Hyphantria moths (Arctiidae) characterized by red (RD)‐ and black (BL)‐headed larvae, of which the taxonomic status is unresolved. Genetic divergence of 26 populations, based on 710 bp of the mtCOI sequence, showed two phylogenetic lineages, which could not be connected in the haplotype network with 95% confidence. The two lineages are separated by 3.1% sequence divergence and should be considered for full species status. The estimated split occurred 1.2–1.6 million years ago. The range of the RD type covered most of the continent, whereas that of the BL type was limited to eastern deciduous forests. Several biological characteristics were differentiated in the zone of cohabitation where BL had more annual generations than RD. Spring emergence of BL precedes that of RD in the field by at least 1 month, because the diapause in BL was shallow, whereas it was deep in RD. Voltinism requires discreteness of numbers, which functions as a sink of hybrids between the two parental lines that have distinct but equally adaptive reproductive strategies; BL may be more r‐strategist‐like and RD more K‐strategist‐like, because fast‐developing BL has multivoltine life cycle, investing less silk proteins as the round‐the‐clock feeder, and slow‐developing RD univoltine one investing more silk as the nocturnal feeder. Also, intensity of diapause, deep in RD and weak in BL, was grossly different, which may enforce segregation of spring adults. Allochronic speciation avoiding coincidental occurrence of adult stages is therefore the most likely scenario. Because the adults never meet in nature, large morphological differentiation is not required.North America has distinct types of  Subsequently a sibling species H. textor (Harris) was proposed based on spotless adults as the spotless fall webworm, although it was not described formally  and a black‐headed type (BL). Their formal taxonomic status is not fixed .The fall webworm, lly Fig. . Morris lly Fig.  observedMorris and Fulton  and Ito DNA barcoding, based on a partial sequence of the mitochondrial COI, has been widely used to: characterize intraspecific genetic diversity  Mammalian Genomic DNA Miniprep Kit  in accordance with the manufacturer's protocols. The primers used to amplify 710 bp of the COI region were C1‐J‐1718 5′‐GGAGGATTTGGAAATTGATTAGTTCC‐3′ and TL2‐N‐3014 5′‐TCCAATGC ACTAATCTGCCATATTA‐3′ . A multiple alignment of all the sequences was conducted using ClustalW and edited manually in BioEdit Hall, . Based oFs  upon pupation when they were exposed to a temperature of 5°C in constant darkness (DD). The diapause pupae of RD were chilled at 5°C for 20, 60, 90, 120, and 260 days, while 125, 200, and 260 days for BL populations. After chilling treatment for the designated period, diapause pupae were incubated at 25°C LD 16:8, and the number of days until 50% of individuals became adults was recorded.To compare diapause intensity between BL and RD, mature larvae were collected from the field in 1982, fall and brought in the laboratory and feeding was continued in the laboratory. RD are all from New Haven, CT but collection of BL larvae is difficult and only one nest was found in New Haven, two nests from New York City (BL population), and Washington, D.C. (BL population) were used to supplement the experiment. Diapause pupae were divided into groups , RD‐W (west coast) populations.Additionally, diapause intensity was examined nation‐wide. Diapause intensity changes during cold storage, which is expressed as the number of days till 50% of adults emerged after variable periods of chilling at 5°C. Fall generation field‐collected larvae of two types were kept in the laboratory and reared until pupation. The period of chilling treatment was 17, 31, 47, 68, 82, 96, 110, 130, 151, 165, and 179 days for BL populations collected in Baton Rouge, Louisiana; 154 and 206 days for RD‐E (east coast) collected in Woodville, Mississippi; 29, 97, 178, 206, and 224 days for RD‐W (red‐headed and maroon‐headed from west coast) collected in Sand Point, Idaho and 123, 137, and 157 days for RD‐W (west coast) collected in Pullman, Washington. Each group (20–30 pupae) was treated for a different period without mixing. Curve fitting adjustments and the logistic regression were performed with MATLAB when the relationship of two variables was not linear. Mann–Whitney To compare their morphological characteristics between populations showing sympatry and allopatry, adults specimens of BL, RD‐E, and RD‐W were measured for the forewing length using a micrometer. The comparison was made in two ways: first using adults grown in the laboratory on the same artificial diet in the last instar and second using field‐collected pinned specimens stored in the museum. The second comparison was made for east and west at almost the same latitudinal zone. Size can be compared between different voltinism zones. All data were analyzed by analysis of variance with the Tukey–Kramer multiple comparisons tests.33.1The survey map showed that BL larvae spun a loose web Fig. e, and th3.2Figure Two unconnected networks in TCS showed two haplotypes in BL populations (BL1 and BL2) and 17 haplotypes in the RD populations . The mismatch distribution of BL populations was distinctly unimodal  and bimodal mismatch distribution analysis  than the BL populations, whose haplotype and nucleotide diversity values were 0.00004 and 0.032 ± 0.022, respectively Table . The val3.4The two types of adults, with spotted wings and spotless wings , emerged3.5Figure Figure 3.6The body size of West Coast USA RD (RD‐W) was significantly larger than those in Eastern USA RD (RD‐E) and BL populations Table .44.1Hyphantria, and no easily observable break point had been found between them. However, a critical breakpoint for the demarcation of species should not always be sought in external morphology. Here, we first investigated genetic differentiation mainly based on mitochondrial COI sequences. Molecular phylogenetic analysis of Hyphantria populations collected from 26 localities  resulted in two major clades represented by BL and RD. MR was nearly identical to RD and, therefore, belongs genetically to the RD clade. The average K2P distance between the BL and RD (plus MR) (3.1%) was slightly greater than the interspecific threshold of 3% observed for Lepidoptera by Hebert, Cywinska, and Ball .Jaenike and Selander  investigFs value and unimodal distribution indicated historical instability in the BL populations. The low level of genetic diversity in BL was perhaps related to population expansion after amelioration of climatic or biotic stresses. The BL population rapidly expanded its territory to the west but failed to penetrate the western territory of maternal RD species, probably because it preferred plants adapted to the more humid climate prevailing in eastern North America. However, without fossil records and age identification of the ecological background, reconstruction of colonization history is not possible within North America.Congruence between larval morphology and wing patterns showed that the BL and RD types were both sympatrically and allopatrically distributed in North America Figure . The RD 4.2The substitution rate for insect mitochondrial DNA was estimated to be 2.3% per million years Brower, . Using tRhagoletis flies  have been presented (Johnson & Gullberg, es Bush, . Howevern Loewy, , which dGryllus species. G. pennsylvanicus and G. veletis, which overwinter at egg and nymphal stages, respectively. This split in overwintering stage inevitably splits the reproductive season between the two types, through which speciation was realized, according to the authors. This hypothesis was, however, refuted by Harrison (Rhagoletis flies (Bush, Hyphantria moths have potentially multivoltine capacities, and the mechanism to maintain the two types requires more sophisticated tuning.The allochronic speciation hypothesis was first presented by Alexander and Bigelow  for the Harrison  based onHarrison  analyseses Bush,  implicates Bush, , 2012. TObservation for sympatric populations in central Missouri suggested that differentiation in developmental rate and critical photoperiod resulted in the separation of two species, favoring univoltinism for the RD species and bivoltinism for the BL species Takeda, . Univolt4.3Initial congeneric competition may have resulted in sympatric speciation allochronically that reduced competition (Dieckmann & Doebeli, 4.4Hyphantria more efficiently if it was supported by a demographic strategy. Seasonal niches exert differential r/K selection. Bivoltinism conforms an r‐like strategy, that is facilitated by fast development, comparatively less spinning, and the round‐the‐clock feeding. Univoltinism results from intensive investment for silk spinning, nocturnal feeding that slow development, conforming a K‐like strategy (MacArthur & Wilson, Any new phenotype must be sustained in the ecological background. Seasonal segregation in mating time is an effective mechanism for assortative mating for distinct ecological adaptations. Allochronic speciation could occur in None declared.Makio Takeda contributed to the conception of the study. Fan Yang, Eriko Kawabata, and Makio Takeda contributed significantly to analysis and manuscript preparation; Fan Yang and Makio Takeda performed the data analyses and wrote the manuscript; Makio Takeda and John J. Brown collected live materials and field data in the US; Fan Yang and Makio Takeda helped perform the analysis with constructive discussions. Click here for additional data file. Click here for additional data file.


