Neurology 2006;66:366-372). The objective of this study was to assess the long-term safety and effectiveness of TBZ for chorea in HD.Tetrabenazine (TBZ) selectively depletes central monoamines by reversibly binding to the type-2 vesicular monoamine transporter. A previous double blind study in Huntington disease (HD) demonstrated that TBZ effectively suppressed chorea, with a favorable short-term safety profile  score from the Unified Huntington Disease Rating Scale.Of the 75 participants, 45 subjects completed 80 weeks. Three participants terminated due to adverse events (AEs) including depression, delusions with associated previous suicidal behavior, and vocal tics. One subject died due to breast cancer. The other 26 subjects chose not to continue on with each ensuing extension for various reasons. When mild and unrelated AEs were excluded, the most commonly reported AEs (number of subjects) were sedation/somnolence (18), depressed mood (17), anxiety (13), insomnia (10), and akathisia (9). Parkinsonism and dysphagia scores were significantly increased at week 80 compared to baseline. At week 80, chorea had significantly improved from baseline with a mean reduction in the TMC score of 4.6 (SD 5.5) units. The mean dosage at week 80 was 63.4 mg (range 12.5-175 mg).TBZ effectively suppresses HD-related chorea for up to 80 weeks. Patients treated chronically with TBZ should be monitored for parkinsonism, dysphagia and other side effects including sleep disturbance, depression, anxiety, and akathisia.Clinicaltrials.gov registration number : NCT00219804 Huntington disease (HD) is a hereditary, progressive neurodegenerative disease clinically characterized by a triad of chorea, cognitive symptoms and behavioral changes. Although there is no established treatment to delay the onset or forestall the progression of HD, symptomatic treatment of chorea may be beneficial in some individuals as it may have a favorable impact on motor function, quality of life and safety ,2.Many agents and surgical procedures have been evaluated in HD for their anti-choreic efficacy including dopamine depleting agents, dopamine antagonists, benzodiazepines, glutamate antagonists, acetylcholinesterase inhibitors, dopamine agonists, anti-seizure medications, cannabinoids, lithium, deep brain stimulation, and fetal cell transplantation -5. A preTBZ is a reversible dopamine depleting agent that is highly selective for the central vesicular monoamine transporter type 2 (VMAT2) . TBZ depFar too little evidence is available to guide long term symptomatic treatment in HD. Double-blind and long-term studies assessing various treatment strategies in HD are urgently needed . The obj2 receptor blockers, selective and non-selective monoamine-oxidase inhibitors, amantadine, levodopa, or dopamine agonists. Participants were permitted to be on antidepressants, antianxiety agents and other psychotropic medications at stable doses. Clinically, subjects could not have disabling dysarthria, dysphagia or depression present at screening or have an unstable or serious medical or psychiatric illness, untreated depression or lack of a caregiver.Patients with HD who were ambulatory, had a Total Functional Capacity (TFC) score of greater than 5 and a Total Maximal Chorea (TMC) score of greater than 9 from the Unified Huntington Disease Rating Scale (UHDRS), were initially enrolled in the thirteen-week, double-blind, placebo-controlled study . SubjectThe subjects completed the double-blind study within eight weeks of enrollment but were subsequently excluded if they had suffered from a serious adverse event (AE) judged to be possibly or probably related to study drug. There were three possible lengths of enrollment in the open-label study Figure . The iniWritten informed consent was obtained from all study participants and accompanying caregivers. In compliance with the Declaration of Helsinki, this study was approved by the Research Subjects Review Board at the University of Rochester as the coordinating site and by the ethics review boards at all individual sites that enrolled subjects.TBZ was titrated over a maximum of 12 weeks every 3-7 days to the best individual dose (maximum of 200 mg/day). All subjects started at 12.5 mg per day and were titrated upward at the end of each week by 12.5 mg increments to doses equal to or lower than 125 mg/day, and then by 25 mg increments for doses higher than 125 mg/day. If at any time during the titration phase, moderate to severe, possibly or probably drug-related AEs occurred, the dose of TBZ was decreased to the patient's previous well-tolerated dose. Study drug titration (up or down) was permitted only during the first 11 weeks of the study.Participants were examined at the end of weeks 2, 6, 12, 24 and then every 12 weeks; and had a safety follow-up visit one week after the end of treatment. Characteristics of participants and non-participants were compared using chi-square tests and Kruskal-Wallis tests, as appropriate.The primary efficacy endpoint was the TMC score from the UHDRS at week 80 compared with the baseline TMC score. TMC score at week 80 was also compared to week 81 (after washout) to determine the degree of re-emergent chorea. To determine if TBZ may have worsened overall underlying chorea over time, TMC at week 81 was compared with baseline. T-tests and analysis of covariance (ANCOVA), adjusted for site and baseline value, were used to determine significance. Secondary endpoints included the Clinical Global Impression scale and the individual sections of the UHDRS.Tolerability was assessed using adverse events, UHDRS parkinsonism score , Barnes Akathisia Scale (BAS) , UnifiedTreatment emergent AEs were designated to be those AEs that emerged after the start of the open-label study, excluding those for which subjects had a prior history and those that had been present during the double-blind study, including those without resolution. Those AEs that carried over from the double-blind portion of the study were not considered new events as they have been previously reported . The timThe total number (%) of subjects experiencing at least one AE was calculated separately for the titration and maintenance periods. McNemar's test was used to compare the number of subjects experiencing at least one AE during the titration phase with the number experiencing at least one AE during the maintenance phase. AEs starting in the period from the date of baseline visit to week 12 were designated as having occurred during the titration period. AEs starting in the period from the first day of week 12 to week 24 were designated as having occurred during the maintenance period. AEs designated as mild and/or not related to study medication were excluded, and three subjects with fewer than 13.5 weeks in this study period were omitted from this analysis.To determine if 12 additional weeks of exposure to study medication impacted chorea or HD, efficacy and safety measures were compared between subjects originally assigned to placebo and TBZ in the double-blind study using ANCOVA.Of the 84 subjects enrolled in the double-blind study, 6 subjects were ineligible to enroll in the open-label study due to early terminations or serious AEs. Four subjects decided not to participate due to moving away from the study site, decision with the caregiver not to enroll due to lethargy, intensity of protocol, and lost to follow-up. Although one subject who withdrew from the double-blind study early due to pre-existing breast cancer was initially excluded, once medically cleared, she was permitted to enroll, making a total of 75 subjects who enrolled in the open-label study Table . Three oExcluding subjects who had stopped medication, the mean daily dosage at week 24 (n = 66) was 74.2 mg , week 48 (n = 54) was 71.5  and at week 80 (n = 41) was 63.4 mg . For the 44 subjects with complete dosage data at week 80 (including three subjects with zero dose), 24 (55%) of participants were taking either 37.5 mg or 50 mg per day , have higher CAG repeat length , and have a lower total functional assessment score at baseline . There were no differences in race, affected parent, history of depression, chorea score, TFC, total motor score, age, years of education or duration of illness.There were 12 serious AEs including two falls, two cancer diagnoses, a single suicide attempt, pneumonia, hip replacement (elective) with post-op agitation, agitation, anxiety, akathisia and one abnormal CA 27-29 titer in a participant who later died due to metastatic breast cancer. During the study, 56 subjects reported 170 AEs. Seventeen subjects reported depressed mood as an AE during this study with a prior history of depression in 15 of those subjects. All AEs with an incidence of 5% or more are listed in Table When mild or unrelated events were excluded, 39 subjects reported at least one AE during titration while 20 subjects reported at least one AE during maintenance (p < 0.001). Insomnia, somnolence and diarrhea emerged during titration and subsequently resolved during maintenance. The number of subjects with somnolence decreased from 36 to 11 (p < 0.0001), insomnia from 14 to 2 (p < 0.003) and diarrhea from 5 to 1 (p < 0.05) when comparing titration to maintenance phases.Between baseline and week 80, the mean parkinsonism score increased 2.1 (SD 4.3) UHDRS units (p = 0.002) and the mean UPDRS dysarthria score increased 0.4 (SD 0.8) UPDRS units (p < 0.002). There were no significant changes in the HAM scale scores, UPDRS dysphagia score, or BAS. Marked or severe akathisia  was experienced by only one participant, who terminated due to elevated bilirubin, rather than akathisia. Three participants (4%) had mild or moderate akathisia at baseline. Ten other participants (13.3%) developed mild or moderate akathisia during the course of the study.There were 3 participants with isolated elevation of AST, greater than 3 times the upper limit of normal. Two occurrences were at baseline and one at week 24. All abnormal liver tests returned to normal by week 80 or at the end of study participation except in one participant with >2 times upper limit of normal AST in isolation. No participant experienced clinical liver dysfunction, but one participant was terminated early due to elevated AST and ALT at baseline and one subject terminated due to elevated bilirubin without clear etiology.When TMC at week 80 was compared to baseline, there was a reduction in mean TMC score by 4.6 (SD 5.5) UHDRS units (p < 0.001). At week 81 after washout, chorea re-emerged with a mean TMC score increase of 5.3 (SD 3.2) UHDRS units (p < 0.001 compared to week 80) .At week 80, there was a significant change in the CGI score of 0.3 . Of the 45 completers, there was improvement in 16 (36%) and worsening of 4 (9%) with the remainder unchanged compared to baseline.In the 45 participants who completed 80 weeks, there was no significant change in total motor score, but there were significant declines in cognitive measures, including verbal fluency, symbol digit and Stroop  and functional measures as measured by the functional checklist, independence score and TFC Table .When the groups initially randomized to placebo and TBZ were compared, there were no differences in numbers of subjects with AEs, change in TMC score, change in chorea after withdrawal, best dosage, or any of the secondary measures. There was a reduction in the total number of AEs (p < 0.002) and somnolence (p < 0.01) in maintenance for those subjects initially assigned to TBZ.The double-blind, randomized controlled trial preceding this study demonstrated that TBZ reduced chorea associated with HD for up to 12 weeks with the main AEs including