FHIT), a tumor suppressor that induces apoptosis. Subsequent association tests on 16 SNPs spanning approximately 381 kb surrounding D3S1234 in Americans of European descent revealed significant evidence of association for a single SNP within intron 5 of FHIT. In the current study, re-sequencing and genotyping within a 28.5 kb region surrounding this SNP further delineated the association with prostate cancer risk to a 15 kb region. Multiple SNPs in sequences under evolutionary constraint within intron 5 of FHIT defined several related haplotypes with an increased risk of prostate cancer in European-Americans. Strong associations were detected for a risk haplotype defined by SNPs 138543, 142413, and 152494 in all cases  and for the homozygous risk haplotype defined by SNPs 144716, 142413, and 148444 in cases that shared 2 alleles identical by descent with their affected brothers . In addition to highly conserved sequences encompassing SNPs 148444 and 152413, population studies revealed strong signatures of natural selection for a 1 kb window covering the SNP 144716 in two human populations, the European American  and the Japanese , as well as in chimpanzees . These results strongly support the involvement of the FHIT intronic region in an increased risk of prostate cancer.Previously, a candidate gene linkage approach on brother pairs affected with prostate cancer identified a locus of prostate cancer susceptibility at D3S1234 within the fragile histidine triad gene ( The genetic complexity of prostate cancer has been well-demonstrated by independent, large-scale, genome-wide association studies that identified multiple risk loci throughout the human genome FHIT in prostate cancer risk FHIT gene, which encodes a 16.8 kD triphosphatase, comprises 10 short exons spanning approximately 1.5 Mb. It resides at the most frequently observed fragile site in the human genome, FRA3B (3p14.2); and it is one of the earliest and most frequently deleted regions in multiple cancer types FHIT gene in prostate cancer tissue has not been widely reported, loss of heterozygosity (LOH) has been reported in 2 of 15 tumors through the use of microsatellite markers located in introns of FHITFHIT was also detected in an in vitro model of a prostate cancer tumor cell line that was established by using HPV-18 to immortalize a normal adult human prostate epithelium cell line, followed by malignant transformation through exposure to a chemical carcinogen We recently used linkage analysis of candidate genes and subsequent association tests to implicate a 30 kb region within intron 5 of FHIT that is associated with cancer risk has not been reported, possibly because of limitations of previous studies that focused only on exons, untranslated regions of mRNA, and promoters. Characteristic landmarks of a fragile region, such as aphidicolin-induced hybrid breaks, HPV16 integration sites, pSV2neo integration sites, and deletion end points in cancer cell lines, have been identified within introns of FHITAlthough FHIT protein expression is lost or reduced in many types of human cancers FHIT intronic region. We conducted a re-sequencing survey and analyzed linkage disequilibrium (LD) and haplotype structure in sequences from intron 5 of FHIT in European American, Yoruban, and Japanese populations, and several non-human primates. Based on these data, we refined the region associated with prostate cancer risk to a 15 Kb LD block and revealed strong signatures of selection in multiple human populations and, possibly, other primate species.The evolutionary forces of mutation, natural selection, genetic drift, and recombination have shaped the pattern of variation in the human genome. Natural selection, which acts on functionally important genetic variations that result in alteration of fitness, such as adaptation to local environment and disease susceptibility, may leave specific signatures on affected loci FHIT for association with prostate cancer risk and detected a significant association with one of the SNPs, rs760317. A less significant association with prostate cancer risk was found at a closely linked SNP, rs722070, located 13 kb from rs760317 on the centromeric side; no association was detected with SNPs on the telomeric side. To map the association with prostate cancer risk at high resolution and look for evidence of selection, we conducted a re-sequencing survey using 13 randomly chosen cases and controls of European-American descent. The total surveyed sequence length was 28.5 kb, excluding two un-amplifiable sequence gaps of 487 bp and 263 bp. One of the gaps contained an AT repeat and a long poly A tract, and the other contained AT and AG repeats. Two fragments of this region with lengths of 19 kb  and 7 kb  were also sequenced in 16 Yoruban and 16 Japanese individuals, respectively.Larson et al. Across the entire region, we identified 216 SNPs and 9 indels, ranging from 1 to 24 bp, in the 13 European-American individuals . Within 2 based on SNPs with a minor allele frequency greater than 0.05 (196 SNPs for the 13 European-Americans samples and 178 SNPs for the 16 Yorubans samples) using Haploview  was observed for several SNPs and maximized at 137302  1, P = 0.0231) for all cases  for the 2 IBD subgroup  and several other genotyped SNPs, 135240, 137261, 139813, 144716, 147904  with SNP 152494 among all genotyped SNPs. Replacing SNP 152494 with 148444 in the three-SNP combinations also defined a haplotype for which homozygotes were especially enriched in cases .We observed a striking increase of nucleotide diversity that spanned multiple LD blocks for the three human populations . The maxP = 0.003 assuming standard neutral model I; P = 0.006 assuming neutral model II; P = 0.021 assuming neutral model III) corresponded to the window of maximum π. In the 14 CEPH individuals, the same window exhibited a Tajima's D of 3.11. Only one small ENCODE region, less than 0.6% of all ENCODE regions, displayed a higher maximum Tajima's D. A significant Fay & Wu's H value  was detected for the same window in the Japanese population. A 14-SNP sliding window analysis for all ENCODE regions in the Japanese population found 237 of 6265 windows with a Fay & Wu's H value greater than 8.05 (P = 0.038).We also detected a significant increase of Tajima's D in the European-American population . The 1 kTo determine if the greater nucleotide diversity was due to an increase in the local mutation rate, we evaluated the nucleotide differences in the 28.5 kb sequenced region by comparing one human sequence with one chimpanzee sequence retrieved from the UCSC genome browser. For each 1 kb window across the 28.5 kb region, the divergence between the human and the chimpanzee sequences ranged from 0.004 to 0.026 and averaged 0.0145 . For theTwo SNPs, 144716 and 144552, within the 1 kb window that showed the maximum signal of natural selection, were in strong LD with 135181 and demonstrated a comparable level of association to prostate cancer risk in combination with SNPs 142413 and either 152494 or 148444 . The regP = 0.0001 assuming the standard neutral model) suggested a hitchhiking effect under a recent positive selection. For the bonobo sequence, two rare SNPs, each observed only once in the 6 individuals, and no fixed nucleotide change were present in the 1 kb window  compared to chimpanzee sequence.Sequencing data in the same 1 kb window in common western chimpanzees and bonobos also revealed potential natural selection. Although the chimpanzee sequence possessed a completely different collection of SNPs compared to the human sequence, the haplotype distributions exhibited a pattern similar to that of the Japanese population: predominantly one haplotype with extremely high frequencies of the derived allele for multiple SNPs . A significantly high Fay & Wu's H  in western Africa, Pan troglodytes troglodytes (Ptt) in central Africa, and Pan troglodytes schweinfurthii (Pts) in eastern Africa. Previous studies suggested distinct demographic histories for the three subspecies, resulting in a slightly positive average value of Tajima's D for western chimpanzees (Ptv) and a significantly negative average value of Tajima's D for central chimpanzees (Ptt). To establish a genome-wide distribution of population statistics for the common chimpanzees, we retrieved and reanalyzed sequence data from two previous studies: 50 intergenic regions (Genebank acc.# AY276396 to AY277244) sequenced in 17 common chimps  Three subspecies are recognized among common chimpanzees based on their geographic distribution: Previous linkage and association studies identified an approximately 30 kb region associated with prostate cancer risk. In this report, detailed analysis of local LD structure and additional association tests refined the maximum signal to within a 15 kb region, possibly involving a haplotype defined by three or more SNPs within sequences under strong evolutionary constraint.FHIT. The risk haplotypes defined by major alleles of several SNPs in combinations were not in complete LD with any single SNP discovered in the 28.5 kb region and exhibited much stronger associations with prostate cancer than any single SNP tested. Among the 9 SNPs that delineated risk haplotypes, four  were located within or near sequences that are highly conserved among mammals; and one (144716) was located within a sequence that exhibited significant and distinct signals of natural selection within diverse human and primate populations. The alleles of 5 SNPs  in risk haplotypes were ancestral. The alleles of both SNPs 148444 and 152494 in risk haplotypes were derived and reached very high frequency (>0.8) in all three human populations tested; therefore, they appeared to be under positive selection, especially in the Yoruban population. For example, SNP 148444 overlapped with 1-kb windows of minimum Tajima's D (−1.804 for 10 SNPs) and elevated Fay and Wu's H (9.31 for 17 SNPs) in the Yoruban population.Evidence of both association and selection supported important and interactive functions for sequences within the 15 kb intronic region of 2<0.3 for all possible pairs), consistent with the hypothesis originally proposed by Lin et al. Levin et al. Detection of signatures of natural selection has been proposed to map genes and regulatory elements involved in human diseases FHIT gene is known for its responsiveness to environmental factors, such as smoking FHIT coding region between the human and the chimpanzee and found 4 synonymous and 2 nonsynonymous changes within its 441 bp coding region. Both nonsynonymous changes altered the chemical properties of amino acids involved, implying that FHIT might be one of the fast-evolving genes subjected to positive selection.Population genetics in this region suggested that diverse selective forces may have been acting on different populations of humans and primates. It is, therefore, intriguing that the FHIT expression/function or through unrelated intronic functional elements remains to be investigated.Conventional association studies have largely focused on known coding sequences, which account for only approximately 1.5% of the human genome. However, recent studies have revealed large populations of previously unknown RNA transcripts, most of which are non-coding, within introns and intergenic regions The case and control samples have been described previously We obtained the primate DNA panel (PRP00003) from the Coriell Cell Repositories. The sample included one individual from each of the following species: common chimpanzee, bonobo, gorilla, Sumatran orangutan, pigtailed macaque, rhesus monkey, black-handed spider monkey, common woolly monkey, red-chested mustached tamarin, and ring-tailed lemur. We also obtained DNA of another 12 unrelated common western chimpanzees  from the Coriell Cell Repository, as well as DNA from five additional unrelated bonobo individuals (identifiers available on request).Genomic DNA was extracted as described previously Genomic DNA was amplified using overlapping PCR primers and re-sequenced using PCR and internal primers. SNPs were detected using PolyPhred 4.0 2 tests of Hardy-Weinberg equilibrium (HWE) for each marker genotyped in cases and controls and found no extreme deviation. We also used Haploview to calculate and visualize r2 between each pair of markers , and to compare allele frequencies of cases and controls. Recombination rates were calculated using rhomap We used Haploview http://genapps.uchicago.edu/slider/index.html), to obtain several population statistics, including the average pair-wise difference, π We used an online program, SLIDER ; and a neutral model assuming a recent bottleneck, in which a population was reduced to 0.35 of its original size at T2 = 0.0375 in units of 4N0 generations, followed by a population expansion starting at T1 = 0.000375 and reaching its current size of 1.5 times its original size . Sample sets were always simulated with the same fixed number of segregating sites as observed in the region being simulated. A Bonferroni correction was applied whenever multiple windows were considered.We tested the significance of observing a given statistic by simulating sample sets under neutral models using MS, as described by Hudson Sample sets of natural selection models were simulated using SelSim Table S1FHIT intron 5SNPs identified within the sequenced region of (0.05 MB XLS)Click here for additional data file.Table S2Association tests for single SNPs(0.03 MB XLS)Click here for additional data file.Table S3Association tests for SNP combinations(0.05 MB XLS)Click here for additional data file.


