed using NCBI primer design tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). As endogenous controls the geometric mean of the ct of the genes ssr2, ankyf and smntl2 were used. For the PCRs, the Sybr Power Plus Master Mix kit (Thermo®) was used following the manufacturer instructions in a Applied 7500 real-time PCR equipment (Applied Biosystems®). Following PCR amplifications, a melting curve was analysed for each gene, and a single TM peak was observed.Total RNA that was previously extracted from microdissected tissue samples (200 ng per sample) was used for cDNA synthesis using Vilo cDNA synthesis kit .


Considering limitations of liver biopsy for diagnosis of nonalcoholic liver disease (NAFLD), biomarkers’ panels were proposed. The aims of this study were to establish models based on serum adipokines for discriminating NAFLD from healthy individuals and nonalcoholic steatohepatitis (NASH) from simple steatosis.This case-control study was conducted in patients with persistent elevated serum aminotransferase levels and fatty liver on ultrasound. Individuals with evidence of alcohol consumption, hepatotoxic medication, viral hepatitis, and known liver disease were excluded. Liver biopsy was performed in the remaining patients to distinguish NAFLD/NASH. Histologic findings were interpreted using “nonalcoholic fatty liver activity score.” Control group consisted of healthy volunteers with normal physical examination, liver function tests, and liver ultrasound. Binary logistic regression analysis was applied to ascertain the effects of independent variables on the likelihood that participants have NAFLD/NASH.Decreased serum adiponectin and elevated serum visfatin, IL-6, TNF-a were associated with an increased likelihood of exhibiting NAFLD. NAFLD discriminant score was developed as the following: [(−0.298 × adiponectin) + (0.022 × TNF-a) + (1.021 × Log visfatin) + (0.709 × Log IL-6) + 1.154]. In NAFLD discriminant score, 86.4% of original grouped cases were correctly classified. Discriminant score threshold value of (−0.29) yielded a sensitivity and specificity of 91% and 83% respectively, for discriminating NAFLD from healthy controls. Decreased serum adiponectin and elevated serum visfatin, IL-8, TNF-a were correlated with an increased probability of NASH. NASH discriminant score was proposed as the following: [(−0.091 × adiponectin) + (0.044 × TNF-a) + (1.017 × Log visfatin) + (0.028 × Log IL-8) − 1.787] In NASH model, 84% of original cases were correctly classified. Discriminant score threshold value of (−0.22) yielded a sensitivity and specificity of 90% and 66% respectively, for separating NASH from simple steatosis.New discriminant scores were introduced for differentiating NAFLD/NASH patients with a high accuracy. If verified by future studies, application of suggested models for screening of NAFLD/NASH seems reasonable. Ischemic heart disease, cerebrovascular accident, chronic kidney disease, diabetes mellitus, and liver-related morbidities (comprising liver transplantation and hepatocellular carcinoma) are among the NAFLD complications.2Nonalcoholic liver disease (NAFLD) is a common cause of cirrhosis and liver-related morbidity and mortality. It is already considered the hepatic manifestation of metabolic syndrome. The prevalence of NAFLD is increasing worldwide due to the alterations in lifestyle and the epidemic of obesity and insulin resistance (IR). Accumulation of visceral adipose tissue (VAT) and the development of IR seem to be pivotal to the steatohepatitis (NASH) and concomitant fibrosis.2 A combination of clinical data and serum markers that may be related to the pathogenesis of NAFLD was applied to design these panels. There is still paucity of evidence on accuracy of these models. Adipokines that are hormones secreted by VAT are implicated in development of obesity and pathogenesis of IR.1 Recently, there is great enthusiasm in studying their potential role in the pathogenesis of NAFLD. Notably, previous studies showed the relationship of mentioned adipokines with NAFLD/NASH.9 In this research, we aim to evaluate the association between some important adipokines, in particular adiponectin, visfatin, resistin, hepcidin, as well as inflammatory cytokines of IL-6, IL-8, and TNF-α with NAFLD.Liver biopsy remains the gold standard for precise diagnosis of NAFLD in patients. However, application of this procedure is limited in clinical setting due to invasiveness, cost, concerns about sampling error, and potential serious complications. Therefore, unraveling an alternative to accurately diagnose patients with NAFLD is crucial. In this regard, various panels of serum biomarkers have been proposed in different cohorts of NAFLD populations.The aims of this study were: to assess the correlation of clinical parameters, mentioned serum adipokines, and inflammatory cytokines with the presence of NAFLD/NASH; to establish a model to discriminate NAFLD from healthy subjects and NASH from simple liver steatosis.The study was conducted according to ethical standards for human experimentation (Helsinki Declaration). The ethics committee of the hospital approved the study protocol (No: 8861). The purpose of the study was explained to the participants and they were enrolled in the study after filling out the written consent.11 The upper normal limit for serum aminotransferases was considered 40 units per liter.12 Individuals with evidence of alcohol consumption, hepatotoxic medication, viral hepatitis, and known liver disease were excluded (Phase 2).13 The remaining patients in Phase 2 were presumed to have