In humans, meiotic chromosome segregation errors increase dramatically as women age, but the molecular defects responsible are largely unknown. Cohesion along the arms of meiotic sister chromatids provides an evolutionarily conserved mechanism to keep recombinant chromosomes associated until anaphase I. One attractive hypothesis to explain age-dependent nondisjunction (NDJ) is that loss of cohesion over time causes recombinant homologues to dissociate prematurely and segregate randomly during the first meiotic division. Using Drosophila as a model system, we have tested this hypothesis and observe a significant increase in meiosis I NDJ in experimentally aged Drosophila oocytes when the cohesin protein SMC1 is reduced. Our finding that missegregation of recombinant homologues increases with age supports the model that chiasmata are destabilized by gradual loss of cohesion over time. Moreover, the stage at which Drosophila oocytes are most vulnerable to age-related defects is analogous to that at which human oocytes remain arrested for decades. Our data provide the first demonstration in any organism that, when meiotic cohesion begins intact, the aging process can weaken it sufficiently and cause missegregation of recombinant chromosomes. One major advantage of these studies is that we have reduced but not eliminated the SMC1 subunit. Therefore, we have been able to investigate how aging affects normal meiotic cohesion. Our findings that recombinant chromosomes are at highest risk for loss of chiasmata during diplotene argue that human oocytes are most vulnerable to age-induced loss of meiotic cohesion at the stage at which they remain arrested for several years. In humans, chromosome segregation errors during meiosis are the leading cause of birth defects and miscarriages. Moreover, as women age, these errors increase dramatically. For accurate segregation during the first meiotic division, homologous chromosomes must remain physically associated until anaphase I. Normally, attachments along the arms of sister chromatids keep the recombinant homologues together. Human oocytes complete meiotic recombination during fetal development and arrest until ovulation. Therefore, accurate segregation of homologous chromosomes during the first meiotic division requires that recombinant chromosomes remain associated for decades. One hypothesis to explain why segregation errors increase as women age is that the connections between sister chromatids deteriorate over time and allow recombinant homologues to dissociate prematurely. Here, we address this hypothesis using Drosophila as a model system. We find that when Drosophila oocytes undergo experimentally induced aging, recombinant homologues missegregate during meiosis I. Furthermore, the meiotic stage at which Drosophila oocytes are most vulnerable to age-induced errors is analogous to the stage at which human oocytes remain arrested for decades. Together, our data argue that aging does cause premature loss of the connections between meiotic chromosomes and that this is a major determinant of segregation errors in both Drosophila and human oocytes. In humans, meiotic chromosome segregation errors that give rise to aneuploid gametes are the leading cause of fetal loss and birth defects The link between increased maternal age and meiotic segregation defects in humans is well established. At maternal age 25, the risk of a trisomic pregnancy is approximately 2% but increases to approximately 35% for a woman at age 42 Accurate segregation during meiosis I requires that homologous chromosomes undergo recombination and remain physically attached to one another until they segregate to opposite poles during anaphase I. In the absence of a stable connection, homologues will segregate randomly resulting in meiosis I NDJ. Cohesion between the arms of sister chromatids provides an evolutionarily conserved mechanism for maintaining the association of recombinant homologues . NormallGiven that human oocytes undergo meiotic recombination during fetal development and remain suspended in a prolonged dictyate (diplotene) arrest until ovulation, the continuous association of homologous chromosomes demands that meiotic sister-chromatid cohesion remain intact for decades. One attractive hypothesis to explain age-dependent NDJ is that deterioration of cohesion with age causes recombinant homologues to dissociate prematurely and segregate randomly during the first meiotic division . HoweverUsing Drosophila as a model system, we have developed an experimental regimen to study the mechanisms that contribute to increased levels of meiotic NDJ in oocytes as a result of aging smc1 gene +/−smc1 females  in 6–12% of Drosophila oocytes +/−smc1 oocytes, the majority of tetrads (74%) had at least one exchange event  disrupts the segregation of achiasmate chromosomes in Drosophila oocytes +/−mtrm oocytes mtrm in +/−smc1 females.To examine the effect of SMC1 