drowsiness and insomnia . In thisIn this study, there were no completed suicides, but one attempt listed as a serious AE and an additional subject who expressed suicidal ideation. Depression was not assessed by formal psychiatric criteria, but depressed mood was screened for using the Hamilton Depression Scale and item 25 of the UHDRS. During the double-blind phase of this study, there was a completed suicide in a subject randomized to the TBZ group, despite scoring within the normal range on the Hamilton Depression Scale two weeks prior to the event . CliniciAt the conclusion of this study, the majority of participants were taking either 50 or 75 mg of TBZ. Notably, even after subjects had been on TBZ for over one year, dosage adjustments continued. In two other open-label, long term studies of TBZ used for a variety of hyperkinetic conditions including HD, the mean doses after more than two years were 60.4 and 106.2 mg, respectively [Discontinuation of TBZ after 80 weeks of treatment appears to be safe and is associated with the return of chorea, without significant worsening compared to baseline. Participants with higher baseline chorea scores experienced a greater reduction in chorea. When TBZ was discontinued, no subject developed signs consistent with neuroleptic withdrawal such as nausea, excessive sweating, tachycardia or akathisia. We did not identify AEs related to the withdrawal of TBZ, but other studies have specifically examined subjects when TBZ is stopped ,28. One Participants did not demonstrate a faster than expected rate of motor or functional decline due to HD. Although parkinsonism and dysphagia were increased in subjects at week 80, this finding was likely a result of the natural progression of disease. Had the parkinsonism been due to a pharmacological impact of tetrabenazine, we would have anticipated that the signs would have emerged more quickly and found on exams throughout the study. TBZ does not appear to have an effect on cognition or function after two years of therapy. Participants experienced an overall improvement on TBZ as demonstrated by the CGI at week 80, but there was no correlation to support the association of global improvement with either motor or chorea change. The UHDRS measures of cognition and function while taking TBZ declined at a rate consistent with the natural history of HD. The TFC scale declined 1.6 ± 0.4 points over almost two years in those initially assigned to TBZ, consistent with previously published measures of TFC ,32. AlthThe main limitation of this open-label study was the loss of subjects at each extension phase. The reasons for the attrition were related to administrative issues and a decision not to continue rather than any specific AEs. The majority of patients elected to complete 80 weeks on therapy, and in fact, once the study concluded, some subjects refused to stop taking TBZ and found other sources of the study medication prior to its availability on the US market. The findings support the clinical impression that chorea suppression in this large cohort of patients can be valuable to patients and families. The unblinded nature of this study limits the degree to which we can make conclusions about effectiveness, but the reduction in chorea was consistent with that seen in the double-blind trial. Further investigation is needed to determine the effectiveness and adverse event profile, particularly regarding cognition, when TBZ is combined with other commonly used medications.Chorea, the most striking physical manifestation of HD, may be disabling and stigmatizing. Frequent or large amplitude involuntary movements can cause embarrassment or frustration and physical harm due to direct injury or falls as well as impact quality of life. The impact of suppressing chorea on weight, gait, behavior and functioning deserves further study, as do the indications for suppressing chorea in the ambulatory high functioning patient and the more advanced patient in whom chorea may be injurious. In lieu of discovering therapy that modifies the global course of the disease, identifying targeted symptomatic treatments for HD, such as those for chorea, is a way to palliate some of the more disabling and stigmatizing aspects of the disorder. This study confirms efficacy in long term use and may guide clinicians in dosing and treatment emergent, long-term adverse effects.http://www.huntington-study-group.org/. None of the HSG investigators or staff had equity interests with Prestwick Pharmaceuticals, Inc. Dr. Fahn received consulting fees of less than $10,000 from Prestwick Pharmaceuticals. After the study was completed, Dr. Frank received consulting fees of less than $10,000 from Ovation Pharmaceuticals. Dr. Marshall presented data at meetings for which he received travel reimbursement from Prestwick Pharmaceuticals, Inc. Dr. Stamler and Ms. Wilson were employees of Prestwick Pharmaceuticals, Inc. The HSG Coordination and Biostatistics Centers at the University of Rochester independently compiled and analyzed the data for this study.This study was funded by a grant from Prestwick Pharmaceuticals, Inc.  to the University of Rochester and in turn through subcontracts to the participating research sites. The Huntington Study Group (HSG) is a non-profit consortium of HD investigators The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/9/62/prepub


Tobacco smoking is a recognized risk factor for many chronic diseases and previous study evidences have indicated that smokers receive smoking cessation service after the diagnosis of chronic diseases increases successful rate in quitting. But the prevalence of tobacco related chronic diseases (TCD) among smokers, as well as the role of TCD diagnosis in smoking cessation is still unclear in China.From June 2016 to December 2017, we sampled 36, 698 residents aged over 18 years by a three stage sampling in Songjiang district, Shanghai. We conducted a cross-sectional study to understand the prevalence of TCD among smokers, and the role of TCD diagnosis in smoking cessation among ex-smokers as well as the smoking cessation attempt among current smokers.Over all, the prevalence of current smoking is 19.78% . 15.93% of smokers have stopped smoking successfully . The prevalence of ten selected TCDs among smokers range from 0.63%  to 36.31% (hypertension). All of 1, 376 ex-smokers had at least one kind of TCD, and 52.33% of them stop smoking after the diagnosis of TCD, the time interval between TCD diagnosis and smoking cessation ranges from 0 to 65 years, with a median of 9 years. Smokers with TCD had higher prevalence of quit smoking, and current smokers with TCD had higher smoking cessation attempt proportion.The prevalence of current smoking is still very high among male residents in rural area of Shanghai, and the occurrence of TCD even non-lethal one could provide an opportunity for doctors to assist the smoking cessation among smokers. Smoking is a practice in which substance like tobacco is burned and the resulting smoke inhaled in to be tasted and absorbed into the bloodstream . GeneralChina is the largest producer and biggest consumer of tobacco in the world, which produce a heavy burden of tobacco related chronic diseases (TCD). The Global Adult Survey conducted in 2010 indicates that 300 million adults in China are current smokers, with 72.40% of non-current smokers are exposed to second hand smoke (SHS), and 1 million deaths are attributed to tobacco consumption each year . Due to In this paper, we conducted a cross sectional study among residents in a rural area of Shanghai China. We aim to understand the prevalence of smoking and current smoking, the prevalence of TCD among smokers, and the role of TCD diagnosis in smoking cessation among ex-smokers as well as the smoking cessation attempt among current smokers.This cross-sectional study was conducted in Songjiang District, a rural area of Shanghai from June 2016 to December 2017. Songjiang District is located in the southwest of Shanghai, with a population size of 1.76 million in the year of 2016 . The TCD for each patients were then verified by the diseases information extracted from the local HIS . Part C covered 36 health-related behavior information , we designed 16 questions to collect tobacco use information . Part D covered personal contact information both for the investigator and the participant.Data were collected by a questionnaire designed by School of Public Health, Fudan University. The questionnaire includes four parts. Part A covered 8 demographic questions including age, gender, ethnicity, education level, monthly income, working status, marital status and occupation. Part B covered 10 tobacco related chronic diseases (TCD) information including hypertension, coronary disease, cerebral apoplexy, diabetes, chronic bronchitis, asthma, COPD, chronic gastritis, chronic hepatitis and cancer. We designed 3 questions to collect information for each TCD . Education is recorded as completed years of schooling and categorized to 5 categories of 0 year (illiterate), 1–6 years (primary school), 7–9 years (junior high school), 10–12 years (senior high school), and > 12 years (college and above). Monthly income is collected in Chinese Yuan (RMB) and classified into four groups .p-value of less than 0.05 (two-tailed) was considered as statistically significant.Data analysis was performed by SAS software (version 9.2). We described the data by using frequency counts and proportions (rate) for qualitative variables and means as well as standard deviations (SD) for quantitative variables. We applied Chi-squire test to examine the difference of smoking prevalence, current smoking prevalence and quit smoking prevalence among participants with different demographic characteristics, and the differences of TCD prevalence between non-smokers and smokers, as well as between current smokers and ex-smokers. We also applied Chi-squire test to compare the difference of quit smoking prevalence among smokers with TCD and without TCD, as well as the difference of smoking cessation attempt proportion among current smokers with TCD and without TCD. Weighted logistic regression was applied to calculated the odds ratios (OR) and 95% confidence interval (95% CI) of quit smoking prevalence among smokers with TCD compared with smokers without TCD, as well as the smoking cessation attempt proportion among current smokers with TCD compared with current smokers without TCD. Co-variables selected as potential confounders by Directed Acyclic Graph (DAG) were adjusted in the weighted logistic regression, including gender, age (age groups was changed into dummy variable and set ‘18–29’ as the reference), education (education was changed into dummy variable and set ‘illiterate’ as the reference), working status, and alcohol consumption. A box plot was produced by calculating the years for TCD diagnosed before quit smoking among ex-smokers. A In this study, we investigated 36, 698 residents in Songjiang district, including 14, 912 males ) and 21, 786 females ). The age ranged from 18 to 92 with an average age of (56.37 ± 11.29) years old. The majority of residents were married ), and over 80% of residents had an education under junior high school (less than 12 years), more than 50% of residents’ monthly income were over 2500 RMB and 58.58% (95% CI: 58.13–58.84%) of residents were retired at the investigation time, see Table Among 36, 698 investigated residents, 8, 636 were smokers, the prevalence of smoking was 23.53% (95% CI: 23.10–23.97%). The prevalence of smoking was significantly higher in males (57.48%) than in females (0.30%). Residents aged 18 to 29 years had the lowest prevalence