NAFLD. Liver biopsy was performed in this group to document NAFLD before final enrolment (Phase 3). Control group consisted of age and sex-matched healthy volunteers who accompanied the patients to the gastroenterology clinic. The gastroenterologist examined the controls to evaluate their health status. They were enrolled into the study if there was no evidence of fatty liver at their liver ultrasound examination and abnormal routine laboratory investigation including liver function tests. A statistician who was unaware of participants’ data used the block-matching method (with control to case ratio of 1:1) for the matching of the controls.This case-control study was conducted in a general university hospital between 2012 and 2014. The patient group consisted of subjects with persistently elevated serum aminotransferase levels and well-defined criteria of fatty liver in ultrasound examination. They were consecutively admitted to the hepatology clinic (Phase 1).11Fatty infiltration in liver scatters the beam of ultrasound more than normal liver tissue; therefore, the fatty liver appears hyperechogenic in ultrasound examination. The comparison of echogenicity is required with internal structures known to be void of fat, such as the kidney. In this study, the radiologist obtained the sagittal view of liver right lobe and right kidney for the evaluation of fatty liver.P = 28%) according to the previous studies, α=0.05, and d = 0.12, the sample size was calculated as 54 in NAFLD group.11The sample size was determined by a statistical power analysis. Considering the mean prevalence of NAFLD , adiponectin, visfatin, hepcidin, resistin, IL-6, IL-8, and TNF-α concentrations. All of the measurements were performed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. To minimize the laboratory errors, the same operator performed the whole assay, from the beginning to the end, and room temperature, air humidity, and incubator temperature were controlled. All the measurements were performed in duplicate. The intra-assay coefficient variations were less than 12%. The laboratory investigators were blinded to the case or control status of participants. The following ELISA kits were used in this study: human adiponectin and visfatin ELISA kits , resistin , hepcidin , IL-6 , IL-8 , and TNF-α . Other serum assays including fasting blood glucose, insulin, lipid profile , and LFT  were performed based on methodology of previous studies.15Percutaneous liver biopsy was performed in patients with NAFLD using true cut needle (G14). The acceptable liver biopsy sample size was considered sample with 10-mm length or containing at least 5 portal tracts after fixation in formaldehyde (10%) and staining. Hematoxyline-eosin stain was used for the evaluation of necroinflammation. A pathologist who was blinded to the patient data interpreted the slides. The degree of liver steatosis, lobular inflammation, and fibrosis were defined based on “nonalcoholic fatty liver activity score” (NAS). The patients with score equal to 5 or higher were presumed to have NASH. Those with scores equal to 2 or lower were considered to have simple liver steatosis.t test and χ2 tests were applied to assess differences between groups, where appropriate. Binary logistic regression analysis using standard model was applied to ascertain the effects of independent variables  on the likelihood that participants have NAFLD/NASH. Standardized correlation coefficients (beta) with 95% confidence intervals (CI) were calculated. The biomarkers that were independently associated with NAFLD/NASH were selected for discriminant function analysis (DFA). Kolmogorov–Smirnov test was used for the assessment of normality distribution of biomarkers. In order to transform the distribution of non-normal variables to normal, the logarithm of non-normal distributed biomarkers was calculated and entered for DFA. This analysis determined the weight of the biomarkers that discriminated between study groups by defining the standardized canonical discriminant function coefficients. Finally, equations were developed based on DFA to calculate the discriminant scores, by weighted combination of discriminating biomarkers. Receiver operating characteristic (ROC) analysis was performed to determine the threshold values of discriminant scores for differentiating NAFLD from control group and NASH from simple liver steatosis. The best cut-off values were determined in order that the sum of sensitivity and specificity was the highest. All statistical analyses were performed by SPSS, version 17 . The probability of difference between variables was considered statistically significant if 2-sided P value was less than 0.05.Continuous variables were reported as mean (± standard deviation) and categorical variables were shown as count (percent). For univariate analysis, Seventy participants suspected of having NAFLD were evaluated from September 2012 to September 2014 (Step 1). Reasons for leaving out were patient unwillingness to participate in the study (n = 8), normalization of ALT during the lead-in phase (n = 6), autoimmune hepatitis (n = 1), and viral hepatitis (n = 1) (Step 2). Finally, 54 biopsy proven NAFLD patients were included in the study (Step 3). The comparisons of clinico-demographic and laboratory data between patients and controls are demonstrated in Table The mean (±SD) NAS score was 4.87 ± 1.71) in the NAFLD group. The percent of the patients with an NAS score of 5 or more (those with steatohepatitis) was 31.7%. The percent frequency of patients with steatosis grade of less than 33% was 