CWD was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA). The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrPCWD levels by traditional assays  and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg(CerPrP) mice . These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids . The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role. Here we report the presence of infectious prions in the urine and saliva of deer with chronic wasting disease (CWD). Prion infectivity was detected by bioassay of concentrated, dialyzed urine and saliva in transgenic mice expressing the cervid PrP gene (Tg[CerPrP] mice). In addition, PrP Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids  and is the only prion disease affecting free-ranging, non-domestic animals. The origins of CWD are uncertain, but the disease has been present in wild cervid populations of northern Colorado and southern Wyoming for at least 40 years The mechanisms of CWD transmission are not well understood, although there is evidence that infection is transmitted horizontally and can be acquired from environmental sources Infectious CWD prions have been detected in saliva and blood, e.g. “prionsialia” and “prionemia,” suggesting a role for specific body fluids in transmission and dissemination Samples of urine and saliva were collected at terminal disease from five experimentally infected white-tailed deer. Deer in this cohort had been inoculated intracranially with brain, intravenously with blood, or orally with saliva from CWD-infected deer. All source deer were in the terminal stages of CWD infection, and demonstrated moderate to severe neurologic signs, paradoxical polyphagia with declining body condition, polydypsia and polyuria, and were confirmed CWD+ by western blot and immunohistochemistry, as previously described tg1536) mice were generated in the Telling laboratory at the University of Kentucky CWD at the completion of bioassay studies were rescreened to reconfirm the presence of cervid PrP gene. Animals were treated according to Colorado State University guidelines.Tg[CerPrP] line 1536  was used as the positive control material. A 1% w/v homogenate of brain from a single CWD-negative white-tail deer from outside the CWD-endemic zone was used as a negative control and was provided by David Osborne, University of Georgia. Urine and saliva samples were collected from five symptomatic whitetail deer as described above.Spiked positive and negative control samples consisted of 1% homogenates of positive or negative deer brain prepared using 10 ml of negative control saliva or urine as diluent. For the study groups, 10 mls of either urine or saliva were collected from each of five symptomatic deer, 50 ml total volume, and homogenized. All urine and saliva homogenates were then prepared for bioassay by lyophilization followed by resuspension in 0.1 volumes of phosphate-buffered saline (PBS) (e.g. a 10-fold concentration) and dialyzed against 2000 volumes of PBS to return the sample to isotonicity.Mice were anesthetized with ketamine and xylazine and inoculated intracerebrally into the left parietal lobe with 30 µl of inoculum. Incubation time was defined as the number of days from inoculation to the onset of clinical neurological signs consistent with a TSE Brain tissue was initially prepared as a 10% (w/v) suspension in homogenization buffer . Eleven µl of sample homogenate were mixed with 7 µl of sample buffer (0.1% [v/v] triton-X 100 and 4%(w/v) SDS in PBS) and digested with 2 µl proteinase-K at 500 µg/ml  for 20′ at 37°C followed by 10′ at 45°C. Seven µl of 4× running buffer were then added to the sample, followed by denaturation for 5′ at 95°C. Twenty µl of this preparation were run on a pre-cast 12% SDS-PAGE gel (Invitrogen) in a Bio-Rad electrophoresis apparatus for 2 hours at 110 mV. Samples were then transferred to a PVDF membrane for 1 hour at 110 mV in a Bio-Rad transfer apparatus. PVDF membranes were subsequently blocked for 1 hour in 5%(w/v) powdered milk in TBST, followed by application of the primary antibody, BAR224-HRP, diluted 1∶20,000 in 5% powdered milk in TBST, for 1 hour. Following washing, immunoreactivity was detected using an enhanced chemiluminescent detection system  in an LAS 3000™ imaging system. tg1536 neural tissues were fixed in formalin overnight, treated with 88% formic acid for one hour, washed in tap water for two hours, and then stored in 60% ethanol prior to paraffinization. Paraffin-embedded tissue sections (6 µm) were mounted onto positively charged glass slides, deparaffinized, and rehydrated through graded ethanol. Tissues were subjected to Heat Induced Epitope Retrieval (HIER) using an automated antigen-retrieval system (Retriever™) and a proprietary buffer solution . Tissues were then stained with an automated immunostainer, using PrP monoclonal antibody BAR-224 conjugated to HRP as the primary antibody . Detection was completed using HRP-mediated hydrogen peroxide immunostaining , with haematoxylin as a counterstain.Cervid renal tissues and CWD by both WB and IHC were further analyzed by PMCA. The amplification protocol, described below, was similar to those described by Soto and colleagues in vitro, was prepared in a room that had not previously been used for prion research as follows: naïve tg1536 mice were euthanized intraperitoneally with 15 mg of sodium pentobarbital and perfused with 25 ml of 5 mM EDTA in PBS via intracardiac catheterization. The calvarium was removed and the entire brain excised and placed on ice. Brain homogenate was then prepared at a 10% (w/v) solution in PMCA buffer  with the addition of Complete Protease Inhibitors  in a dounce homogenizer. Homogenates were then centrifuged for 1 minute at 2000 rpm to remove bulk brain material, and the supernatant frozen in single-experiment aliquots at −70°C in a “prion-free” room until use in PMCA. Experimental handling protocols were identical to protocols commonly used for PCR: NBH was added to a plate in a room not previously used for prion research and then transferred to a room used exclusively for prion research, where samples were added in a biosafety hood prior to sonication. Twenty-five µl of either source inocula or WB and IHC negative brain homogenate was added to 25 µl of NBH in individual wells of a 96 well PCR plate , placed in an ultrasonic processor  and incubated at 37°C. Samples were sonicated for 40 s at power setting 7.0, followed by 30 minutes of incubation. Ninety six cycles of sonication were preformed over 48 hours, with a 25 µl aliquot transferred to a fresh NBH preparation for serial amplification. Following three rounds of amplification, samples were evaluated by western blotting, as described above, for the presence of PrPCWD.Source inocula as well as mice negative for PrPCWD in saliva, we pooled and concentrated urine or saliva from five terminally infected CWD+ deer and inoculated two groups of tg1536 mice. Following inoculation, mice were monitored for clinical signs of prion infection and were euthanized when terminal disease was apparent. Central nervous system tissues were evaluated for PrPCWD using western blotting (WB), immunohistochemistry (IHC) and, when negative by conventional assays, protein misfolding cyclic amplification (PMCA).To investigate whether urine may play a role in natural CWD transmission, and to confirm the presence of PrPIn a group of 9 mice inoculated with lyophilized urine, 2 animals developed neurologic disease consistent with a TSE , includiCWD was absent by both WB and IHC in mice not displaying clinical disease, including those in negative control groups.Brains of all mice demonstrating terminal neurologic disease, including 2 of 9 inoculated with urine and 8 of 9 inoculated with saliva, had evidence of protease-resistant prion protein by both WB and IHC. PrPCWD plaques, with no apparent relationship between deposition pattern, lesion severity, and source inoculum  over three rounds of amplification. In our experience, three rounds of amplification permits an approximate 4000-fold increase in sensitivity as compared to traditional western blotting detection, while avoiding both cross-contamination and generation of any spontaneously formed protease-resistant PrP, thereby maintaining 100% specificity CWD was identified in lyophilized urine homogenate from CWD+ deer.  may also have played a role in this variation.While our findings point to urine as an additional vehicle for CWD transmission, only 2 of 9 inoculated CWD was repeatedly identified in test urine and spiked urine and saliva used as positive control, but was not detected in test saliva after three rounds of amplification. The reasons for our inability to identify PrPCWD in saliva – given the definitive bioassay findings – remain unknown, and we propose the presence of as-yet unidentified inhibitors such as mucin or salivary proteases which are thought to negatively affect other in vitro assays Using sPMCA, PrPCWD in urine and saliva calls for the identification of the pathological processes and cellular associations of the prion protein involved in shedding. Previous studies have related renal pathology to prionuria CWD has been identified by immunostaining in renal tissue of prion-infected deer The finding of PrPC-converting activity has been identified by sPMCA in the urine of scrapie-infected sheep, hamsters and mice Evidence for excretion and shedding of infectious prions is also accumulating in the scrapie system. PrPmay serve as a model for other protein-misfolding diseases. For example, feces, but not urine, from both mice and cheetahs affected with systemic amyloidosis A (SAA) was recently shown to induce SAA in a mouse model, although negative controls were not available in those studies As CWD transmission may model communicability of other TSE's, the transmissible nature of prion diseases In summary, we confirm prionsialia in CWD-affected deer by bioassay in cervidized mice and demonstrate for the first time infectious prions in the urine of these cervids by both bioassay and sPMCA. We are currently evaluating urine and saliva from individual animals in hopes of identifying predisposing factors, such as genotypic background and underlying pathology, which may contribute to prionuria and prionsialia. Concurrently, we have begun to explore the tissue origins and protease sensitivity of the infectious prions as well as the onset and duration of shedding in these bodily fluids.