of smoking, whereas residents aged 60 to 69 years had the highest prevalence of smoking, and the prevalence of smoking in other 4 age groups ranged from 16.58 to 23.66%, the prevalence of smoking was statistically significant in different age groups. Residents with education of illiterate and over college had lower prevalence of smoking than residents with education of primary and high school (junior and senior), and the difference was statistically significant. Married as well as divorced residents had higher prevalence of smoking than unmarried and widow/widower, and the differences was statistically significant. Residents with monthly income over 2500 RMB had higher smoking prevalence, but the difference was not statistically significant. Retired residents had lower smoking prevalence than non-retired residents, and the difference was statistically significant. The different prevalence of smoking in six age groups was still statistically significant, after the adjustment of gender, marriage, working status and education. And the difference in smoking prevalence among residents with different education was still statistically significant even with the adjustment of gender, working status and marriage. See Table For current smoking, the prevalence was 19.78% (95% CI: 19.38–20.19%) in 36, 698 residents. The prevalence of current smoking in male (48.36%) was significantly higher than in female (0.22%); and the prevalence of current smoking increased with the increase of age among residents. For education, marriage and working status, retired residents and residents with illiterate and above college had lower prevalence of current smoking, and married as well as divorced residents had higher prevalence of current smoking. See Table In this study, 1, 376 out of 8, 636 smokers had stopped smoking, the prevalence of quit smoking was 15.93% (95% CI: 15.16–16.71%). The prevalence of quit smoking was higher in older residents than younger residents, and retired residents had higher quit smoking prevalence than non-retired residents, the differences were all statistically significant; residents with lower education level and higher monthly income were prone to quit smoking. See Table p < 0.01) and chronic bronchitis  among smokers were higher than non-smokers, but smokers had a lower prevalence of coronary diseases than non-smokers . See Table In this study, the prevalence of TCDs among 36, 698 residents ranged from 0.53% for COPD to 33.05% for hypertension. Table The prevalence of the ten selected TCDs ranged from 0.48 to 34.45% among current smokers, and ranged from 1.38 to 46.15% among ex-smokers. For each TCD, the prevalence among ex-smokers was higher than current smokers, and the differences of TCD prevalence between current smokers and ex-smokers were all statistically significant except for chronic hepatitis. See Table In this study, smokers with TCD had higher prevalence of quit smoking than smokers without TCD. Tables In this study, all of 1, 376 ex-smokers had at least one kind of TCD. We divided ex-smokers into two groups according to chronological order of TCD diagnosis time and smoking cessation time. In total, 720 ex-smokers stopped smoking after the diagnosis of TCD, accounting for 52.33%. Table Figure In this study, current smokers with hypertension (14.83% vs 15.68%) as well as cancer (14.81% vs 15.39%) had slightly lower smoking cessation attempt proportion than current smokers without the TCD, but was not statistically significant. For coronary diseases, cerebral apoplexy, diabetes, chronic bronchitis, asthma, COPD, chronic gastritis and chronic hepatitis, current smokers with these TCDs had statistically significant higher smoking cessation attempt proportion than current smokers without these TCD after the adjustment of age, gender, education, working status and alcohol consumption, the OR value ranged from 1.25 to 2.06. See Table Morbidity and mortality related to smoking is a major public health challenge worldwide . In thisHammond D, etc.  reportedJha P  previousPrevious studies identified age as one of the factors related to smoking cessation. In studies that conducted in Western countries, age was mostly reported as negatively related with smoking cessation . WhereasA key strength in this study is the local HIS  verified TCD information. The HIS is a web-based system collecting disease information in real-time from hospitals of all levels in Shanghai. The HIS covers diseases information including but not limited to demographic information, onset of diseases, symptoms, diagnosis time, treatment, prescription information and cost, etc. In this study, we use the HIS to verify the self-report TCD information that collected by questionnaire for two reasons. First, the HIS verified TCD information is accurate without recall bias, it improves the study quality in the aspect of TCD prevalence calculation and the ascertainment of time interval between disease diagnosis and smoking cessation. Second, the consistency between HIS and the self-reported TCD information could reflect the data quality collected by the questionnaire, and the consistent rate in this study is finally proved as 95.88%. Meanwhile, we sampled 36, 698 residents that accounting for about 6% of total local population in Songjiang district, the large study population size is other strength of this study, study results could be generalized to total residents in Songjiang district, or even other rural areas of Shanghai.There are some limitations in this study. First, the cross-sectional study design only allow the calculation of prevalence but not the incidence rate, which limited the estimation of causal relation between cigarette smoke and TCDs. Second, the time for TCD diagnosis was verified by the HIS, but the time for smoking cessation was self-reported by residents which could induce recall bias, leading to inaccuracy of time interval calculation between disease diagnosis and smoking cessation. Third, in this study, approximately 16% current smokers had smoking cessation attempt in a recent year but all relapsed, but reasons for smoking cessation relapse were not collected which impeded the establishment of targeted tobacco control measure in this group. Forth, this study was implemented in Songjiang district, a rural area of Shanghai, since smoking issue is not only a rural topic, we need select some urban residents in future studies. Fifth, for TCD diagnosis is correlated with age, it is better to describe the relationship between TCD diagnosis time and smoking cessation time with the adjustment of age, but due to the small number of ex-smokers with TCDs in this study, it is impossible to analyze the data by different age groups, which might induce some bias. Sixth, we only adjusted age, education, gender, working status and alcohol consumption to evaluate the role of TCD diagnosis in smoking cessation, the other features includes diseases severity, family relationships, marital status may also influence the study results as well, so incorporation of some improvements should be considered in further studies.The prevalence of current smoking was still very high among male residents in rural area of Shanghai, China, and the occurrence of TCDs even non-lethal one could provide an opportunity for doctors to assist smoking cessation among smokers.


Understanding the genetic function of the forage quality-related traits, including crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose (HC), and cellulose (CL) contents, is essential for the identification of forage quality genes and selection of effective molecular markers in sorghum. In this study, we genotyped 245 sorghum accessions by 85,585 single-nucleotide polymorphisms (SNPs) and obtained the phenotypic data from four environments. The SNPs and phenotypic data were applied to multi-locus genome-wide association studies (GWAS) with the mrMLM software. A total of 42 SNPs were identified to be associated with the five forage quality-related traits. Moreover, three and two quantitative trait nucleotides (QTNs) were simultaneously detected among them by three and two multi-locus methods, respectively. One QTN on chromosome 5 was found to be associated simultaneously with CP, NDF, and ADF. Furthermore, 3, 2, 2, 5, and 2 candidate genes were identified to be responsible for CP, NDF, ADF, HC, and CL contents, respectively. These results provided insightful information of the forage quality-related traits and would facilitate the genetic improvement of sorghum forage quality in the future. Sorghum bicolor L.) is a popular crop worldwide, which is used a food source, animal fodder, and raw material for alcoholic beverages and biofuels in industries  content are the main focus for forage sorghum breeding , which is based on linkage disequilibrium (LD) and provided sufficient genetic background information, have become a powerful alternative for the investigation of quantitative traits. There are three main strategies for GWAS. Firstly, a generalized linear model (GLM) was proposed for the genetic analysis of the quantitative traits  and phenotyped them in the four environments. The data were analyzed by the multi-locus GWAS software, mrMLM.The 245 sorghum accessions Table  includedSeven hundred and thirty-five sorghum samples (3 replicates) were measured for CP, CL, HC, NDF, and ADF using the traditional chemical methods, and simultaneously scanned for near-infrared (NIR) spectra with an Antaris™ II FT-NIR Analyzer . A model was established using TQ Analyst software based on the NIR spectra and the results of the chemical analysis. The samples were then scanned for NIR spectra, and their CP, CL, HC, NDF, and ADF were calculated using the model. The mean of the phenotypic data and the correlation coefficients were calculated using Microsoft Excel.Total DNA was extracted using the DNAsecure Plant Kit . All the samples were standardized to 50 ng/μL, and 10 μL of each sample was digested with the enzymes, PstI (CTGCAG) and MspI (CCGG), at 37°C for 2 h and then at 65°C for 20 min. The digested samples were ligated with the adapters from Illumina . The ligated samples were then pooled using the same volume (10 μL) for PCR-amplification in a single tube. The fragment length was analyzed using a Bioanalyzer (Agilent), and the PCR products were quantified by a Qubit3.0 fluorometer (Invitrogen). The GBS library was run on an Illumina Hiseq2500 .http://hannonlab.cshl.edu/fastx_toolkit/). The high-quality sequencing data were aligned using BWA MEM  of fastx_toolkit-0.0.13.2  is an n × c matrix of covariates (fixed effects), including a column vector of 1; the population structure or principal components can be incorporated into W. Moving on, α is a c × 1 vector of fixed effects, including the intercept, and X is an n × 1 vector of marker genotypes. Z is an n × m design matrix, and m × 1 vector of polygenic effects. K is a known n × n relatedness matrix. n × 1 vector of residual errors, and In is an n × n identity matrix, and MVN denotes multivariate normal distribution. An LOD score of 3 was used as the critical threshold for significant QTNs for all the six methods.The GWAS for the five forage quality-related traits  was performed using six methods, including mrMLM, FASTmrMLM, FASTmrEMMA, pLARmEB, pKWmEB, and ISIS EM-BLASSO in the mrMLM software. The main model used in this study in the mrMLM software is as follows :https://www.ncbi.nlm.nih.gov/genome/108). The detailed functions of the corresponding genes were annotated by performing BLASTP search at the NCBI website, and the candidate genes were assigned to different biological processes based on the function of their homologs in other