dosage on age-dependent NDJ, we subjected tion see . Howeverere aged . Becauseere aged . Therefoere aged . Hawley +/−smc1 oocytes following the four-day aging regimen when the achiasmate pathway was also compromised (+/−mtrm) . Although the absolute level of NDJ may vary between experiments, we have repeatedly observed higher levels of NDJ in +/− mtrm+/−smc1 aged oocytes in the first two 24 hour broods  . Signifimtrm+/−) . We havest brood ; we did st brood . The fin+/− mtrm+/−smc1 oocytes, we assayed the recombinational history of the missegregating chromosomes. For this analysis, we collected “Diplo-X” female progeny that received two maternally contributed X chromosomes due to meiotic missegregation (see X chromosomes (car) allowed us to assess whether NDJ events involved homologous chromosomes (MI NDJ) or sister chromatids (MII NDJ). The genotype of the Diplo-X females suggested that the majority of NDJ events (70/72) occurred during meiosis I  in both aged and non-aged oocytes dl-49) harboring an inversion that severely suppresses crossovers is not completely random in +/−mtrm oocytes (+/−mtrm oocytes (compare NR/N for aged and non-aged). These data implicate the cohesin complex in accurate disjunction of achiasmate as well as chiasmate bivalents. However, the low number of MII NDJ events that we observe  argues that the increased missegregation of non-recombinant chromosomes is not due to loss of centromeric cohesion between sister chromatids.Although the achiasmate pathway is compromised in  oocytes  but we o oocytes . The dat+/− mtrm+/−smc1 oocytes indicates that broods 1 and 2 are most susceptible to age effects. Each Drosophila ovariole consists of multiple oocytes at progressive stages of development. At the posterior end of the ovariole (stages 13/14), the mature oocytes undergo nuclear envelope breakdown and spindle assembly. Stage 14 oocytes remain arrested at metaphase I until passage through the oviduct triggers resumption of the meiotic divisions. After an aging regimen, the most mature oocytes are laid first and therefore will contribute to the progeny in brood 1  following aging  are not vulnerable to age-related defects. Similarly, oocytes that undergo aging during pachytene (sub-broods 5 and 6) also appear to be refractory to age effects. Therefore, only oocytes that age during a very specific window of oogenesis (post-pachytene/pre-metaphase I) exhibit age dependent NDJ.Following the aging regimen, age-dependent NDJ was observed in sub-broods 3 and 4, which arose from oocytes that were fertilized 16–32 hours after the aging regimen was completed . Our cyt oocytes . These rX progeny for a subset of sub-broods obtained from the NDJ analysis shown in X chromosomes in sub-broods 1 and 6, which represent opposite ends of the developmental spectrum examined in this study (P = 0.0029) or sub-brood 6 (P = 0.0179). In addition, this trend holds true when the NDJ of recombinant chromosomes in sub-brood 4 aged oocytes is compared to that for sub-broods 1 or 6 aged oocytes, although the difference is not statistically significant. These data contrast strongly with those for non-aged oocytes, in which recombinant chromosomes missegregate at similar frequencies in the four sub-broods analyzed.To determine what fraction of missegregating chromosomes were recombinant in the aged versus non-aged oocytes, we genotyped the Diplo-is study , S8. Agais study , S8. In is study . NDJ of The data shown in +/− mtrm+/−smc1 oocytes missegregate more frequently when aging is induced in oocytes prior to metaphase I (P<0.27). However, non-recombinant chromosomes in sub-brood 3 are significantly more vulnerable to missegregation after aging (P = 0.029). These results support the model that the connections that keep achiasmate bivalents together depend at least in part on meiotic cohesion proteins Our sub-brood analysis also suggests that non-recombinant chromosomes in aphase I , althoug+/−smc1 oocytes are sensitized to the effects of aging, meiotic cohesion is initially intact. Therefore our regimen allows us to observe the effect of aging upon the normal cohesin complex that functions during Drosophila meiosis. In this respect our analysis differs markedly from previous studies using knock-out mice in which the meiosis-specific subunit SMC1-β was eliminated Together, our data support the hypothesis that weakening of meiotic cohesion with age is a significant determinant of age-dependent nondisjunction in both fly and human oocytes. One major advantage of these studies is that we have lowered but not eliminated the level of the cohesin subunit SMC1. Although +/− mtrm+/−smc1 aged and non-aged oocytes, we have not observed any obvious differences in SC morphology, or the timing of its assembly or disassembly . Therefore, we speculate that the synaptonemal complex may play a role in protecting sister-chromatid cohesion from age-induced deterioration. The differential response of