Sleep is modulated by several factors, including sex, age, and chronotype. It has been hypothesised that contemporary urban populations are under pressure towards shorter sleep duration and poorer sleep quality. Baependi is a small town in Brazil that provides a window of opportunity to study the influence of sleep patterns in a highly admixed rural population with a conservative lifestyle. We evaluated sleep characteristics, excessive daytime sleepiness, and chronotype using the Pittsburgh Sleep Quality Index, Epworth Sleepiness Scale and Morningness-Eveningness Questionnaire questionnaires, respectively. The sample consisted of 1,334 subjects from the Baependi Heart study . Average self-reported sleep duration was 07:07 ± 01:31 , sleep quality score was 4.9 + 3.2, chronotype was 63.6 ± 10.8 and daytime sleepiness was 7.4 ± 4.8. Despite a shift towards morningness in the population, chronotype remained associated with reported actual sleep timing. Age and sex modulated the ontogeny of sleep and chronotype, increasing age was associated with earlier sleep time and shorter sleep duration. Women slept longer and later, and reported poorer sleep quality than men (p < 0.0001). This study provides indirect evidence in support of the hypothesis that sleep timing was earlier prior to full urbanisation. Sleep has several physiological functions relevant to health. Several reports have suggested that poor sleep quality, short sleep duration, or inappropriate sleep timing are related to cardiovascular diseases (CVD)It has been proposed that electricity and urbanisation have changed human sleep behaviour and sleep patterns, such as timing and duration14152/day . The traditional lifestyle, moderate and ongoing urbanisation, and very limited inward migration of this rural town make it a location of interest for the study of sleep quality and sleep characteristics. Since the beginning of this study in December 2005, a significant number of discoveries have been reported, such as the heritability of cardiovascular risk factorsversus municipal)The Baependi heart study is a family-based cohort study designed to investigate cardiovascular disease and associated factors, specifically factors typical of Brazilvs 4.5 ± 2.9; p < 0.001, Cohen’s d value = 0.18), accounting for a small difference between the sexes. Consistent with this, more women had a PSQI score above 5 than men . Women also presented later wake up times (means ± standard deviation (06:26 ± 01:17) vs (06:04 ± 01:34); p < 0.01, Cohen’s d value = 0.17), meaning that this difference between sexes was small. Women spent more time in bed than men (means ± standard deviation (07:52 ± 1:26) vs (07:32±01:31) hour:min ± hour:min; p < 0.005, Cohen’s d value = 0.15). Self-reported sleep duration was longer in women than men (means ± standard deviation (07:13 ± 1:33) vs (06:57 ± 1:27) hour:min ± hour:min; p < 0.01); however, the difference was small according to the Cohen’s d value of 0.10.Χ2 = 20.80; p < 0.001) number of women presented scores above 5 in PSQI, meaning poor sleep quality. In order to investigate the relationship between sex and sleep quality, we analysed the score of the seven different components of the PSQI: Subjective sleep quality, sleep latency, self-reported sleep duration, sleep efficiency, sleep disturbances, use of sleep medication, and daytime dysfunction. Each component is derived from one or more questions and receives a score from 0 to 3, a lower the score indicates better sleep quality. To further explore the sleep quality in this population, we first evaluated the frequency distribution of PSQI scores by sex . CompareDuring the past month, how often you had trouble sleep because you…”) The frequency distribution analysis of PSQI categories also permit identification of the components sleep latency, sleep duration and sleep disturbances as the main contributors to high PSQI total scores. However, not a single subject described their sleep as “very bad”. Conversely, 23.3% of subjects presented sleep disturbances at least once in the past month, and 10.2% of the sample reported not having a single sleep disturbance episode. In order to identify the most prevalent sleep disturbance in this population, we evaluated the specific answers to the question regarding sleep disturbances . Consistent with this, M-types also reported longer sleep duration (07:46 ± 1:24) than N-types (07:07 ± 1:28) and E-types (06:59 ± 1:42 h) . We classified chronotype using a variation of the method of Robilliard To further evaluate the relationship between age and sex on sleep patterns and chronotype ontogeny, the sample was binned in 5 age groups . Detailed information about the age groups is presented in post hoc test, p = 0.01)  . Sex mod = 0.01) .Age and sex comparisons also were performed for sleepiness. Sleepiness scores did not change according to age (p = 0.076) or differ between sexes (p = 0.91); consequently, no interaction between factors was found (p = 0.09)  one could accept our hypothesis. However, 34.9% of subjects were poor sleepers (scores >5 on PSQI). According to % of subjects presented with excessive daytime sleepiness and the mean Epworth Sleepiness Score (ESS) was 7.8 ± 4.8, below the threshold for excessive sleepiness. However the mean of PSQI score for the entire sample was 4.9 ± 3.2 and 34.9% were categorized with poor sleep quality. Age played a role in sleep quality — the older the subjects, the poorer sleep quality. Women presented poorer sleep quality than men, but no subject ranked their sleep quality as “very bad” on the Pittsburgh Sleep Quality Index. One of the main factors influencing the total PSQI scores may be sleep latency, which also increased with age. As presented in We set out to provide a quantitative and qualitative description of sleep patterns, and describe in detail the relationships between age, sex, and chronotype with sleep quality and timing in this rural population. We found that the self-reported mean bedtime and wake up times were 22:32 ± 01:27 (SD) and 06:17 ± 01:25 respectively, which was consistent with the previously reported morning preference of this population. Sleep duration was 07:07 ± 01:31(hh:min). Additionally, 31.7The Baependi Heart Study population presented a high prevalence of self-reported poor sleep quality. The mean PSQI score for the full sample was 4.9 (3.2), which is higher than studies elsewhere including the control group in the validation of the Brazilian version for the PSQI questionnaire32935Sex also modulated sleep quality in our study sample. Women presented poorer sleep quality than men. Post-menopausal women presented higher prevalence of sleep disturbance . Men and women did not differ significantly in diurnal preference until age of 50 years old when men presented higher morningness. A similar pattern of chronotype ontogeny was described in a very large Brazilian study showing that, although morningness increases with age in both men and women, it does so with different slopes, so that men (who start life with higher average eveningness) have a higher average morningness tendency from age 50 onwardsSleep timing and sleep duration were modulated by age and sex as mentioned before. Furthermore, these variables were also strongly associated with chronotype categories. Subjects from Baependi presented considerably earlier wake up and bed times similar to what have been reported in rural populations1314419935This study presents strengths and limitations worth mentioning. We report subjective data based on questionnaires. Although these instruments have been used for sleep research and in sleep clinics worldwide, they contain measures that are subjected to considerable variation in the subject’s personal interpretation. The life experience of Baependi residents may be uniquely reflected in some of their answers. Once supplemented with objective measures, such as actimetry and circadian phase markers, these datasets can be combined with the multiple layers of phenotype data from the Baependi study, and be related to health outcomes and major risk factors. The findings from this interesting cohort show that the presence of electrification does not obligately (or at least not immediately) lead to shifted chronotype distributions towards eveningness and chronotype and delayed sleep and wake timings. They also indicate that an earlier sleep phase does not equate to a perception of better sleep. Taken together, they justify the further investigation of the environmental and lifestyle factors associated with these observations. The longitudinal nature of the Baependi Heart Study will make it possible to investigate whether and how these parameters will change over future years.The methodology for recruitment has been described in detail previouslyet al.The PSQI was developed to assess the quality and disturbances related to sleep over a period of 1 month. It is composed of seven different components: subjective sleep quality (C1), sleep latency (C2), sleep duration (C3), habitual sleep efficiency (C4), sleep disturbances (C5), use of sleeping medication (C6) and daytime dysfunction (C7). All the seven components are graded on scores from 0 to 3, and the sum of these components scores yields a global score from 0 to 21. The higher the global score, the worse the sleep quality; a score higher than 5 is associated with poor sleep qualityΧ2 tests were applied. Age was considered as continuous variable for correlational analysis and the sample was binned into five age groups  to perform mean comparisons.From the entire sample, data from 1,334 individuals were considered complete and eligible for statistical comparisons, the results of which are presented here. Data from subjects with missing one or more answers for more than one questionnaire were excluded. Data were analysed by comparison of means using Student’s t tests, one-way analysis of variance, and multiple factors analysis of variance. To evaluate distributions, How to cite this article: Beijamini, F. et al. Timing and quality of sleep in a rural Brazilian family-based cohort, the Baependi Heart Study. Sci. Rep.6, 39283; doi: 10.1038/srep39283 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This study aimed to describe trends in prevalence, awareness, treatment, and control in hypertension in Chengdu from 2000 to 2010. Two community-based cross sectional surveys were conducted among those aged 40–79 years in 2000 (n = 4850) and 2010 (n = 5456). Demographic characteristics, blood pressure (BP) and associated risk factors were examined. Mean systolic and diastolic BP increased from 117.8 ± 33.9 to 132.1 ± 21.2 mmHg (P < 0.001), and 75.3 ± 19.1 to 79.3 ± 11.2 mmHg (P < 0.001) over past decade, respectively. The prevalence of hypertension increased from 27.7 to 29.4% (P < 0.001). Awareness increased from 37.7 to 42.5% (P < 0.001). The overall treatment rate increased from 20.9 to 28.0% (P < 0.0001), while among subjects aware of hypertension, treatment increased from 46.9 to 65.8% (P < 0.001). In hypertensives, control increased from 6.8 to 6.9% (P = 0.6684). Nevertheless, in hypertensives aware and treated, control decreased from 32.5 to 24.3% (P = 0.020). Hypertension prevalence increased in the last decade, while awareness, treatment and control remained considerably low in Chengdu. In the Chinese population, more than a quarter of adults have two cardiovascular risk factors , and at least 17% adults have three risk factors3. As a major risk factor of cardiovascular diseases, hypertension has become a common health challenge with rising prevalence and morbidity in China4, of which