species in literature or with the help of data in the Conserved Domains Database. The selected candidate genes were associated with the main QTNs of the five traits if they made a contribution (r2) greater than 5%.Genes that were hit directly by the associated QTNs within a 50-kb stretch were selected to choose the candidate genes as described in Upadhyaya et al. . The phyExtensive phenotypic variations of CP, CL, HC, NDF, and ADF were observed in the 245 sorghum samples in the four environments, including two locations in 2 years  across the four environments using six methods Table . There wr2 > 5%). Besides, 3 QTNs  were detected simultaneously by 3 methods, and another 5 QTNs  were identified simultaneously by 2 methods. The remaining QTNs were detected by a single method, but most of them were considered as reliable because of the high thresholds at which they were detected.Among the above six methods, pLARmEB was the most powerful and accountable for the identification of the 24 QTNs that mainly contributed to the NDF content trait (17 QTNs); however, their contributions were less than what were detected by other methods, except for one major QTN RSS17673), whose contribution was greater than 5%  encoded a serine/threonine-protein kinase (Sobic.002G217100), which was consistent with a previous study that concluded that serine/threonine-protein kinases are involved in signal cascade for nitrogen metabolism in plants (Champigny, Genome-wide association study is an important alternative for mapping quantitative traits. It has been applied rapidly and extensively in plant research. These methods have been widely adopted, but only a few QTNs for each complex trait have been identified. In this study, we implemented the latest multi-locus GWAS methods available in mrMLM (Wang S. B. et al., According to the GWAS analysis, 5, 3, 3, 7, and 24 QTNs were identified for CP, CL, HC, ADF, and NDF content, respectively. Of the 5 candidate loci for the CP content, 2 were already identified in the previous studies. The locus on chromosome 9 was mapped in the same region by Murray et al.  and Li eThe QTLs for the NDF or ADF content co-localized with those for the CL or HC content, which has been reported previously in sorghum. Cardinal et al.  reportedBoth NDF and ADF include CL and lignin. There are a series of reports about the biosynthesis and signaling pathways of CL and lignin in plants (Kim et al., Lignin, CL, and HC are the main components of secondary cell walls (Zhong et al., JL, LW, and S-QX designed and conceived the experiments. Y-WZ, K-NC, and S-BW performed the computational analysis. WT and JL extracted the DNA and performed the experimental analysis. CCW and YL assisted with experiments in data collection and analysis. QZ and CW participated in the design and supervised the study. S-QX and JL discussed the results and interpretation of the final data. S-QX and JL drafted the manuscript. All authors read and approved the final manuscript.The reviewer ML declared a shared affiliation, though no other collaboration, with several of the authors WT, CW to the handling Editor. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Land-use change can have profound effects on forest communities, compromising seedling recruitment and growth, and long-term persistence of forests on the landscape. Continued forest conversion to agriculture causes forest fragmentation which decreases forest size, increases edge effects and forest isolation, all of which negatively impact forest health. These fragmentation effects are magnified by human use of forests, which can compromise the continued persistence of species in these forests and the ability of the forests to support the communities that depend on them. We examined the extent and influence of human disturbance  on the ecological status of sacred church forests in the northern highlands of South Gondar, Ethiopia and hypothesized that disturbance would have a negative effect. We found that disturbance was high across all forests (56%) and was negatively associated with tree species richness, density, and biomass and seedling richness and density. Contrary to expectation, we found that forests < 15.5 ha show no difference in disturbance level with distance from population center. Based on our findings, we recommend that local conservation strategies not only protect large forests, but also the small and highly used forests in South Gondar which are critical to the needs of local people, including preserving large trees for seed sources, removing exotic and weedy species from forests, and reducing clearings and trails within forests. Isolation and degradation can be the silent demise of forests as towering canopy trees give the impression of a healthy forest ecosystem, while understory species composition can be dominated solely by pioneer species, completely void of seedlings from old growth species. This phenomenon has been written about in numerous ways, most famously by Janzen  who charEucalyptus; [Land use change (LUC) continues in tropical countries, with a conservative estimate from the Food and Agricultural Organization of 8 million ha deforested annually from 2000–2010 , plantinalyptus; ), and lialyptus; , 15, decalyptus; , increasalyptus; , and losalyptus; . Land usalyptus; . These eForest size, the degree of disturbance, the presence of roads and distance to population center can also contribute to forest degradation. In tropical regions, roads are cited as the “first of a hundred cuts” that degrade intact forests, as they allow the initial penetration of the forests, which is quickly followed by increased population density, logging, and minor road development . As such2) there are 1022 of these forests that span elevations and biomes making up ~ 5500 ha of tree cover . These forests have a church at their center and are considered critical in protecting the sacredness of the church and as such their association with the church protects them [While landscapes characterized by large, intact forests are best suited to sustain high levels of biodiversity and provide a constant supply of multiple ecosystem services , 24, 25,cts them . This prcts them , 29. Thects them . Howevercts them .-1), and biomass (ha-1) of standing live trees, as well as the richness and density of tree seedlings in 44 church forests across South Gondar, Ethiopia. Previous research indicates that the presence of a wall, established for various reasons including demarcating land and blocking human and animal access, can have a protective effect on forests as their presence increased seedling richness and density, but did not affect tree species richness or density [This study principally aimed to determine the impacts of human disturbance  on church forest integrity and regeneration potential. In addition, we examined the factors of forest size, elevation, distance to population center, and the presence of an exterior wall on species richness, density . The distribution and abundance of the church forests in the region are detailed in another paper . In brieThese forests are sacred as they surround a Christian Orthodox Tewahido Church , 28, 30.Prunus (Rubiaceae) and Mimusops (Sapotaceae) and the upper montane region (2400–2700 m a.s.l) characterized by Juniperus (Cupressaceae) and Euphorbia (Euphorbiaceae) [-1; and seedling richness and density.The church forests of Ethiopia span elevations and biomes, with montane region (1800–2050 m a.s.l.) characterized by evergreen broadleaf taxa biaceae) . Sample Modified Gentry transects , 32 wereNative taxa were noted along each transect in addition to the type and location of any form of disturbance across four categories . While o2 quadrats for a total of 3 seedling plots per transect and 9 seedling plots per site.Along each transect at three points , we measured seedling richness and density in 1 mp > 0.05).We used Model 1 Fixed Effects Analysis of Variance (ANOVA) to determine if size, distance to population center, elevation, and the presence of a wall were significantly associated with each of our dependent variables, namely: tree species richness, trees species density, tree biomass, seedling density, seedling richness, and disturbance . We usedTo compare tree species richness among church forests, we used rarefied species richness to control for the variation in the number of individual trees found among our sites. We used the rarefy function in the vegan package  in R  in 105 genera and 69 families and found an average species richness, excluding exotic and planted taxa, of 10.8 (± 0.5). The most species rich plant families were: Fabaceae, Asteraceae, Rubiaceae, Celastraceae, Euphorbiaceae, Lamiaceae and Malvaceae.F3,180 = 33.02, p < 0.001; F4,39 = 1.888, p = 1.34). However, disturbance had a weak but significant, negative linear relationship with forest area .Human disturbance was pervasive across sites, with an average total disturbance of 56.2% ± 3.2; . When brWe found that species richness was significantly associated with forest size and disturbance, but not elevation, distance to population center or the presence of a wall . Species-1) was significantly associated with forest size, disturbance, and elevation, but was not associated with distance to population center or the presence of a wall (-1) was significantly higher in the montane region (65.5 ± 9.2) than the upper montane region . While disturbance did not vary significantly between elevations , tree density in montane forests did not vary with disturbance whereas upper montane forest tree density varied significantly with disturbance  in the full ANOVA model .Disturbance was the only factor that significantly explained biomass  and a marginally significant effect of wall and wall * size interation, but no effect of forest size, on seedling density . In church forests with a wall, we found a significant positive relationship between seedling richness and density with forest size , but no significant effect of either without a wall .Seedling density (2.1 ± 0.33) and richness (0.66 ± 0.09) were low across all sites. We found that there was a significant effect of a wall, forest size, and a significant wall * size interaction on seedling richness (Model: -1) and density (ha-1) of species. The forest clearings are important for human gatherings and other activities [Justica and Acanthus (Acanthaceae), have been shown to outcompete tree species hindering regeneration [Our research on 44 sacred Ethiopian church forests across elevations and distances from population center indicate that more than half of the forest area showed human disturbance, which was the key factor, followed by size, influencing species richness, biomass  and tree density (ha-1) decreased significantly and this was exacerbated by decreasing forest size. In a survey of 78 church forests in the northern highlands of Ethiopia, the average size was even smaller than found in our study , an, an-1) a-1) was the only variable influenced by elevation, with upper montane forest density decreasing with increasing disturbance while montane forest density did not vary significantly (Teclea (Rutaceae) and Mimusops (Sapotaceae), whereas the upper montane region has more sparcely dispersed taxa such as Juniperus (Cupressaceae) and Euphorbia (Euphorbiaceae).Tree density  due to their increasing isolation combined with high disturbance and low seedling recruitment [While these church forests have exhibited remarkable persistence over the last 50–80 years, on average only 0.42 ha smaller 50 years ago , recent ruitment , 45, 46.2 area of our study, there are 1022 forests with 93% of them < 15 ha Click here for additional data file.


