Pinus radiata. In the absence of full reference genomes for large megagenomes such as in conifers, RNA sequencing in a range of genotypes and tissue types, offers a rich source of genetic markers for downstream application. We compared nine different assembler and variant calling software combinations in a single transcriptomic library and found that Single Nucleotide Polymorphism (SNPs) discovery could vary by as much as an order of magnitude . The assembler with the best realignment of the packages trialled, Trinity, in combination with several variant callers was then applied to a much larger multi-genotype, multi-tissue transcriptome and identified 683,135 in silico SNPs across a predicted 449,951 exons when mapped to the Pinus taeda ver 1.01e reference.There is a wide diversity of bioinformatic tools available for the assembly of next generation sequence and subsequence variant calling to identify genetic markers at scale. Integration of genomics tools such as genomic selection, association studies, pedigree analysis and analysis of genetic diversity, into operational breeding is a goal for New Zealand’s most widely planted exotic tree species,  Pinus radiata D.Don) is New Zealand’s most widely planted exotic forestry species   performed sequencing on the RNA extracted from all trees (Trees 1–8). RNA quality (18S and 26S ratios) and RNA concentration were determined using the RNA 6000 LabChip in an Agilent 2100 Bioanalyzer . Thereafter, samples progressed through sequencing on the next-generation Illumina Hiseq 2000 sequencing platform . The total number of reads and RNA input metrics are summarised in Trinity v r2013-01-25 [All programmes and software used in this analysis are listed in 13-01-25 . Dataset13-01-25 .To evaluate a different variant calling workflows, different software for the various steps were tested in a range of combinations . For theSAMtools (Sequence Alignment/Map) was used to reformat the outputs and identify sequence variants. All defaults were used, with the following exceptions: 1) to reduce the mapping quality for reads with a high number of mismatches, the coefficient to reduce the mapping quality of reads that had a higher number of mismatches was set to 50 as recommended for BWA alignments, 2) Bayesian inference was used to call variants using maximum-likelihood inference for the priors, 3) genotypes were called at the variant site, and 4) for areas of high coverage, , variants with a read depth greater than 100 were removed, as there is a known problem assembling abundantly expressed genes .https://broadinstitute.github.io/picard/) was user to reorder files prior to running the GATK variant calling tool.Default parameters were also used for GATK, however, some file reformatting was required. For BWA alignment files, SAMtools was first used to merge, sort and convert outputs into binary sequence alignment/map format (BAM) prior to performing local realignments in GATK. Picard . Therefore, to identify SNPs that were variable between genotypes, the raw reads from each genotype were mapped to the multi-genotype transcriptome using Bowtie2 version 2.1.0 , and polPinus taeda ver 1.01e genome [Neves et al 2013  reportede genome  using the genome .After SNP discovery via the mapping of raw reads back to the assemblies, additional filtering was performed to increase the likelihood of detecting biologically real SNPs and not sequencing or alignment errors. Firstly, SNPs had to be located more than 10 bp from the edge of contig. Secondly, read depth at SNP locations had to be greater than or equal to 10. Thirdly, as each genotype was from a diploid individual, heterozygous SNPs within an individual should display approximate allele frequencies of 0.5 across all reads; we widened our criteria to allow minor allele frequencies of no less than 0.25 within an individual.www.ncbi.nlm.nih.gov/bioproject/482145).A total of 18 transcriptome libraries were sequenced , and assPinus taeda ver 1.01e genome assembly, 144,007 (74%) of the radiata contigs aligned to the P. taeda genome. This enabled the prediction of 449,951 putative exons, corresponding to 46,342 P. taeda scaffolds.The 8 individual genotypes, previously assembled independently, were combined into a large “multi-genotype radiata transcriptome” containing 194,299 contigs, ranging in size from 201 bp to 16,575 bp, with an N50 of 1,434. When mapped to the denovo assembly. However, many of the raw ‘clean’ reads could not be remapped onto the unigene sequence, suggesting a problem with this approach. The best result was achieved with Mosaik (combination 9), although only 36% of the raw reads remapped to the unigene sequence. This unexpectedly low rate of realignment meant that a high number of polymorphisms were likely missed. Therefore, the decision was made to perform a new assembly using Trinity, and a combined transcriptomic library contig set was created for Tree 1, comprising 137,228 different contigs sequences, ranging in length from 201 to 9,175 bp.Three different combinations of global and/or local alignment, followed by variant calling  to 93% (BWA and Mosaik). For this reason, no further analyses were performed using the SOAPdenovo assembly, and all subsequent reporting of SNPs were identified solely from the Trinity assemblies. SNPs were regarded as high confidence when all of the following criteria were satisfied: 1) more than 10 bases from the edge of the unigene, 2) allele frequency between 0.25 and 0.75, 3) a read depth of 10 or more sequences at that SNP position, and 4) at least 60 bases clear of other polymorphic features on at least one side of the SNP.Variant calling was undertaken using all nine different combinations of global alignment, and/or local alignment, and SNP identification software packages  for the The number of high confidence SNPs  ranged fSNPs predicted multiple times using different software package combinations were expected have a higher likelihood of being a true polymorphic event, therefore, we aimed to distinguish SNPs common to multiple pipelines from those predicted in only one pipeline. Using a pair-wise combination approach, each pipeline was compared to the others . PipelinA total of 164,145 different SNPs were predicted across all pipelines, with only six SNPs predicted in all 9 software combinations . There wFor SNP discovery within each of the genotypes, we selected the three best SNP prediction tools identified in the pilot study: rtg-GA, GATK and SAMtools . Using iFor the final round of SNP discovery within the multi-genotype Trinity v 2.0  assemblywww.affymetrix.com) [in silico SNP predictions became less critical with these alternative genotyping platforms as preselected SNP markers were not required. However, the prediction of high confidence SNPs, described here, did influence the selection of genomic sites to target for an exome capture GBS assay.SNPs identified from tissue-specific transcriptomes can be an ideal resource  for candrix.com) , 33. Durrix.com) , or targrix.com)  became mWe tested a number of sequence assembly software packages, including Trinity, SOAPdenovo, and Velvet/Oases , 49 , and highlights the extent of variation among SNP calling pipelines, with 54% of the 164,145 SNPs discovered in the Tree 1 pilot study being unique to a single pipeline. A similar study comparing SNP discovery pipelines in Antarctic fur seals  showed tThere are two processes that current NGS algorithms can employ in the discovery of polymorphic loci: (a) global and local sequence alignment, and (b) polymorphism detection. The Burrows-Wheeler alignment (BWA) can be used to assemble short sequence reads  then SAMin silico SNP markers and 449,951 exome templates, the first large-scale SNP resource reported for this species. In addition, the P. radiata multi-genotype transcriptome assembly is proving to be a valuable resource and being utilised in multiple downstream projects, including facilitating the assembly of a P. radiata reference genome, various gene discovery programmes, pedigree reconstruction and genomic selection.The purpose of this work was to evaluate bioinformatic workflows and combinations of software for identification of polymorphic loci and the development of a resource for a number of genomic tools for the radiata pine industry, with a requirement for varying SNP densities. A large, complex genome and incomplete reference resources precluded whole genome resequencing for SNP marker discovery in radiata pine. Nine different pipelines applied to a single pilot transcriptome identified SNPs at a rate the ranged over an order of magnitude. However, the utilisation of transcriptomic RNA sequencing in combination with several variant calling pipelines and quality filtering, has identified 683,135 S1 Table(DOCX)Click here for additional data file.S1 File(TGZ)Click here for additional data file.