the overall prevalence is about 30%5.Cardiovascular disease is the leading cause of death in Chinese people aged 40 years and olderChengdu is the capital city of Sichuan, which is in southwestern China with a population of 132 million. Accompanied by industrialization and urbanization of Chengdu, unhealthy changes in lifestyle have increased the risk of chronic cardiovascular disease. However, evidence in support of hypertension trend is still not available in Chengdu.Two epidemiological surveys were conducted from 1998 to 2000 and from 2008 to 2010, separately, in the community population aimed to investigate the impact of lifestyle changes on hypertension and related cardiovascular risk factors. The present study is a retrospective analysis of two surveys on prevalence, awareness, treatment, and control, as well as related risk factors for adults aged 40–79 years in Chengdu over past decade.A total of 4850 and 5456 adults were included in two surveys, respectively. Of these included individuals, 243 and 251 (5.0% and 4.6%) refused to accept examination or had missing data. 4607 and 5205 (95.0% and 95.4%) were eligible for analysis, respectively.P < 0.001) were included in 2010. Current smokers decreased over the past ten years (P < 0.001). And participants with DM increased sharply (P < 0.001). Mean BMI increased nearly 4 units in past ten years (P < 0.001). And the prevalence of being overweight increased more than 6% (P < 0.001). Moreover, the prevalence of being obese doubled over time (P < 0.001). Mean levels of total cholesterol(TC), low-density lipoprotein cholesterol(LDL), triglycerides(TG) and fasting plasma glucose (FPG) increased in 2010 compared with those in 2000 (All P < 0.001).The characteristics and cardiovascular risk factors of all participants are summarized in Tables P < 0.001). The mean levels of SBP/DBP were 117.8 ± 33.9/75.3 ± 19.1 mmHg  in 2000, and 132.1 ± 21.2/79.3 ± 11.2 mmHg  in 2010, respectively. Mean levels of SBP and DBP were higher in 2010 than it in 2000 by age and sex (All P < 0.001). In both years, there was a trend that the levels of SBP and DBP were increasing with age . In both surveys, the highest hypertension prevalence was among subjects aged 70 years and older.The overall age-standardized prevalence of hypertension has increased nearly 2% in this population over past 10 years . And the overall pre-hypertension prevalence was higher among men than women  in 2010 was double that of the 2000 Table . The oveP < 0.001). In both surveys, there was a trend that the prevalence decreased with age. Moreover, the prevalence was the highest among subjects aged 40–49 years in both years and the prevalence in each age group in 2000 was higher than that in 2010 . In both surveys, the SDH prevalence increased with age and the prevalence of SDH was higher for men than women in each age group .In 2000, the prevalence of IDH and SDH were higher than that of ISH. However, the prevalence of ISH and SDH were much higher than that of IDH in 2010 (All P < 0.001). This descent was present in both sexes, and especially noticeable in women (Table P < 0.001). The increase was larger in women than in men.The overall awareness slightly increased from 37.7% in 2000 to 42.5% in 2010  over the past ten years. Among treated hypertensives, control (BP < 140/90 mmHg) was 32.5% in 2000, compare to 24.3% in 2010  to 65.8% (in 2010) significantly . SBP and DBP levels increase over past decade, 2) prevalence of hypertension and pre hypertension increase steadily with slowly increasing awareness, while treatment and control remain considerably low.6, rich fat food intake , and higher BMI7. Smoking9 and work related stress are associated with hypertension development10. Accompanying with the industrialization and urbanization, increased stressful workload may involve in the hypertension prevalence increasing. Limited knowledge of hypertension prevention, diagnosis treatment and its relevant risk factors among community physicians and general population may contribute to the low awareness. Treatment of hypertension was improved with economic development in past decade, especially apparently among those were aware of their hypertension. Higher socioeconomic status could positively improve the awareness and control of hypertension, and people would also have a better compliance of medicine treatment. Accompanying with the rapid economic development of Sichuan, people changed their dietary habits with more unhealthy fatty and salty food, while tended to be more dependent on morden transportation daily. Meanwhile, more people gradually became to care more about their health and generally could relatively afford the disease cost during past decade. It could be one reason for the increasing hypertension prevalence accompanying by increasing awareness and treatment. While during past decade, no apparent control improvement in smoking and drinking, increasing overweight and obesity, and increasing lipid and glucose levels could interact and potentially contribute to the poor hypertension control. Besides, poor compliance with medication and lack of health eduction could also be important reasons for poor BP control.There were more women in survey 2010, mainly because many younger and middle aged men moved to work in more developed east coastal areas  over past decade. The steadily increased BP level and prevalence may be related to lifestyle changes accompanying with industrialization and urbanization, such as high salt food12 during the past two decades and in Japan13, while there was a consistent decreasing of BP in Germany14, Spain15, Italy16 and Korean17. From 1988 to 2008, there was an increasing trend of hypertension prevalence in Americans aged 40 years and older11, however, the prevalence of hypertension in Japan13, Germany14, Italy16 and Korea17 decreased. Other Chinese studies in other areas20 also have found that there is an increasing trend of prehypertension and hypertension prevalence. A recent study in Chongqing20, which is a neighbor city of Sichuan with similar geographic, culture, and dietary background, reported that hypertension prevalence in local adults (23.4% in 2012 and 12.0% in 2002) doubled during past decade. The increasing trend of hypertension prevalence in our study is in accordance with studies in other Chinese cities, especially cities with similar background, and other developing countries, such as India21 and Thailand22. During the same period, the hypertension awareness and treatment in America12, Germany14 and Korean17 increased substantially, which contributed to a better BP control. And awareness of hypertension in other areas in China tended to increase consistently20. Nevertheless the treatment in Chengdu is unluckily lower than in Shandong18, Beijing19, while higher than in Chongqing20, India21 and Thailand22. In past decade, the hypertension control doubled in Germany14, Spain15 and nearly doubled in America11 and it increased steadily in developed couriers, such as Italy16 and Korean17. Unfortunately, the hypertension control in Chengdu was low, which was in accordance with studies in other Chinese cities20. It is apparently that improvements in hypertension awareness, treatment contributed to better control in european countries16.During past decade, there was no increasing trend of SBP and DBP levels in AmericaLifestyle modification contributing to BP decrease is recommend to general population in Chengdu. People should increase their physical activity, improve levels of fruit and vegetable intake, consume less fatty diet and quit smoking. A better community based primary health care and primary prevention system focused on early diagnosis and treatment, patients management, hypertension knowledge education and lifestyle risk factors modification in general population is needed for better BP control.This study has following limitations. First, results of this study should be interpreted with caution, since both surveys only focused on population in Chengdu, although main results are comparable to studies in other Chinese cities. Second, the gender constituents in two separate surveys were different and quantity of physical activity was not measured in 2000. Third, due to the different economical support by the government, the methods for diabetes mellitus diagnoses were different in both surveys. Fourth, smoking and alcohol consumption data are based on self-report with the possibility of misclassification of exposure.In conclusion, hypertension prevalence increased steadily among residents aged 40 to 79 years in Chengdu from 2000 to 2010, while awareness, treatment and control remained considerably low. More efforts should be taken to improve hypertension awareness, treatment and control among middle aged and older residents. Future enhanced public health care program focused on these problems is needed.Two cross-sectional surveys were conducted from 1998 to 2000 and from 2008 to 2010 separately in communities of Chengdu, using a multistage cluster sampling. The study population included 4850 and 5456 subjects aged 40–79 years (All subjects were Han ethnicity) living in Chengdu more than 3 years in survey 2000 and survey 2010, respectively. The response rates for the subjects participating in the surveys of 1999 and 2009 were 95.1% and 95.4%, respectively. Ethics approval was obtained from the Ethics Committee of the Second People’s Hospital of Chengdu, China. The methods in the study were in accordance with relevant guidelines and the Declaration of Helsinki. All participants gave informed consent.Data were collected through a standard household questionnaire interview with additional interviewing and a physical examination in community hospitals in both 2000 and 2010, separately. In both years, all researchers were trained for questionnaire administration, correct cuff size selecting, BP measurement and other techniques needed in surveys with standard techniques. The information collection included demographic characteristics, smoking, alcohol consumption, history of hypertension, cardiovascular disease, diabetes, and administration of antihypertensive medication, physical examination, waist circumference, body weight, and height. In both years, participants had their blood pressure (BP) measurements with a validated mercury sphygmomanometer by trained researchers. Three consecutive BP readings were taken on the right arm with an appropriate cuff size (22 to 26 cm long and 12 to 14 cm wide), in a sitting position after a 10 minutes rest. Participants were not allowed to smoke cigarettes, drink tea/coffee, or do physical exercise half an hour before BP measurements. The room temperature was required to be between 18 °C and 25 °C for measurements. Overnight fasting blood specimens of participants were collected and tested in the central laboratory of our hospital in both years. The oral glucose tolerance test (OGTT) was tested in all subjects in 2010, while not in 2000.24. Hypertension subtypes in surveys include: isolated systolic hypertension(ISH), defined as 140 mmHg ≤ SBP and DBP < 90 mmHg; isolated diastolic hypertension(IDH), defined as SBP ≤ 140 mmHg and 90 mmHg ≤ DBP; systolic - diastolic hypertension(SDH), defined as 140 mmHg ≤ SBP and 90 mmHg ≤ DBP24.In both years, BP level was defined as the mean value of three measurements and hypertension was defined as systolic blood pressure (SBP) ≥ 140 mmHg, or/and diastolic blood pressure (DBP) ≥ 90 mmHg, and/or normotensives treated with antihypertensive medications. Prehypertension was defined as 120 ≤ SBP < 140 mmHg or/and 80 ≤ DBP < 90 mmHgBased on the 1999 World Health Organization standards, diabetes mellitus (DM) was defined as fasting plasma glucose > 7.0 mmol/l (in 2000), and by OGTT .24.Awareness of hypertension was determined by definite response to question “Have you ever been told that your blood pressure was high or you had a hypertension” by a physician/heath care. Treatment of hypertension was confirmed by saying “yes” to this question, “Are you taking antihypertension medication during last three months, because of your high BP/hypertension?”, Control of hypertension was defined as the measured SBP < 140 mmHg and DBP < 90 mmHg in patients with hypertension under antihypertension treatment2). Overweight was define as 25 kg/m2 ≤ BMI < 28 kg/m2. Individuals with a BMI of 30 kg/m2 or greater are defined as obese.Body mass index (BMI) was defined as weight (kg)/height . Continuous variables were expressed by mean ± standard deviation. Categorical variables were expressed by percentage (%). Rate of awareness, treatment and control of hypertension were standardized by the China national census. Comparisons between groups used the Chi - Square Test for categorical variables and one way ANOVA for continuous variables. The Cochran - Mantel - Haenszel test was used to analyze for the trend of prevalence over various age groups. A two-sided