Controversies exist between the previous two prognostic nomograms for patients with bone metastatic prostate cancer (PCa), and a nomogram applied to western patients has yet to be established. Thus, we aimed to build a reliable and generic nomogram to individualize prognosis.The independent prognostic factors were identified in a retrospective study of 1556 patients with bone metastatic PCa registered in the Surveillance, Epidemiology and End Results (SEER) database. Besides, the prognostic nomogram was developed using R software according to the result of multivariable Cox regression analysis. Then, the discriminative ability of the nomogram was assessed by analyses of receiver operating characteristic curves (ROC curves). We also performed 1-, 2-, and 3-year calibrations of the nomogram by comparing the predicted survival to the observed survival. Furthermore, the model was externally validated using the data of 711 patients diagnosed at different times enrolled in the SEER database.Age ≥70 years, Gleason score ≥8, PSA value of 201 to 900 ng/ml, stage T4, stage N1, with liver metastases, and Asian/Pacific ethnicity were identified as independent prognostic factors. In the primary cohort, 1-, 2-, and 3-year area under the ROC curve (AUC) of the nomogram for predicting cancer-specific survival (CSS) were 0.71, 0.70, and 0.70, respectively. Besides 1-, 2-, and 3-year AUC were 0.70, 0.68, and 0.69, respectively, in the external validation cohort. Moreover, calibration curves presented perfect agreements between the nomogram-predicted and actual 1-, 2-, and 3-year CSS rate in both the primary and external validation cohorts. In other words, our nomogram has great predictive accuracy and reliability in predicting 1-, 2-, and 3-year CSS for patients with bone metastatic prostate cancer.This study established and validated a prognostic nomogram applied to not only Asian patients but western patients with bone metastatic PCa, which will be useful for patients’ counseling and clinical trial designing. With the occurrence of bone metastases, patients experience bone pain, pathological fracture, and other symptoms that negatively affect their quality of life.,5 Worse still, the 5-year survival rate of patients with bone metastases was significantly lower than that of those without bone metastases (3% vs 56%).Prostate cancer (PCa) is the second most common malignancy and the fifth leading cancer-related cause of death in men worldwide, with nearly 1.3 million newly diagnosed cases and about 359,000 deaths in 2018. At present, the independent prognostic factors for bone metastatic PCa remain controversial and there are significant differences in OS between studies.,9 It should also be noted that prostate-specific antigen (PSA) values were >20 ng/ml in over 90% patients with bone metastatic PCa, which means almost all patients enter the D’Amico high-risk stage when bone metastases are present. Therefore, nomograms based on the equation derived from the regression coefficients of each variable always integrates many prognostic factors may better predict their survival. Unfortunately, noticeable differences and even wholly contrary opinions exist regarding the contribution of some factors in the previous two prognostic models (namely the Indonesian and Japanese models) for patients with bone metastatic PCa, which may lead to confusion among urologists and patients.,11 Furthermore, in view of geographic and ethnic differences, these two models can only be applied within Indonesia or Japan. For these reasons, there is an urgent need to obtain accurate information on factors correlated with survival and to develop a prognostic model that can be applied to not only Asian patients but also western patients.In the past two decades, androgen axis therapies have made remarkable advances; however, the overall survival (OS) and cancer-specific survival (CSS) of patients with metastatic PCa have not improved.The present study represents one of the most extensive series to investigate prognostic factors for patients with bone metastatic PCa. Moreover, this is the first nomogram that can be applied to multi-racial patients with bone metastatic PCa.22.1 After obtaining approval from the NCI, we searched a total of 316,724 cases of PCa registered between 2010 and 2015. We included only cases with a histological subtype of adenocarcinoma ; PSA values of 20 to 900 ng/ml; and non-Hispanic white (NHW), non-Hispanic black (NHB), Hispanic (HIS), and Asian/Pacific Islander (A/PI) ethnicities. Patients were excluded from our cohort if PCa was not the first tumor for patients, if the cases were diagnosed without or with unknown bone metastases, if the cases were diagnosed through death certificate or at autopsy only, if the cases were missing clinical or demographic data , and if the cases were T0 stage. Furthermore, cases diagnosed after January 1, 2015, were excluded to ensure that all cases had undergone observation of survival status for more than 1 year when the last follow-up was conducted on December 2015. Finally, 2267 cases were eligible for our study. The 1156 patients who were diagnosed between January and August of each year from 2010 to 2014 were included in the primary cohort, while the 711 patients diagnosed between September and December of each year from 2010 to 2014 constituted the validation cohort. The present study conformed to the 1964 Helsinki Declaration and its later amendments, and was approved by the research ethics board of Xijing Hospital of the Air Force Medical University.The SEER database supported by the National Cancer Institute (NCI) is a population-based cancer registry covering approximately 30% of the United States population.2.2 Finally, continuous variables  were transformed into categorical variables according to the median number in the analysis.The covariates extracted from the SEER database included the patients’ demographic characteristics , PSA value, Gleason score, T stage, N stage, and the presence or absence of liver/lung metastases. As we were aiming to establish a pre-treatment nomogram, we did not consider treatment variables. In addition, we only included cases with PSA value between 20 and 900 ng/ml because the SEER database does not provide specific values over 980 and patients with PSA levels <20 ng/ml only account for about 3% of all patients with bone metastatic PCa.The starting point of follow-up was the date of diagnosis with bones metastatic PCa. The endpoint was cancer-specific death or the last follow-up in December 2015. The data of patients who were lost to follow-up, died due to other causes, or who survived to the last follow-up were censored.2.3P < .1 in univariate Cox regression were entered into multivariate Cox proportional hazard models for further analysis.Statistical analyses to identify the prognostic factors were performed in SPSS 22.0 . The Kaplan–Meier method was used to estimate the CSS and OS. The significance of differences in CSS was assessed by log-rank tests. Variables that achieved significance at http://www.r-project.org/). The predictive accuracy of the nomogram was measured by ROC curve analysis. Besides, 1-, 2-, and 3-year calibrations of the nomogram were performed by comparing the predicted survival to the observed survival. Bootstraps with 1000 resamples were used for these evaluations. The data were extracted using SEER∗Stat Software version 8.3.5. Differences with P ≤ .05 (two-sided) were considered statistically significant.The nomogram was developed according to the results of multivariate Cox analysis and using the rms package in R version 3.5.1 , 46.7% (726/1556) of patients died, and 39.2% (610/1556) died due to PCa up to the last follow-up conducted in December 2015. The median CSS was 51 months , with 1-, 2-, and 3-year CSS rates of 88.1%, 71.6%, and 60.5%, respectively. The median OS was 41 months , with 1-, 2-, and 3-year OS rates of 85.6%, 67.5%, and 55.0%, respectively.In the validation cohort, after a median follow-up of 24 months (interquartile range: 13–38 months), 41.4% (294/711) of patients died, and 33.3% (237/711) died due to PCa. The median CSS was 54 months , with 1-, 2-, and 3-year CSS rates of 87.6%, 73.4% and 60.4%, respectively. In addition, the median OS was 40 months  and the 1-, 2-, and 3-year OS rates were 84.8%, 68.2%, and 54.2%, respectively.3.2P < .001, hazard ratio [HR] = 1.436, 95% CI, 1.219–1.691); PSA of 201-900 ng/ml ; Gleason scores of 8 , 9 , and 10 ; T4 stage ; N1 stage ; and combined with liver metastases  were independent risk predictors for CSS in patients with bone metastases PCa. In addition, A/PI ethnicity  was an independent protective factor for CSS compared to NHW as the reference. As shown in Table Through univariable analysis and subsequent multivariable Cox analysis, patient age ≥70 , and its superiority was further verified by comparing with the Gleason system, which made the most significant contribution to survival in our nomogram. In the primary cohort, the nomogram showed strengths in the discriminative ability compared with the Gleason system  at 1, 2, and 3 years after the first diagnosis in both the primary cohort and the external validation cohort. In other words, perfect agreements were achieved between the nomogram-predicted and actual 1-, 2-, and 3-year CSS rates, which guaranteed the reliability of the nomogram, as indicated by the calibration plots  focused exclusively on patients presenting with bone metastases.,19 Unfortunately, significant differences regarding the contribution of age, and opposite opinions related to the contribution of PSA exist between the two models. Moreover, both models can only be used for Asian populations, in view of geographic and ethnic differences, a nomogram applied to westerners has yet to be established. For these reasons, the main aim of our study was to develop a generic model on the basis of large samples, which could be applied to not merely Asian patients but also western patients with bone metastatic PCa.A nomogram is a graphical representation of a multivariable prognostic model that integrates many prognostic factors and can be used to evaluate individual probabilities of survival at a certain time accurately. Nomograms have been built for several cancers and have been shown to be more accurate than traditional tools in predicting prognosis.Through univariable analysis and subsequent multivariable analysis, we identified Gleason score ≥8 and T4 stage as independent risk factors for CSS, concordant with the findings of the previous Indonesian and Japanese studies. Age also played a crucial role in our model, similar to that in the Japanese model. However, age was not included in the Indonesian nomogram because it was not an independent predictor for OS. This difference may be caused by the insufficient sample size of the Indonesian cohort.More interestingly, with the increase in PSA valve, the prognosis worsened