66.7%; meanwhile, the frequency of those with steatosis grade of more than 33% was 33.3%  + (0.022 × TNF-α) + (1.021 × Log visfatin) + (0.709 × Log IL-6) + 1.154].The classification result of DFA showed that 86.4% of original grouped cases were correctly classified. ROC analysis depicted that the discrimination score threshold value of −0.29) yielded a sensitivity and specificity of 91% and 83% respectively, for separating NAFLD from healthy subjects  + (0.044 × TNF-α) + (1.017 × Log visfatin) + (0.028 × Log IL-8) − 1.787].DFA showed that 84% of original and 77% of cross-validated grouped cases were correctly classified. ROC analysis demonstrated that the discrimination score threshold value of −0.22) yielded a sensitivity and specificity of 90% and 66% respectively, for separating NASH from simple steatosis  in the pathogenesis of NAFLD is of great value. Designing therapeutic approaches by the modulation of associated adipokine might have great impact on disease burden. Noteworthy, aminotransferase levels fluctuate in the course of NAFLD; therefore, the results of this study need to be evaluated in further prospective trials.There is currently no defined “normal range” for serum adipokines. Moreover, adipokine levels might also fluctuate over-time according to the metabolic environment. These concerns might impact the accuracy of the proposed models in this study.Several models have been proposed based on clinical and laboratory parameters for screening of NAFLD/NASH. Assessment of variables in the introduced models in the current project is simple and safe. However, cost-effectiveness of such panels needs to be further studied. The classification results of DFA in these models documented that a high percentage of original grouped cases were correctly classified. ROC curves also showed a great accuracy with respect to the high area under curve (AUC) of both developed models. The characteristics of previous developed models are summarized in Table 44 In order to construct our panel, we selected the biomarkers with the high evidence for their role in NAFLD  using a “knowledge-based” approach.The development of NASH biomarkers can be theoretically achieved via 2 different strategies. The first strategy can be defined as “knowledge-based” (deductive method based on the current knowledge of NASH pathophysiology), while the second one is more “unbiased” (inductive strategy). The “knowledge-based” approach relies on a direct understanding of the pathophysiological processes that underlie the development of NASH as well as the evolution of its sequelae. It may consist of biochemical assays aiming to assess attractive novel candidate markers informed by the biology of disease process. For instance, the understanding of the role played by hepatocyte apoptosis and insulin resistance in the pathobiology of liver injury has enabled the development of promising biomarkers of NASH, such as caspase-cleaved cytokeratin 18 fragments or numerous different adipokines. On the other hand, the “unbiased” approach involves the use of modern techniques including proteomics, metabolomics, and bioinformatics that have allowed unbiased investigations of numerous putative markers that may be informative with regard to the various stages of NAFLD, including overt NASH and its sequelae.9 In this research, several key adipokines together with metabolic profiles and LFT were evaluated, providing an advantage to previous studies. To improve the power of study, the cases were selected from a cohort of biopsy-proven NAFLD/NASH subjects. In this research, we used the NAS system for histologic description of NAFLD/NASH. This model is a validated scoring system for NAFLD that interpret the spectrum of disease with an excellent reliability and degree of agreement.15The relationships between few serum adipokines with NAFLD/NASH were evaluated in individual reports previously.46 Justification of the proposed biomarker panels with regard to follow-up and response to treatment in NAFLD patients is recommended.The individuals with known liver disease were excluded from this study and the mean age of participants was 37. These facts indicate the early stage of NAFLD in the studied cohort. Consequently, the current results could not be generalized to all NAFLD patients. Well-controlled studies are recommended for the validation of the new proposed formula for NAFLD/NASH determination in patients with advanced disease including cirrhotic and pretransplantation. Liver histology is now considered the gold standard method for detection of NAFLD/NASH; however, there are controversies on type 2 error. Since only small amount of liver  is evaluated in liver biopsy, sampling error is a concern. The distribution of necroinflammation throughout the liver is not homogenous especially in the early stage of NAFLD. It was proposed that scoring systems would potentially characterize a more reliable sign of global liver damage severity than is obtained by the liver biopsy.Our suggested models for predicting NAFLD and NASH based on serum adipokines show promising accuracy to detect patients with NAFLD/NASH.The association between a panel of serum biomarkers  and clinical data with presence of NAFLD/NASH was evaluated in this study. We introduced new models for discriminating NAFLD from healthy subjects as well as patients with NASH from those with simple liver steatosis based on a panel of serum markers. The models demonstrated great accuracy with regard to their significant AUC. The defined threshold values based on these discriminant scores showed decent sensitivity and specificity.