pachytene and diplotene-like Drosophila oocytes to the aging process has profound implications regarding the vulnerability of human oocytes to age-induced loss of meiotic cohesion. Our data argue that the meiotic stage (dictyate) at which human oocytes remain suspended as women age is also the stage during which recombinant chromosomes are at highest risk for loss of chiasmata.Here we demonstrate that deterioration of normal meiotic cohesion during the aging process causes loss of chiasma maintenance. Loss of arm cohesion in aged oocytes accounts for the increased frequency with which recombinant chromosomes missegregate during meiosis I. In contrast, low levels of MII NDJ indicate that centromeric cohesion remains intact, at least for the duration of our aging regimen. Recombinant chromosomes in Drosophila oocytes that undergo aging after they have disassembled their synaptonemal complex (diplotene-like) are most vulnerable to age effects and these oocytes bear a striking resemblance to the stage at which human oocytes remain arrested for decades. Interestingly, Drosophila oocytes that are subjected to aging during pachytene do not exhibit significant age-dependent nondisjunction. Using epifluorescence microscopy to compare matrimony (KG08051mtrm) as a genetic tool to disrupt the achiasmate pathway. However, we do not think that heterozygosity for mtrm contributes to age-dependent loss of arm cohesion. Recent work has demonstrated that Mtrm protein physically interacts with and inhibits the activity of Polo kinase in Drosophila oocytes from stage 11 until nuclear envelope breakdown (NEB) at stage 13 +/−mtrm), NEB can occur prematurely (at stage 12) and the spindle assembles around a karyosome that is less compact than in wild type. Although this may account for the eventual missegregation of achiasmate chromosomes in +/−mtrm heterozygotes, sister chromatid cohesion remains intact in these oocytes as evidenced by the striking chiasmata visible in the individualized bivalents +/−mtrm oocytes still occurs much later than the stages that exhibit sensitivity to aging in our experiments (stages 7–8). Therefore, we conclude that reducing the dosage of Mtrm is not causing the increased missegregation of recombinant chormosomes that we observe in aged oocytes.Because the achiasmate pathway in Drosophila oocytes will ensure the accurate segregation of recombinant chromosomes that lose chiasmata due to deterioration of arm cohesion during the aging process, we needed to dismantle this system for our analysis. Therefore, we used a P-element insertion in KG08051mtrm heterozygotes, we also have been able to assess the effect of aging on the segregation of non-recombinant chromosomes. Interestingly, our data demonstrate that when the dosage of SMC1 is reduced, MI missegregation of non-recombinant chromosomes also increases with age. This result is reminiscent of our previous findings that NDJ of achiasmate chromosomes increases with age when activity of the meiotic cohesion protein ORD is compromised by a hypomorphic missense mutation ord mutations, even in the absence of aging . Together, these data suggest that in addition to holding sister chromatids together, cohesion proteins play an important role in maintaining the heterochromatic pairing of achiasmate homologues in Drosophila oocytes. Whether cohesion proteins function directly to “glue” homologues together or play a more indirect role, such as influencing the structure of heterochromatin, remains to be determined. Regardless of the mechanism, loss of heterochromatin-associated cohesion proteins with age could account for the increased missegregation of non-recombinant chromosomes that we observe.Because the achiasmate segregation pathway is not completely disrupted in Our data support the hypothesis that age-dependent NDJ in women is caused at least in part by progressive loss of sister-chromatid cohesion over time. One unresolved issue regarding meiotic cohesion in human oocytes is whether the original cohesin molecules used to establish cohesion are maintained for decades or continually replaced as oocytes age. Addressing this question will be essential in understanding the specific mechanisms that lead to loss of meiotic cohesion and chiasma maintenance during the aging process. Early experiments in yeast led to the widely accepted model that cohesion between sister chromatids can only be established during DNA replication The power of genetic manipulation combined with the ability to age oocytes makes Drosophila an ideal model system to address whether re-establishment of meiotic cohesion occurs during prophase I. These future studies will be pivotal in understanding how cohesion dynamics during meiosis govern chiasma maintenance and ultimately, why and how loss of cohesion occurs as oocytes age.ex46smc1 allele is a deletion that removes the gene −smc1. The [KG08051]mtrm allele results from a P-element disruption