in the Indonesian nomogram, completely opposite to the finding that patients with a higher PSA value have a more favorable prognosis in the Japanese study. The completely contrary opinions between the previous two models for patients with bone metastatic PCa are confusing when predicting patient survival. Fortunately, based on large samples, we confirmed that high PSA is associated with inferior prognosis; however, the predictive contribution of PSA in the bone metastases PCa-specific nomogram is very weak. However, to our knowledge, our study is the first one to include N stage and liver and lung metastasis status into models for patients with bone metastasis PCa. In our nomogram, the presence of liver metastases, which is probably because of lymphovascular invasion, made a decisive contribution to an inferior prognosis. Therefore, we strongly recommend that patients with bone metastatic PCa undergo immediate upper abdominal CT examination to determine whether liver metastasis is also present to better predict prognosis and adopt a reasonable treatment regimen. In the present study, only 10 PCa patients had bone plus brain metastases after excluding cases with incomplete clinical or demographic data; thus, as this sample did not meet statistical requirements, we did not include information on brain metastases as a variable in our analyses.In addition, we identified N1 stage and the presence of liver metastases as independent risk predictors for CSS, consistent with another study also using data from the SEER database.,22 The results of our study confirmed that NHW had an approximately two-fold increased risk of death compared to that of A/PI and that there were almost no survival differences among NHW, NHB, and HIS. Petrovics et al reported different spectrums of genomic alterations between African-American and Caucasian-American patients with PCa; thus, the differences in survival between A/PI and other ethnicities may also be due to different spectrums of genes. Because of the ethnic differences in survival, there are considerable limitations in the use of the Indonesian and Japanese models. With broader applicability, our model is the first nomogram for western patients with bone metastatic PCa and can be used in almost all patients with bone metastatic PCa worldwide by incorporating ethnicity as a variable in the nomogram, which may be the greatest strength of the present study.Ethnic differences in survival have been reported in patients with PCa.The present study was based on data from the SEER database, with a large sample size and sufficient data. However, our study has several limitations. First, as we aimed to develop a pre-treatment nomogram, we did not include treatment variables, by adding which might increase the predictive accuracy of the nomogram. In addition, we only included patients who had presented with bone metastases at first diagnosis and did not include those who developed bone metastases at later times, who were not enrolled in the SEER database. Furthermore, although they may also be strong independent prognostic factors, we did not analyze alkaline phosphatase and hemoglobin levels, as these variables are not available in the SEER database. Moreover, patients with missing data with respect to each of the variables were excluded from our cohort, which may have increased the bias. Despite these limitations, the performance of our model are ensured, as evidenced by the agreements between the nomogram-predicted and actual 1-, 2-, and 3-year CSS, with 1-, 2-, and 3-year AUC of 0.71, 0.70, and 0.70, respectively.5Age ≥70 years, Gleason score ≥8, PSA value of 201 to 900 ng/ml, T4 stage, N1 stage, and the presence of liver metastases were identified as independent risk factors and that A/PI ethnicity was an independent protective factor for survival in patients with bone metastatic PCa. Furthermore, we established a reliable and generic prognostic nomogram for application to not only Asian but Western patients with bone metastatic PCa, by which 1-, 2-, and 3-year CSS can be predicted individually and accurately. However, further validation using external data is required to generalize the applicability of our model in clinical practice.Study designed: Jianlin Yuan. Searching of data: Jiarui Yuan. Statistical analysis: Guangdong Hou and Zheng Zhu. Analysis and interpretation of data: Yu Zheng, Fuli Wang, Geng Zhang, Fei Yan and Zhibin Li. Drafting of the manuscript: Guangdong Hou. Figs designed: Xi’an Li and Jingyang Tian. Searching of literature: Di Wei, Bin Zhang, and Zibao Xing. Obtained funding: Ping Meng and Ming Gao.Data curation: Jiarui Yuan.Formal analysis: Yu Zheng, Fuli Wang, Geng Zhang, Fei Yan.Funding acquisition: Ping Meng, Ming Gao.Methodology: Jianlin Yuan.Resources: Di Wei, Zhibin Li, Bin Zhang, Zibao Xing.Software: Xi’an Li, Jingyang Tian, Zheng Zhu.Supervision: Fuli Wang.Writing – original draft: Guangdong Hou.


NUCB2) gene might be associated with milk production traits due to its expression being significantly increased in early lactation or peak of lactation as compared to dry period . Hence, in this study, we detected the single nucleotide polymorphisms (SNPs) of NUCB2 and analyzed their genetic associations with milk yield, fat yield, fat percentage, protein yield, and protein percentage. We re-sequenced the entire coding and 2000 bp of 5′ and 3′ flanking regions of NUCB2 by pooled sequencing, and identified ten SNPs, including one in 5′ flanking region, two in 3′ untranslated region (UTR), and seven in 3′ flanking region. The single-SNP association analysis results showed that the ten SNPs were significantly associated with milk yield, fat yield, fat percentage, protein yield, or protein percentage in the first or second lactation . In addition, we estimated the linkage disequilibrium (LD) of the ten SNPs by Haploview 4.2, and found that the SNPs were highly linked in one haplotype block (D′ = 0.98–1.00), and the block was also significantly associated with at least one milk traits in the two lactations . Further, we predicted the changes of transcription factor binding sites (TFBSs) that are caused by the SNPs in the 5′ flanking region of NUCB2, and considered that g.35735477C>T might affect the expression of NUCB2 by changing the TFBSs for ETS transcription factor 3 (ELF3), caudal type homeobox 2 (CDX2), mammalian C-type LTR TATA box (VTATA), nuclear factor of activated T-cells (NFAT), and v-ets erythroblastosis virus E26 oncogene homolog (ERG) . However, the further study should be performed to verify the regulatory mechanisms of NUCB2 and its polymorphisms on milk traits. Our findings first revealed the genetic effects of NUCB2 on the milk traits in dairy cows, and suggested that the significant SNPs could be used in genomic selection to improve the accuracy of selection for dairy cattle breeding.We previously used the RNA sequencing technique to detect the hepatic transcriptome of Chinese Holstein cows among the dry period, early lactation, and peak of lactation, and implied that the nucleobindin 2 ( NUCB2) gene was significantly increased in early lactation  and the peak of lactation  as compared to dry period, respectively [NUCB2 might be associated with milk production traits.Milk provides rich nutrients, protein, fatty acids, and vitamins, etc., for human , so the 2+ binding protein, and it processes to generate a terminal peptide, termed nesfatin-1. Nesfatin-1 regulates the appetite and glucose metabolisms in humans and domestic animals [NUCB2 exhibits a high conservation among species, and its mRNA is ubiquitously expressed [NUCB2 mRNA and nesfatin-1 protein were highly expressed in the liver of mouse fetus at embryonic day (E) 10.5, and their expression were extensively decreased at E17.5, which suggests that NUCB2 might play a critical role in liver development and physiological functions in the developmental process of mouse fetus [NUCB2 is involved in human mammary epithelial cell proliferation and migration [NUCB2 might be beneficial to the increase of milk production. In addition, the NUCB2 gene was located on chr.15:43.5418 and it had a distance of 0.86 and 2.54 cM to the peak of reported quantitative trait locus (QTL) regions that have large effect on protein percentage [NUCB2 was also near to three significant SNPs for milk traits, BFGL-NGS-116109, BTB-00590405, and BTB-00590603, with the distance of 0.93–5.99 Mb [NUCB2 may be involved in milk production traits.Nucleobindin 2 (NUCB2) is a Ca animals ,6,7. NUCxpressed . Sun et se fetus . NUCB2 iigration , which ircentage  and protrcentage , respect–5.99 Mb . These dNUCB2 and milk production traits in dairy cattle. In addition, the studies have revealed that haplotype blocks that are formed by the SNPs have important implications for identifying associations with complex traits [NUCB2 gene and estimated the haplotype block that was formed by the identified SNPs, and then analyzed their associations with five milk traits, including milk yield, fat yield, fat percentage, protein yield, and protein percentage.To date, rare studies have been reported to uncover the genetic association between the single nucleotide polymorphisms (SNPs) of x traits ,16,17. HWe selected a total of 1067 Chinese Holstein cows of 40 sire families from 22 dairy farms of the Sanyuan Lvhe Dairy Farming Centre  on the same feeding conditions. The cows in each sire family were distributed in different dairy farms. These 1067 cows have three-generation pedigree information at least and Dairy Herd Improvement (DHI) records for 305-day milk yield, protein yield, protein percentage, fat yield, and fat percentage. Ethics Approval: The Institutional Animal Care and Use Committee (IACUC) of China Agricultural University approved all of the protocols for sample collection (permit number: DK996).http://www.bdcc.com.cn/), and the descriptive statistics of them are shown in The Beijing Dairy Cattle Centre provided the phenotypic data of 305-day milk yield, fat yield, fat percentage, protein yield, and protein percentage in the first and second lactations , according to the manufacturer’s instructions. Subsequently, we measured the quantity and quality of the extracted DNAs by the NanoDrop 2000 spectrophotometer  and gel electrophoresis, respectively.NUCB2 by Primer3  (http://bioinfo.ut.ee/primer3-0.4.0/), and then synthesized them in the Beijing Genomics Institute . We randomly mixed 40 semen DNAs into two pools (20 sires per pool) with equal concentrations (50 ng/µL) for each DNA. The reaction conditions and amplification procedures for PCR are provided in the We designed the primers  to ampliP value, divided by number of genotypes or haplotype combinations. In addition, the additive (a), dominant (d), and substitution (α) effects were calculated, as follows: The extent of LD between the identified SNPs was estimated while using Haploview 4.2 . Subsequently, we used the Statistical Analysis System (SAS) 9.13 software  to analyze the associations of the SNPs and haplotype blocks with milk production traits with the following animal model: http://www.genomatix.de/cgi-bin/welcome/welcome.pl?s=d1b5c9a9015b02bb3b1a806f9c03293f) to predict the changes of the TFBSs that are caused by the identified SNPs in the 5′ flanking region of the NUCB2 .We used the MatInspector  , and the quantity and quality of the RNA were measured with a NanoDrop 2000 spectrophotometer  and gel electrophoresis, respectively. We used a PrimerScriptH RT reagent Kit  for the reverse transcription. The qPCR primers of NUCB2 and two reference genes  were presented in ® 480 II  with the procedures that are shown in RPS9 and UXT with 2-ΔΔCt method.Three healthy lactating Chinese Holstein cows were selected from the Sanyuanlvhe Dairy Farming Center , and the heart, liver, spleen, lung, kidney, ovary, mammary, and uterus from each cow were collected. Subsequently, the expression of NUCB2, including one in the 5′ flanking region, two in 3′ UTR, and seven in the 3′ flanking region , nine with fat percentage , and eight with protein percentage , and no association was found between the SNPs and milk and protein yields . In the second lactation, ten SNPs had strong associations with milk yield , five with fat yield , one with fat percentage , ten with protein yield , and six with protein percentage , respectively. Two SNPs, c.