Consumption of sugar-sweetened beverages (SSBs) is associated with increased risk of obesity, diabetes, heart disease and dental caries. Our aim was to assess the effects of plain packaging, warning labels, and a 20 % tax on predicted SSB preferences, beliefs and purchase probabilities amongst young people.A 2 × 3 × 2 between-group experimental study was conducted over a one-week period in August 2014. Intervention scenarios were delivered, and outcome data collected, via an anonymous online survey. Participants were 604 New Zealand young people aged 13–24 years who consumed soft drinks regularly. Participants were randomly allocated using a computer-generated algorithm to view one of 12 experimental conditions, specifically images of branded versus plain packaged SSBs, with either no warning, a text warning, or a graphic warning, and with or without a 20 % tax. Participant perceptions of the allocated SSB product and of those who might consume the product were measured using seven-point Likert scales. Purchase probabilities were measured using 11-point Juster scales.F  = 54.4, p <0.001; warning label: F  = 19.8, p <0.001; 20 % tax: F  = 11.3, p <0.001). Plain packaging and warning labels also had a significant negative impact on reported likelihood of purchasing SSB’s (p = <0.001). A 20 % tax reduced participants’ purchase probability but the difference was not statistically significant (p = 0.2).Six hundred and four young people completed the survey  years). All three intervention scenarios had a significant negative effect on preferences for SSBs (plain packaging: Plain packaging and warning labels significantly reduce young people’s predicted preferences for, and reported probability of purchasing, SSBs. Consumption of sugar-sweetened beverages (SSBs) is associated with increased risk of obesity, diabetes, heart disease, and dental caries –4. InterLess is known however regarding the effects of warning labels and plain packaging on diets. In Finland, warning labels are mandatory on foods high in salt and their introduction led to a decrease in availability of high salt foods , thus reWARNING: Drinking beverages with added sugar(s) contributes to obesity, diabetes, and tooth decay” on advertisements for SSBs [In 2014, a bill was proposed for introduction of warning labels on SSBs in California, although it was subsequently rejected . The citfor SSBs .Although a number of studies have shown that branding influences children’s food preferences , 21, theA 2  × 3  × 2  between-group experimental study design was used, with the intervention scenarios delivered online and outcome data collected entirely via online survey. The study was undertaken in New Zealand over one week in August 2014. Ethical approval was received from the University of Auckland Human Participants Ethics Committee on August 1st 2014 (reference number 012200) and all study participants provided informed consent.People were eligible for inclusion in the study if they reported consuming SSB in the previous two months, and were aged between 13–24 years. Recruitment of 16–24 year olds was undertaken by a market research company, Research Now, which invited a random sample of their panel members aged 16 or older to participate in the study. Younger participants (aged 13–16 years) were additionally recruited via parents who were panel members. Recruitment continued until at least 600 eligible respondents consented to take part and were randomised. All participants received a nominal monetary reward  for 16–24 year olds, and NZ$3,  for parents who consented to their 13–16 year old children participating) on completion of the study.Participants were randomly allocated using a computer-generated algorithm to view one of 12 experimental conditions, specifically images of branded versus plain packaged SSB cans, with either no warning, a text warning, or a graphic warning, and price with or without a 20 % tax .The text warning was presented in an orange-coloured octagon and covered approximately one eighth of the front side of the can. It stated “uly 2016 . The gral caries , and it The tax/no tax scenarios were operationalised by presenting the usual (average) price of a can of the displayed SSB  or the price of a can with 20 % tax applied  directly below the images.Prior to the survey, data were collected on participants’ key demographic characteristics, usual consumption of common foods and beverages, and frequency of consumption of a range of beverages including carbonated drinks, energy drinks, milk, and juices. These also served as screening questions to ensure respondents were in the target age range and had consumed SSBs in the previous two months.Following randomisation, participants viewed the allocated image, and used a seven-point rating scale to answer questions regarding attitudes and predicted product preferences. Specific semantic differential attitude statements assessed whether participants believed the displayed SSB was: expensive/cheap, unattractive/attractive, low quality/high quality, uncool/cool, unhealthy/healthy, and tasted bad or good. As adolescents and young adults often consume SSBs in public settings, and brands are used to communicate aspects of consumers’ identities , we alsoThe 11-point Juster Scale was used to measure the “in the moment” probability of purchasing the displayed drink if it were one of the options available at a convenience store, where 0 represented “no chance or almost no chance”, 5 represented “fairly good possibility”, and 10 represented “certain or practically certain” , 25. FivAssuming a standard deviation of 1.0, power calculations indicated that a sample size of 600 participants  would provide 80 % power to detect a minimum one-point difference in purchase probability on the Juster scale.t-tests were used to assess differences in mean predicted preference scores as a result of packaging or taxes, and a one-way Analysis Of Variance (ANOVA) was used to assess differences in product preferences as a result of warning labels. A three-way ANOVA was conducted to determine whether the three experimental scenarios were associated with significant effects on participants’ likelihood to buy SSBs. Multivariate Analyses Of Variance (MANOVA) were undertaken to assess the relationships between the interventions.Independent sample n = 350, 58 %) reported consuming SSBs between one and three times per month, while 49 (8.1 %) consumed SSB at least once a day.Six hundred and four panel members responded to the study invitation, met study inclusion criteria, and completed the online survey. Three hundred and three were recruited directly from the panel (age range 16–24 years) and 301 were recruited via parent members (age range 13–16 years). All questions were completed by all participants. Key demographic characteristics are detailed in Table F  = 54.4, p <0.001; warning label: F  = 19.8, p <0.001; and 20 % tax: F  = 11.3, p <0.001) (Table p = <0.001), tasting worse (p = 0.025), and appearing less ‘cool’ (p = 0.034). The tax had no significant effect on other measures however. A significant interaction was found between packaging and warning labels for the attractiveness of the can itself  = 14.9, p = <0.001), and between packaging and a 20 % tax for how interesting/boring a typical user was perceived to be  = 4.13, p = 0.04).Compared with the control of a branded SSB can with no warning label or tax applied, all three interventions had a significant negative effect on predicted preferences for SSBs (plain packaging: p = <0.001). Text and graphic warning labels also significantly decreased likelihood of buying SSBs compared with no warning label: 3.3 (text warning) and 2.7 (graphic warning) on Juster scale versus 3.9 (no warning), p = <0.001. The 20 % tax had no significant effect on probability of purchasing   and warning labels  had a significant effect on purchase probability, while a 20 % tax did not .Compared with branded packaging, plain packaging significantly decreased reported likelihood of buying SSBs  agreed/strongly agreed that SSBs should carry a text warning label while half (50 %) agreed/strongly agreed with introduction of graphic warning labels. Fewer participants agreed with application of a tax to SSBs .To the best of our knowledge this is the first research to examine the effects of plain packaging and warning labels on young people’s predicted preferences for, and likelihood to buy, SSBs. The online experiment suggests that plain packaging and warning labels reduce young people’s preferences for, and reported likelihood to buy, SSBs. Of the experimental conditions examined, plain packaging had the most significant negative impact on predicted product preferences, and was associated with less positive perceptions of those who might consume the product. These results align with findings from studies of plain packaging for tobacco products , 9, 26. Although graphic warning labels have not yet been proposed for SSBs, this research suggests they would be more effective than text warning labels. Furthermore, warning labels had a greater effect when placed on beverages with plain packaging compared with branded packaging, suggesting that attractive branding and colours on SSBs may reduce effectiveness of warning labels. These results are again consistent with tobacco research , 9, 27.Given the success of tobacco taxes in reducing consumption , 10 and The online survey method was convenient and quick for participants, and likely had a low level of social desirability bias given no interviewer was present and anonymity was maintained. The study also had a sufficiently large sample size to detect a minimum one-point difference in purchase probability measured on an 11-point Juster scale. Some limitations are however noted. First, the scenarios were hypothetical and respondents did not actually purchase the drinks in question; as such their responses reflect self-predictions rather than actual behaviour . Whilst Plain packaging and warning labels could significantly reduce adolescents’ and young adults’ preferences for and likelihood to purchase SSBs, and may therefore reduce consumption. These labelling measures warrant consideration as part of a comprehensive portfolio of strategies to reduce young people’s SSB intakes and reduce rates of childhood obesity.