Subjects were also split into three subgroups according to their body mass index (BMI). The new predictive equations were generated using two models: Model 1 with age, weight, height, and BMI as predictors, and Model 2 in which raw BIA variables (bioimpedance-index and phase angle) were added. Our results showed that REE was directly correlated with all anthropometric and raw-BIA variables, while the correlation with age was inverse. All the new predictive equations were effective in estimating REE in both sexes and in the different BMI subgroups. Accuracy at the individual level was high for specific group-equation especially in subjects with BMI > 50 kg/m2. Therefore, new equations based on raw-BIA variables were as accurate as those based on anthropometry. Equations developed for BMI categories did not substantially improve REE prediction, except for subjects with a BMI > 50 kg/m2. Further studies are required to verify the application of those formulas and the role of raw-BIA variables for predicting REE.This study aimed to develop and validate new predictive equations for resting energy expenditure (REE) in a large sample of subjects with obesity also considering raw variables from bioimpedance-analysis (BIA). A total of 2225 consecutive obese outpatients were recruited and randomly assigned to calibration ( An accurate prediction of resting energy expenditure (REE) is crucial for assessing energy requirements in individuals with obesity, since REE accounts for 60–70% of total energy needs in sedentary people ,2. Indir2; while Madden et al. [2), with underestimated values in approximately 50% of subjects [Generally, predictive equations for REE provide acceptable results when applied in the population from which they are derived. To date, several predictive equations for REE have been developed for subjects with obesity, resulting not always suitable for predicting REE as far as those obtained in the general population ,4,5,6. Tsubjects ,4.It is commonly accepted that fat-free mass (FFM) is the major determinant of REE in normal-weight subjects, including both highly and moderately metabolically active tissues and organs, such as brain, liver, heart, kidney, and muscle. Specifically, in subjects with obesity, the increase in body weight may not strictly reflect changes in FFM due to an excessive presence of fat mass (FM) ,11,12, wHence, it is still uncertain to which extent the use of body composition (BC) variables could effectively enhance REE prediction, since data are still controversial ,15,16,172; (2) to assess whether including raw BIA variables into the model enhances the prediction power of the regression; and (3) to establish if specific-BMI equations could improve the reliability of prediction.Based on this background, the objectives of the present study were: (1) To develop and validate new predictive equations for REE specific for subjects with obesity, especially those with a BMI > 40 kg/mThe present study was a retrospective analysis of data collected between 2005 and 2017 from consecutively adult outpatients with obesity undergoing routine procedures to evaluate nutritional status  at the Internal Medicine and Clinical Nutrition Unit of the Federico II University Hospital in Naples, Italy. The study was carried out in accordance with the Helsinki Declaration and received the approval of the Ethical Committee of Federico II University. Informed consent was obtained from all patients. 2. Exclusion criteria were as follows: current participation in a weight loss program, presence of active inflammatory diseases, dysthyroidism, type 2 diabetes mellitus, pregnancy, lactation, or daily use of prescription medication or drugs known to influence energy metabolism. A total of 2225 Italian caucasian patients with obesity, 1428 females and 797 males were selected, according to the following inclusion criteria: Both sexes, age 18–65 years and BMI ≥ 30 kg/mAll measurements were performed early in the morning after a fasting period of 8–10 h according to standardized conditions, i.e., abstention from alcohol, smoking, and vigorous physical activity for 24 h prior to the assessment. According to the protocol, smoking was not allowed for occasional and current smokers on the morning of the test, until the end of measurements; however, current smokers were asked to maintain their smoking habits on the day before . Data weBody weight and height were measured to the nearest 0.1 kg and 0.5 cm, respectively. Measurements were taken while the subject wore light clothes and no shoes using a platform beam scale with a built-in stadiometer . BMI was calculated as body weight expressed in kilograms divided by squared height reported in meters. The coefficient of variation on three consecutive measures (in 20 subjects) was always <0.1% for body weight and <0.3% for height. 2/resistance (cm2/Ω). The coefficient of variation on three consecutive measures (20 subjects) was always <2% for R and < 4% for PhA.BIA  was perfResting Energy Expenditure (REE) was measured by indirect calorimetry  using a Measurement conditions for IC were defined following the suggestions made by Compher et al.  and Fullp < 0.05.Statistical analyses were performed using IBM SPSS  for males and females separately (sex-specific whole sample groups). Data are presented as mean ± standard deviation (SD), and statistical significance was defined as 2; BMI subgroup 2 = 40–49.9 kg/m2; BMI subgroup 3 ≥ 50 kg/m2. In addition, as outlined in n = 783 for r = 0.100, n = 198 for r = 0.200, and n = 85 for r = 0.300.Within each sex, patients were also split into: BMI subgroup 1 = 30–39.9 kg/mR2) and standard error of the estimate (SEE) were considered for assessing the predictive power of formulas.The Kolmogorov-Smirnov Test and the Shapiro-Wilk Test were used as tests of normality, which is to examine if variables were normally distributed. Data were compared between sexes using one-way ANOVA, while linear correlation was applied for evaluating associations between variables. Multivariate linear regression analysis was performed to develop the new predictive equations, with measured REE bi IC (MREE) as dependent variable. We generated sex-specific models as follows: in Model 1, age, weight, height, and BMI were set as predictors, while in Model 2, we added to the model the raw BIA variables BI-index and PhA. Coefficient of determination  and MREE, was used as a measure of accuracy at the group level and found acceptable if within ± 5% ,29. ConcFinally, PREE values of these new formulas were compared to those of other equations as the Harris-Benedict , Henry , Mifflinn = 1680 and 545, respectively). Anthropometric, raw BIA variables, and MREE data are summarized for the calibration and validation groups in As already mentioned, sex-specific whole sample groups and BMI subgroups were randomly split into a calibration subset and a validation subset (2) did BI-index emerge as a better determinant of REE than body weight. Pearson correlation coefficients for the association of MREE with individual characteristics or raw BIA variables are shown in As a further step, multiple regression analysis was performed to assess the relationships between MREE and different combinations of potential predictors. Age and basic anthropometric characteristics  were considered first (Model 1) to develop the following equations:β) were much higher for body weight  compared to age (β = −0.070 and β = −0.089). Height entered into the model only in females (un-standardized regression coefficient −2.43), leading to a very small decrease in SEE values, while BMI did not emerge as a significant predictor in either sex. In both males and females, the standardized regression coefficients , leading to the following equations:R2 and a small decrease in SEE. While MREE was significantly related to the BI-index , the main determinant was still body weight . Compared to Equations (1) and (2) (Model 1), in both sexes, the inclusion of raw-BIA variables (Equations (3) and (4)) determined an increase in 2. In this latter group, BI-index emerged as the best determinant of MREE ; thus, model 2 improved R2 and decreased SEE values of approximately 15 kcal/d in males and females compared to Model 1. Predictive equations derived in BMI subgroups are shown in To evaluate the accuracy of the new predictive equations in the whole sample as well as each BMI subgroup, 545 individuals with obesity  were assigned to validation groups. Prediction accuracy at the group level, assessed by the difference between PREE and MREE (bias), is reported in 2.All new developed predictive equations were accurate at the group level for either the whole sample groups or each BMI subgroup, since mean bias was always within the range ± 3%. As an index of accuracy at the individual level, the percentage of subjects with a PREE within ± 10% of MREE is shown in When REE was predicted using selected equations from the literature, we found that the bias was acceptable for the Harris-Benedict equation only , and more negative for the Henry , Mifflin , Muller , and Lazzer equations . Overall, accuracy at the individual level was low (<57%) in both sexes and for all the equations considered.Finally, the Bland-Altman plots of PREE-MREE differences vs mean PREE-MREE values were shown in 2, whereas an inverse correlation emerged with age. Interestingly, when the BMI exceeds 50 kg/m2, the BI-index is the best determinant of REE in both sexes. Finally, we found that prediction accuracy was good for all equations, resulting significantly higher in males with BMI above 50 kg/m2. However, from a clinical point of view, the inclusion of raw BIA variables in the equation model did not significantly improve the prediction power of equations. The aim of this study was to investigate the relationships of REE with age, main anthropometric and raw BIA variables, and to develop new predictive equations for REE in individuals with moderate or severe obesity. Our results showed that REE is largely determined by body weight, at least if the BMI is lower than 50 kg/m2 [2 [The accuracy of predictive equations is expected to be higher when they are applied in individuals sharing the same characteristics. For instance, equations developed in the general population should not be applied in individuals with severe obesity, unless specifically validated. Previously, we have assessed prediction accuracy in our outpatients, reporting that only the Muller  equation2 , suggest2 [2 ,35.In the present study, we developed new predictive sex-specific equations, based on basic individual parameters alone  (Model 1) or in combination with raw BIA variables (BI-index and PhA) (Model 2). Our new predictive equations showed similar SEE values between males and females in the calibration groups. Body weight resulted the best predictor of REE in both sexes. On the other side, we found an inverse association between REE and age in all predictive models, as previously reported ; howeverR2 = 0.343, SEE = 341 kcal; females: R2 = 0.439, SEE = 279 kcal) or in combination with age . The prediction power of these equations is much lower compared to those including basic individual characteristic (Model 1). Secondly, when we included, in the model, both individual characteristic and raw BIA variables, we observed an increase of R2 and a decrease in SEE values in both sexes. Beyond the use of