of the gene −mtrm. Descriptions of the other genetic markers and chromosomes used in this study can be found at http://www.flybase.org.Flies were reared at 25°C on standard cornmeal molasses media. The mtrm allele to be the ideal choice for our studies nod, achiasmate segregation is severely affected in the mtrm heterozygote and chromosome loss is not observed Axs oocytes, spindle defects and premature onset of anaphase I have not been observed in mtrm heterozygotes ald mutations given the role of this protein in the spindle assembly checkpoint; ald oocytes also exhibit low levels of NDJ of the exchange bivalents +/−mtrm oocytes combined with the observation that sister-chromatid cohesion appears to be normal convinced us that this was the best approach to disrupt the achiasmate segregation pathway.Although several proteins  are required for the accurate segregation of achiasmate chromosomes in Drosophila oocytes, we considered a X-chromosome crossover frequency and distribution in +/−smc1 oocytes, 5–7 females were crossed to 3 yw males per vial. Crossover frequency and distribution was measured by assaying sc, cv, v, f, car markers in the male progeny. The recombination data was used to estimate tetrad exchange ranks To assay et al. was modified To age Drosophila oocytes, the aging regimen described by Jeffreys A schematic of the aging regimen is shown in X∧Y, v f B males to measure meiotic nondisjunction in the oocytes . The parents were transferred to new vials every 24-hours and three broods of progeny were analyzed for NDJ.At the end of the four-day aging regimen, the experimental females (with aged oocytes) and the control females (with non-aged oocytes) were crossed to ytes see . To geneX∧Y, v f B males . The parents were transferred to new vials every 8-hours for a total of 48 hours and six sub-broods of progeny were analyzed for NDJ.For simplicity, we have used the term “sub-broods” to differentiate 8-hour broods of progeny from the 24-hour broods of progeny. To generate sub-broods of progeny at the end of the aging regimen, 21 experimental or control females were crossed to 10 Because Drosophila can tolerate certain sex chromosome aneuploidies, segregation errors during meiosis can be monitored in the viable progeny by using differentially marked sex chromosomes see . To compX female progeny obtained from NDJ tests were genotyped. Each Diplo-X female was crossed to two yw males and the genotype of the X chromosomes of the Diplo-X female was inferred from the sc, cv, v, f, car markers in her male progeny  for 10 min. The ovaries were rinsed in PBS containing 0.01% Tween-20. Images were captured using a SMZ1500 stereo-microscope (Nikon) equipped with a Pixelink camera (Diagnostic Instruments).After female flies were subjected to the four-day aging regimen, ovaries from experimental and control females were hand-dissected in modified Robb's buffer To quantify the relative number of ooctyes at specific developmental stages, a single fixed ovary from three females was teased to generate individual ovarioles. Oocyte stages were tabulated and averaged for aged and non-aged ovaries at each time point. Oocytes were staged based on the standard morphological criteria described by Mahowald and Kambysellis smc1 heterozygous females were fattened with yeast for two days. 30 sets of ovaries from each genotype were dissected in modified Robb's buffer 2. Each frozen tissue sample was homogenized in 240 µL of buffer containing 8 M Urea, 2% SDS, 100 mM Tris HCl pH 6.8 and 5% Ficoll containing 4 mM AEBSF . The extract was cleared by centrifugation at 13,000 rpm for 10 min at room temperature and the supernatant was aliquoted and frozen in liquid N2. The protein concentration for each extract was determined using a Bradford assay (Biorad). For each genotype, 10 and 20 µg of total protein was separated by SDS-PAGE on a 7.5% gel and transferred to PVDF membrane. The blot was cut horizontally and anti-SMC1 guinea pig serum Newly eclosed wild-type and e = # of exceptional progeny from aged oocytes, An = # of normal progeny from aged oocytes, NAe = # of exceptional progeny from non-aged oocytes, and NAn = # of normal progeny from non-aged oocytes To compare the relative frequency of missegregation occurring in aged and non-aged oocytes, an odds ratio (OR) was calculated. The odds ratio provides a method for determining whether the frequency of a certain event (missegregation) is equal for two different treatments (aged and non-aged). In our analysis, it is calculated as: logOR retransformed back into non-logarithmic units.Standard errors (SE) are calculated on the logarithm of the odds ratio according to: P values were calculated using a 2×2 χ2 Contingency Test (two-tailed). For these calculations, raw data values  for a specific brood or sub-brood were compared for aged versus non-aged treatments . A 2×2 χ2 Contingency Test also was used to analyze the recombinational history