We examined the influence of overweight and obesity on pulmonary function, exercise tolerance, quality of life and response to pulmonary rehabilitation in COPD.261 patients with COPD were divided into three groups: normal body mass index (BMI), overweight and obese. Baseline and post rehabilitation pulmonary function, 6-min walking test (6MWT), endurance time during a constant workrate exercise test (CET) and St. George's Respiratory Questionnaire (SGRQ) scores were compared between all three classes of BMI.At baseline, obese and overweight patients had less severe airflow obstruction compared to normal BMI patients. There was no baseline difference in CET performance or SGRQ scores across BMI classes and 6MWT was reduced in the presence of obesity (p < 0.01). Compared to baseline, post-rehabilitation 6MWT, CET performance and SGRQ scores improved significantly in each group (p < 0.01), but 6MWT was still significantly lower in the presence of obesity.Walking, but not cycling performance was worse in obese patients. This difference was maintained post rehabilitation despite significant improvements. Weight excess may counterbalance the effect of a better preserved respiratory function in the performance of daily activities such as walking. However, obesity and overweight did not influence the magnitude of improvement after pulmonary rehabilitation. Chronic obstructive pulmonary disease (COPD) is associated with dyspnea and exercise intolerance, two major impediments to quality of life. Although low body weight and muscThe effects of obesity in combination with COPD on exercise tolerance and dyspnea have received little attention. In one study, obese patients with COPD had a greater peak exercise capacity and reduced dyspnea perception at a standardized ventilation during incremental cycling exercise compared to their lean counterparts. These cPulmonary rehabilitation addresses the systemic consequences of COPD, beyond the impairment in lung function. As summarized in a recent meta-analysis pulmonarBased on the existing data suggesting that overweight and obesity may interact with COPD, our hypothesis was that increasing BMI in COPD would reduce exercise tolerance, increase exertional dyspnea and reduce functional status during walking but not cycling and compromise the response to pulmonary rehabilitation in patients with COPD. This study was thus undertaken to investigate the effects of overweight and obesity combined with COPD on 1) resting pulmonary function; 2) 6-min walking distance and endurance time during a constant workrate cycling exercise test (CET time) 3) health-related quality of life and 4) improvement of these parameters following pulmonary rehabilitation. To address these issues, we took advantage of a prospective cohort of patients with COPD entering pulmonary rehabilitation in Canada.1) <70% predicted and FEV1/forced vital capacity (FVC) <70%. Exclusion criteria were: participation to pulmonary rehabilitation in the preceding 12 months, living in a long term care facility and a diagnosis of asthma, congestive heart failure or dementia. All patients gave informed consent to participate in the study. Ethics committee from all 10 study sites approved this research project.Patients with COPD about to take part in pulmonary rehabilitation were recruited in 10 study centers across Canada. Inclusion criteria were: stable COPD, post-bronchodilator forced expiratory volume in one second (FEV-2), overweight (25 - 29.99 kg·m-2) and obese (>30 kg·m-2).The data for this study was collected as a part of a prospective observational study of pulmonary rehabilitation in Canada. The main objective of this cohort was to compare home versus hospital-based pulmonary rehabilitation. The secSpirometry and lung volumes were measured according to recommended procedures. ResultsCET was performed on a cycle ergometer with a workload set at 80% of peak work capacity achieved during incremental cycle ergometry. Patients were asked to cycle for as long as possible. The minThe 6MWT was administered in an enclosed corridor in accordance to the procedures recommended by the American Thoracic Society (ATS). The MCIHealth status was evaluated using French or English versions of the SGRQ. This diRatings of perceived exertion were reported by patients at the end of exercise tests (CET and 6MWT) on a 10-point Borg scale, for dyspnea and leg fatigue. The MCID for Borg scores was set at 1 unit.Rehabilitation program consisted of 6 to 12 weeks of tri-weekly 90-minute exercise sessions that integrated stationary bicycle endurance training, resistance exercises, and patient education, which has been described extensively elsewhere,30. The 1% predicted, FEV1/FVC ratio, functional residual capacity (FRC)% predicted, residual volume (RV)% predicted, total lung capacity (TLC), inspiratory capacity (IC)/TLC ratio). The normality assumption was verified using the Shapiro-Wilk's statistic while the homogeneity of variances was verified graphically with the residuals plot. Univariate and multivariate regression analyses were carried out to identify possible correlates of the response to pulmonary rehabilitation using age, sex, BMI, length of rehabilitation program and all the pulmonary function tests reported in table Results are reported as mean ± SD. A p value < 0.05 was considered as statistically significant. One-way ANOVA was used to compare baseline characteristics, except gender for which we used the Pearson's chi-square. Post rehabilitation data was compared to baseline using repeated measures two-way ANCOVAs  using baseline spirometric data and lung volumes as covariates , lost to follow-up (1.5%) and death (0.5%). We report here data for the 261 patients who have completed pulmonary rehabilitation and whose baseline characteristics are presented in table 1 (L and %), FEV1/FVC ratio and inspiratory capacity (IC) to TLC ratio (IC/TLC) were significantly lower in the normal BMI group than the other two groups (p < 0.05). Residual volume , total lung capacity , and BODE scores[Sixty percent of the study population was either obese or overweight, a proportion reflective of the Canadian population aged 40 years or older. FEV1  and 49 m shorter compared to overweight (p < 0.01) (table  and 49 mBorg dyspnea and leg fatigue scores after 6MWT were higher in the obese group at baseline (p < 0.05) Figure  and 3b. The duration and modality  of pulmonary rehabilitation programmes were similar across the three BMI categories table . Albeit MCID for the 6MWT, CET and SGRQ was reached by 19%, 46%, and 60% respectively in the entire study population. Table 2 (range: -6.26 to 3.94 kg/m2). There was a significant reduction in BODE scores in the 3 groups with rehabilitation with a post-rehabilitation BODE scores of 3.1 ± 1.8, 2.5 ± 1.6, 2.6 ± 1.6, for normal BMI, overweight and obese, respectively, p < 0.01 versus pre-rehabilitation.The changes in BMI after rehabilitation were small and not statistically significant averaging -0.03 ± 0.98 kg/m1, lower static lung volumes and higher peak incremental exercise capacity at baseline, ii) despite this, their CET time was not longer than that of patients with normal BMI, iii) obese patients had a reduced walking capacity compared to overweight and normal BMI patients, iv) BMI did not seem to affect SGRQ scores in the present population, finally v) overweight and obesity did not reduce the magnitude of improvement in exercise capacity and quality of life after pulmonary rehabilitation and BMI had no effect on outcomes on univariate or multivariate regression analyses taking account differences in baseline pulmonary function.This study reports on the impact of obesity and overweight in a large prospective cohort of patients with COPD participating in pulmonary rehabilitation. The results can be summarized as follow: i) obese and overweight patients had higher FEVIt is interesting to observe that, in this cohort, the proportion of overweight and obese patients was greater than normal BMI patients, a likely reflection of the obesity epidemic that afflicts industrialized countries ,33. Thes1 and FEV1/FVC ratio than their lean counterparts, an observation that was previously reported [1[2 was associated with a slower decline in FEV1. Another possibility for the differences in baseline lung function relates to the influence of obesity on ventilatory function. Decreased chest wall and lung compliance in obesity[Obese and overweight patients had higher FEVreported ,4,34,35.ported [1 as illusported [1, BMI >25n obesity would teAt baseline, resting hyperinflation was reduced and the IC/TLC ratio increased in the obese population. This finding is consistent with those of Ora et al.. One novWe found that obesity had a significant impact on walking capacity but not on the endurance time during cycling exercise. This is likely the result of the increase in energy expenditure associated with weight bearing exercise as shownAs indicated by similar SGRQ total scores, there was no difference in health status between groups, both at baseline and post-rehabilitation, even though patients in obese group had a more limited walking capacity. This could be related to the fact that obese patients might compensate by adapting their environment and diminishing the amount of activity they perform.2. One limitation of that study is that it did not include overweight patients. This appears to be relevant given that overweight is even more common than obesity. Our prospective study therefore adds to this information in showing that dyspnea, quality of life and exercise tolerance improve as much in the obese and overweight COPD patients as their normal BMI counterparts. BODE scores improved significantly in our population within each group to an extent that is consistent with the literature[In a retrospective analysis, Ramachandran and colleagues reportediterature.We did not observe significant reductions in BMI after pulmonary rehabilitation. Exercise in itself is usually not sufficient to adequately manage obesity and it sThis study provides some novel information. First, it is, to our knowledge, the only study looking prospectively at the effect of BMI on pulmonary rehabilitation outcomes. We also report on improvements in terms of MCID for 3 different outcomes, the 6 MWT, CET and SGRQ. Although it is generally suspected that walking capacity is compromised in obese COPD patients, this study is the first to systematically investigate the impact of obesity on specific exercise modalities. Finally, the number of patients enrolled in our trial also provides sufficient statistical power to make valid conclusions.The impact of comorbid conditions on rehabilitation outcomes is currently being investigated ,42. In gAlthough the improvement in 6MWT following pulmonary rehabilitation was less than typically reported,43, we f2), it would be important to see how such patients fare in the context of pulmonary rehabilitation. Secondly, it is well recognized that reduced fat free mass is associated with muscle weakness[1 was greater in patients normal BMI and the question may be raised as to whether this difference in disease stage between groups could explain our results. We do not believe that this is the case for the following reasons: i) the main analysis consisted in an analysis of covariance that took into account any differences in FEV1 and lung volumes at baseline; ii) since obese patients had milder airflow obstruction and resting hyperinflation, their performance during the 6MWT should have been better, not worse; and iii) the magnitude of improvement following pulmonary rehabilitation is independent from baseline lung function[Our study has potential limitations. First there were only 3% of morbidly obese patients (BMI >40 kg/m weakness, decreas weakness and poor weakness,48 in CO weakness. Thirdly function.2 than normal BMI patients, had more severe walking impairment. Pulmonary rehabilitation was still beneficial in improving several clinical outcomes despite the presence of excess weight. It would be of great interest to study the impact of weight reduction strategies in conjunction to exercise training in this specific patient population.Obesity and overweight are frequently associated with COPD. As the prevalence of excess weight is increasing, this association will be more and more frequent in clinical practice. Obese patients with COPD, despite having less severe airflow obstruction, resting hyperinflation and better peak VOL. Laviolette is recipient of a research training award from the Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec. J. Bourbeau is the recipient of a John R. & Clara Fraser Memorial Award from the faculty of Medicine, McGill University. This work was supported by the Respiratory Health Network of the FRSQ and by a grant from GlaxoSmithKline, Canada. F. Maltais holds a GSK/CIHR Research Chair on COPD at Université Laval.FS and LL carried out the analysis of the database and drafted the manuscript. SB and MJB recruited the patients, performed the exercise tests and the questionnaires and help revising the draft. JB and FM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/10/55/prepub