BIA-derived FFM and FM, we investigated whether and to what extent the addition of raw BIA variables, PhA and BI-index, may improve REE prediction in subjects with obesity. Although raw data can be device-dependent, values for Z, R and Xc , measured at 50 kHz, are usually made available by almost all scientific BIA devices (single or multifrequency). Raw BIA variables have gained considerable attention in recent years because they are not only associated with FFM, but they are also markers of extracellular/intracellular distribution of water, BMC, integrity and muscle quality , withoutIn the validation group, accuracy at the population level was acceptable since it ranged within ± 3%, with similar bias using either Model 1 or Model 2. Likewise, accuracy at individual level was higher (~65%) than that observed in previous studies ,34,38. T2) is continuously increasing, it is crucial to provide a reliable tool for assessing REE in planning adequate nutritional advice for weight management and bariatric surgery procedures. According to the literature, most studies reported that the accuracy of prediction decreased considerably with an increasing BMI (>40 kg/m2) [2; subgroup 2 = 40–49.9 kg/m2; and subgroup 3 > 50 kg/m2). Body weight was the major predictor of REE, as previously observed in the whole study sample; however, in Group 3, we found that BI-index was a slightly stronger predictor than body weight.Finally, since the number of people with an extreme BMI (>50 kg/m0 kg/m2) ,4; hence2 .In the validation samples, prediction accuracy at the group level was comparable between BMI-specific equations and those developed for the whole study sample, as shown in 2. According to the literature, no previous work has developed predictive formulas for different classes of obesity. Muller et al. [2; while Lazzer et al. [2; 45–50 kg/m2 and > 50 kg/m2) without formulating specific-BMI equations. Furthermore, none of studies published so far used raw BIA variables to predict REE in subjects with obesity. This new approach could be helpful to avoid issues related to different equations for BC measurement that can modify the prediction of the formulas and alter their validity. This is, to our knowledge, the first study that specifically develops and cross-validates predictive equations for subjects with severe obesity and opts for using raw BIA variables to estimate REE in individuals with BMI > 50 kg/mr et al.  generater et al.  describeOverall, we performed a cross-sectional protocol in a large sample of individuals with obesity using known and documented methods and in line with previous studies that have derived predictive equations for REE from healthy subjects as well as from patients with different diseases. However, some limitations need to be considered. Firstly, this is a single-center study including adult outpatients, therefore our findings need to be substantiated in other subgroups or in different clinical settings. Secondly, due to the aim of the study and inclusion/exclusion criteria, the effects of comorbidities cannot be evaluated from our data and no information can be provided on elderly subjects with obesity. Then, unfortunately, no data were available on BC by other techniques such as DXA. Last, but not least, other proxy measures of obesity and/or fat distribution need to be considered in the future, being widely-available variables, for improving the predictive power of the formulas. 2. Finally, accuracy at the individual level was higher compared to those assessed by previous studies, especially in subject with severe obesity; nevertheless, we still recognized the importance of measuring REE by IC. Further studies are necessary to verify the application of these equations, especially in the group of subjects with severe obesity, as well as to investigate the role of raw BIA variables in predicting REE.In conclusion, this study showed that new predictive equations were accurate at the population level; although, the inclusion of raw BIA variables did not substantially enhance the prediction accuracy from a practical point of view in clinical settings. Regarding the use of predictive equations based on BMI categories, our results highlighted that these equations were not strictly necessary for REE prediction in subjects with mild/moderate obesity but can be useful in those with a BMI higher than 50 kg/m


The aim of this study was to develop and evaluate the psychometric properties of a questionnaire for the measurement of reproductive health among female adolescents in Iran.This cross-sectional study was conducted among 289 female students aged 12–15 yr in Tehran, Iran from 2015–2016. The participants were selected using multi-stage random cluster sampling. In addition, the Belief-Based Reproductive Health Questionnaire (BBRHQ) was designed using the properties of the Theory of Planned Behavior (TPB).Exploratory factor analysis of 104 items identified a six-factor solution. These factors jointly accounted for 67% of the observed variance of outcome variable. The confirmatory factor analysis indicated a good fit to the data. In addition, the Cronbach’s alpha coefficient showed an excellent internal consistency .Belief-Based Reproductive Health Questionnaire (BBRHQ) is a valid and reliable instrument for measurement of reproductive health behaviors of adolescents. Reproductive health is “a state of complete physical, mental, and social well-being and not merely the absence of disease or infirmity in all matters relating to the reproductive system and to its functions and processes” . Since mIn addition, girls compared to boys suffer from poor knowledge and information on sexual and reproductive health . Hence, There are a few studies  assessinIn the current study, we investigated the influence of perceived parental control within the TPB.This cross-sectional study was conducted among 289 female students aged 12–15 yr in Tehran, Iran from 2015–2016. The adolescents were recruited from 6 female schools in three districts in Tehran, Iran. The schools were selected from among 26 female middle schools in three districts of Tehran , which comprise a mixture of working-class and lower-middle-class families in one of Tehran’s most populated inner-city areas. From each school, three classes were selected randomly; one class from each grade. Therefore, 18 classes were enrolled in this study.A self-administered questionnaire was developed using direct measures of TPB theory . Some pa1.Thirteen items were included in the questionnaire to elicit personal information on age, place of residence, etc.2.Here, 28 items were used and each was scored using 3 categories . Each correct answer was given score one, and wrong and “do not know” responses were scored zero.3.1.3Twenty-one items regarding attitude towards reproductive health, derived from relevant literature, were employed.2.3Nineteen items were used to assess the influence of important people (parents …) on their opinion about reproductive health.3.3Twenty-five items were used to measure the students’ perceptions of behavioral control about behaving in a way that preserves their reproductive health. Answers were rated on a 5-point Likert differential scale ranging from 1 (very difficult) to 5 (very easy). PBC questions were designed , 17.4.3Eight items were used to assess this aspect of the study .5.3Eighteen items about attitude towards reproductive health, derived from relevant literature, were employed to measure this item.6.3Thirteen items were used to evaluate reproductive health skills. Items of the questionnaire reversed included items 2,9,10,11,14,15,16,19,20,21,34 and 36. The maximum total point was 100.Ten adolescent girls aged 12–15 were recruited using convenience sampling to determine the ambiguity, relevance, and difficulty of each item. At this stage, none of the items was removed, but 3 items were changed as a result of the adolescents’ suggestions.First, 10 specialists in health education and health promotion, public health, and reproductive health areas reviewed the questionnaire to check its grammar, wording, item allocation, and scaling. In order to calculate the CVR, 13 other specialists were asked to assess each item on a 3-point Likert scale  during the quantitative stage. Then, based on Lawshe’s table , items tThis validity was investigated using exploratory factor analysis with varimax rotation. The Kaiser-Meyer-Olkin (KMO) and Bartlett’s Test of Sphericity were used to assess the appropriateness of the sample for the factor analysis. Eigenvalues above 1 and scree plot were used to determine the number of factors. Factor loadings equal to or greater than 0.3 were considered appropriate . Data weInternal consistency was evaluated by Cronbach’s α coefficient . A sub-sThe Ethics Committee of Tehran University of Medical Sciences approved the study .The socioeconomic and demographic characteristics of the studied participants are shown in In the qualitative face validity, participants stated that they have had no problems in reading and understanding the items. In the quantitative content validity phase, items with CVR and CVI less than 0.62 and 0.80 were respectively omitted (24 items). The mean of content validity ratio was 0.64.Moreover, the mean of the content validity index (CVI) was 0.74. Exploratory factor analysis (EFA) was used to evaluate construct validity .2/df, TLI, IFI, NFI, CFI, AGFI, GFI and SRMR)  .This study was prospected to develop and validate an instrument for assessing the adolescents’ reproductive health and behaviors. In this study, the constructs of the modified theory of TPB were evaluated using the direct method . EFA wasMoreover, according to the results of CFA, the questionnaire with 4 given domains is a good instrument for measuring the reproductive health among adolescents in Iran.In this study, a ratio of 0.64 was calculated for the content validity and the mean of the content validity index (CVI= 0.74) was likewise calculated. The mean of the CVR and CVI was reasonable and satisfactory. However, they obtained low values in some questions rooted in differences in the cultural context of the countries, so they have amended again in writing.Results of the internal consistency suggest that the provided questionnaire had acceptable reliability. The study revealed an internal consistency of 0.85 to 0.91 for TPB . In addiMoreover, internal consistency of the final scale indicates a desirable reliability. In addition, ICC showed appropriate stability for the scale as it was examined by 45 participants with a 2-week interval (0.92) .This instrument had a number of strengths. Adding item 28 to the instrument was one of them. Another important feature of our questionnaire lies in the way it was worked out; in spite of other questionnaires with short statements, we used long and complete sentences. The main feature of the BBRHQ was the fact that it was developed for middle age students and contained items on nutrition and exercise, adolescents’ health, menstrual health, and AIDS. Notably, a previous study among adolescent boys aged 15–18 yr assessed their sexual and reproductive health knowledge, attitude, and behaviors in Tehran . The advThe BBRHQ is a valid and reliable instrument for evaluating the reproductive health, attitude, and behavior among female adolescents in Iran.Ethical issues  have been completely observed by the authors.