of missegregating chromosomes recovered in Diplo-X progeny. For all tests, a P value of <0.05 was considered statistically significant (rejection of the null hypothesis that the two groups are the same).For the NDJ tests, Figure S1smc1+/−mtrm+/− control and experimental females dissected at 8-hour intervals after the aging regimen. Mature oocytes (stage13/14) at the posterior end of the ovariole can be distinguished (in part) by their chorionic appendages (yellow arrows). The scale bars are 100 microns. At time-point 0 after the aging regimen, mature oocytes are far more abundant in the aged ovary than in the non-aged ovary. It is not uncommon to observe two mature oocytes per ovariole in aged ovaries at this time-point. When both experimental and control females are allowed to lay eggs for 8 hours after the regimen, the difference between the number of mature oocytes in the aged ovary and non-aged ovary is reduced. By 16 hours, the excess mature oocytes have been cleared from the aged ovaries, and the distribution of stages found in experimental and control females is very similar (see Excess of mature oocytes in the aged ovaries is cleared by 16 hours after the aging regimen. This figure shows representative images of whole ovaries and single ovarioles from ilar see .(9.77 MB TIF)Click here for additional data file.Figure S2P<0.76 for pair-wise comparisons). Non-recombinant chromosomes in aged oocytes missegregate more frequently in sub-broods 3, 4 and 6 than in sub-brood 1, although this difference is not statistically significant (0.10<P<0.27). However, a comparison of sub-brood 3 aged and non-aged oocytes shows that aging causes a significant increase in the frequency at which non-recombinant chromosomes missegregate (P = 0.029). These data reinforce the model that the achiasmate chromosome segregation pathway is vulnerable to age effects and that sister-chromatid cohesion proteins play a role in this process (see Discussion).Non-recombinant chromosomes exhibit increased missegregation when oocytes undergo aging. The number of non-recombinant (NR) Diplos recovered in sub-broods 1, 3, 4 and 6 is shown for aged oocytes and non-aged oocytes Click here for additional data file.Table S1X chromosome nondisjunction in +/− and mtrm+/−smc1oocytes (no aging regimen).(0.03 MB DOC)Click here for additional data file.Table S2+/−smc1 aged oocytes when the achiasmate pathway is functional.Higher meiotic missegregation is not observed in (0.03 MB DOC)Click here for additional data file.Table S3+/−smc1 oocytes when achiasmate segregation also is disrupted.Age-dependent segregation errors arise in (0.07 MB DOC)Click here for additional data file.Table S4Segregation errors do not increase with age when the achiasmate pathway is compromised but cohesion is wild type.(0.04 MB DOC)Click here for additional data file.Table S5X females arise from missegregation of both recombinant and non-recombinant chromosomes in aged and non-aged +/− mtrm+/−smc1 oocytes.Diplo-(0.06 MB DOC)Click here for additional data file.Table S6+/− mtrm+/−smc1 oocytes that give rise to sub-broods 3 and 4 are prone to age-dependent NDJ.(0.06 MB DOC)Click here for additional data file.Table S7X females arising from aged +/− mtrm+/−smc1 oocytes.Genotype of Diplo-(0.07 MB DOC)Click here for additional data file.Table S8X females arising from non-aged +/− mtrm+/−smc1 oocytes.Genotype of Diplo-(0.08 MB DOC)Click here for additional data file.


It is well known that hypoxic exercise in healthy individuals increases limb blood flow, leg oxygen extraction and limb vascular conductance during knee extension exercise. However, the effect of hypoxia on cardiac output, and total vascular conductance is less clear. Furthermore, the oxygen delivery response to hypoxic exercise in well trained individuals is not well known. Therefore our aim was to determine the cardiac output (Doppler echocardiography), vascular conductance, limb blood flow (Doppler echocardiography) and muscle oxygenation response during hypoxic knee extension in normally active and endurance-trained males.2max = 46.1 and 65.5 mL/kg/min, respectively) performed 2 leg knee extension at 25, 50, 75 and 100% of their maximum intensity in both normoxic and hypoxic conditions . Results were analyzed with a 2-way mixed model ANOVA (group × intensity).Ten normally active and nine endurance-trained males  brought about a 3 fold increase in limb blood flow but decreased stroke volume compared to normoxia. In the trained group there were no significant changes in stroke volume, cardiac output and limb blood flow at sub-maximal intensities (compared to normoxia). During maximal intensity hypoxic exercise limb blood flow increased approximately 300 mL/min compared to maximal intensity normoxic exercise.Cardiorespiratory fitness likely influences the oxygen delivery response to hypoxic exercise both at a systemic and limb level. The increase in limb blood flow during