Marine reserves have been advocated worldwide as conservation and fishery management tools. It is argued that they can protect ecosystems and also benefit fisheries via density-dependent spillover of adults and enhanced larval dispersal into fishing areas. However, while evidence has shown that marine reserves can meet conservation targets, their effects on fisheries are less understood. In particular, the basic question of if and over what temporal and spatial scales reserves can benefit fished populations via larval dispersal remains unanswered. We tested predictions of a larval transport model for a marine reserve network in the Gulf of California, Mexico, via field oceanography and repeated density counts of recently settled juvenile commercial mollusks before and after reserve establishment. We show that local retention of larvae within a reserve network can take place with enhanced, but spatially-explicit, recruitment to local fisheries. Enhancement occurred rapidly (2 yrs), with up to a three-fold increase in density of juveniles found in fished areas at the downstream edge of the reserve network, but other fishing areas within the network were unaffected. These findings were consistent with our model predictions. Our findings underscore the potential benefits of protecting larval sources and show that enhancement in recruitment can be manifested rapidly. However, benefits can be markedly variable within a local seascape. Hence, effects of marine reserve networks, positive or negative, may be overlooked when only focusing on overall responses and not considering finer spatially-explicit responses within a reserve network and its adjacent fishing grounds. Our results therefore call for future research on marine reserves that addresses this variability in order to help frame appropriate scenarios for the spatial management scales of interest. As a response to declining fish stocks and threats to marine ecosystems, marine reserves (areas closed to fishing) have been widely advocated as conservation tools and means to achieving more sustainable use of marine resources Models that inform effects of marine reserves via enhanced larval export rely on (a) assumptions about recruitment limitations in unprotected populations, and (b) connectivity between reserves and non-reserve sites in situ field oceanography and monitoring of densities of individuals recruited since the establishment of a reserve network in Northwest Mexico. The Puerto Peñasco reserve network was established in summer 2002 primarily as a means to protect declining stocks of two commercial species of mollusks: rock scallop  and black murex snail (Hexaplex nigritus). The network includes an offshore reserve (San Jorge Island), and two coastal reserves (Las Conchas and Sandy)  . It coveTo assess the effects of the reserve network, we generated a larval export model and tested its predictions through observations of currents and bi-annual density counts of juvenile rock scallops and murex snails within reserves and fishing grounds prior and after reserve establishment (summer 2002-summer 2004). We developed a three-dimensional baroclinic numerical model that was based on the circulation pattern for the summer (the spawning season for both species), which is cyclonic overall In case (a) released particles showed a median south-northwest travel distance of 148 km in four weeks . Larvae Particles released in the area surrounding San Jorge Island (case b), the southern portion of the reserve network, showed a marked flow toward the coast and northwestern reserve sites . Direct 4, 41 = 2.53, P = 0.05; black murex: Pillai's Trace F4, 41 = 3.02, P = 0.02). Density of juvenile rock scallop had increased by up to 40.7% within coastal reserves and by 20.6% in fished areas . Changes were also evident for black murex, with more than a three-fold increase in density of juveniles within fished areas . The pattern of increase in juveniles, however, was variable in space, evident only for the northwestern portion of the network   were consistent with predictions of our larval transport model. Only two years after establishment of the reserves, both species showed evidence of changes in density as a function of time, protection from fishing (reserve effects) and site effects as a whole   and S2.Observed increase in recruitment was spatially-constricted to the northern portion of the reserve network and consistent with predictions of our larval transport model and field oceanographic observations. This recruitment pattern reflects effects of larval dispersal within the reserve network rather than other effects such as density-dependent adult spillover, a good year of high overall regional recruitment, or wider oceanographic processes. The restricted adult movement of both species  constrains our inference to larval dispersal rather than density-dependent adult spillover. Furthermore, high overall recruitment and regional oceanographic processes would likely have resulted in changes throughout the study area, not only in the northern portion of the reserve network.4, 45 = 4.46, P<0.01) and those of black murex remained relatively constant . However, although density of juveniles did not increase, even the lowest average numbers in five monitoring seasons were 80% higher than those of coastal reserves. Overall densities (adults and juveniles) were also six times higher than those of all coastal reserves and fishing sites combined, reaching up to 1.6/m2 and exceeding any others reported for the Gulf of California Surveys on San Jorge Island, as well as our modeling and current measurements  and 2B sOur findings provide needed insights for theory and empirical understanding of effects of marine reserves. First, we show evidence of rapid effects of reserve networks on adjacent fisheries via larval dispersal. Second, we also show that local retention of larvae within a network can take place with enhanced but spatially variable recruitment to local fisheries. Hence, effects should not be expected across an entire reserve network. Rather, they can be markedly variable within a local seascape.These results have important implications for management. Reserves reduce the total area available for fishing, likely causing an initial economic cost to fishers. Therefore, in situations where there is local support for reserve establishment, evidence of rapid positive reserve effects, as we have here shown, could play a crucial role in reinforcing cooperation among fishers for further compliance Finally, effects of marine reserves, positive or negative, may be overlooked when only focusing on overall responses and not considering finer spatially-explicit responses within a reserve network and its adjacent fishing grounds. Our results therefore call for future research on marine reserves that addresses this variability in order to help frame appropriate scenarios for the spatial management scales of interest. Not doing so could lead to false expectations among stakeholders.We released 2000 passive particles in two areas (between 0–60 m deep): San Jorge Island, and the nearest substantial rocky reef south of the marine reserve network. Particles were tracked for four weeks and the temporal scales resolved by the model (due to forcing) are tidal and seasonal. The model is described in detail for the Gulf of California by Marinone www.pacificgyre.com/Lagrangian.aspx) Microstar surface drifters which drogues were centered at 1 m below the sea surface. These drifters provided GPS positions every 10 minutes. One PacificGyre ARGOS SVP (Surface Velocity Program) drifter was used to observe the current field offshore (west) of San Jorge Island; it was drogued with a 4.8 m- tall Holey Sock centered at 15 m depth.We used two bottom-mounted acoustic current profilers moored at the sites marked ADCP and ADP in Site ADCP , equippeSpondylus calcifer) and black murex snail (Hexaplex nigritus) in reserve and fishing sites for two consecutive years beginning in May 2002, one month preceding reserve establishment. The region monitored encompassed the reefs of San Jorge Island and those found near the fishing town of Puerto Peñasco (within 3 km from highest tide line) in the eastern part of the northern Gulf of California, Mexico. This region extends from 31,22,18.1 N; 113,39,09.4 W to 31,15,03.8 N; 113,20,48.1 W.We estimated changes in density of rock scallop .We subdivided the region into 5 sampling areas: a) two coastal reserves (replicates), Las Conchas and Sandy; b) two coastal fishing areas (“controls”), Los Tanques and La Cholla; c) one offshore island reserve, San Jorge Island . We pairTo reduce heterogeneity associated with depth, we restricted all sampling to depths ranging from 40–65 ft. This also reduced health risks associated with diving and facilitated overall monitoring as we were able to remain underwater for longer periods of time. In all cases except San Jorge Island, this depth as well as the established constrained distance from the tide line covers the entire extension of the reefs. We restricted sampling in San Jorge Island to the reefs found on the eastern part of the island, as these are shallower and more similar to those found on the mainland coast.We selected plots from within these five areas through simple random sampling. In the event that a specific plot selected happened to fall where at least 50% of sand was present, that plot was replaced by another one by swimming underwater in a straight line along the reef until reaching sufficient (>50%) rocky substrate. Once selected, all plots were permanently marked underwater.Plots were 10×10 m subdivided into 16 quadrats of 2.5×2.5 m for ease of observation. Testing other sampling methods such as the use of 5×50 m or 5×30 m transects, distance sampling or others typically used for sessile organisms did not prove adequate for this region given the highly variable visibility of the region, the strong currents, and the overall patchiness of the reefs . We counted all individuals visible within each 2.5×2.5 m quadrat (subplot). For rock scallop, we estimated size of each individual to fall within one of three categories: small juveniles (up to 5 cm of height), medium-sized juveniles and young adults (>5 and <10 cm of height), and large adults (>10 cm of height). For black murex, sizes fell into two categories: juveniles and reproductively mature adults.To reduce variation in detectability, the same person counted organisms on each sampling occasion and in the same designated plots while another diver assisted setting and maintaining the plot lines in place. To support this work, ten commercial divers with extensive experience searching for benthic mollusks (>5 years) were trained to participate in the monitoring process. We calculated variations in the detection of monitored species  and incorporated this variation to calculate statistical power of our sampling design (see below).We established a total of 58 sampling plots: San Jorge = 10, Las Conchas = 10, Los Tanques = 10, Sandy = 10, La Cholla = 18. Power analyses from baseline data on density of rock scallops found on these 58 plots gave us a high probability of detecting at least a 10% increase in their density . Given that rock scallops are harder to detect than black murex (when closed they resemble rocks), we assume an even higher statistical power for detection of changes in population densities of black murex. We monitored each plot twice every year (Spring and Summer) for two consecutive years . These months provide some of the best visibility underwater and are also usually devoid of algae beds covering the rocky reefs, which reduce detectability of species monitored.This is a longitudinal study with a repeated measures research design and various levels of analysis. We first generated profile plots of baseline data and applied square root transformations to improve homogeneity of variance. We then addressed “between subject” and “within subject” variability of baseline data graphically, determined the coefficient of variation, and tested for independence of plots and sampling sites in order to avoid pseudo-replication. We used multivariate analyses of variance (MANOVA) and relied on Multivariate Pillai's Trace P values to help assess time, protection from fishing, and treatment effects independently as well as combined factors. Univariate estimates were also obtained and analyzed to further understand observed patterns see  and S2.Table S1Univariate and multivariate tests for the analysis of temporal changes in density of juvenile rock scallops found within monitored reserve and fishing areas.(0.05 MB DOC)Click here for additional data file.Table S2Univariate and multivariate tests for the analysis of temporal changes in density of juvenile black murex found within monitored reserve and fishing areas.(0.05 MB DOC)Click here for additional data file.Figure S1Progressive vector diagrams (PVD) calculated from the ADCP velocity data at 3.6, 9.6, 15.6 and 20.6 m above the bottom  for the period July 6 to August 18 2006. For clarity, successive diagrams are shifted to the left by 5 km.(0.13 MB TIF)Click here for additional data file.Figure S2Tracks and velocities of the Microstar surface drifters for the period 19:00 (UT) July 12 to 00:10 July 16 2006. Four of the five drifters shown were redeployed during the period; the exception is the green trace, which is used in (0.02 MB TIF)Click here for additional data file.