Gastrodia that has been reported to improve memory and other cognition. And the TBMCZG has been approved clinical trial with patients with VaD by center for drug evaluation of China (CFDA). To evaluate the efficacy, safety, and tolerability of TMBCZG tablets in the treatment of mild to moderate VaD, we designed and reported the methodology for a 24-week, randomized, double-blind, parallel, placebo-controlled, multicenter trial.Vascular dementia (VaD) is the 2nd most common subtype of dementia after Alzheimer disease. Currently, there are no medications approved for treating patients with VaD. Tianmabianchunzhigan (TMBCZG) tablet is an active ingredient extracted from A total of 160 patients with mild to moderate VaD will be enrolled. After a 2-week run-in period, the eligible patients will be randomized to receive either TMBCZG high-dose group ; TMBCZG middle-dose group (TMBCZG 2 tablets and placebo 1 tablet twice per day); TMBCZG low-dose group ; placebo group  for 24 weeks, with a follow-up 12 weeks after withdrawn drug treatment. The primary efficacy measurement will be the vascular dementia assessment scale-cognitive subscale and the Clinical Dementia Rating-Sum of the Boxes scale. The secondary efficacy measurements will include the mini mental state examination and activities of daily living. Adverse events will also be reported.This randomized trial will be the 1st rigorous study on the efficacy and safety of TMBCZG tablets for treating cognitive symptoms in patients with VaD using a rational design.ClinicalTrials.gov NCT03230071. Registered on July 26, 2017. Although most have shown a small but significant benefit of cholinesterase inhibitors on cognition, the magnitude of this effect has been slight and benefits on global functioning, activities of daily living (ADL), and behavior have not been consistently reported; thus, cholinesterase inhibitors or memantine were not authorized for patients with VaD. Additionally, it has not yet been possible to establish disease-modifying effects in VaD syndrome. Thus, the development of an effective therapy for VaD is important.Vascular dementia (VaD) is a very frequent form of dementia, it had reported that dementia develops in around 15% to 30% of subjects 3 months after a stroke.Gastrodia (Tianma in Chinese) by Peking Union Second Pharmaceutical Factory. Preclinical studies have shown that TMBCZG can improve the learning and memory ability of rats with VaD and reduce the neuropathologic damage of model rats. A human tolerability test in a healthy person showed that the maximum tolerated dose of the drug was 84 mg/d, and the excessive adverse reactions were elevated urinary N-acetyl-β-d-glucosaminidase enzyme, hypotension, and diarrhea. In addition, toothache, sinus tachycardia, and elevated microalbuminuria for other suspected adverse events are further recommended for observation in expanded population samples. And the TBMCZG has been approved clinical trial with patients with VaD by center for drug evaluation of China (CFDA). To further investigate the safety, efficacy and effective dose of TMBCZG tablets in the treatment of mild to moderate VaD, we designed and report the methodology for a 24-week randomized, double-blind, parallel, multicenter study.Tianmabianchunzhigan tablet (TMBCZG) is an active ingredient extracted from 22.1This ongoing study is designed as a randomized, double-blind, parallel, placebo-controlled, multi-central trial. It involves a single-blind run-in period using placebo only (2 weeks) and a double-blind treatment phase after randomization (24 weeks). It has been approved by the China Food and Drug Administration code: 2012L02385). Ethics approval was requested and approved through the Institutional Review Board of Dongzhimen Hospital, Beijing University of Chinese Medicine (DZMEC-JG-2016-108) and has been registered with ClinicalTrials.gov  on July 26, 2017. The present study protocol  is designed in accordance with the Good Clinical Practice Guidelines and the principles of the Declaration of Helsinki. The protocol design is based on the guidelines of standard protocol items: recommendations for interventional trials (SPIRIT), and the study results will also be reported according to these guidelines. The patients and responsible caregivers will provide written informed consent. The study will be carried out at Dongzhimen Hospital and some other centers in China. A schematic of the trial protocol is detailed in Figure 2L02385. 2.2: dementia defined by clinical core criteria; cerebrovascular disease, defined by history of stroke and through a magnetic resonance imaging (MRI) scan, including infarct in the main blood vessels, single strategic infarct , multiple lacunar infarcts, and/or extensive white matter damage surrounding ventricles , hemorrhage; a relationship between dementia and cerebrovascular disease, manifested, or inferred by the presence of one or more of the following: onset of dementia within 3 months following a recognized stroke; abrupt deterioration in cognitive functions; or fluctuating, stepwise progression of cognitive deficits.This trial will enroll both Chinese-speaking males and females, participants’ inclusion criteria are described as follows: Patients meeting the clinical diagnosis of probable VaD established according to the National Institute of Neurological Disorders and Stroke and the Association Internationale pour la Recherche et l’Enseignement en Neurosciences (NINDS-AIREN)were eligible to participateOther inclusion criteria including:1.;mild to moderate dementia, defined with mini mental state examination (MMSE) score of ≤26 and ≥112.aged ≥55 and ≤80 years old; weighing of ≥45 and ≤90 kg;3.adequate vision and hearing ability to complete all study tests;4.have a stable caregiver;5.can read simple articles and write simple sentences;6.informed consent, signed informed consent by legal guardian.They will also be assessed for the following exclusion criteria:1.A medical history of other dementia types, like Alzheimer disease, frontotemporal dementia, Parkinson disease dementia, dementia with Lewy bodies, Huntington disease, etc;2.Subdural hematoma, traffic hydrocephalus, brain tumor, thyroid disease, vitamin deficiency, or other diseases which can lead to cognitive impediment;3. or major anxious ;Major depression 4.Subject cannot complete related test due to severe neurologic deficits, such as hemiplegia, aphasia, audio-visual disorder, and so forth;5.Severe cardiovascular disease ;6.Severe liver or kidney dysfunction ;7.Uncontrolled diabetes ;8.Asthma, chronic obstructive pulmonary disease, multiple neuritis, myasthenia gravis, and muscle atrophy;9.Severe indigestion, gastrointestinal obstruction, gastric and duodenal ulcers, and other gastrointestinal disorders that can affect drug absorption;10.A medical history of epileptic history, glaucoma, alcoholism, or psycho-substance abuse;11.Subject has been taking cholinesterase inhibitors, memantine, nimodipine, or herbal medicine which have function to improve cognition in the past 1 month;12.Use of sympathomimetic or antihistamines drugs within 48 hours before assessment;13.Allergic constitution or allergic reactions to experimental drug;14.According to the assessment of the investigator, subject cannot complete the study due to poor compliance or other reasons;15.Subject is participating in other clinical trials or participated in the past 1 month.2.3Gastrodia organic benzyl polyglycolate 14 mg. The TMBCZG tablets were produced in a single batch  in strict compliance with standards of good manufacturing practice. During the 2-week placebo washout period, all patients will receive 3 placebo tablets twice times per day. During the double-blind, the patient will be randomized to TMBCZG high-dose group ; TMBCZG middle dose group ; placebo group . To preserve blinding, the placebo tablets have an identical taste and appearance to the TMBCZG tablets.The TMBCZG tablet will be supplied by the Peking Union Second Pharmaceutical Factory. Each tablet weighs 0.1 g, which contains Gastrodia will be forbidden. The investigator must record the concomitant drugs, including the name of the drug, daily dose, reason for using, and date of termination.Concomitant use of anticonvulsants, antipsychotics, cholinomimetic drugs, anticholinergic agents, anti-Parkinson drugs, cholinesterase inhibitors, memantine, nootropic drugs, nimodipine, other cognition enhancers, traditional Chinese medicine tonic, or any drugs containing 2.4 and the clinical dementia rating-sum of the boxes (CDR-SB) scale from baseline to the 24th week after treatment. VADAS-cog is a revision of the ADAS-cog to be a better measure in vascular conditions, and a higher score indicates higher impairment. In addition to ADAS-cog, the VADAS-cog comprises additional frontal lobe tests reflecting attention, working memory, executive function, and verbal fluency. It was suggested that the VADAS-cog may be a more sensitive endpoint in patients with white matter load and vascular burden of the brain than ADAS-cog.The primary efficacy measurements are changes of the vascular dementia assessment scale-cognitive subscale (VADAS-cog/17 items)The CDR-SB is a standard for disease grading in clinical studies of dementia and is used for overall endpoint assessment in clinical trials. It comprehensively assesses the cognitive and functional aspects of dementia patients, including memory, orientation, judgment and problem-solving skills, social affairs, family and hobbies, and personal cooking. The CDR-SB scores from 0 to 18 points, and a higher score indicates higher impairment.,13 The MMSE is used to assess the global cognition. ADL was used to measure the physical self-maintenance ability and instrumental ADL ability.,14The secondary efficacy measurements include the MMSE and ADL scale.2.5The safety assessment will include the following: vital signs: physical examination of vital signs, including rate of breathing, heart rate, and blood pressure; electrocardiography; the laboratory parameters included complete blood count, urine routine test, fecal routine and occult blood test, hepatic and renal function, coagulation function and electrocardiogram (ECG); and any adverse events that may occur, including the types of adverse events, time of occurrence, duration, treatment measures, and evaluation of the correlation between the tested drugs and the adverse event ; the severity of the adverse event  must be evaluated.2.6The efficacy measurement will be assessed at baseline (week 0), at the midpoint (week 4 and week 12), and at the endpoint (week 24), 12 weeks after withdrawal (week 36).The safety assessments will be evaluated at the baseline (week 0), at the midpoint , and at the endpoint (week 24).2.7There are no similar studies using TMBCZG tablets to treatment patients with VaD for the proposed study duration (24 weeks). The sample size was calculated based on the primary endpoints, VADAS-cog. It is hypothesized that the efficacy of TMBCZG in the treatment of mild to moderate VaD is similar to that of donepezil. Previous studies reported that the mean changes of ADAS-cog scores after 5 and 10 mg donepezil treatment were (5.75 ± 7.44) points and (2.25 ± 7.44) points, respectively. We assume that the placebo control group has a certain progression after 24 weeks’ treatment (△ = −0.25). The mean scores of the 3 groups of VADAS-cog scale improvement were set at equal intervals, that is, the changes before and after the scores of each group were −0.25, 0.75, 1.50, 2.25, and the variance