GC). Thus, we aimed to develop a reliable prognostic signature that could assess the prognosis risk in GC patients. Two microarray datasets of GSE662254  and GSE15459  were included into analysis. Prognostic genes were screened to construct prognosis‐related gene pairs (PRGPs). Then, a penalized Cox proportional hazards regression model identified seven PRGPs, which constructed a prognostic signature and divided patients into high‐ and low‐risk groups according to the signature score. High‐risk patients showed a poorer prognosis than low‐risk patients in both the training set  and test set (1.773 [1.107–2.840]). The PRGPs signature also achieved a higher predictive accuracy  than two existing molecular signatures (0.706 [0.667–0.744] for a 11‐gene signature and 0.684 [0.642–0.726] for a 24‐lncRNA signature) and TNM stage (0.764 [0.715–0.814]). In conclusion, our study identified a novel gene pairs signature in the prognosis of GC.Current prognostic signatures need to be improved in identifying high‐risk patients of gastric cancer ( Gastric cancer (GC) is one of the most common cancers around the world, with an estimated 951,600 cases and 723,100 deaths per year The availability of large‐scale gene expression profiles brings the chance to identify more reliable prognostic signatures in various cancers. Several studies have proposed gene‐expression prognostic signatures in GC http://www.ncbi.nlm.nih.gov/geo/). Microarray datasets were selected if fulfilling the following criteria: (1) based on the chip platform of Affymetrix Human Genome U133 Plus 2.0 Array (GPL570); (2) availability of related clinical data, especially follow‐up time and survival status; (3) sample size of more than 150. Finally, two datasets (GSE62254 and GSE15459) were included in this study. GSE62254 (n = 300) was used as a training set for signature identification, while GSE15459 (n = 192) was used as a test set for signature validation.Normalized gene expression profiles of GC were downloaded from Gene Expression Omnibus (GEO) database (http://www.affymetrix.com). When multiple probes matched to an identical gene symbol, we selected the probe ID with the largest inter‐quartile range (IQR) of expression values among all multiple probe IDs to represent the gene, which was biologically more reasonable and robust than the average method The probe IDs were matched to gene symbols using the Affymetrix annotation file (n = 300).Prior to analysis, we calculated mean intensities of each gene across all samples, and filtered out the un‐expressed genes  and un‐informative genes (the lowest 20% rank sum of standard deviations) to decrease the false discoveries. Then, survival analysis was conducted in the training set to screen prognostic genes in GC, using the log‐rank test and permutation method  The patients were divided into high‐ and low‐risk groups according to the PRGPs score cutoff, which was determined by a time‐dependent receiver operating characteristic (ROC) curve  at 5 years http://software.broadinstitute.org/gsea/index.jsp) was conducted to detect whether a series of priori defined biological processes were enriched in the gene rank derived from differentially expressed genes (DEGs) between the high‐ and low‐risk groups. False discovery rate (FDR) <0.05 was chosen as the cut‐off criteria.To identify potential biological processes related with the risk based on the PRGPs signature, Gene set enrichment analysis (GSEA) (https://www.r-project.org/). For use with GSEA software, the collection of annotated gene sets of h.all.v5.2.symbols.gmt in Molecular Signatures Database  was chosen as the reference gene sets. A two‐sided P value <0.05 was considered statistically significant.All statistical analyses were performed using R . A total of 518 prognostic genes were identified to construct 133903 PRGPs. Then, we constructed a risk score consisting of seven PRGPs using L1‐penalized Cox proportional hazards regression . According to the same signature score and cutoff value with the training set, high‐risk patients also showed a poorer prognosis than low‐risk patients in the test set  , which proved the methodological reliability in this study Fig. .P = 0.026) and the 24‐lncRNA signature , and moderately higher than the TNM stage signature  , as well as for TNM stage (AUC at 5 years = 0.737)  Fig. . The setWith the development of high‐throughput gene detection technology, we were entering a new era of big biological data. A tremendous amount of genomic information was detected in individual samples, which promoted the identification of novel diagnostic, prognostic, predictive or pharmacodynamic biomarkers Prognostic signatures were baseline measurement to provide information about the long‐term outcome for cancer patients. Currently, the microarray or RNA‐sequencing data of gene mutation and expression were usually used to construct novel prognostic signatures by a Cox proportional hazards regression model In this study, we constructed a risk score consisting of seven GRGPs using L1‐penalized Cox proportional hazards regression. The calculated score by this gene pair‐based method was based entirely on the gene expression profile of one GC patient, and could be used without the need for eliminating batch effects. Thus, the formula and cutoff value could be used across multiple datasets, which was an important advantage. When taking the same formula and cutoff value in the test set, we also reached a consistent result. This indicted the robustness of the method and the PRGPs signature. Accordingly, the PRGPs signature showed a higher predictive efficiency and accuracy than other prognostic signatures.The seven GRGPs consisted of 12 genes, among which two genes (CES1 and TNFRSF11A) were also part of the 11‐Gene signature. Of these 12 genes, only PBX1 has been investigated for potential mechanism in GC The limitations should be acknowledged for our study. First, this was a retrospective designed study, rather than a prospective cohort study. Second, the sample size was relatively small, although the method we developed could eliminate the batch effects. Third, we only considered the microarray data based on GLP570 and ignored other unusual platforms, which might lead to selection bias.In conclusion, our study identified a novel gene pairs signature in the prognosis of GC.None.Table S1. Sensitivity analysis for the prognosis‐related gene pairs signature.Figure S1. Time‐dependent receiver operating characteristic (ROC) curveof the pseudo prognosis‐related gene pairs signature.Figure S2. Protein–protein interaction network of genes inthe enriched set of “oxidationand phosphorylation”.Click here for additional data file.


MTD), dose‐limiting toxicities (DLT), safety profile, and anti‐tumor efficacy of RAF265. We conducted a multicenter, open‐label, phase‐I, dose‐escalation trial of RAF265, an orally available RAF kinase/VEGFR‐2 inhibitor, in patients with advanced or metastatic melanoma. Pharmacokinetic (PK) analysis, pharmacodynamics (PD) and tumor response assessment were conducted. We evaluated metabolic tumor response by 18[F]‐fluorodeoxyglucose‐positron‐emission tomography (FDG‐PET), tissue biomarkers using immunohistochemistry (IHC), and modulators of angiogenesis. RAF265 has a serum half‐life of approximately 200 h. The MTD was 48 mg once daily given continuously. Among 77 patients, most common treatment‐related adverse effects were fatigue (52%), diarrhea (34%), weight loss (31%) and vitreous floaters (27%). Eight of 66 evaluable patients (12.1%) had an objective response, including seven partial and one complete response. Responses occurred in BRAF‐mutant and BRAF wild‐type (WT) patients. Twelve of 58 (20.7%) evaluable patients had a partial metabolic response. On‐treatment versus pretreatment IHC staining in 23 patients showed dose‐dependent p‐ERK inhibition. We observed a significant temporal increase in placental growth factor levels and decrease in soluble vascular endothelial growth factor receptor 2 (sVEGFR‐2) levels in all dose levels. RAF265 is an oral RAF/VEGFR‐2 inhibitor that produced antitumor responses, metabolic responses, and modulated angiogenic growth factor levels. Antitumor activity occurred in patients with BRAF‐mutant and BRAF‐WT disease. Despite low activity at tolerable doses, this study provides a framework for the development of pan‐RAF inhibitors and modulators of angiogenesis for the treatment of melanoma.To establish the maximum tolerated dose ( Melanoma remains the most common cause for skin‐cancer related mortality with over 10,000 deaths anticipated in 2016 BRAF gene in several cancers BRAF gene, most commonly BRAFV600E, result in dramatically enhanced activity of the BRAF kinase and constitutive activation of the MAP kinase (MAPK) pathway enabling tumor growth. Targeting the MAP kinase pathway in patients with advanced melanoma was first attempted with sorafenib, either alone or in combination with chemotherapy with ultimately disappointing results The discovery of somatic activating mutations in the RAF265 is a novel, orally active, small molecule kinase inhibitor with potent activity against mutant and wild‐type BRAF, CRAF and additional anti‐angiogenic activity through inhibition of vascular endothelial growth factor type 2 (VEGFR‐2) in preclinical models Patients with histologically confirmed melanoma; measurable locally advanced or metastatic disease; age ≥ 18 years; an Eastern Cooperative Oncology Group performance status of 0 or 1; available tumor to determine BRAF mutation status; no prior treatment with MEK, VEGF/VEGFR, or RAF inhibitors; no history or presence of brain metastases; a minimum of 4 weeks elapsed since any major surgery or prior anticancer/investigational therapy; and adequate bone marrow, liver and renal function were eligible. Patients signed an institutional review board–approved informed consent form.This study was an open‐label, multicenter, nonrandomized, phase I/II, dose‐escalation study of orally administered RAF265. The study was designed primarily to determine the maximum tolerated dose (MTD) and/or or recommended phase II dose (RP2D), dose‐limiting toxicities (DLTs), and safety of oral dosing of RAF265 in patients with locally advanced or metastatic melanoma using different dosing schedules. The study was comprised of two phases: A dose escalation phase and an expansion phase at the MTD/RP2D. The dose‐escalation phase was guided by pharmacokinetics (PK), pharmacodynamics (PD) and safety. In the dose escalation phase, cohorts 1‐7.1 were administered RAF265 as a single oral dose on Day 1 of the PK run‐in period followed by one loading dose of RAF265 on Cycle 1 Day 1, and by lower once daily doses of RAF265 starting on Cycle 1 Day 2. In the expansion phase (cohort 7.1) the PK run in was omitted and patients