It has been demonstrated that brief episodes of sublethal ischemia-reperfusion, so-called ischemic preconditioning, provide powerful tissue protection in different tissues such as heart, brain, skeletal muscle, lung, liver, intestine, kidney, retina, and endothelial cells. Although a recent study has claimed that there are no protective effects of ischemic preconditioning in rat testis, the protective effects of ischemic preconditioning on testicular tissue have not been investigated adequately. The present study was thus planned to investigate whether ischemic preconditioning has a protective effect on testicular tissue.Rats were divided into seven groups that each contained seven rats. In group 1 (control group), only unilateral testicular ischemia was performed by creating a testicular torsion by a 720 degree clockwise rotation for 180 min. In group 2, group 3, group 4, group 5, group 6, and group 7, unilateral testicular ischemia was performed for 180 min following different periods of ischemic preconditioning. The ischemic preconditioning periods were as follows: 10 minutes of ischemia with 10 minutes of reperfusion in group 2; 20 minutes of ischemia with 10 minutes of reperfusion in group 3; 30 minutes of ischemia with 10 minutes of reperfusion in group 4; multiple preconditioning periods were used  in group 5; multiple preconditioning periods were used  in group 6; and, multiple preconditioning periods were used  in group 7. After the ischemic protocols were carried out, animals were sacrificed by cervical dislocation and testicular tissue samples were taken for biochemical measurements  and histological examination.Although decreased tissue malondialdehyde levels were detected in the groups of 2, 3, 4, and 5 compared to group 1, significant decreases were observed in only group 2 and group 5 (p < .05). Nitric oxide levels were numerically decreased in all groups compared to the control group but was statistically significant only in group 5 (p < .05). Histopathological examination demonstrated that all groups subjected to ischemic preconditioning had less tissue damage than group 1 (p < .05).These results suggest that ischemic preconditioning provides tissue protection in testicular tissue. Testicular torsion is an acute clinical condition in humans that must be treated promptly to avoid loss of the ipsilateral testis. Long-term follow-up has shown subsequent late atrophy of the affected testis. Testicular damage after spermatic cord torsion is related to the duration of ischemia and to the severity of the torsion ,2. The mIt is well-known from studies of different tissues such as heart, brain, skeletal muscle, lung, liver, intestine, kidney, retina and endothelial cells -9 that bA recent study  has claiThe present study was planned to investigate whether ischemic preconditioning has a protective effect on subsequent testicular ischemic tissue injury by using biochemical parameters and histolological examination.ad libitum food and water at 20–24°C. On the day of the experiment, animals were anesthetized with ketamine (50 mg/kg) and the surgical operation described below was performed. Our experimental research on animals followed internationally recognized guidelines. Ethic committee of our university gave approval to the study, with a reference number of 2007/01.Prepubertal, male, albino rats (180–200 g) were acquired from university vivarium sources and maintained on a 12 h light/12 h dark cycle with After the induction of anesthesia, a left scrotal incision was made. The tunica vaginalis was opened, and the testicle was delivered to the surgical field. The testicle was rotated 720° in a clockwise direction and maintained in this torsion position by fixing the testicle to the scrotum with a 5–0 silk suture. At the end of testicular ischemia period the testicle was released for the perform reperfusion period.By the same incision and surgical procedure described above, the main ischemia period lasting 180 min was performed just following ischemic preconditioning periods. After all procedures were completed, the incision was closed and the animal was kept on a heating pad to maintain its body temperature. At the end of the procedures, the testes were removed and divided longitudinally into two halves for biochemical measurements , nitric oxide (NO)) and histological examinations. Then all animals were sacrificed by cervical dislocation.Animals were divided into seven groups each containing seven rats. Each group underwent the procedure described below.Group 1: No preconditioning + 180 min ischemia (control group)Group 2: 10 min transient ischemia + 10 min reperfusion + 180 min ischemiaGroup 3: 20 min transient ischemia + 10 min reperfusion + 180 min ischemiaGroup 4: 30 min transient ischemia + 10 min reperfusion + 180 min ischemiaGroup 5: Three consecutive "10 min transient ischemia + 10 min reperfusion" cycles + 180 min ischemiaGroup 6: Three consecutive ''5, 10, and 20 min, respectively, transient ischemia + 10 min reperfusion" cycles + 180 min ischemiaGroup 7: Three consecutive ''10, 20, and 30 min, respectively, transient ischemia + 10 min reperfusion" cycles + 180 min ischemiaThe testes were homogenized separately in phosphate buffer and malondialdehyde (MDA) and nitric oxide (NO) were measured in the specimens. MDA detection was based on the measurements of the absorbance of thiobarbituric acid-malondialdehyde . NO was The testicles immersed in Bouin's fixative were dehydrated in alcohol and embedded in paraffin blocks, sectioned at 5 μm, and stained with hematoxylin and eosin. The light microscope histological examination of the slides prepared from middle portion of each hemi-testis was done by a pathologist in a blinded fashion. The 4-level grading scale of Cosentino et al. , was useμmol/mg protein, respectively. The results were given as sum or mean ± SD. Statistical analysis was done by Kruskall Wallis analysis of variance, Mann-Whitney U test and Fischer's exact Chi-square test where appropriate. The level of statistical significance was p < 0.05.MDA and NO levels were expressed as nmol/mg protein and No animals died because of the procedure.The results of testicular MDA levels in all groups are shown in table NO levels were numerically decreased in all groups compared to the control group but this was statistically significant in only group 5 (p < .05) Table .The findings of the histopathologic evaluation for each group are shown in Fig. The protective effects of ischemic preconditioning, a phenomenon by which a traumatic or stressful stimulus confers protection against subsequent injury, have been well documented in many organs other than testis such as heart, brain, skeletal muscle, lung, liver, intestine, kidney, retina and endothelial cells -9. HowevIt has been shown that ischemic preconditioning is a biphasic phenomenon, with an early and late phase of protection. There is increasing evidence that the ischemic stres hormones adenosine and norepinephrine are involved in the early phase of preconditioning, acting through the activation of protein kinase C -18. CellLipid peroxidation stimulated by free radical formation is one of the most important mechanisms involved in cellular damage and death. Free oxygen radicals induce peroxidation of unsaturated fatty acids of the cell membrane, if severe enough, destroying membrane integrity. MDA level measurements are widely used as an indicator of lipid peroxidation -24. SeveOzokutan et al suggested that NO plays an important role in damaging the testis with ischemia-reperfusion . AlthougThere was no certain correlation between biochemical test results and histological observations in our study. In contrast to biochemical test results, histological examination of the testes showed significant improvement in all experimental groups subjected to ischemic preconditioning compared to controls. While grade 3 injury was not observed in any preconditioned group, it was seen in group 1. All our histopathological findings support that ischemic preconditioning has a protective effect on testicular tissue. The reason why biochemical test results could not reach the level of statistical significance in all groups unlike histological observations might be related to the small number of rats used in each group. Therefore, further studies with an increased number of rats and different preconditioning protocols are needed to confirm our suggestion that ischemic preconditioning has a protective effect on testicular ischemic tissue injury.We conclude that our results suggest that ischemic preconditioning performed just before main ischemia provides tissue protection in testicular tissue.malondialdehyde (MDA), nitric oxide (NO)The author(s) declare that they have no competing interests.TS and UKO participated in the design of the study, carried out animal model and drafted the manuscript. HC carried out the pathological examination and participated in the sequence alignment. FIT and SSI carried out the biochemical examination. All authors read and approved the final manuscript.