of the 4 groups of VADAS-cog means 1.14. The standard deviation within is 7.44 points. For this phase IIa study, the 2-sided test level α = 0.20 and the power 1-β = 0.50. This clinical trial adopts a balanced design and uses nQuery Advisor 7.0 to calculate, 32 cases are needed for each group. Considering a greater rate of discontinuation of 20%, the sample size was increased to 39 per group to ensure adequate patients to complete the study. The total number of cases was 160 cases, that is, 40 cases in the placebo control group and 40 cases in the 3 dose groups of the TMBCZG tablets. Note also that because this study is a pilot efficacy study (phase IIa), a larger sample size to reach a higher degree of statistical power is not required.2.8Two methods are being used to recruit participants with VaD. The 1st source of subjects is from memory clinics of different research centers, and the 2nd source is those who respond to advertisements published in local newspapers. Advertisements will be placed in local papers in surrounding the research centers; internet advertisements will be several website placed on different research centers.Participants will undergo an initial face-to-face interview after written informed consent is obtained from both the participant and her informant, the participant's medical history and demographics will be taken with a semi-structured clinical interview, and participants will be estimated cognitive function using MMSE, HAMD, HAMA. Researchers will assess them against the study's inclusion and exclusion criteria, and the appropriate subjects will be initially selected. The subjects who decided to enroll will be assigned to run-in period. After 2 weeks run-in period, the subjects will be evaluated MMSE, VADAS-Cog, CDR-SB, ADL and vital signs, 12-lead ECG, laboratory tests, skulls MRI. Inclusion and exclusion criteria will be assessed again. And then subjects will be randomized to different treatment groups. Schedule of interventions and assessments at screening baseline, midpoint, and endpoint sessions is shown in Table After baseline measurements, the first 4 weeks of medication will be dispensed. And the medication will be dispensed every 4 weeks. Participants will be required to return any unused medication at midpoint and endpoint; this will be used to determine compliance.2.91)Prior to the maximum treatment period of 24 weeks, study drug will be discontinued if any of the following conditions are met:2)Patient or informant request termination of study drug;3)Patient or informant withdraws consent for the study;4)Noncompliance with study drug as determined by the investigator (patients who have taken 80% or more of the expected number of tablets will be considered compliant);5)Decision by the investigator that the study drug should be discontinued on safety grounds;6)The reason for study drug termination should be recorded and any unused medication returned to pharmacy.After the protocol is approved by the Ethics Committee, if modified, it must be reported to the Ethics Committee before execution and also recorded in the drug administration department.2.10To maximize subjects’ compliance, we will try to prevent dropouts by providing ongoing support to patients. A direct telephone line set up for this clinical trial will enable the study team to communicate personally with the patients. Participants will be offered reimbursement to cover travel expenses for their study visits: up to ¥100/visit will be provided and total travel expense will be up to ¥700. If a patient is lost to follow-up, we will call to ask the reason and attempt to schedule a meeting at the patient's convenience.2.11In this trial, a complete randomization is adopted. Eligible subjects are randomly assigned to groups A, B, C, or D. The specific method is as follows:1.The numbers of the observed cases are each labeled “No. 001-160.”2.Using SAS 9.0, a biostatistics expert develops a computing procedure statement with set seeds, and a random number table is generated. Based on the table, a series of random numbers emerges and these are matched individually with each case number.3.Subjects are divided into groups A, B, C, and D at the ratio of 1:1:1:1.4.According to the case number and grouping, each subject is provided with the appropriate kit, with a drug number matching the case number.5.Based on the case number, random number and grouping, emergency sealed envelopes are prepared and sent to the hospitals involved in the trial. The outside of each envelope is marked with the case number.6.When a qualified subject is enrolled into the trial, the kit with the appropriate case number, in the order of subject enrollment, is provided.The 1st blinding is case number matching groups, that is, group A, B, C, or D. The 2nd blinding is the disposal among the 4 groups. The blinding are sealed separately, duplicated, and stored in the research unit and the pharmaceutical factory. Once the blinding is broken, the patient will be managed as off-trial.2.12Statistical analyses will be conducted in 3 populations. The statistical analyses are planned to conduct in 3 populations. The intent-to-treat (ITT) population consist all randomized population who take at least 1 dose of medication and at least 1 primary efficacy evaluation on treatment. The fully evaluated (FE) population include all randomized patients who have receive at least 80% assigned 24 weeks’ double-blind medication with complete record of efficacy variable, with no major protocol violations. The safety set population included all randomized population and receive at least 1 dose of the study medication, with at least 1 safety record post baseline.The efficacy analysis will be conducted with the ITT population and FE populations, for the VADAS-cog and CDR-SB will be analyzed by using the Last Observation Carried Forward method for the replacement of missing observations.Multivariate analysis of covariance (ANCOVA) models that included baseline score as covariate, treatment, and center were used to assess differences between the treatment groups for linear efficacy measures. Categorical efficacy assessments were analyzed with a Cochran–Mantel–Haenszel test. The least squares mean changes from baseline scores to endpoint were presented for variables analyzed with the ANCOVA models. All values were 2-tailed, and all analyses were significant if the value was ≤0.05.3Gastrodia elata (Tianma in Chinese), usually used to treat ischemic stroke, epilepsy, dizziness, and dementia for centuries in China in traditional Chinese medicine. TMBCZG is the active ingredient, which is extracted from G elata; studies have showed that gastrodin is effective in the treatment of bilateral common carotid artery occlusion-induced VaD via targeting Aβ-related protein formation and inhibiting autophagy and apoptosis of hippocampus neurons, ameliorates sub-acute phase cerebral ischemia reperfusion injury by inhibiting inflammation and apoptosis in rats. However, there is no clinical trial on the treatment of VaD with TMBCZG tablets.Studies showed that the most impaired cognitive domains in patients with VaD were attention/psychomotor speed, executive function, visuo-spatial , and language . So we used the VADAS-cog scale as the primary efficacy measurement. And the neuropsychologic battery in this study covered these important domains comprehensively.This ongoing randomized, double blind, parallel Group, placebo controlled, multicenter, 24-week clinical trial will be the 1st rigorous testing of TMBCZG tablets for the treatment of patients with VaD. Success in this clinical trial will provide evidence for the use of TMBCZG tablets in the treatment of VaD, and supply evidence of effective dose of TMBCZG in VaD.Conceptualization: Jinzhou Tian, Jing Shi, Mingqing Wei, Ting Li, Jingnian Ni, Xuekai Zhang, Yongyan Wang.Funding acquisition: Jinzhou Tian, Mingqing Wei, Meng Zhang, Yang Li.Methodology: Jinzhou Tian.Writing – original draft: Jinzhou Tian, Jing Shi.Writing – review & editing: Jinzhou Tian.


BOLL), an ancestral member of the DAZ (deleted in azoospermia) gene family, is required for testicular function, maintenance, and spermatogenesis in males. However, in sheep, little is known about the expression profiles and molecular function of BOLL. In In this study, obvious seminiferous tubule lumens and various spermatogenic cells, including spermatogonia, primary and secondary spermatocytes, round spermatids, elongated spermatids, and spermatozoa, were observed in the testes of sheep aged 1 year and older. Our results showed that BOLL was expressed exclusively in sheep testes. Moreover, significant BOLL expression at the transcript and protein levels were discovered in 1- and 2-year-old sheep testes, in comparison with testes from 0-day-, 2-month-, and 5-month-old sheep. BOLL protein was located in spermatogenic cells, ranging from primary spermatocytes to round spermatids, as well as in spermatozoa with intensive immunoexpression. A preliminary study demonstrated that the sheep BOLL gene is critical for meiosis and sperm maturity. This study contributes to further understanding the regulatory mechanisms of the BOLL gene during spermatogenesis.Boule homolog, RNA binding protein , 2 months (2M), 5 months (5M), 1 year (1Y), and 2 years (2Y), by qPCR, Western blot, and immunohistochemistry methods. Firstly, morphological studies revealed that, in addition to spermatogonia, ordered and clear spermatocytes, as well as round and elongated spermatids and sperm, were found in the 1Y and 2Y testicular seminiferous tubules of the sheep testes, compared with the D0, 2M, and 5M testes, as analyzed by hematoxylin and eosin (H&E) staining. The diameter and area of the seminiferous tubules, epithelial thickness, and the area and perimeter of the tubule lumens gradually increased with age. BOLL was specifically expressed in testes and upregulation of BOLL transcript expression was higher in the testes of the 1Y and 2Y groups than in those of the D0, 2M, and 5M groups. Similarly, BOLL protein was expressed mainly in the 1Y and 2Y testes, ranging from primary spermatocytes to round spermatids, as well as in the spermatozoa. This study is the first demonstration that sheep BOLL might serve as a key regulator of the spermiogenesis involved in sperm maturity, in addition to its role as a crucial meiotic regulator. DAZ family genes are germ cell-specific RNA-binding proteins (RBPs) which are implicated in male spermatogenesis [DAZ family consists of three known members, including two autosomal genes, BOLL and DAZL(DAZ-like), and one Y chromosome gene, DAZ [Spermatogenesis in mammals is a complex, continuous, and tightly coordinated physiological process that is largely regulated by many genes whose expression varies at the levels of transcription and translation during different stages ,2,3. Theogenesis ,5. Durinogenesis ,7. The Dene, DAZ ,9,10,11.BOLL (Boule), an ancestral gene of the DAZ family, is evolutionarily conserved in all metazoans [BOLL gene plays an important role in testicular function, maintenance, and spermatogenesis and that loss of the gene may cause male dyszoospermia and infertility [BOLL may regulate human embryonic stem cells to enter into meiotic phase and produce meiotic germ cells which are further induced to form ovarian follicle-like cells by expressing DAZL and BOLL with recombinant GDF9 and BMP15 [BOLL mRNA is significantly decreased and BOLL protein is completely lacking in the testes of infertile men, when compared to healthy men [BOLL expression is