*1253A>G and g.35692674A>G, were strongly associated with fat yield  in both of the two lactations. The g.35735477C>T had significant association with fat percentage in the first  and second lactation . There were four SNPs, g.35735477C>T, c.*1276C>T, g.35692145C>T, and g.35691328T>C, which were associated with protein percentage  in both lactations. In addition, as the results show in p values < 0.05).We analyzed the genetic associations between the ten SNPs of actation . In the NUCB2 and found a haplotype block  in the first lactation, milk yield , fat percentage , protein yield , and protein percentage  in the second lactation, respectively , caudal type homeobox 2 , mammalian C-type LTR TATA box , and the allele T in this SNP could create the TFBSs for nuclear factor of activated T-cells  and v-ets erythroblastosis virus E26 oncogene homolog , which might impact the statistical significance. Generally, cows have higher milk production in the second lactation, and the different physiologic status of cows between the two lactations was also one possibility for the different genetic effects across lactations. In addition, the unstable association of the SNPs in two lactations was probably due to the fact that the causal polymorphisms are not very far, but not for these ones. Besides, we observed that the cows with heterozygous genotype showed the dominance for milk traits, ten SNPs for milk yield in the second lactation, for instance, which were caused by the dominance effects of the heterozygotes. We usually do not consider it in breeding, since the dominant effect is not heritable across generations. Hence, we should choose SNPs with significant additive effects on milk traits in all lactations for dairy cattle breeding.In addition, for each identified SNP of NUCB2 was expressed in heart, liver, spleen, lung, kidney, ovary, mammary, and uterus tissues of dairy cows, and relatively highly expressed in liver. In dairy cows, the liver participates in carbohydrate, fat, protein, and other substance metabolisms, through blood circulation, it provides cholesterol, triglyceride, and other ingredients to mammary gland to synthesize milk protein and fat [NUCB2 may play an important role in milk synthesis metabolism in dairy cows.In this study, we used the qPCR and observed that  and fat ,27,28. OSPRR2A mRNA expression through binding to the sites in its promoter during terminal differentiation of the epidermis [KRT12 in the differentiated mouse corneal epithelium [CDH-17 and promoted hepatocellular carcinoma cell proliferation [SRY gene in human and bovine, including the TFBS for VTATA in the TATA-binding protein factors family with the matrix similarity score of 0.923 in bovine [SMAD2/3 to cause endothelial-dependent liver fibrogenesis [NUCB2 by the coactions of transcription factors ELF3, CDX2, VTATA, NFAT, and ERG. However, the further functional validation should be performed to verify the regulatory roles of NUCB2 and its polymorphisms on milk traits in dairy cattle.The specific binding of transcription factors to the regulatory regions of the DNAs is a important regulatory mechanism that affects gene expression ,30. In tpidermis . The oveithelium . ELF3 biithelium . ELF3 diithelium . Zhu et feration . CDX2 trferation . CDX2 biferation . Ross etn bovine . The NFAn bovine . NFAT prn bovine . ERG regogenesis , and it ogenesis . These dNUCB2 gene with milk production traits in dairy cows, and to show that g.35735477C>T might change the TFBSs of NUCB2. Our findings provide valuable molecular information for dairy cattle breeding, and the significant SNPs could be put into the SNP chip for genomic selection to improve the milk production in dairy cows.This is the first study to demonstrate the genetic associations of SNPs of


ISA2) was up-regulated by HT. Actually ISA2 was the only gene up-regulated by all three stress treatments. The expression of 17 genes was up-regulated, while six genes, including granule-bound starch synthase (GBSSI), AGPS2, BEIII, PHOL, ISA1, and AGPL2, were down-regulated by DS. Eleven genes were down-regulated and 12 were up-regulated by HT+DS. The activity of ADP-Glc pyrophosphorylase, starch synthases, GBSS, SS, and starch branching enzymes in the stress treatments  often appeared to peak values in advance and declined significantly to be lower than that in the CK. The genes that coordinated participation in the enzymes formation can serve as an indicator of the enzymes activity potentially involved in starch biosynthesis. HT, DS, and HT+DS altered the timing of starch biosynthesis and also influenced the accumulation of amylose, amylopectin, total starch, and sucrose. Under HT, DS, and HT+DS, the key enzymes activity and their genes expression associated with the conversion of sucrose to starch, was reduced, which was the leading cause of the reductions in starch content. Our study provide further evidence about the effects of stress on starch biosynthesis in wheat, as well as a physiological understanding of the impact of post-anthesis heat and DS on starch accumulation and wheat grain yield.High temperature (HT) and drought stress (DS) play negative roles in wheat growth, and are two most important factors that limit grain yield. Starch, the main component of the wheat [endosperm, accounts for 65–75% of grain weight, and is significantly influenced by environmental factors. To understand the effects of post-anthesis HT and DS on starch biosynthesis, we performed a pot experiment using wheat cultivar “Zhengmai 366” under field conditions combined with a climate-controlled greenhouse to simulate HT. There were two temperature regimes (optimum day/night temperatures of 25/15°C and high day/night temperatures of 32/22°C from 10 days after anthesis to maturity) accompanied by two water treatments . Optimum temperature with optimum water treatment was the control (CK). We evaluated the expression patterns of 23 genes encoding six classes of enzymes involved in starch biosynthesis in wheat grains using real-time qPCR. HT, DS, and HT+DS treatments altered gene expression profiles. Compared to the CK, expression of 22 of the 23 genes was down regulated by HT, and only one gene ( Triticum aestivum L.), grown in many different environments worldwide, is one of the major cereal crops and an important source of calories for human nutrition. Environmental constraints are major influence factors for wheat productivity in many regions. Wheat is particularly susceptible to temperatures above the range of 20–30°C, and mean temperatures during the wheat grain-filling stage often exceed the optimum in most wheat-growing regions, which is considered to be one of the most important factors limiting wheat grain yield (Wheat (in yield . Some whin yield . Becausein yield . Wheat iin yield . Droughtin yield .ADP-Glc pyrophosphorylase (AGPase) and granule-bound starch synthase (GBSS). Amylopectin is mainly synthesized by the coordinated actions of AGPase, starch synthases (SSs), starch branching enzymes (SBEs), and starch debranching enzymes (DBEs) (PHO), metabolizing sucrose to the carbon precursors, is also needed for starch biosynthesis  . In addiynthesis . There aand GBSS . Both hiand GBSS . Temperaand GBSS .SSIIb, SSIIc, SSIIIb, SSIV, BEIIb, BEIII, GBSSII, ISAI, ISA2, PUL, PHOH, and PHOL, have not yet been examined under conditions of high temperature and water deficit. Furthermore, most studies examine the effects of either high temperature or drought stress seperately, thus give insufficient knowledge of the combined effects of high temperature and drought stress. Therefore, in this study, we analyzed the relative transcription of 23 isozyme genes encoding enzymes involved in starch biosynthesis in wheat grains using real-time qPCR. We also evaluated the activities of starch biosynthetic enzymes, and the accumulation of starch and sucrose. Furthermore, we simulated the starch biosynthesis process in response to high temperature and drought stress. The expected results will enable a better understanding of the influence of high temperature, drought stress, and their interaction on starch biosynthesis, and will also extend our knowledge on the impact of post-anthesis heat and drought stress on starch synthesis and grain yield in wheat.High temperatures and drought stress, as the major abiotic stress factors that affect wheat growth, development, and production , occur f2O5, and 1.15 g K2O per pot before sowing. Pots were buried in the field, keeping the soil surface inside the pot at the same level as that of the field. The trial was arranged in a randomized complete block design with three replicates. The plants were thinned to 12 seedlings per pot at the three-leaf stage. An additional 1.1 g N per pot was then added at the stem elongation stage.Seeds of “Zhengmai 366,” a widely cultivated local wheat variety, were obtained from the Henan Academy of Agricultural Sciences. An experiment combining pot culture with a climate-controlled greenhouse to simulate high temperature was conducted at the farm of the Scientific and Educational Station of Henan Agricultural University, Zhengzhou, China  during the 2015–2016 and 2016–2017 growing seasons. Seeds were sown in 24 cm-diameter pots containing 10 kg loam soil. The soil was collected from the top 20 cm soil layer at the experimental site. The total N, rapidly available phosphorus, and potassium were 1.43, 68.14, and 204.87 mg/kg, respectively. Fertilizer was applied at a rate of 1.15 g N, 1.35 g PThe spikes were tagged for sampling when the