received a loading dose in 3 divided doses followed by lower daily dose beginning on Cycle 1, Day 2. The cycle length was 28 days in all cohorts. Here, we report the dose escalation and expansion arm investigating a continuous treatment regimen.Medical history and demographics were collected at screening. The safety of study medication was evaluated on the basis of rate, type, severity graded in accordance with NCI CTCAE version 3.0. Safety assessments consisted of collecting all AEs, serious adverse events (SAEs) with their severity and relationship with study drug, and pregnancies. They included the regular monitoring of hematology, blood chemistry and urine performed at the study center/central laboratory and regular assessments of vital signs, physical condition, body weight, performance status, and cardiac data (i.e. electrocardiogram (ECG)/echocardiogram (ECHO)). Abnormalities in clinical laboratory tests that were considered clinically significant by the Investigator were recorded on the AE CRF. Dose‐limiting toxicities (DLTs) were defined for various organ systems and other clinically relevant adverse effects Table . The DLTCmax), time to reach Cmax (Tmax), area under the curve from time 0 to last measurable concentration (AUClast), time point of the last measurable concentration (Tlast), and terminal half‐life of elimination phase (T1/2). Cycle 1 Day 1 and day 15, reported PK parameters include Cmax, Tmax, AUClast and Tlast. Following log‐transformation, the PK parameters AUC and Cmax from the PK run‐in were analyzed separately using a mixed effects model fitting terms for dose and patient (Patient treated as a random factor)‐ln(PK parameter) = a + b*ln(Dose)+error. In this model, the dose‐exposure relationship is mainly characterized by the slope parameter b. A slope lower (higher) than 1 indicates an under (over) proportional dose‐exposure relationship. The point estimate and associated 90% CI for the slope parameter were determined for safety considerations.Blood samples for plasma RAF265 concentration determination were collected on cycle 1, day 1 and day 15, and on cycle 2, day 1. All PK analyses were based on concentrations of unchanged RAF265 in plasma. PK parameters included the maximum observed concentration  and tumor imaging. BRAF mutation status was determined by Sanger sequencing. Pharmacodynamic markers analyzed, in available tumor/nevus samples obtained before treatment or archival tissue and during treatment on cycle 1, day 8 or day 15, including cytoplasmic p‐MEK, p‐ERK, BIM, c‐KIT, p‐AKT473, pS6 and PTEN, and nuclear p‐ERK, Ki‐67, PARP, cyclin D1, MITF, p27, and p53. Biomarker expression was determined by calculating the percentage of change in the H‐score (14) between on‐treatment and fresh/archival pretreatment specimens. Blood levels of VEGFR‐2 and were assessed before and at multiple time points while on treatment using enzyme‐linked immunosorbent assay (ELISA). Tumor imaging using 18[F]‐FDG‐PET to determine effects of RAF265 on tumor metabolic activity was performed within 2 weeks prior to treatment start (baseline), and on‐treatment on C1D15 and C1D28. Changes in standard uptake value (SUV) were determined by a blinded, independent central imaging review panel. Complete metabolic response (CMR) was complete resolution of tumor FDG‐PET uptake so SUV is the same as background. Partial metabolic response (PMR) decrease in sum of the tumor SUV of ≥25% from the baseline scan. Progressive metabolic disease (PMD) was defined as an increase in tumor SUV of ≥25% from the baseline scan, or the appearance of new FDG‐PET uptake in metastatic lesions. Stable metabolic disease (SMD) was defined as a change in tumor SUV between the PMR and PMD criteria.A Bayesian Logistic Model with five‐parameter overdose control methodology A total of 77 patients were enrolled in this study at four sites in the US and one site in Europe, and received one or more doses of RAF265. Patient demographics and baseline characteristics are listed in Table Treatment–related AEs that occurred in ≥10% of patients are listed in Table Ceff) was 4127 ng/mL, which corresponded to estimated plasma Ceff between MTD of 48 mg (dose level 6) and 67 mg (dose level 7/7.1). In general, the mean observed maximum plasma concentration (Cmax) and area under the curve (AUC) increased nearly proportionally with dose.RAF265 had an extensive distribution phase and a long half‐life of approximately 200 h or 8 days Table . PreclinBRAFV600E mutation and two PR and one CR in 33 patients with BRAF‐WT melanomas and one PR in a patient with an unknown BRAF mutation status  of 12.1% across all dose levels Table . These ius Table . The medP = 0.028; two‐tailed Fisher's test) and there was a trend toward higher metabolic responses in these patients on C1D28 . Five of eight patients with RECIST‐defined responses also had partial metabolic response  patients exhibited a partial metabolic response (PMR)  as vemurafenib. However, in comparison to vemurafenib, response rates were lower in BRAFV600E patients. A distinguishing feature of RAF265 from vemurafenib and dabrafenib is the clinical activity in BRAF‐WT patients. In contrast, RAF265 seems to achieve plasma levels that are commensurate with active exposures in preclinical models; however, the agent may not have sufficient selectivity for BRAFV600E in most patients, as indicated by the increased incidence of thrombocytopenia and visual side effects—both more consistent with inhibition of wild‐type BRAF and CRAF, and decreased incidence of skin squamous cell carcinoma. In line with this, we observed an increase in p‐ERK expression in patients treated at DL1‐5, and moderately decreased p‐ERK abundance in patients treated at DL 6‐7.1. In comparison, vemurafenib suppresses pERK by well over 80% at the recommended phase II dose Preclinical data indicated that RAF265 had a similar an EC50 (=14 nmol/L for μmol/L) for the wild‐type BRAF protein, it is conceivable that these responses were not due to BRAF inhibition. Inhibition of wild‐type BRAF with vemurafenib, dabrafenib and related BRAF inhibitors results in transactivation of alternative RAF‐dimers and downstream activation of ERK We observed responses in patients with BRAF‐WT melanoma, including a complete response. This is consistent with preclinical observations of responses in BRAF‐WT patient‐derived xenografts treated with RAF265 Responses to RAF265 in BRAF‐WT patients may also be associated with alternative mutations that were not systematically evaluated in this study, such as mutations in NRAS, which occur mutually exclusive from BRAF mutations in ~25% of patients, non‐V600E BRAF mutations in ~5–10% of patients, NF1 in ~10–15% of patients and PTEN loss, which is found in ~10% of melanoma and occur with or without concomitant BRAF mutation We observed partial metabolic responses in 20.7% and stable metabolic disease in 43% of patients. Previous work found a correlation between metabolic and RECIST‐defined responses in patients with BRAF‐mutant melanoma treated with vemurafenib Treatment options for patients with BRAF WT patients primarily include immune checkpoint inhibitors and HD‐IL2. Immune checkpoint inhibitors, such as ipilimumab, nivolumab and pembrolizumab either as single agents, or nivolumab and ipilimumab in combination, produce durable responses in 10 to over 50% of patients In conclusion, we have shown that RAF265, an orally available RAF/VEGFR‐2 inhibitor, produces clinical and metabolic responses in a subset of patients with locally advanced or metastatic melanoma, including patients with BRAF‐WT disease. The MTD was determined to be 48 mg PO daily as higher doses produced visual problems and prolong thrombocytopenia. Furthermore, RAF265 significantly modulates key regulators of angiogenesis, including VEGFR‐2 and PGIF. A long half‐life and prolonged toxicities along with a relatively low response rate indicate that RAF265 has limited selectivity or activity for BRAF in vivo. This marginal therapeutic index together with the advent of other much more effective and selective BRAFV600 inhibitors resulted in the phase II dose expansion study being cancelled. Responses in patients with BRAF‐WT melanoma indicate that the development of more specific pan‐RAF inhibitors may provide a feasible therapeutic avenue for these patients. This study also provides rationale to continue ongoing efforts investigating the combination of VEGFR‐2 targeted therapies with immunotherapies.W. Sharfman serves as consultant for Merck, Novartis and Castle Bioscience; received research funding from BMS and Merck. F. S. Hodi served as consultant for Novartis. K. T. Flaherty served as consultant for Novartis and receives research funding from Novartis. J. Sosman served on advisory boards for Merck, Genentech and Array. R. Dummer serves as consultant or has advisory board relationship with Novartis, Merck Sharp & Dhome, BMS, Roche, GSK, Amgen; received research funding from Novartis, Merck Sharp & Dhome (MSD), BMS, Roche, GSK. S. M. Goldinger has advisory board relationship and received travel funding from BMS, Novartis and Roche. D. F. McDermott participated at an advisory board for Novartis. M. B. Atkins serves as consultant for Novartis, Genentech, BMS, Merck, Amgen, Pfizer, Astra Zeneca, Nektar, Agenus and Celldex. L. P. Lam, S. Kakar, Z. Tang are employed by Novartis. B. Izar, D. Lawrence, R. Amaravadi, K. B. Kim and I. Puzanov have nothing to disclose.Table S1. Definition of dose limiting toxicities (DLTs).Table S2. Scheme of eight dose levels. At each dose level, with the exception of DL 8, patients received a 7–10 days run‐in dose, followed by a single loading dose (LD) and a daily maintenance dose (MD). *administered as three doses.Table S3. Pharmacokinetics of RAF265.Table S4. Changes in expression of cytosolic biomarkers. Patients included in this analysis had a fresh or archival tissue scored as described in the methods section. Changes of biomarker abundance were evaluated in on‐treatment specimens. Shown is the number of patients with tissue available for each biomarker analysis, mean and median % changes compared to the pretreatment specimen, and the range for each biomarker in each dose level and in the entire evaluable population. **pMEK, phosphorylated MAPK/ERK kinase; pERK, phosphorylated extracellar signal‐regulated kinase; Ki67, proliferation‐associated antigen Ki‐67; BIM, a pro‐apoptotic member of the BCL‐2 family; PARP, poly(ADP‐ribose)polymerase; Cyclin D1, cell cycle gene, MITF, microphthalmia‐associated transcription factor; CKIT, c‐KIT; P53, tumor protein 53/TP53; PAKT473, phospho Akt S 473; PS6, phosphoserine 240‐S6 ribosomal protein; PTEN, phosphatase and tensin homolog.Table S5. Changes in expression of nuclear biomarkers.Click here for additional data file.