maximal exercise in hypoxia  suggests a hypoxic stimulus that is not present in normoxic conditions. A number of investigators have reported that during hypoxic sub-maximal knee extension (KE) exercise an increase in limb blood flow, leg oxygen extraction and enhanced limb vascular conductance occurs compared to the equivalent work rate in normoxia -4. The eHighly trained athletes have a greater stroke volume, total vascular conductance and leg oxygen extraction during normoxic exercise compared to normally active individuals ,9. Howev2 delivery it was reasonable to assume that a differential response to acute hypoxia would occur based on training status of the participants. However, how this differential response might be manifested remains unclear. Thus our aim was to determine the changes in the central (lungs and heart) and peripheral (limb blood flow and muscle oxygenation) cardiovascular response to acute hypoxia in incremental knee extension exercise between active and well trained males. To assess the full O2 cascade muscle oxygenation was measured to provide an assessment of O2 delivery and utilization . Significance was set 2 condition and intensity and intensity and fitness for oxygen consumption. Specifically, hypoxia decreased VO2 at every workload in the trained group but VO2 only decreased at rest and 100% relative intensity in the active group  and at 25 and 50% relative intensity (active group)  hypoxia elicits an increase in limb blood flow as well as a decrease in stroke volume and total vascular conductance in active males but does not significantly affect limb blood flow, conductance or Q in trained males; 2) at maximal exercise the trained group had similar Q and stroke volume in hypoxia and normoxia, but the active group decreased Q and stroke volume in hypoxia compared to the normoxic condition. In addition, both groups increased limb blood flow at maximum indicating a potential hypoxic stimulus during small muscle mass exercise. Overall this study provides a unique comparison between trained and active males to a hypoxic stimulus in localized muscle mass exercise and reflects different compensatory responses to hypoxic exercise that is dependent on the aerobic fitness of individuals.2 = ~11–12%) [2 = 15%). Additionally, compensation in limb blood flow may not occur in well trained males during small muscle mass sub-maximal exercise (VO2max > 65 mL/kg/min) despite a similar decrease in oxygen saturation   in activn Figure  between 2 conditions, unlike the results from other investigations [2 concentrations during cycling at the same intensity [There was no significant difference in muscle oxygenation between FIOigations ,32 reporntensity .Comparison of systemic vascular conductance and heart function measures to the hypoxic condition reveals that active males decreased Q and total vascular conductance but trained males essentially maintained Q at rest and all sub-maximal intensities.In the active males this equated to a 22% reduction in Q at rest in hypoxia compared to normoxic rest conditions paralleled by a decrease of 19% for SV. Considering that HR did not increase, the reduced SV predicated on a decreased TVC of 29% would explain why Q is reduced at rest in hypoxia in the active males. This is contrary to other who have found small increases in Q at rest in hypoxia compared to the normoxic condition  although2 during sub-maximal exercise was reduced throughout sub-maximal exercise in the trained group (p < 0.05) with a trend towards reduced VO2 in the active group  and 125 mL/min (active). There was a trend towards decreased Q (0.8 L/min at maximum) in the active group attributed to a non-significant decrease in stroke volume (-7.3 mL). Reduced stroke volume was due in part to a reduced total vascular conductance (-6.8 mL/min/mmHg) which has been shown to affect stroke volume by increasing left ventricular afterload [At maximal exercise intensity in the hypoxic condition compared to the normoxic condition , oxyhemoglobin saturation was reduced approximately 4.7% (trained) and 3.4% (active); with a reduced VOfterload . Conversfterload , these ffterload .2 = 11–12%) than the level used in this study. Severe acute hypoxia (FIO2 = 11%) reduces work rate due to excessive fatigue such that the magnitude of the oxygen delivery is reduced [2 = 15%). Thus, the increased limb blood flow at maximum may be indicative of additional compensation to equalize oxygen delivery compared to normoxia. Within the muscle, this enhanced blood flow appears to moderate TOI so that oxygenation is not significantly changed when hypoxic conditions exist  [2 conditions, it can be assumed that metabolic demand within the muscle was consistent between normoxia and hypoxia, thus factors beyond metabolic control of muscle blood flow likely caused the increase in limb blood flow during the hypoxic condition. Thus the reduced blood saturation found in this study, suggests