progressively reduced with the increasing severity of testicular failure [BOLL expression in male gonads is related to its methylation degree and, consequently, with meiotic arrest and infertility. A long CpG island in the BOLL promoter was hypermethylated in the testes of cattle–yak hybrids  with extremely low BOLL expression, compared with cattle testes [BOLL promoter is also inversely associated with its expression level. For example, the methylation level of the BOLL promoter in normal human testes, with high BOLL expression, is significantly lower than that in somatic tissues with lacking or extremely low BOLL expression [etazoans ,12. Numeertility ,14,15,16nd BMP15 . Expresslthy men . Of note failure . The dowe testes . For malpression , pigs [9BOLL gene during adult mice spermatogenesis is regulated by insulin-like growth factor 1 (IGF1), through extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling and T-independent pathways [BOLL may block production of functional sperm, thereby leading to infertility [Drosophila melanogaster, for example, the mutation of Boule results in meiotic arrest during spermatogenesis [Boule null mice testes, meiotic divisions proceed normally, but spermatogenic cells undergo arrest at the subsequent stage of round spermatid, prior to deformation, and do not further differentiate into mature spermatozoa [BOLL is able to promote the development and differentiation of male germ cells. Li et al. [BOLL in male dairy goat germline stem cells can elevate the expression of meiosis-associated genes, such as STRA8, SCP3, CDC25A, CDC2, and VASA, promoting meiosis and spermatogenesis. In vitro, overexpression of BOLL may effectively promote the transdifferentiation of goat bone marrow mesenchymal stem cells into early germ cells through spermatogenesis [Boll is involved in regulating the formation of stress granules (SGs) in germ cells under heat stress, to protect those cells from heat-induced apoptosis [The transcription of the pathways . In maleertility ,14,21. Hogenesis . Similarogenesis ,18 and cogenesis . Howeverrmatozoa . By conti et al.  reportedogenesis . Additiopoptosis . SGs arepoptosis ,29), whiBOLL gene in male mammals have focused on human, cattle and mouse, but the regulatory mechanisms are not exactly the same in different species, considering the entire range and wide variety. Sheep are an economically important livestock animal with considerable agricultural significance, however, little is known about the expression profiles and biological functions of BOLL in sheep testes. Accordingly, it is important to elucidate the mechanisms of sheep spermatogenesis by investigating the expression patterns and regulatory roles of BOLL in developing sheep testes.Until now, most reports on the n = 3), 2 months old , 5 months old , 1 year old , and 2 years old —were provided by Sanyang Sheep Breeding Farm . Samples from the same section of the right testis, heart, liver, spleen, lung, kidney, and longissimus dorsi muscle were obtained from each sheep. Duplicated samples were collected for all tissues: one sample was immediately placed in liquid nitrogen and stored at −80 °C for the preparation of total RNA and protein, and the other was fixed with 4% paraformaldehyde for up to 48 h, then embedded in paraffin for hematoxylin and eosin (H&E) staining and immunohistochemistry.All of the animals in this investigation were managed according to the animal care and experimental procedure guidelines established by the Ministry of Science and Technology of the People’s Republic of China  and the study was approved by the Animal Care Committee of Gansu Agricultural University. A total of fifteen purebred Small-Tail Han sheep, from five developmental stages—0 days old , first-strand cDNA was synthesized for each sample using 500 ng RNA, according to the manufacturer′s instructions .Total RNA from the selected tissue samples was extracted using TRIzol Reagent , as previously described . The purBOLL gene was amplified  using optimized qPCR assay conditions, specifically, 1 cycle of 94 °C for 30 s; 40 cycles of 94 °C for 5 s, 40 cycles of 60 °C for 30 s. β-actin was used as an internal control for the normalization of BOLL mRNA expression. cDNA (1 μL) was added to 20 μL of the amplification reaction system used for qPCR, comprised of 0.4 μL of forward primer, 0.4 μL of reverse primer, 10 μL of 2 × TransStart® Tip Green qPCR SuperMix , and 8.2 μL of ddH2O. The relative expression level of BOLL mRNA was calculated according to the 2−ΔΔCt method [BOLL mRNA expression results, taken from an average across tissue samples, were presented in the form of bar charts. The qPCR primers, used in the present study, are shown in The t method . Each exTesticular tissues at different development stages were homogenized and lysed using a radio immunoprecipitation assay (RIPA) protein extraction kit , according to the operating instructions. Protein concentrations within the testis samples were determined using a commercial bicinchoninic acid (BCA) Protein Assay kit . Twenty micrograms of the denatured protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) blotting membranes . After blocking in phosphate buffered saline tween-20 (PBST) containing 5% non-fat milk, the membranes were incubated overnight at 4 °C with either rabbit anti-BOLL polyclonal antibody  or anti-beta-actin polyclonal antibody . After washing, the membranes were incubated with goat anti-rabbit IgG/HRP antibody . Enhanced chemiluminescence signals were visualized in an X-ray room. This experiment was biologically repeated three times. Band intensities were quantified using AlphaEaseFC software . The BOLL protein expression results, from an average across tissues, were presented as bar charts.2O2 and then incubated with rabbit polyclonal anti-BOLL antibody  in a wet box, overnight, at 4 °C. The negative controls were generated by replacing the primary antibody with PBS. The positive signals (brown) of the BOLL protein in sections were visualized using a DAB kit  and then observed using a Sunny EX31 biological microscope . This experiment was biologically replicated three times.Immunoreactivity for the BOLL protein was visualized in 5 μm sections from testicular tissues at postnatal development stages using a Histostain™-Plus kit , as previous described . In briep < 0.05 considered as statistically significant. All displayed values, in the form of bar charts, were the mean ± SD.H&E staining and immunohistochemistry images for testicular cross sections were captured using ImageView software . The morphological parameters of 35 randomly selected seminiferous tubules from the 200× magnification of H&E sections were measured using MvImage software . The integral optical density of immunostaining for the BOLL protein was calculated by analyzing four random 400× microscope magnification levels in independently replicated sections using Image-Pro Plus 6.0 software . The data were statistically analyzed using one-way analysis of variance, with p < 0.05).The morphological observation of sheep testes at different ages, using H&E staining, were presented in BOLL transcript and protein in postnatal sheep testes using qPCR and Western blot, respectively. Here, we found that expression of BOLL was relatively low in sheep testes from the D0 to 5M groups, but was dramatically upregulated in the 1Y and 2Y groups , 2M (weaning), 5M (pre-puberty), 1Y , and 2Y (adult). When compared to the D0 and 2M testes, histomorphological observation showed that spermatocytes initially appeared in the seminiferous tubules beside the spermatogonia in 5M testes, indicating that Small-Tail Han sheep have an earlier age of sexual maturity. All levels of spermatogenic cells, from spermatogonia to spermatozoa, could be seen in the seminiferous tubules, most obviously in the tubules from the 1Y and 2Y testes. The measurement of the histomorphological parameters in the cross sections of testicular seminiferous tubules, at different development stages, indicated that the area and diameter of the seminiferous tubules, epithelial thickness, and the area and perimeter of the tubule lumen became gradually larger with testicular development. A significant increase was found in the 1Y and 2Y testes.BOLL is a marker of germ cell development and meiosis, and previous studies in some male mammals have indicated that BOLL expression is restricted to the gonads and implicated in spermatogenesis through regulating spermatocyte meiosis and the male gamete formation required for fertility [BOLL expression was examined in adult testes from cattle and yaks—low expression was detectable in testes of cattle–yaks, but there was no expression in other tissues, including the epididymis, kidney, spleen, stomach, hypothalamus, and pituitary tissues from cattle, yak, and cattle–yak [Boule, namely Boule1 and Boule2, were also found to be exclusively expressed in yak testes, while no expression was observed in other examined tissues, including ovary, muscle, kidney, and spleen [BOLL is also well-documented in male humans [BOLL expression was only present in male and female gonads for fish such as Asian seabass  [Acipenser sinensis) [Oryzias latipes) [BOLL was specifically expressed in the testes of 1-year-old sheep, but was completely lacking in other male somatic tissues, such as heart, liver, spleen, lung, kidney, and muscle, as analyzed by qPCR, which is consistent with previous studies on cattle [BOLL in sheep testes which might be related to its previously reported role in testicular development or spermatogenesis.ertility ,14,33. Bttle–yak . In addid spleen . Similare humans , dairy ge humans , pigs [9carifer) , Chineseinensis) , and medlatipes) . In thisn cattle , goats [n cattle , and micBOLL are not the same in the testes of different species nor in the same species at different development stages. In mice, Zhang et al. [Boll expression is barely detectable from postnatal 3- to 14-day-old testes, but its expression was significantly upregulated at 12-days-old and later. In goats, the expression of BOLL in the testes was observed to be lacking, or low, during the embryonic and pre-pubertal stages, but gradually increased during postnatal progression with a significant expression in mature testes [BOLL expression in adult dairy goat testes with complete spermatogenesis was significantly higher than that in testes with azoospermia or male intersex [BOLL is crucial for male fertility. Herein, we first examined the expression patterns of the BOLL gene at the mRNA and protein level during postnatal sheep testis development, as analyzed using qPCR and Western blot. Consistent with previous studies [BOLL mRNA and protein expression were observed to be at extremely low levels in sheep testes from the D0, 2M, and 5M groups, but dramatically increased expression was detectable in the 1Y and 2Y groups. One can speculate that the BOLL gene is crucial for the testicular development of postnatal sheep and, particularly, for post-pubertal sheep.The expression patterns and regulation mechanisms of g et al.  report te testes ,26. Moreintersex , which d studies ,26, BOLLBOLL is a testis-specific gene that regulates spermatogenesis in males, but its distribution in the testis varies between different species and between different development stages. Positive BOLL protein is restricted to spermatocytes in the testes of normal adult men, but it is completely lacking in the testes of infertile men, which is reported by Luetjens et al. [BOLL might play several important roles in the meiotic phase and spermiogenesis of mature sheep.s et al. . For pris et al. . In aduls et al. ,27. Alsos et al. . Moreoves et al.  and medas et al. . To undes et al. , primates et al. , goats [s et al. , and mics et al. ,27. In cBOLL, an ancestral gene of the DAZ family, in sheep testes at different ages. Our data showed that BOLL demonstrates an extremely low level of expression and is confined to the seminiferous epithelium in testes, from birth through to the pre-pubertal stages. However, its expression demonstrates a significant upregulation in post-pubertal testes, with strong positive signals observed in the spermatocytes, round spermatids, and spermatozoa. These results indicate that BOLL plays a key role in sheep spermatogenesis, especially during meiosis and spermiogenesis. Future studies will be implemented to investigate the specific molecular mechanisms of BOLL gene expression during sheep spermatogenesis.In conclusion, our study investigated the expression patterns and cellular localization of