This study examined the capabilities of an ultraendurance athlete to self-regulate their diet during an attempt on the record for the longest period of stationary cycling. The attempt required the athlete to complete at least 20 km/hr, with a 15 minute break allowed every eight hours. Laboratory tests determined a heart rate-oxygen consumption regression equation enabling calculation of energy expenditure from heart rate during the attempt. Energy intake was determined by a non-weighed dietary record collected at the time of consumption. The athlete completed 46.7 hours, covering 1126 km, at a speed of 24 ± 1.6 km/hr. He expended 14486 kcal and consumed 11098 kcal resulting in an energy deficit  and a weight loss (-0.55 kg). The carbohydrate (42 ± 32 g/hr), water (422 ± 441 ml/hr), and sodium (306 ± 465 mg/hr) intake were all below current recommendations. The athlete was unable to self-regulate his diet or exercise intensity to prevent a negative energy balance. Ultraendurance athletes' are challenged in attempting to balance the high energy requirements of their sport with the high energy consumption required to compete successfully. When the energy intake exceeds 4500 kcal/d, the athlete will commonly have to ingest and digest food while actually competing . In ordeFew athletes have the discipline required to successfully self regulate their energy intake during ultraendurance exercise, but one previous casestudy occurred during the 1000 km running race from Sydney to Melbourne in Australia. Greek ultra marathoner, Yiannis Kouros', self regulated diet across the five days was almost entirely comprised of carbohydrate 95%). Energy dense Greek sweets and honey soaked biscuits were snacked on every 30 minutes, enabling Kouros to consume on average an amazing 11,074 kcal/d [%. EnergyThe purpose of this case study was to assess the capabilities of an experienced ultraendurance athlete to appropriately self regulate his energy intake during an attempt on the world record for the longest period of stationary cycling.The subject was a 35 year old male amateur cyclist  with previous experience in ultraendurance events. The subject gave informed consent to collect data during the record attempt.The subject completed an intense program of endurance training spanning 6 months. He was provided with guidelines about the amounts and types of foods/fluids to consume throughout the world record attempt with specific emphasis on carbohydrate, sodium and fluid amounts. The subject was then responsible for planning and preparing the appropriate items for the ride. He began the attempt with access to ample sports drinks, protein bars and energy gels (Endura™).2) in order to estimate energy expenditure during the record attempt [2max), ventilatory threshold, and peak power.The athlete completed two cycle ergometer protocols a week prior to the record attempt. The first comprised four submaximal workloads, commencing at 0 W and increasing by 25 W every 2.5 minutes. This test was conducted to develop a relationship between heart rate and oxygen uptake (VO attempt . The sec2), minute ventilation (VE), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were calculated. Before testing, the gas analysers were calibrated with a known gas mixture and room air. Heart rate was measured by telemetry  and recorded every 15 seconds.All testing was undertaken on a cycle ergometer  with ventilation and expired gas collected and analysed every 15 seconds using a metabolic cart . Oxygen consumption . The distance covered and the speed maintained was calculated by a calibrated bike computer . Throughout the record attempt, the athlete's heart rate  and blood pressure was monitored, and the self regulated fluid and food intake recorded. Heart rate was recorded every minute and energy expenditure calculated from the equation obtained from the laboratory submaximal workload test. Blood pressure was recorded every hour. Body mass was measured every eight hours and urine output was weighed throughout. The total energy, carbohydrate, protein, fat and sodium consumed by the athlete was analysed using a dietary software program .Throughout the attempt the athlete was provided with verbal encouragement and physical assistance to continue eating and drinking and encouragement to continue cycling.2 max from the incremental test was 60.3 ml/kg/min and he achieved a peak power of 315 W. The relationship between heart rate and VO2 determined with the multiple submaximal workloads was represented by the following equation (VO2 (ml/min) = 26.826 * heart rate – 1434.6, r2 = 0.9974). The energy expenditure measured in kcal was calculated by multiplying the estimated VO2 (in L/min) by 4.948. This conversion factor was calculated from the average non-protein respiratory quotient measured during the multiple submaximal workloads [The subject's VOThe athlete completed 46 hours 44 minutes and 20 seconds of continuous stationary cycling during the record attempt. The ambient temperature ranged from 16.9 to 27.9°C and the relative humidity from 44 to 65%. The total distance covered was 1126 km at an average speed of 24 ± 1.6 km/hr and heart rate of 91 ± 15 bpm , less than the 17430 kcal (72.9 MJ) reported during the 2004 XXAlps ultraendurance cycle race  or the 2Based on an estimated basal metabolic rate (BMR) for a 70 kg male of 1640 kcal/day , the firThe athlete averaged a fluid intake of 422 ml/h, nearly identical to the 417 ml/h recorded during a 24 hour road race in Britain , but subThe total volume of fluid consumed, plus the water contained within food, was 13 litres greater than the volume of urine produced. This positive balance did not result in an increase in unexplained body mass as the intensity of exercise maintained has been shown to produce sweat at a rate of 233 ml/hour , and insThe minimal sweat rate maintained throughout the attempt would also have enabled the athlete to reabsorb, within the sweat duct, the majority of the sodium secreted by the gland. In more humid and hot conditions, and in unacclimatised individuals, the increased risk of exercise associated hyponatremia  resultinThe current recommendations for CHO supplementation during ultraendurance exercise range from 30 – 90 g/h ,27,29. TDespite being provided with appropriate guidelines, the athlete did not pre-plan or prepare his energy intake for the record attempt. He supplemented his intake of sports drinks, protein bars, and energy gels (Endura™) with impulse selections from nearby commercial food outlets. These impulse selections included fried rice, McDonalds™, and bacon and eggs. In any ultraendurance event, requiring the athlete to eat while competing, it would be strongly recommended that the athlete commence the event with all meals and fluids planned and prepared according to nutritional recommendations, likes and dislikes, and ability to store items to be consumed. Alternatives also need to be prepared for when food and flavour fatigue occurs, as is often the case during ultraendurance events . Poor planning and preparation, as well as other common factors such as gastrointestinal discomfort  and lossIt is unknown from the current data exactly why the athlete was unable to complete the record attempt. An energy deficit situation, and therefore loss of body mass, is common in ultraendurance events ,20,27,31The initial eight hour period particularly highlights the inadequacies of self-regulation with a lower than required total energy, and particularly CHO, intake, and an elevated energy expenditure. Overcoming such a large initial energy deficit, while the athlete is still competing, is difficult due to the limited digestive capacity of the gastrointestinal tract . The inaIBS conducted the laboratory testing, physiological data analysis and drafted the manuscript.KLS conducted the nutritional data analysis and contributed to the manuscript.All authors read and approved the final manuscript.


Many studies have evaluated chemical, heavy metal, and other abiotic substances present in cigarettes and their roles in the development of lung cancer and other diseases, yet no studies have comprehensively evaluated bacterial diversity of cigarettes and the possible impacts of these microbes on respiratory illnesses in smokers and exposed nonsmokers.The goal of this study was to explore the bacterial metagenomes of commercially available cigarettes.A 16S rRNA-based taxonomic microarray and cloning and sequencing were used to evaluate total bacterial diversity of four brands of cigarettes. Normalized microarray data were compared using principal component analysis and hierarchical cluster analysis to evaluate potential differences in microbial diversity across cigarette brands.Acinetobacter, Bacillus, Burkholderia, Clostridium, Klebsiella, Pseudomonas aeruginosa, and Serratia in ≥ 90% of all cigarette samples. Other pathogenic bacteria detected included Campylobacter, Enterococcus, Proteus, and Staphylococcus. No significant variability in bacterial diversity was observed across the four different cigarette brands.Fifteen different classes of bacteria and a broad range of potentially pathogenic organisms were detected in all cigarette samples. Most notably, we detected Previous studies have shown that smoking is associated with colonization by pathogenic bacteria and an increased risk of lung infections. However, this is the first study to show that cigarettes themselves could be the direct source of exposure to a wide array of potentially pathogenic microbes among smokers and other people exposed to secondhand smoke. The overall public health implications of these findings are unclear at this time, and future studies are necessary to determine whether bacteria in cigarettes could play important roles in the development of both infectious and chronic respiratory diseases. Cigarette smoking adversely impacts almost every organ system of the human body [International Agency for Research in Cancer ]. It is Bacillus spp.  were purchased from five randomly selected tobacco stores in Lyon, France. Four cigarette brands were included: Marlboro Red , Camel , Kool Filter Kings , and Lucky Strike Original Red . These brands are among the most commonly smoked brands of cigarettes in Westernized countries and represent three major tobacco companies. All of the cigarettes were made in the European Union.In January 2007, cigarettes . Resulting DNA was purified using the NucleoSpin Extract 2 Kit .16S rRNA genes present in purified metagenomic DNA were amplified using universal primers pA and pH′  to obtain = 288) were isolated and analyzed to check for positive clones and to choose clones with different restriction profiles. Each colony was inoculated in 100 μL LB containing 100 μg/mL ampicillin and grown overnight at 37°C. One microliter of each overnight culture was used for a subsequent PCR reaction. Amplification was performed with the universal M13 reverse and M13 forward primer pairs using a Platinum PCR SuperMix 96 (Invitrogen).Purified 16S amplicons from one of the Marlboro Red metagenomic samples were cloned for sequencing using a TOPO TA cloning kit  with a pCR4 TOPO vector and One Shot TOP10 electrocompetent cells (Invitrogen). Resulting colonies  were selected and sequenced with the M13 primers by the ABI3730x/DNA Analyzer system . The DNA sequences were analyzed by Lasergene 7.2 software . For identification of closest relatives, the consensus sequences were compared with 16S rRNA gene sequences in GenBank databases using the NCBI Blast search tool .Each amplification product was digested by 4. The fragmentation reactions were incubated for 30 min at 60°C, and 1.43 μL 500 mM EDTA was added to stop each reaction.Purified 16S amplicons from all samples were reverse-transcribed and labeled with UTP-Cy3. The reaction mixture yielded a final solution of 1X