arterial oxygen saturation may be a mechanism by which vasodilatation occurs, thereby increasing muscle blood flow beyond the normoxic maximum value.One other investigation has alluded to increased limb blood flow brought about by 1 leg KE maximal exercise in hypoxia (FIO 10–11%) . However 10–11%)  and the  10–11%) . Moreove 10–11%) . BecauseThese results reflect an apparent underestimation of both limb blood flow and cardiac output due to a number of potential reasons described below. First, previous investigations have measured aorta diameter at the sinotubular junction and others at the aortic valve hinge points . We usedIn addition the KE protocol required significant muscular effort to extend the knee with little muscular effort on the return phase. This meant that at 75% relative intensity 37 and 53 kg and at 100% relative intensity 50 and 71 kg of weight was lifted at a rate of 40 contractions per minute in active and trained males respectively. This amount of weight required significant stabilization in the upper body which may have influenced the intra-thoracic pressure and atrial filling pressure reducing end diastolic volume and stroke volume. Thus, both of these factors likely influenced the magnitude of increase in Q that was lower than expected compared to other investigations using similar protocols and subject characteristics .Furthermore, exercise limb blood flow values were lower than expected, despite resting leg blood flow data which was similar to others using both echocardiography and other techniques ,4,46. In2max remain limited [2max > 65 mL/kg/min). With this understanding, it may be conjectured that endurance trained males may cope better during endurance performance events at moderate altitude however the comparative changes between trained and active individuals exposed to chronic hypoxia remain less clear.Altitude acclimatization has been a popular method of increasing blood haemoglobin concentration and arterial oxygen saturation,  however  limited . Moreove limited . Recentl limited , decreas limited  as well  limited . These r2. These findings are an important distinction compared to other research  because 15% FIO2 allowed all participants to complete the same workloads in both conditions. This allowed for a direct comparison of a hypoxic stimulus even during very intense exercise, where previous research has not yet adequately described this effect due to reduced maximal workloads compared to normoxia.The effect of hypoxia on the cardiovascular response was evident during sub-maximal and maximal small muscle mass exercise while participants breathed 15% FIOApplication of these results to performance reveals that trained males may cope better at moderate altitude. Future research should confirm the existence of a hypoxic limb blood flow increase during maximal exercise, and the mechanism(s) which regulate this unique phenomenon.The authors declare that they have no competing interests.MDK designed the study, coordinated the study, acquired the data, performed the statistical analysis, and drafted the manuscript. DEW participated in coordination of the study, provided the equipment used in the study and provided important revisions to the manuscript. CAB participated in the design of the study, interpretation of the results, and preparation of the manuscript. BTE participated in acquisition of data and coordination of the study. JMS participated in acquisition of data and helped organize the study. MJH participated in the design of the study, interpretation of the results, and preparation of the manuscript. All authors have read and approved the final manuscript.


P = 0.037). Peripheral blood mononuclear cells (PBMC) contained MCPyV more frequently in patients sampled with disease than in patients in complete remission . Moreover, the detection of MCPyV in at least one PBMC sample during follow-up was associated with a shorter overall survival (P = 0.003). Sequencing of viral DNA from MCC and non MCC samples characterized common single nucleotide polymorphisms defining 8 patient specific strains. However, specific molecular signatures truncating MCPyV LT were observed in 8/12 MCC cases but not in respiratory and urinary samples from 15 patients. New integration sites were identified in 4 MCC cases. Finally, mutated-integrated forms of MCPyV were detected in PBMC of two patients with disseminated MCC disease, indicating circulation of metastatic cells. We conclude that MCPyV molecular features in primary MCC tumour and PBMC may help to predict the course of the disease.Merkel Cell Polyomavirus (MCPyV) is associated with Merkel Cell carcinoma (MCC), a rare, aggressive skin cancer with neuroendocrine features. The causal role of MCPyV is highly suggested by monoclonal integration of its genome and expression of the viral large T (LT) antigen in MCC cells. We investigated and characterized MCPyV molecular features in MCC, respiratory, urine