Although measures to reduce and treat degenerative changes after fusion are discussed, these are still controversial.A retrospective study was conducted on a consecutive series of 3,799 patients who underwent posterior lumbar fusion for degenerative lumbar disease between January 1999 and January 2009. A total of 28 patients with symptomatic adjacent segment degeneration surgery were identified. Another group of 56 matched patients with degenerative lumbar disease without symptomatic adjacent segment degeneration after spinal fusion were marked as the control group. These two groups were compared for demographic distribution and clinical and radiographic data to investigate the predictive factors of symptomatic adjacent segment degeneration surgery by logistic regression.The overall incidence rate of symptomatic adjacent segment degeneration surgery was 0.74%. Strong risk factors for the development of a symptomatic adjacent segment degeneration requiring surgery were preoperative distance from L1 to S1 sagittal plumb line (p = 0.031), preoperative lumbar lordosis (p = 0.005), and preoperative adjacent disc height (p = 0.003). Mean postoperative lumbar lordosis was smaller (p = 0.000) in symptomatic adjacent segment degeneration surgery (SASDS) group compared with in the control group (33.3° vs. 39.8°). Postoperative adjacent disc height was also significantly lower in the former group compared with the latter group (p = 0.002). Logistic regression analysis showed that body mass index (BMI) , preoperative adjacent disc degeneration (ADD) on MRI , and disc bulge in preoperative CT examination  maintained their significance in predicting likelihood of symptomatic adjacent segment degeneration surgery.The occurrence of a symptomatic adjacent segment degeneration surgery is most likely multifactorial and is related to BMI, preoperative ADD on MRI, and disc bulge in preoperative CT examination. Posterior lumbar fusion with pedicle screw fixation has been used widely in such settings as trauma, disc herniation, spondylosis, and tumors because of the advantages pedicle screw fixation offers: initial stability, higher fusion rate, and recovery of normal lordosis while sparing the necessity for external support. In addition, good results with this technique have been reported in literature. However, in spite of the procedure’s success at achieving successful fusion in spinal surgery, a long-term follow-up after a solid fusion has revealed degenerative changes including segmental instability, spinal stenosis, intervertebral disc lesion, retro-spondylolisthesis, and fracture at the adjacent segments -3. AlthoThis retrospective cohort study was undertaken to investigate 1) the overall incidence of adjacent segment degeneration in a large population of patients with a background degenerative lumbar condition treated with spinal fusion and instrumentation and 2) the predictive factors for the development of symptomatic adjacent segment degeneration requiring surgery in patients after posterior lumbar fusion.We examined data from a consecutive series of 3,799 patients who underwent posterior lumbar fusion for degenerative lumbar disease  between January 1999 and January 2009, at one academic hospital—a university-based medical center. Those patients undergoing surgery for nondegenerative disease  were excluded. Criteria of degenerative change at adjacent segments: 1) Anterior or posterior displacement of >3 mm was found on the X-ray of the sagittal plane of the closest upper segment and the closest lower segment at the last follow-up. 2) The height of the intervertebral disc relative to that of the upper interbody had declined by 20%. 3) A segmental motion instability of more than 15° was observed on the X-ray of the sagittal plane with flexion and extension. Those patients who had symptomatic adjacent segment degeneration were defined as degeneration at a segment adjacent to a fusion-causing symptoms. The cohort identified was divided into patients undergoing symptomatic adjacent segment degeneration surgery (SASDS) and those who did not (control). The SASDS group was composed of patients who underwent secondary adjacent segment surgery because of symptoms concordant with adjacent segment pathology. Only those patients who had a minimum of five-year follow-up were reviewed. All patients with SASDS were identified, and these patients in the control group were chosen and best matched for sex, age, approximate date of surgery and diagnosis for the patients with SASDS, and then matched at a 2:1 ratio, respectively.Demographic data included age at primary surgery, gender, smoking history, body mass index (BMI), and diagnosis. Follow-up was defined as the time from primary surgery to reoperation in the SASDS group and the period after surgery in the control group. Surgical