Donor scarcity and the necessity for immunosuppression limit one effective therapy, orthotopic liver transplantation. But in some conditions such as inborn errors of metabolism or transient states of liver insufficiency, patients may be salvaged by providing partial quantities of functional liver tissue. After transplanting multicellular liver organoid units composed of a heterogeneous cellular population that includes adult stem and progenitor cells, both mouse and human tissue‐engineered liver (TELi) form in vivo. TELi contains normal liver components such as hepatocytes with albumin expression, CK19‐expressing bile ducts and vascular structures with α‐smooth muscle actin expression, desmin‐expressing stellate cells, and CD31‐expressing endothelial cells. At 4 weeks, TELi contains proliferating albumin‐expressing cells and identification of β2‐microglobulin‐expressing cells demonstrates that the majority of human TELi is composed of transplanted human cells. Human albumin is detected in the host mouse serum, indicating in vivo secretory function. Liquid chromatography/mass spectrometric analysis of mouse serum after debrisoquine administration is followed by a significant increase in the level of the human metabolite, 4‐OH‐debrisoquine, which supports the metabolic and xenobiotic capability of human TELi in vivo. Implanted TELi grew in a mouse model of inducible liver failure. S The worldwide burden of liver disease is approximately 30 million cases. In 2010, there were more than 1 million global deaths from one cause of liver disease alone, cirrhosis. The only effective therapy for end‐stage liver failure is liver transplantation, which is profoundly limited by scarce donor supply. In some conditions, such as inborn errors of metabolism or transient states of liver insufficiency, patients may be salvaged by providing partial quantities of functional liver tissue. In this study, it was hypothesized that a hardy multicellular cluster, or liver organoid unit, could be extracted from donor livers, which, after transplantation, would generate functional tissue‐engineered liver. This novel cellular therapy would have several proposed advantages: an accessible in vivo hepatic replacement that can be monitored, more efficient stem/progenitor cell production (perfusion is not required and cellular loss is minimized), and durable function. This approach could represent a personalized future therapy that would not require cellular reprogramming or immunosuppression to cure end‐stage liver diseases.The worldwide burden of liver disease is approximately 30 million patients, affecting 1 in 10 people in the United States. In 2010, there were more than 1 million global deaths from one cause of liver disease alone, cirrhosis Additional future approaches include the possible application of human‐induced pluripotent stem cells (hiPSCs) but current progress with hiPSCs has been limited by the immaturity of some of the hepatocyte‐like cells generated from hiPSCs Based on our expertise in generating tissue‐engineered intestine (TEI) from adult stem cell populations All animal experiments were carried out after protocol approval by the Institutional Animal Care and Use Committee. Human liver samples were collected under an institutional review board‐approved protocol from the uninvolved margin of liver tumor resections or the explants obtained in the course of human liver transplantations for indications of liver failure.GFPflox/flox UBC‐Cre/ERT2 g), and then digested in either 800 or 11,000 U/ml of collagenase type I  and 0.12 mg/ml of dispase . LOU were pelleted and washed twice in Dulbecco's modified Eagle's medium (DMEM), the first solution containing 10% fetal bovine serum. After the final wash in DMEM, organoid units (OU) were pelleted by centrifugation at 150g for 5 minutes at 4°C, and the resultant pellet containing LOU was either vitrified or dispersed on a biodegradable scaffold for immediate implantation. Vitrification was performed as described by Spurrier et al. LOU were prepared from human liver or from 2‐week‐old Actin−3 bulk density; porosity >95%; Concordia Fibers, Coventry, RI. http://www.concordiafibers.com). The entire cylinder is sealed with 5% poly‐L‐lactic acid  in chloroform . After sterilization with 80% ethanol, the polymers were coated with 0.4 mg/ml type 1 collagen solution (Sigma‐Aldrich) for 20 minutes at 4°C, rinsed with phosphate buffered saline (Thermo Fisher), and stored dry at room temperature in a desiccator  to avoid premature hydrolysis, degradation or contamination. Fresh cut constructs (3 mm) were used for implantations Scaffolds were prepared as previously published 5 LOU/scaffold were loaded on to biodegradable scaffolds and directly implanted under the abdominal skin of 3‐ to 6‐month‐old mice. NOD/SCID γ host mice  received full‐body irradiation (350 cGy) before implantation. Scaffold loading was consistent with 60 μl of LOU‐containing medium transferred by sterile micropipette onto each scaffold. This volume contains approximately 1× 105 OU, as we have previously determined that this is a range that grows well. Because OU are multicellular, the actual cell number will vary. Under anesthesia and sterile conditions, the abdominal skin was opened approximately 1 cm without violating the rectus muscle. Scaffolds containing LOU were implanted under the skin and the incision closed with 4‐0 Vicryl sutures (Ethicon). Mice were given free access to  and water with Septra 200 mg / 40 mg per 5 ml  at 1:100 dilution. After euthanasia, blood draws were performed for the serum albumin assay and mass spectrometric analysis of drug metabolite at 4‐week time points at the time of TELi harvest via cardiac puncture.Under sterile conditions, approximately 1 × 10g for 2 minutes at 4°C. The sample was returned to the ice bath, supernatant was discarded, and an additional 1 ml of 0.75 M sucrose in HBSS was added. After 5 minutes, 1 ml of plain HBSS was added to the solution, decreasing the sucrose concentration by half. After 5 minutes, another 2 ml of HBSS was added, followed by 4 ml after 5 additional minutes. The solution was again centrifuged at 150g for 10 minutes at 4°C, and the supernatant was discarded.Vitrification was performed as described in LOU were mounted in agarose as in https://www.jacksonimmuno.com). Tissue sections were mounted with Vector shield containing 4′,6‐diamidino‐2‐phenylindole . Images were acquired using an upright fluorescent microscope (Leica DM5500). Staining was performed at least three or more times on independent tissue sections.TELi was harvested at 4 weeks except for the arginase deficiency experiments, when the TELi was harvested 2 weeks after all animals usually succumb from liver failure in this model. Explanted TELi constructs were fixed overnight in formalin (Thermo Fisher) and processed for paraffin embedding. Explanted TELi were also frozen for RNA analysis and stored at −80°C until analysis. Five‐micrometer‐thick cross‐sections were stained with hematoxylin and eosin (H&E) according to standard procedures. Immunofluorescence staining on tissue sections was performed as described http://www.bio-rad.com). Reverse transcription‐polymerase chain reaction (RT‐PCR) was performed to detect the gene expression of albumin, CK18 and HNF4α. Primer sequences are given in supplemental online Table 1.RNA was isolated from frozen TELi with the Qiagen RNA purification kit. cDNA was prepared from 1000 ng of RNA using Bio‐Rad cDNA synthesis kit  according to the manufacturer's instructions.Blood from TELi implanted and control mice were collected by cardiac puncture on time points as describe previously, spun down in a heparin gel containing tube for plasma separation. Human albumin in the plasma was quantified by human albumin enzyme‐linked immunosorbent assay kit  at 1 and 4 hours after drug administration. Plasma was separated by centrifugation. Internal standards  in methanol solution (100 μl) was added to 10 μl of serum and the supernatant after centrifugation  was subjected to liquid chromatography/mass spectrometric analysis. Chromatographic separation was achieved with a Waters Acquity BEH C18 column  with a Thermo Ultimate 3000 UHPLC system (Thermo Fisher). Column temperature was maintained at 50°C and the flow rate was 0.9 ml/min. Mobile phase A consisted 0.1% formic acid in water, and mobile phase B consisted 100% acetonitrile. The gradient started with an initial condition of 3% mobile phase B for 1.5 minutes, followed by a linear gradient to 97% mobile phase B over 3 minutes. This condition was maintained for 2 minutes, returned to the initial condition over 0.1 minute, and maintained until the end of a 9‐minute run. The liquid chromatography was connected to a Thermo Quantiva mass spectrometer (Thermo Fisher) operated in positive electrospray ionization mode. Quantitation was based on multiple reaction monitoring mode with the following parameters: spray voltage, 2,100 V; sheath gas pressure, 55 arb; auxiliary gas pressure, 13 arb; sweep gas pressure, 3 arb; ion transfer tube temperature, 375°C; a vaporizer temperature, 480°C. Debrisoquine was quantified with a mass transition of 176.3 to >134.1 (m/z) and 4‐OH‐debrisoquine was quantified with a mass transition of 192.3 to >132.1 (m/z). Other production ions were also monitored to confirm the identities of the metabolite and parent drug. Percentage metabolite conversion was then calculated and compared.The xenobiotic metabolizing capacity was evaluated with in vivo administration of debrisoquine as described previously flox/flox UBC‐Cre/ERT2 GFP mice flox/flox UBC‐Cre/ERT2 mice rescued with TELi were stained for arginase to confirm that arginase deficiency had been induced as in flox/flox UBC‐Cre/ERT2 mice was performed as in Six Fah−/− Fah−/−Rag2−/−Il2rg−/−Arghttp://www.biochrom.co.uk) as previously described http://www.abcam.com). Briefly, after equilibrating all reagents at room temperature, standards were prepared. Samples were set up using 5 μl of plasma added to 45 μl of assay buffer in duplicate in a 96‐well microplate. The reaction mix was prepared as a master mix with each reaction consisting of 42 μl ammonia assay buffer, 2 μl OxiRed Probe, 2 μl Enzyme Mix, 2 μl developer, and 2 μl converting enzyme. Fifty microliters of reaction mix was added to samples and standard wells, mixed, and incubated for 1 hour at 37°C in a dark container. Optical density was measured at 570 nm using a microplate reader . From the standards, a line was generated and sample results in mM were calculated.Plasma amino acid analysis was performed on a Biochrom 30 HPLC amino acid analyzer , cholangiocytes (CK19‐positive), and mesenchymal cells such as stellate cells (desmin‐positive) and portal fibroblast cells (α‐SMA‐positive) were also positive for GFP, indicating their origin from implanted LOU , which marked our donor cells in LOU when derived from ActinLOU Fig. –5E. FurtLOU Fig. . Few alpIn an effort to generate hTELi, human LOU were prepared from human liver tissue that would otherwise have been discarded in the operating room, under an institutionally approved protocol. After 4 weeks, donor LOU developed into full thickness hTELi, more than twice the size of the original implanted scaffolds Fig. , 6C. Hemn = 6) compared with the mice (n = 4) that had two hTELi implants  deficiency is an autosomal recessive disorder that affects the urea cycle, leading to impaired ureagenesis The multicellular LOU contains multiple liver cell types including hepatocytes, biliary ductal cells, and stellate cells Fig. , 1D. FouTELi contains nonsinusoidal blood vessels and biliary tubules Fig. , 2B confpositiveHNF4αpositive proliferating cells (Fig. The hepatocytes within TELi expressed albumin and HNF4α, which costained with CK8 similarly to the native liver Fig. , 2D, andlls Fig. –4E, althlls Fig. , 4D. AFPhTELi was generated from both fresh and vitrified LOU, with similar key cell types as noted previously, including hepatocytes, biliary epithelium, and vascular structures as noted in murine TELi Fig. . PCNA inGFPflox/flox UBC‐Cre/ERT2 debrisoquine by hTELi. However, a more complete inducible model of liver failure will be required to make an absolute statement.ActinTissue‐engineered liver was successfully generated from both mouse and human tissues and contained all of the key hepatic cell types required for effective liver function. Some of these critical hepatic functions were demonstrated in TELi, including the ability of the tissue to survive and proliferate in the toxic milieu of liver failure from arginase deficiency. Although TELi appears disorganized in comparison with native liver tissue, our model avoids two difficulties of liver cell transplantation: inability to surveil the transplanted cells and cell loss in preparation of the donor cell population. In future work, longer‐term studies of TELi will determine whether this approach might result in a durable future therapy.N.M., A.T., R.S.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; Y.X., X.F., and G.S.L.: collection and/or assembly of data, data analysis and interpretation, final approval of manuscript; X.H., D.J., B.T., and C.W.: collection and/or assembly of data, final approval of manuscript; K.S.W.: conception and design, financial support, data analysis and interpretation, manuscript writing, final approval of manuscript; T.C.G.: conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.The authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.