To determine the psychopathological profile of patients with central sensitization (CS) in a sample of knee osteoarthritis, with and without CS, and fibromyalgia, and to compare their psychopathological profiles.The final sample consists of 19 patients with osteoarthritis and CS (mean 66.37 years ± 8.77), 41 osteoarthritis patients without CS (mean 66.8 ± 7.39 years), 47 fibromyalgia patients (mean 46.47 years ± 7.92) and 26 control subjects (mean 51.56 years ± 11.41). The psychopathological profile was evaluated with the Millon Multiaxial Clinical Inventory.The average score of MCMI-III reflect higher scores in the fibromyalgia and osteoarthritis-CS groups. Patients with osteoarthritis-CS are more likely to report larger scores in Borderline and Major Depression scales. Fibromyalgia patients are more likely to report more increased scores in Somatoform and Major Depression, versus osteoarthritis-CS group. Fibromyalgia patients versus osteoarthritis without CS are more likely to report higher scores in Schizoid, Depression, Histrionic, Sadistic, Borderline, Somatoform, Posttraumatic Stress Disorder and Major Depression scales.Patients with CS have less differences in their psychopathological profiles as well as in both osteoarthritis groups and greatest differences are obtained between the fibromyalgia and osteoarthritis without CS, so perhaps presence of CS is the key to differentiate those groups and not chronic pain. An exhaustive assessment brings more accurate psychopathological profiles, thus better psychological treatment could be applied. Musculoskeletal disorders are one of the most common causes of disability and incapacity due to pain. Two diseases where chronic pain is typically present are osteoarthritis (OA), knee OA as the most frequent type, and fibromyalgia (FM). About 10% of people aged over 55 years have painful disabling knee OA of whom one quarter are severely disabled .Knee OA is a common chronic condition causing disabling symptoms, such as joint pain, physical and psychological dysfunction, and reduced quality of life (QoL) . PsycholFM is a chronic disease characterized by widespread musculoskeletal pain and hyperalgesia after digital pressure in at least 11 of 18 tender points . FM is cOn one hand, OA and FM patients share suffering chronic pain. On the other, they also share the presence of sensitization of Central Nervous System (CNS). CS is a phenomenon that appears normally when there is pain. When it is acute, the sensitization is so. However, in chronic pain patients, sensitization remains present after nociception has resolved . CS maniEven it is known that CS is a relevant phenomenon in the explanation of chronic pain, in our opinion, there are very few investigations regarding emotional and affective symptoms or disease impact or functional disabilities. George et al.  found thSo, the aims of this study are to determine the psychopathological profile of OA patients with and without CS, and to compare these psychopathological profiles with that of FM patients.This study was carried out at the Rheumatology Department of both, Hospital del Mar and Hospital CIMA-Sanitas, in Barcelona (Spain). It was approved by the Local Ethics Committee and was in compliance with the Helsinki Declaration.n = 28) and (b) absence of CS . It is defined by the presence of both spreading sensitization and temporal summation to repeated pressure pain stimulation [The patients were selected by a senior rheumatologist and a senior psychologist during a period of 18 months. The initial sample was composed by 90 patients with osteoarthritis diagnosis and 150 with fibromyalgia diagnosis at the Rheumatology Department of Hospital del Mar and Hospital CIMA Sanitas, in Barcelona. There were also 35 healthy controls (C). Patients with OA were separated into 2 sub-groups: (a) presence of clinical CS  presence of temporal summation  on the most sensitive site of the peripatellar region [The main inclusion criteria for OA patients were: (1) radiological and clinical diagnosis of knee OA based on American College of Rheumatology (ACR) criteria  affectin2 ; (3) pair region .The main inclusion criteria for FM patients were: (1) a diagnosis of FM following the ACR criteria (1990); (2) history of widespread non-articular pain with insidious onset over 3 months; (3) one year minimum of disease evolution; (4) presence of CS; (5) absence of comorbid Chronic Fatigue Syndrome.Inclusion criteria for control group selection were as follows: no history of rheumatic disorder, no history of functional pain or physical widespread pain, no history of Axis I or II psychiatric illness, and no history of neurological disease. In all groups, patients with a history of psychotic disorder or substance abuse, patients with a history or diagnosis of personality disorders and patients with a history of neuropathic pain were not included. The participants signed informed consent to accept the conditions of the study. This written consent was corresponding to a larger protocol which included the present study. We have used a systematic and rigorous process that allows us to ensure the sample fulfill strictly all the inclusion/exclusion criteria, so the sample is very well delimited.The final sample was made up of 19 patients with OA and CS (OA-CS) aged between 44 and 81 (mean 66.37 yrs±8.77), 41 with OA without CS (OA-no CS) aged between 46 and 79 (mean 66.8 yrs±7.39), 47 FM patients aged between 32 and 63 (mean 46.47 yrs±7.9) and 26 participants in the control group aged between 50 and 77 (mean of 51.56 yrs±11.41). Most relevant characteristics of the sample are shown in All patients went first to rheumatologic visit where they were selected and, after check the inclusion/exclusion criteria and the will to participate, they were also enrolled. During the same week, the patient went to the visit of psychological assessment, always done by the same clinical psychologist, which last 2 hours, approximately. If necessary, we visit again the patient in case of too much fatigue that may influence the responses. The completed protocol is wide and in the present research we only use some parts of it.Millon Clinical Multiaxial Inventory, MCMI-III, Spanish version . The MCMFibromyalgia Impact Questionnaire (FIQ) . It is aWestern Ontario and McMaster Universities Osteoarthritis Index (WOMAC) . It is aversus controls, OA-CS versus OA-noCS patients, OA-CS versus FM patients, and OA-noCS versus FM patients) are presented as non-linear logistic regression coefficients (ORadj) with the corresponding 95% confidence intervals (95% CI), and P-values (P). We include also the transformation of ORadj into a percentage of change ((ORadj—1) * 100) to facilitate its interpretation. The initial logistic regression models also included gender, age, academic level and cognitive screening as potentials confounders. Regression backward model selection was conducted, fitted using IBM SPSS Statistics package .We carried out a descriptive analysis to know if there are differences between the 4 groups  in MCMI-III scores, and logistic regression analyses to investigate the most characteristic MCMI-III psychopathological profile of those 4 groups. The results of the association between MCMI-III scores and the four binary responses investigated  and Passive-aggressive (increase in ORadj between 2.4% and 17.2%) subscales, and lower scores in Antisocial (decrease in ORadj between 0.8% and 11.6%) and Compulsive (decrease in ORadj between 1.4% and 16.6%) subscales. Regarding severe personality pathology OA patients are more likely to report lower scores in Schizotypal (decrease in ORadj between 0.3% and 6%) subscale. The clinical syndrome which was different is Somatoform subscale, where OA patients are more likely to show higher scores (increase in ORadj between 0.3% and 7.6%).versus OA-noCS patients. In severe personality pathology subscales, OA-CS patients are more likely to report larger scores only Borderline show significant differences (increase in ORadj between 0.2% and 10.4%). There were no differences in clinical syndrome scales, however, in severe clinical syndrome subscales OA-CS patients report significant differences in Major Depression (increase in ORadj between 0.7% and 9.7%) subscale.versus FM patients. We only found differences in 2 subscales, one from clinical syndromes and another from severe ones. Patients with FM are more likely to report more increased scores in Somatoform (increase in ORadj between 0.2%– 10.5%) subscale. With regard to severe syndromes, Major Depression is more likely to appear in FM patients (increase in ORadj between 1.5% and 11.3%).versus FM patients. FM patients are more likely to report higher scores in Schizoid (increase in ORadj between 0.8% and 12.5%), Depressive (increase in ORadj between 2.5% and 17.1%), Histrionic (increase in ORadj between 1.4% and 15.3%) and Sadistic (increase in ORadj between 1.7% and 13%) subscales of clinical personality pattern. On severe personality pathology, FM patients are more likely to report higher scores in Borderline subscale (increase in ORadj between 0.2% and 7.2%). OA-noCS patients are more likely to show higher scores in Somatoform scale (increase in ORadj between 1.01% and 11.85%) and lower scores in Posttraumatic stress disorder scale (decrease in ORadj between 0.1% and 14%). In severe clinical syndromes, FM patients are more likely to show increased scores in Major depression scale (increase in ORadj between 3.8% and 12.8%).The purposes of this study are to investigate the psychopathological profile of knee OA patients with and without CS, and to compare them with the psychopathological profile of FM patients. In After divide the subjects depending on the psychometric significance scores PREV≥75) we realize that the only conditions that are truly related to group of patients are Anxiety, Somatoform, Dysthymia and Major depression. So, patients show mainly emotional alterations or affective problems and theses could be the first target to assess in CP. If we look at the mean scores, contradicting the main evidence in this field at this moment  we reali found thDue to the absence of clinical significance in psychopathological scales the interpretations always will refer to tendency to feel, behave and/or think in a certain way. Besides, this is an exploratory study as we found little evidences in this field, so the comparisons with others studies are quite difficult.Regarding OA profile (without taking account if there is CS or not) versus Control