anthers had extruded from the florets in 50% of the tillers in each pot. The plants in pots were grown under field conditions until they were transferred to the climate chamber for high temperature treatment beginning at 10 days after anthesis (10 DAA). In the controlled climate chambers, two temperature regimes consisting of optimum day/night temperatures of 25/15°C and high day/night temperatures of 32/22°C were accompanied by two water treatments; an optimum water treatment with ∼75% of the relative soil water content, and a mild drought stress treatment with ∼50% of the relative soil water content, was set to wheat maturity. The optimum day/night temperature combined with the optimum water treatment was the control (CK), and the plants were irrigated regularly to maintain the soil relative water content at ∼75%. High day/night temperature with optimum water treatment was defined as the high temperature (HT) treatment. For the drought stress (DS) treatment, the water content was controlled from ∼3 DAA prior to transfer to the climate chambers to ensure that the soil relative water content was maintained at the objective level of ∼50%. The soil water content was determined by weighing the pots, combined with time domain reflectometry measurements . The treatment consisting of high day/night temperature and DS was defined as the combined HT and drought stress (HT+DS) treatment.Tagged heads were initially collected before plants were transferred to the climate chamber. Sampling was then performed every 4 days until seed maturity. One portion of the grains sampled were frozen immediately in liquid nitrogen for 30 min and then stored at °80°C for RNA extraction and cDNA synthesis as well as for determination of the activity of starch biosynthesis enzymes, while another portion of the grains sampled were dried to constant weight for measurement of sucrose, soluble sugar, and starch contents as well as for determination of the starch accumulation rate. All the determination was in triplicate for each biological replicate and each sampling date contained three biological replicates.RNA was extracted from wheat grains at each sampled time point during grain development. Total RNA from grain endosperm was extracted using a test kit from Takara and purified with gDNA Eraser (Takara) to remove contaminating genomic DNA. cDNA was synthesized from 2 µg of total RNA using the ThermoScript Reverse Transcriptase Reagent Kit (Takara). The integrity of the RNA samples was examined by gel electrophoresis.Expression profiles of the wheat genes were determined by quantitative real-time PCR analysis. cDNAs for all of studied genes were amplified using primers designed from known wheat nucleotide sequences (SS), GBSS, soluble starch synthase (SSS), AGPase, and starch branching enzyme (SBE), respectively, using the reagent kits SS-2-Y, GBSS-2A-Y, SSS-S-Y, AGP-2A-Y, and SBE-2-Y obtained from Su Zhou Keming Biotechnology Incorporated. The activities of GBSS, SSS, and AGPase were determined as nmol min–1 of NADPH produced g–1 grain FW; SS activity was measured by quantifying the production of sucrose from fructose and glucose (µg min–1 g–1 grain FW); and SBE activity was determined as the percentage decrease in absorbance at 660 nm of that decrease of iodine blue value as an enzyme activity unit.We assayed the activities of five starch biosynthesis enzymes, including starch synthase (The contents of soluble sugars and sucrose in wheat grains were determined using the anthrone colorimetric and resorcinol colorimetric methods as described by The amylose and amylopectin contents in wheat grains were determined with a coupled spectrophotometric assay as described by Determination of starch accumulation rate was referenced to the method of http://bioinformatics.psb.ugent.be/webtools/Venn/).Data from three biological replicates in the experiment were statistically analyzed for variance and correlation using SPSS 21.0. The line graphs were prepared and edited with SigmaPlot 13.0. Venn diagrams of gene expression were obtained using the Bioinformatics & Evolutionary Genomics online analysis software  expression in DS treatment peaked by 4 days (apart from BEIII by 8 days) in advance, while AGPS1-b and SSIIIa expression peaked by 4 days late. For the HT, HT+DS treatments, the genes expression, including SSIIb, SSIIc, SSIIIb, SSIV, BEI, BEIII, PUL, and PHOH, peaked by 4 or 8 days earlier than the CK. However, the expression of genes  in HT treatment, and the expression of genes  in HT+DS treatment, all had the maximum values at 10 DAA, and showed gradually declining trend during grain filling stage.In the CK, the expression of 23 genes all displayed a unimodal curve with the peak times varying for the different genes Figure 1.AGPS2, AGPL2, GBSSI, ISA1, PHOL while significantly increased the expression of AGPS1-a, AGPS1-b, AGPL1, BEI, BEIIb, ISA2, PUL, PHOH; HT significantly reduced the expression of AGPL1, AGPL2, AGPS1-a, AGPS1-b, AGPS2, SSI, SSIIa, SSIIc, SSIIIa, SSIIIb, ISA1, ISA2, PHOL; HT+DS significantly reduced the expression of AGPL1, AGPL2, AGPS1-a, AGPS1-b, AGPS2, SSIIa, SSIIIa, SSIIIb, ISA1, PHOL, while only led to the increase of ISA2 expression  showed up-regulated expression; ISA2 was the only gene in which expression was up-regulated by the HT, DS, and HT+DS treatments  was down-regulated  and the CK, the positive and negative values represent up-regulated and down-regulated gene expression, respectively. Of the 23 genes examined, relative expression in 22 was down-regulated by HT  with the expression of their isozyme genes as a whole at 22–26 DAA.The expression of isozyme genes is obviously related to the activities of the enzymes they encode during different phases of grain filling, especially at the late stage (The expression of genes for isozymes involved in starch biosynthesis was closely correlated with the contents of starch and its components (amylose and amylopectin) and with the contents of sucrose and soluble sugars. The expression of genes involved in starch biosynthesis mainly affected the accumulation of amylopectin and total starch, and the expressions of most of the 23 genes showed significant correlations (p < 0.05 or p < 0.01) with amylopectin and total starch content in most of the different sampling periods Table 4.SS activity with the amylopectin and starch content were significant (p < 0.05 or p < 0.01) in different sampling times. The correlations of SBE and AGPase activities with the amylopectin and starch content were significant (p < 0.01) from 22 to 26 DAA. The correlations of GBSS and SS activities with the amylopectin and starch content were significant (p < 0.05 or p < 0.01) except for 18 DAA. The SSS activity was more closely related to the dynamic accumulation of amylose, amylopectin, and total starch than the other key enzymes. And the activities of starch biosynthetic key enzymes were more closely related to the amylopectin and total starch content than to the amylose content, sucrose content, and soluble sugar content. The correlations between the activities of starch biosynthetic key enzymes and starch accumulation rate were significant (p < 0.05 or p < 0.01) (except for SBE and AGPase activity at 14 DAA). Which indicated that the activity of starch biosynthetic key enzymes directly influenced starch accumulation in wheat endosperm.The enzyme activities during different phases of grain filling were found to be closely related to the dynamic accumulation of starch and sucrose Table 5.AGPase, SSS, GBSS, and SBE, all of which are key enzymes associated with the conversion of sucrose to starch, were reduced, and this could explain the decline in starch accumulation. Transcription of the genes encoding AGPase isoforms  was reduced by the HT and HT+DS treatments, leading to the repression of the conversion of sucrose from glucose-1-phosphate (G-1-P) to ADP-glucose (ADPG). The expression of all the main genes involved in the conversion of ADPG to amylopectin  was reduced to some extent in response to HT, DS, and HT+DS, although the expression of ISA2 increased. This led to a significant decrease in the amylopectin content and was also the primary cause of the decrease in total starch content. Under DS, the activities of AGPase, SSS, and SBE were reduced, although the expression of AGPS1-a, AGPS1-b, AGPL1, BEI, BEIIa, BEIIb, and SS isozyme genes increased, which implied that starch biosynthetic enzymes did potentially display the coordinated participation of many genes not depend on one or some genes expression. The conversion of sucrose from G-1-P to ADPG and of ADPG to amylopectin was also probably repressed, leading to a significant reduction in the amylopectin and starch content. In the HT, DS, and HT+DS treatments, the decrease in starch accumulation was strongly associated with the change in starch biosynthesis in the wheat grain endosperm, which is attributed to the dynamic change in the activities of key enzymes and the expression profiles of their isozyme genes involved in starch biosynthesis.The simplified model of the starch biosynthetic pathway was demonstrated (PUL and up-regulation of SSIIIb expression in response to HT was previously observed in barley (ISA2) expression was down-regulated by HT, while of the 23 genes expression, 17 was up-regulated by DS. For the HT+DS treatment, almost half-and-half of the 23 genes expression was down-regulated and up-regulated, respectively. High temperature significantly decreased the relative expression of the 23 genes and the negative effect on genes expression was greater than DS.There are few reports which describe the expression of genes involved in starch synthesis in wheat under high temperature and DS , althougn barley . High ten barley . In the SS was found to be the most sensitive enzyme in starch biosynthesis to drought. The activity of SS is highly correlated with starch synthesis rate -linked glucan primer is catalyzed by SSs  in wheat  between HT and DS.The results of our study showed that the HT, DS, and HT+DS treatments caused earlier maturation of wheat by 8, 4, and 12 days, respectively, compared with the CK. During the grain filling stage, the dynamic change in the expression of genes and enzyme activity involved in starch biosynthesis in wheat endosperm was all altered, and often appeared to peak values earlier. Of the 23 genes expression, 22 was negatively influenced by HT, while only 6 and 11 was down-regulated by DS and HT+DS, respectively. The expression levels of genes encoding enzymes involved in starch biosynthesis can possibly serve as indicators of the activities of starch biosynthetic enzymes during grain filling. The activity of HL and CW conceived the original research plan. YH and WL performed most of the experiments and analyzed the data. DM and QH supervised the experiments. YH and GM performed the real-time qPCR analysis. HL wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.