T7 RNA buffer; 10 mM DTT; 0.5 mM concentrations of dATP, dCTP, dGTP, and dUTP; 20 units of RNasin (Invitrogen), 1 μL T7 RNA polymerase (Invitrogen), 0.25 mM UTP-Cy3, and 400 ng purified PCR amplicons. Each reaction mixture was incubated in the dark at 37°C for 4 hr. Resulting RNA was purified using the RNeasy Mini Kit . Purified RNA was then quantified, and frequency of incorporation (FOI) of Cy3 was calculated: FOI of Cy3 = (OD550/0.15)*(324)/OD260*40). Purified RNA was then fragmented in a reaction mixture yielding a final solution of 25 μM Tris Cl and 10 mM ZnSOThe 16S rRNA-based taxonomic microarray slides  and probes (positive controls and targets)  were custom designed and synthesized as previously described , 2006b. Labeled and fragmented RNA was prepared in a hybridization mixture yielding a final solution of 0.1% SDS, 1X Denhardt’s solution , 6X SSC, and 300 ng RNA. Slides were prehybridized, hybridized, and washed in an A-Hyb Hybridization Station . Prehybridization was performed at 57°C for 10 min, hybridization was performed at 57°C for 240 min, and a four-step wash cycle was performed at 20°C for 4 min. Slides were dried by centrifugation in a microcentrifuge for 2 min at top speed. Scanning was performed using a GenePix Personal 4100A scanner , and data were analyzed using GenePix Pro 6 Microarray Image Analysis (Molecular Devices Corporation).http://www.r-project.org/). Data filtration and normalization were performed as described by http://pbil.univ-lyon1.fr/ADE-4/).Filtration, normalization, and data analysis were performed using the R Project for Statistical Computing . As expected, Nicotiana tabacum 16S chloroplast rRNA genes also were detected in every sample.All cigarette samples were positive for 16S bacterial rRNA  and withAmaracoccus, Legionellales, Methylobacterium, Nostoc, Paracoccus, Pseudomonas chlororaphis, and Pseudomonas cichorii, to name a few. A broad range of gram-positive and gram-negative bacterial genera and species medically important to humans and/or potential human pathogens were also detected in the cigarette samples  may be the cigarettes themselves. However, it is important to note that some bacterial organisms recovered from smokers commonly colonize the nasopharynx, and increased susceptibility to colonization, as well as the increased risk of lung infections associated with smoking, could be due to immunosuppressive activities by chemicals and particulate matter present in cigarette smoke. Long-term smokers may have less-effective mucociliary clearance mechanisms , chronicBacillus subtilis, which was detected in 90% of our cigarette samples, is a possible degrader of pyrene and benzo[a]pyrene, major chemical carcinogens found in cigarettes (B. subtilis are more toxic than their parent compounds (Bacillus and others that may be deposited in the lungs as a result of cigarette smoking—could alter cigarette-associated chemicals into greater or lesser carcinogens.Beyond the issue of bacterial colonization and acute respiratory illnesses, another avenue to explore is whether bacteria in cigarettes could also contribute to human carcinogenesis. The role of microbes in the development of some human cancers is being explored . Althouggarettes . Althougompounds , it is wOne limitation to this study is that the microarray platform used was primarily developed to explore bacterial diversity in environmental samples. Therefore, the probes present on the microarray slides more heavily represented soil and water microorganisms versus potential human pathogens, such as those included in the Enterobacteriaceae family. The implication of this limitation is that there could be even more potential bacterial pathogens present in the cigarette samples compared with the number that we detected with our current microarray system.Another limitation of this study is that we analyzed cigarette tobacco alone and did not include analyses of cigarette smoke. Future studies are necessary to fully characterize the microbiology of cigarette smoke. Beyond the smoke, additional studies will be necessary to determine whether organisms present in the cigarettes and smoke can colonize smokers and other exposed individuals and ultimately cause human illnesses.Nearly every scientific paper concerning the acute human health effects associated with cigarette smoking mentions that smoking is associated with increases in bacterial infections among smokers. However, to our knowledge no one has comprehensively evaluated whether the actual cigarettes themselves may be the source of exposure to bacterial organisms that subsequently cause infection and other potential illnesses. Here we describe that multiple brands of cigarettes are rich in bacterial diversity, harboring a broad array of microorganisms from environmental bacteria and commensals to potential human pathogens. The overall public health implications of these findings are unclear at this time, and future studies are necessary to determine whether microbes originating from cigarettes could play a considerable role in the development of both infectious and chronic diseases among smokers and other exposed populations.


AD and CD measures and performed a principal component analysis, as well as a clustering analysis by structure. Results showed that we successfully recovered the genetic structure of analyzed populations formed by historical migrations. Aggregation patterns of these populations accord well with their linguistic affiliations, suggesting that deciphering of genetic relationships does in fact offer clues for study of ethnic differentiation.Population migrations in Southwest and South China have played an important role in the formation of East Asian populations and led to a high degree of cultural diversity among ethnic minorities living in these areas. To explore the genetic relationships of these ethnic minorities, we systematically surveyed the variation of 10 autosomal STR markers of 1,538 individuals from 30 populations of 25 ethnic minorities, of which the majority were chosen from Southwest China, especially Yunnan Province. With genotyped data of the markers, we constructed phylogenies of these populations with both  Other than the majority Han Chinese, there are 55 ethnic minorities living in China, composing 9.44% of the Chinese national population . Most of these minorities inhabit peripheral regions of China, especially border provinces such as Yunnan, Guangxi, and Tibet, where special landforms like the Hengduan Mountains vastly influenced their lives and history Recent researches on East Asian populations have benefited from STR markers In addition, close relationship between language and nationality has long been observed Microsatellite markers have been broadly used for analyzing relationships between human populations In this study, we surveyed the variation of 10 STR markers dispersed on Chromosome 3 for a total of 1,538 individuals in 30 populations, most of which come from southwestern provinces of China. Through analysis of the variations, we assessed the genetic diversity and interrelationships of these populations and tried to evaluate the reciprocal influences of languages and genetic structure among populations mostly living in contiguous regions.30 populations of 25 ethnic minorities from 9 provinces of China were surveyed in this study . Among tAll 1,538 DNA samples used in this study were obtained from 30 ethnic panels of immortalized cell lines created by the Chinese Human Genome Diversity Project (CHGDP) Ten microsatellite markers on Chromosome 3 included in ABI Prism Linkage Mapping Set (v2.5) were selected to be genotyped. They are D3S1297, D3S1304, D3S1263, D3S1266, D3S1285, D3S1278, D3S1292, D3S1279, D3S1614, and D3S1580 See  document2, 1 µL primer, 1520 ng DNA, and 0.25U Taq DNA polymerase  was adopted to perform polymerase chain reactions (PCR). Thermal cycling on the GeneAmp PCR System 9700 (Applied Biosystems) included a 5 min denaturation at 94°C, followed by 10 cycles of 30 s at 94°C, 30 s annealing at 55°C, and 30 s extension at 72°C as well as another 25 modified cycles with a denaturation temperature of 89°C, and a final extension at 72°C for 10 min.Dye-labelled primers from the aforementioned mapping set were 1∶10 diluted to 1pM for subsequent amplification reactions. After optimization, a 5-µL final volume with 0.5 µL PCR buffer , 1.25nmole dNTPs, 12.5nmole MgClElectrophoresis of amplified products was conducted on an ABI 3730 XL DNA Analyzer (Applied Biosystems). For each marker, a size-call panel was trained using the software GeneMarker (SoftGenetics), with data from a random successfully typed 96-well plate. Fragment sizes of each reaction were automatically determined with established panels and then manually checked and adjusted. Output data were then readied for further analysis. Unsuccessful reactions were retried until either success or three failures.EH) were calculated using Arlequin version 3.11 EH to present because it is considered a superior estimator of populational genetic variability Allele frequencies and expected heterozygosities  and the stepwise mutation model (SMM), with or without a bottleneck effect Previous comparison of different distance measures had shown that Nei et al's AD distances averaged over all loci were deployed to reconstruct a neighbor-joining (N-J) tree CD distance matrices by MICROSAT To investigate the genetic relationships of populations in a phylogenetic way, Z-score process is similar to the one advocated by Cavalli-Sforza A principal component analysis (PCA) based on allele frequencies was performed in MATLAB 2007a (MathWorks Inc.) to explore the extent of correlation between genetic relationships and geographical distribution of the populations. Ahead of the analysis, frequency data were normalized for each allele by dividing the offset from mean with standard deviation. This structure program K for the studied populations; clustering results were then visualized by the program CLUMPP K = 2 to 7 were carried out with both a burn-in and a run length of 50 000. The most likely K was then determined by comparing posterior probabilities of data under different K settings.In addition, the AD), geographic and linguistic distance matrices and assessed for significance by 2-way and 3-way Mantel tests vegan. In addition, contributions to correlation by different linguistic groups were assessed by running Mantel tests on the data that excluded relevant populations. To assess the correlation between the distributions of populations on the PCA plot and on the Earth surface, we also ran a Mantel test for the PCA distances against the geographical distance matrix.Lastly, the correlations between genetic relationship and linguistic affiliations as well as geographical distribution were assessed in a quantitative way. Linguistic distances of populations were determined according to the ‘least controversial phylogeny’ proposed by Sagart on the basis of literature As previously mentioned, some PCR reactions might fail 3 times and introduce missing data. Over 90% of loci for all populations have a success rate above 80%, and most of them are greater than 90% . HoweverEH) for the loci of all populations (EH of 0.883 (±0.021). D3S1266 is the least polymorphic, with a mean EH of 0.702 (±0.040). The 10th and 90th percentiles of EH for all markers are 0.677 and 0.886, respectively, with the highest being 0.913 (D3S1263 of Kirgiz) and the lowest being 0.598 (D3S1266 of Dai). Mean heterozygosities of D3S1297, D3S1278, D3S1279, and D3S1614 are much lower than expected when compared to that of CEPH individuals contained in the panel guide of ABI Prism Linkage Mapping Set v2.5. Such differences are not unexpected; though, EH values indicate that selected markers are highly diversified.To examine the diversity of selected markers, we