subjects, there is a clear and differentiated profile. Patients are more likely to score higher in Histrionic and Passive-Aggressive, and lower in Antisocial and Compulsive clinical personality patterns scales. They also rate lower in Schizotypal personality scale. In clinical syndromes, the score of Somatoform scale is more likely to be higher. So, we could say that patients with OA are more likely to show gregarious self-image, dramatic speech, being interpersonally submissive, irritability, express resentful, felling blamed and shamed, and show physical weakness, fatigue, exaggeration of physical symptoms and health worries. On the other hand, insensitivity with others, suspicion, being demanding and perfectionist, social isolation and eccentricity are not distinctive features. Thus, the combination of positive and negative emotions  and atteThe second comparison shows the characteristic features of FM versus OA-noCS. In this case, FM profile is composed by schizoid, depression, histrionic and sadistic patterns of personality, borderline personality disorder, somatoform and posttraumatic stress disorder and major depression as clinical syndromes. So, they are more likely to reflect apathy, lack of pleasure, pessimism, hopelessness, need of affection, avoidance of disapproval, covert hostility, emotional lability, expression of psychological distress trough physical and health complaints and worries, trauma-related emotions associated to anxious activation and avoidance of the environment of that stressful event and devaluation, feelings of blame, food alterations, sleep alterations, depreciation and lack of concentration and motivation . So, in The third profile is to differentiate OA-CS from OA-noCS where OA-CS patients are characterized for borderline and major depression tendency. These scales are related to being temperamentally labile, combination of anger and sadness, uncertain self-image or identity, anxiety, and hopelessness, apathy, psychomotor delay or agitation, problems of sleep, food, weight, cognitive alterations like attention and concentration and feelings of blame. So, the profile focuses on emotional alterations .And finally, the comparison between both groups with CS, FM and OA-CS, is composed by somatoform and major depression traits. That means, somatic complaints due to emotional discomfort, tiredness, weakness, worry about health, disease oversize and feelings of indifference, desperation, psychomotor agitation or delay, sleep and food difficulties, weight loss or gain, concentration alterations and guilt. In sum, mood alterations and health complaints are more likely to appear in FM patients.As found in previous works regarding FM patients , our proBeyond the specific mood alterations that FM and OA patients actually show, this study also takes into account coping styles and certain personality disorders. In periods of stress, the traits intensity rises and cause more maladaptive behaviors than in normal and routine situations. This fact implies an increase of strength of psychological symptoms . Characteristic behaviors are dramatic, emotional, attention-seeking and fluctuating mood for all patients, however, FM use to show higher scores.Our study has some limitations: 1) the sample is reduced, especially for OA-CS group, because of the accuracy in inclusion/exclusion criteria; 2) Although a conservative cut-off score of 75 or more for each of the 10 clinical syndrome scales has been established as a criterion for the presence of symptomatology clinically relevant, the likelihood of Type II error due to the small sample size of our study groups may have hidden other differences in the differential MCMI-III psychopathological profiles analysed; 3) in FM group there are only women, with high educations levels and most of them were taking antidepressants, so it is challenging to generalize the results.Hence, this study highlights in the differentiated pattern of FM and OA-CS groups, both with CS, which is quite specific and focus on somatic symptoms, guilty feelings, resignation, agitation or delay and ruminative thoughts, mainly. The strategies to affront illness in patients with CS are the same. It is not clear if there is a specific psychopathological profile for CS syndromes, but FM and OA-CS are more similar than with OA-noCS, and they only show differences in somatic symptoms and mood.With a detailed and comprehensive study of the psychopathological characteristics of patients suffering from CP, a more precise and rich description is obtained that will help a better description of the patient and consequently the treatment may also be more individualized . CliniciIn conclusion, we found that presence of CS in OA patients is related to larger presence of psychopathology. So, perhaps, CS is a risk factor to suffer more psychopathological symptoms, which brings to a more complicated clinical prognostic of these patients, as Galvez-Sánchez Duschek and Reyes del Paso found . This isFurther studies are necessary to replicate, or not, our findings and to fully investigate the whole psychopathological profile of OA, FM and CS syndromes. Maybe this investigation encourages researchers to look for new ways to treat OA, FM and CS patients and they can have less pain and better quality of life.


In this paper, we investigate the state estimation of systems with unknown covariance non-Gaussian measurement noise. A novel improved Gaussian filter (GF) is proposed, where the maximum correntropy criterion (MCC) is used to suppress the pollution of non-Gaussian measurement noise and its covariance is online estimated through the variational Bayes (VB) approximation. MCC and VB are integrated through the fixed-point iteration to modify the estimated measurement noise covariance. As a general framework, the proposed algorithm is applicable to both linear and nonlinear systems with different rules being used to calculate the Gaussian integrals. Experimental results show that the proposed algorithm has better estimation accuracy than related robust and adaptive algorithms through a target tracking simulation example and the field test of an INS/DVL integrated navigation system. As the benchmark work in state estimation problems, the linear recursive Kalman filter (KF) has been applied in various applications, such as information fusion, system control, integrated navigation, target tracking, and GPS solutions ,2,3,4. IHowever, when the measurements are polluted by non-Gaussian noise, such as impulsive inference or outliers, GF will have worse estimation results and even break down ,13. Besi∞ filter , are alsan noise . The Huban noise ,19,20. Ran noise ,26,27,28an noise , and it an noise ,26,28. San noise ,24,25,26Traditionally, the unknown noise statistic can be estimated by the adaptive filter, such as the Sage–Husa filter  and fadiIn this paper, we proposed an adaptive MCGF based on the VB approximation, which is especially useful for estimating the system state from the measurements with unknown covariance non-Gaussian noise. Typical applications include low-cost INS/GPS integrated navigation systems  and maneThe rest of this paper is given as follows. In X and Y is defined as  [25]6) [ [25]6) [Multiplying Equation , we obtak-th element of Then, the optimal estimation Equation  can be sNote here that the Based on the above equation and using Equation , MCGF caThen, We easily find that the main difference between MCGF and GF is the modified measurement noise covariance, and MCGF shows excellent estimation performance when measurement is polluted by outliers or shot noise ,25,26. HThe main idea under state estimation is to obtain the posterior probability density function ximation ,32:(22)ximation ,32:(23)According to the VB method, ribution ,32:(25)The integrals in  and 23)23) can bBesides this, the expectation can be rewritten asSubstituting  and 31)31) into Step 1:Predict: through  and 6),,x^i− andStep 2:Update:First, set d Pxz by  and 21)x^i(1)=x^For End For. In addition, set The VB based GF works well for unknown measurement noise covariance. However, when the measurement contains outliers or shot noise, their estimation will degrade, as will be shown in our simulation results. To overcome the shortcomings of MCGF and VBGF, we take the advantages of VB and MCC by the fixed-point iteration method, and design the so called VBMCGF algorithm, which is summarized as follows:The main difference between the proposed VBMCGF and existing GFs lies in the modified estimation error covariance r method ,25,26. Ar method ,32.As the general framework, our filter can be easily implemented according to the real requirements. For linear systems that are described by In addition, the When it comes to the nonlinear systems, the Gaussian integrals contained in x and y directions. The position of radar is set as cth Monte Carlo experiment, respectively. The RMSE and ARMSE of velocity are similar.To illustrate the performance of the proposed algorithm, we first give the simulation results using a typical target tracking model, where cubature rules are used to calculate the integrals. We compare the estimation accuracy of seven filters: CKF , MCCKF-1We here consider the following five kinds of measurement noises:Case A: Gaussian distributionCase B: Time varying measurement noise covarianceCase C: Gaussian mixture noise with time varying measurement noise covarianceCase D: Time varying measurement noise covariance and shot noiseCase E: Gaussian mixture noise with time varying measurement noise covariance and shot noiseIn these simulations, we use h are infinity). VBCKF and VBMCCKF work slightly worse as compared with CKF because they only use their online estimated measurement noise covariance instead of the real one. In particular, the VBHCKF has the worst performance since the commonly used parameter Under the Gaussian measurement noise with known noise covariance, as given in j denotes the subscribe e and n present the east and north directions in the local-level frame, and x, y, and z are the directions of three axises in the body frame. Then, the continuous system model is given as follows:t is the continuous system time, and To further illustrate the effectiveness of the proposed algorithm, we compare our algorithm and existing related methods using the real data collected by a self-made fiber optical gyroscope inertial navigation system (INS) together with a doppler velocity logger (DVL). The integrated navigation results of photonics inertial navigation system (PHINS) and GPS are used as the reference system. We adopt the loosely coupled method to fusion the information of INS and DVL. The state vector is chosen as refer to . The meaP0 = diag, where g is the gravitational acceleration. The process noise covariance and measurement noise covariance are set as −3g, 1 × 10−3g, 0.025°/h, 0.025 °/h, 0.025 °/h, 0, 0, 0, 0, 0) and We compare the estimation results of KF, VBKF, HKF, MCKF, and VBMCKF, where we choose In this experiment, the system is first tested in anchorage for about 50 min, then the ship starts to move. The real velocities of the ship are shown in It can be seen from In this paper, a novel adaptive MCGF based on VB approximation is proposed. The MCC is used to reduce the effect of non-Gaussian measurement noise and outliers, while we use VB to estimate the unknown measurement noise covariance. Experimental results based on simulation examples and real data show that the proposed algorithm has better estimation accuracy than related robust and adaptive filters.