data collected included levels of surgery, number of fusion levels, and type of bone graft . Radiographs at the primary surgery and at the final follow-up or before an additional surgery were assessed in each group to determine the lumbar lordotic angle, the distance between the L1 and the S1 sagittal plumb lines, the sagittal slope angle of the superior end plate of S1, adjacent disc angle, and adjacent disc height. In addition, magnetic resonance imaging (MRI) was used to investigate whether there had been a degree of preoperative adjacent disc degeneration (ADD). Patients shown to have grade ≥III in the five-grade classification of Pfirrmann et al.  based onRoutine lateral radiographs were obtained using standard techniques. The patient stands upright, his or her head facing forward. The X-ray tube is positioned 72 in from the patient. The lumbar lordotic angle was measured by Cobb’s angle made by the upper endplate of the first lumbar vertebra and the upper endplate of the sacrum. The L1 sagittal plumb line was drawn with a lateral gravity plumb line from the center of L1 . All significance tests were two-tailed, with p <0.05 representing statistical significance. In addition, a multivariate logistic regression analysis was performed to identify which factors helped predict the probability of a SASDS.Factors associated with SASDS were identified using univariate analysis. The data analysis was performed using SPSS version 19.0 . Continuous data were compared between the two groups using the student A summary of the clinical data before spinal fusion for the SASDS and control groups is presented in Table The average body mass index (BMI) for the SASDS and control groups was 27.9 ± 2.3 and 25.2 ± 3.3, respectively (p = 0.000). No significant differences were observed between the two groups in age, sex ratio, number of fused segments, or the fusion method.2 p = 0.000).The preoperative MRI and CT of L1–S1 levels were measured preoperatively; Of the 28 patients with SASDS, 16 patients (57.6%) were noted to have a degenerative change. However, in the control group, only 16 patients (28.6%) were noted to have a degenerative change preoperatively (p = 0.011). The disc bulge present in preoperative CT examination was also statistically associated with increased risk of SASDS: It was found in 27 of 28 in the group with SASDS, as compared with 8 of 56 in the matched cohort , preoperative ADD on MRI , and disc bulge in preoperative CT examination  maintained their significance in predicting likelihood of SASDS (Table A thorough understanding of the development of clinical adjacent-segment pathology in degenerative population treated with spinal fusion and instrumentation is a critical component in avoiding revision surgery for symptomatic disease. Although previous reports have described degenerative changes adjacent to the fusion site, the predictive factors of SASDS in patients after posterior lumbar fusion have not been identified -6. This Many studies have focused on the altered biomechanics at the adjacent levels after fusion that result in increased mobility -11, incrHa KY et al.  reviewedIt has been previously suggested that alteration of sagittal plane anatomy was associated with an increased prevalence of SASDS ,23. KumaThe findings of this study should be viewed after considering the following limitations. Firstly, these data represent the experience at a single institution that is an academic tertiary care center with trainees in the anesthesia, orthopedic, neurosurgical, and nursing departments. Secondly, our data collection system only captured symptomatic adjacent segment degeneration information on patients who returned to our hospital for treatment. Patients with asymptomatic ASD or symptomatic ASD who were managed successfully as an outpatient would not be included in this study. However, we consider these ASDs to be clinically significant as these patients required further follow up and/or additional surgeries to treat their ASD. Moreover, the results are limited by a relatively small sample size and the broad time period covered. In addition, changes in anesthetic and surgical practices over time may have affected patient outcomes. Finally, we also acknowledge the limitations introduced by our patients’ clinical heterogeneity.The risks for SASDS for degenerative lumbar diseases are multifactorial. Multivariate logistic regression analysis suggests that BMI, preoperative ADD on MRI, and disc bulge in preoperative CT examination may be reasonable predictions for an individual likelihood of SASDS. It is our hope that surgeons might be able to improve surgical planning, advise the patient accordingly during the consent taking process, and apply strategies that would help to reduce the risk of SASDS from occurring through these predictive measures.