calculated allele frequencies and expected heterozygosities (ulations . D3S1263Exact tests of Hardy-Weinberg Equilibrium (HWE) were applied to all markers to evaluate the extent of inbreeding within each population, given that sampling of ethnic individuals was restricted to typical habitats of corresponding populations and thus unlikely to introduce complex inner-population stratifications. Test results are summarized in P values of each AD matrix test with the full-loci dataset are shown in P values are all less than 0.05, suggesting distance measurements by different markers are overall positively correlated. For the 4 coefficients that are below 0.20, P values of 3 pairs with the marker D3S1266 are around 0.10, and that for D3S1263 with D3S1266 is as high as 0.215. Altogether, these results indicate that the performance of different marker distances is well in consistency. Therefore, it is reasonable to combine all the data for further distance-based analysis despite the existence of slight departures from HWE in our data.As a few marker-population pairs departed from HWE, Mantel tests on marker distance matrices were performed to ensure the legitimacy of joint loci analysis. Correlation coefficients and respective AD matrices reveals the relationships of populations in the full-loci dataset. The populations of Tajik, Uyghur, and Kirgiz from Northwest China, together with those of Dongxiang, Salar, and Mongolian from North China compose a solid branch bearing a high bootstrap value of 77%. Within these populations, Uyghur, Kirgiz, and Salar belong to the Turkic language family, while Dongxiang, Mongolian, and Tu belong to the Mongolian language family. These two language families are all branches of the Altaic languages. Maonan, a population of the Tai-Kadai language family, also appears in this northern cluster instead of clustering with other southern populations. Repeated genotyping and examination of individual genotypes were performed for samples of the Maonan and Mongolian populations and ruled out the possibility of sample mix-up during experiment stages. Although Mongolian also appears in Yunnan as the result of military migrations and war affairs in Yuan Dynasty Genetic relationships of the studied populations were firstly depicted by phylogenetic reconstructions. In , and Lisu, represents the regions of West Yunnan; the other, including Li, Mulam, Zhuang, and HanGuangdong, stands for Southeast China. Bootstrap values for the clusters of West Yunnan and Southeast China are 74% and 86%, respectively. Drung and Mulam are the innermost populations of each cluster. Similar to northern populations, southern ones also have agglomerative linguistic affiliations. The West Yunnan populations belong to the Tibeto-Burman language family of the Sino-Tibetan languages; as for the other cluster, Deang and Blang belong to the Mon-Khmer languages, whereas Li, Mulam, Zhuang, and Dai are members of the Tai-Kadai languages . Only the Maonan population, which also falls within the Tai-Kadai languages, appears at a position outside of its linguistic affiliation.With several populations situated at intermediate places, the remaining ones form two distinct parts. One, including Drung, NuThe three Han Chinese populations included in this study possess distinct positions in constructed phylogeny. HanGanshu and HanShangdong reside at the interface of the north and south clusters, as suggested by their branching sites and bootstrap values. On the contrary, HanGuangdong shows significantly close relationships to Tai-Kadai populations such as Zhuang and Dai., and Tibetan populations, another phylogenetic reconstruction was carried out using the full-population dataset, which has information of only 8 markers. As shown in CD matrices for both the full-loci and the full-population datasets (data not shown).In order to determine the positions for the WaCangyuan, WaXimeng, Jinuo. Positions of Drung and Mulam populations in the plot show evidence of extreme geographic isolations. The Altaic populations take up the upper part, and populations of the Tai-Kadai languages (except Maonan) appear in the lower right quadrant, while populations of the Tibeto-Burman language family occupy the lower left region. All these clusters of populations and linguistic affiliations resemble those in previous phylogenetic analysis. It is noteworthy that the distribution of populations in this plot well approximates their geographical locations in r = 0.418, p = 0.0054) of the Mantel test between PCA distances by the first two components and the geographical distance matrix can be found. Patterns revealed by PCA of the full-population dataset do not alter much; the Wa populations and the Tibetan population show affinity to the clusters of Southeast China and West Yunnan, respectively (data not shown).Besides phylogenetic reconstructions, we applied a principal component analysis (PCA) to allele frequency data of typed markers. structure program was utilized to study relationships of the populations from a different point of view. The results under different K settings for the full-loci dataset are shown in X|K)) from different batches suggested K = 3 as the most appropriate configuration according to the rules set out in the structure manual  than the remaining ones. This suggests that they are ordered at the innermost and the outermost locations in the regional phylogeny, which is not surprising since migrations of these Muslim populations like Tajik and Uyghur into their nowadays areas between Central Asia and East Asia happened much later than the contribution of Central Asians' ancestors to the formation of East Asian populations K = 3, the three previously identified cores, Northwest China (light purple), Southeast China (green), and West Yunnan (orange), constitute individual clusters, leaving populations like Pumi, Bai, HanShandong, HanGansu, and Tu to fall between distinct clusters. When K is equal or greater than 4, no new evident cluster can be introduced and proportions of this newly added part don't vary as much across most populations as those of the other three sources. Complexity of membership coefficients of populations like HanGanshu, HanShandong, Aini, Bai, and Pumi can be best illustrated by their undefined affiliations to any distinct clusters.A clustering analysis by the e manual . When K structure analysis was applied to populations of the three cores separately to examine any additional decomposable stratification. For all three of the cores, structure clustering didn't yield any separation of populations within each of them (data not shown). This result was expected, as previous overall clustering had suggested that with only 10 markers employed in this study, certain level of differentiation among these populations cannot be resolved by the structure program. Increased number of markers might help to produce finer separation for populations of these three cores.As a next step, r = 0.239) for the full-loci dataset is slightly weaker than that between genetics and geography (r = 0.287). Permutation shows that the two correlations are both significant ; though, the significance is greater for the former pair no matter geographical distances were controlled (in 3-way tests) or not (for 2-way tests), which is possibly because the quantification of geographical distances with arch lengths for populations in Yunnan could not take into account the effects of special landforms in that area, thus making the distances among these populations shorter than they would otherwise be. The correlation between linguistic affiliations and geography is both the highest (r = 0.346) and the most significant (p = 0.0002) among all the three pairs. Tests for the full-population dataset gave similar conclusions to the above comparisons (data not shown).As has been seen in previous phylogenetic, factor decomposition, and clustering analyses, the genetic relationships of populations shows a strong correlation with their linguistic affiliations. To explore such a correlation in a quantitative way, we statistically compared genetic, geographic and linguistic distance matrices for our population datasets  level when Tajiks (speakers of Iranian languages) were excluded, which reminds us the special role of this population as revealed in previous PCA analysis (p = 0.017) and its coefficient is even slightly increased (r = 0.165) under 3-way Mantel test. Such a contrast suggests that even though the geographical dispersion may not significantly resemble the genetic relationship when Tajik was excluded, there still be a certain correlation between genetic and linguistic affiliations for these populations.When populations of a specific language were excluded from the full-loci dataset, changes in coefficients and analysis . Howeverp<0.05) correlation for all partial datasets. However, when Mongolian or Turkic populations were excluded, the coefficients between genetics and linguistics of 3-way tests for respective partial datasets reduced to an insignificant level , while it is not the case for other partial datasets that excludes populations of a language like Tai-Kadai and Tibeto-Burman. This change relative to the full-loci dataset is because the exclusion of Mongolian or Turkic populations results in a reduction of averaged genetic distances among the remaining populations, and thus the correlation between genetics and linguistics with geographical distances controlled in 3-way tests is more easily confounded by regional migration events as well as the quantification method of geographical distances for closely distributed populations.2-way Mantel tests yielded significant Click here for additional data file.Figure S2Contributions by PCA components in real and random datasets. The barplot as well as the labelled numbers stands for the contributions by the first 10 components in PCA of our real dataset. Solid line stands for the mean contributions in 10,000 random datasets and dashed lines are corresponding 95% upper bound and 5% lower bound. Contributions by the first two components, though only 24.33% in total, are much higher than that by the first component in random datasets.(0.01 MB EPS)Click here for additional data file.Figure S3structure clusterings. Plotting follows conventions, where the central mark is the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points not considered outliers, and outliers are plotted individually.Boxplot of posterior probabilities of the (0.01 MB PDF)Click here for additional data file.Table S1Information of 30 sampled populations. Lat and Long stand for latitude (north) and longitude (east), respectively.(0.07 MB DOC)Click here for additional data file.Table S2EH) for all markers of each population. Values are crossed out for the 5 loci where missing rates within respective population are higher than 40%.Expected heterozygosities ((0.02 MB XLS)Click here for additional data file.Table S3Detailed frequency data of 10 loci for studied populations. Numbers in header line represent fragment sizes called in the GeneMarker environment. Contained in the last column of each table are the allele numbers that were successfully called for each population.(0.09 MB XLS)Click here for additional data file.Table S4P values of exact tests for Hardy-Weinberg equilibrium. Values<0.05 mean significant departure from equilibrium and are labeled as bold face. Values are crossed out for the 5 loci whose missing rates within respective population are higher than 40%.(0.02 MB XLS)Click here for additional data file.Table S5AD distance; linguistic distances were constructed according to the ‘least controversial phylogeny’ proposed by Sagart Matrices of genetic, linguistic and geographical distances used for Mantel tests. Genetic distances used here is the (0.10 MB XLS)Click here for additional data file.