Pantholops hodgsoni) is one of the most endangered mammals native to the Qinghai-Tibetan Plateau. The population size has rapidly declined over the last century due to illegal hunting and habitat damage. In the past 10 years, the population has reportedly been expanding due to conservation efforts. Several lines of evidence suggest that the Tibetan antelope has undergone a demographic bottleneck. However, the consequences of the bottleneck on genetic diversity and the post-bottleneck genetic recovery remain unknown. In this study, we investigate the genetic variation of 15 microsatellite loci from two Tibetan antelope populations sampled in 2003 (Pop2003) and 2013 (Pop2013). A higher level of genetic diversity  was detected in Pop2013, compared to Pop2003 . We observe that despite passing through the bottleneck, the Tibetan antelope retains high levels of genetic diversity. Furthermore, our results show significant or near significant increases in genetic diversity  in Pop2013 compared with Pop2003, which suggests that protection efforts did not arrive too late for the Tibetan antelope.The Tibetan antelope (chiru,  Pantholops hodgsonii) is a member of the Bovidae family (order Artiodactyla) native to the Qinghai–Tibet Plateau (QTP). It is distributed primarily throughout central Tibet, Qinghai, Xinjiang and west Sichuan in China, occupying a total area of approximately 880,000 km2 at an altitude of 3700 to 5500 mth century, with a population size ranging from 500,000 to 1,000,000 individuals during peak yearshttp://www.iucnredlist.org/details/15967/0) and as a Category I species under the Wild Animal Protection Law of China. To protect the Tibetan antelope and restore the population, the Chinese government has set up seven Nature Reserves since 1993The Tibetan antelope or chiru . Placental tissues were collected from the Zhuonaihu Lake area in Hoh Xil National Nature Reserve in 2013 . All samples were washed three times with deionized water, sucked with 95% ethanol and stored at −80 °C. All necessary approvals for collection and experimentation were acquired for the described field study from the Forestry Department of Qinghai Province, China. All procedures were in accordance with the guidelines of the regulations of experiments on animals and were approved by the China Zoological Society.http://pgrc.ipk-gatersleben.de/misa/misa.html) for extracting the microsatellite DNA sequences from genome DNA of chiru . The forward microsatellite primers were labeled by FAM and synthesized by Sangon Biotech (Shanghai). The primers specific for 15 microsatellite loci are shown in A total of 15 polymorphic microsatellite loci were screened. Seven loci  were obtained using the FIASCO methodTaq DNA polymerase and 1 μL DNA template. The PCR programme was set at 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, x °C  and Shannon index (I) were calculated by Microsatellite Analyzer V4.05Ho), expected heterozygosity (He) and analyzed the Hardy-Weinberg equilibrium (HWE) by implementing Arlequin V3.5PIC) was estimated by modified PowerStats Worksheet17Θ (theta) = 4Ne × Mu, where Ne is the effective population size and Mu is the microsatellite mutation ratio.Original data were analyzed and manually corrected to validate the accurate peak shape and allele size using GeneMarker V1.75. Micro-Checker was used to validate the availability of genotype data12NA varied from 8 to 21 in Pop2013 (P < 0.001). However, significant deviations from HWE were confirmed only at locus P73 and locus P154 in Pop2003 and locus P17 and locus P73 in Pop2013 after Bonferroni correction (adjusted α = 0.05/15).For the 15 polymorphic loci surveyed, a total of 223 alleles were observed in both Pop2003 and Pop2013, 192 alleles were found in Pop2003, and 197 alleles were found in Pop2013. Overall microsatellite variability was high  in Pop2003, and  Pop2013 . Among aP < 0.05). We therefore excluded this locus in subsequent analysis. Over 14 microsatellite loci, microsatellite loci pairs P1/P9, P9/P78 and P24/P96 were in LD (P < 0.05). However, no LD was detected between these locus pairs after Bonferroni corrections (adjusted α = 0.05/14). In Pop2013, there was significant LD between 55 locus pairs . However, only 22 locus pairs were found to be in significant LD after applying Bonferroni corrections for multiple tests  was 12.92 (range 8–19), the average Ho was 0.832 (range 0.660–0.979), and the average He was 0.810 (range 0.636–0.925). In Pop2013, the average of NA, Ho and He across 14 loci was 13.133, 0.810 and 0.840, respectively; NA varied from 8 to 21, Ho varied from 0.556 to 0.957 and He varied from 0.721 to 0.920. The PIC at each microsatellite locus was always larger than 0.5 (range 0.561 to 0.909), a threshold value considered to be highly informative. Shannon information index ranged from 1.240 to 2.678, which also indicated a high genetic diversity.Microsatellite data of 14 microsatellite loci revealed abundant genetic diversity in both Pop2013 and Pop2003 . In Pop2P = 0.923; Pop2013: P = 0.985). The sign test, standardized differences test and Wilcoxon test all showed a significant heterozygosity excess in either Pop2003 or Pop2013 under the IAM (P < 0.05). However, this was not necessarily indicative of true heterozygosity excess, as the IAM is thought to be a less appropriate model for microsatellites than the SMMThe infinite allele model (IAM), TPM and stepwise mutation model (SMM) were applied when BOTTLENECK was used to test for population bottlenecks in this study. Populations exhibiting a significant heterozygosity excess would be considered to have experienced a recent genetic bottleneck. Under the TPM, the results displayed no genetic bottleneck effect in either Pop2003 or Pop2013 . FurtherHe, PIC, I and Θ at 9 out of 14 loci . In comparison with Pop2003, paired t-test demonstrated that Pop2013 had significantly higher values of average He  and PIC  but differences in average MNA  and Ho  were not found  to 5.305 (Pop2013) over an 11-year period (P < 0.05).A tendency toward recovery of genetic diversity was observed in the Tibetan antelope. Pop2013, compared with Pop2003, exhibited higher values in ot found . There wr period , showingMNA, He and PIC. Although the results are not directly comparable because different microsatellite loci were used, comparisons of the Tibetan antelope to other Bovidae species suggest that genetic diversity in the Tibetan antelope ranks highest 217MNA and He, respectively.Demographic bottlenecks can result in a loss of genetic variation22232425Studies have indicated that factors such as dispersive capabilities30313233353638We conclude that ample genetic diversity may still exist in the Tibetan antelope. Furthermore, the Tibetan antelope has shown a slight increase in genetic variation during the past 11-year period. In this sense, the results of the present study suggest that protection efforts did not arrive too late for the Tibetan antelope and provide molecular evidence for the effectiveness of conservation strategies.How to cite this article: Du, Y. et al. Microsatellite Loci Analysis Reveals Post-bottleneck Recovery of Genetic Diversity in the Tibetan Antelope. Sci. Rep.6, 35501; doi: 10.1038/srep35501 (2016).