and blood samples from 33 patients by quantitative PCR, sequencing and detection of integrated viral DNA. We examined associations between either MCPyV viral load in primary MCC or MCPyV DNAemia and survival. Results were interpreted with respect to the viral molecular signature in each compartment. Patients with MCC containing more than 1 viral genome copy per cell had a longer period in complete remission than patients with less than 1 copy per cell  is a recently discovered virus highly associated with a rare skin cancer, Merkel cell carcinoma (MCC). The causal role of MCPyV in cancer is suggested by integration of viral sequences into the cell genome and by a specific molecular signature. We looked for and compared molecular species of MCPyV in tumour and non tumour samples of 33 MCC patients. We showed that a tumour viral load greater than 1 copy per cell was associated with a better outcome, and that detection of the virus in blood but not in urine correlated with a shorter overall survival. A tumour–specific molecular signature was found in the blood of two patients with metastatic disease, but did not occur in their respiratory nor urine samples. We propose that molecular analysis of MCPyV in tumour and blood be used as a biomarker of infection and cancer progression in MCC patients. Polyomaviruses are small, non enveloped double stranded DNA viruses which infect many species with a restricted host range. The initial discovery of Murine polyomavirus (MPyV) and Simian vacuolating 40 (SV40) was closely linked to the demonstration of their experimental tumorigenic properties MCC is a carcinoma of neuroendocrine cells which affects principally elderly and immunocompromised patients. The close association between MCPyV and MCC has been confirmed in several case series worldwide This study was conducted according to the principles expressed in the Declaration of Helsinki. According to French regulation, information was delivered to the patients on research performed on biological samples, and written informed consent was obtained for participation in the study, which was approved by the institutional review board of the Comité de protection des personnes Ile de France 3.All patients with Merkel cell carcinoma who attended the Dermatology Departments of Cochin and Bichat hospitals from March 2008 to April 2010 were prospectively included in the study. Histopathologic confirmation of MCC diagnosis relied on tumour morphology consistent with MCC on hematoxylin-eosin-stained tissue sections, paranuclear dot immunostaining pattern for CK20 or positive immunostaining for synaptophysin and chromogranin A. Clinical data were retrieved from hospital case records, and included sex, age at diagnosis, site and size of primary MCC, and stage of the disease at diagnosis, according to Allen's classification For FFPE samples, two consecutive 10 µ-thick sections were retrieved. Frozen fragments were cut and mechanically dissociated. DNA was extracted using the QIAmp DNA tissue extraction kit (Qiagen) according to the manufacturer instructions. Nasal swabs discharged in 200 µl sterile saline buffer and urine were immediately frozen and kept at –20°C until analysis. PBMC were obtained by Ficollpaque centrifugation (Eurobio) of 3 mL EDTA blood, suspended in 200 µl of PBS, immediately frozen and kept at –20°C until analysis. DNA was extracted using the QIAmp blood DNA extraction kit (Qiagen). For tissue material, lysis was extended to 24 h. DNA was eluted in 200 µl elution buffer (Macheret), and concentration was measured by UV-spectrometry .6 copies) of known concentrations of plasmid MCCIC13 which contains 1 copy of the MCPyV genome inserted in the pCR-XL-TOPO vector To detect MCPyV sequences, a sensitive real time PCR assay was designed using primers which encompass a 91 bp fragment of the LT oncoprotein gene, located upstream of the Rb-binding encoding sequence  [7]. AmpTo detect BKPyV and JCPyV sequences, real time PCR assays were designed to amplify a 79 and a 110 bp fragment of the LT antigen coding sequence in BKPyV and JCPyV genome respectively . AmplifiTo characterize integration of MCPyV, we used the DIPS-PCR technique which amplifies junctions between the host and MCPyV genomes as previously described To discriminate integrated versus non integrated forms of the MCPyV genome in patients' samples, we amplified short fragments bracketing each of the characterized integration sites. DNA (100 ng) was subjected to PCR using a forward MCPyV-specific primer located 100 to 300 bp upstream of the integration site, and a reverse downstream primer, specific either to the human integration locus or to the MCPyV non-integrated genome . PCR wasOverlapping fragments of the MCPyV LT encoding gene were amplified using four primer pairs in order to cover the whole second exon of LT , and overall survival relative to the presence or absence of MCPyV DNA in PBMC were estimated by the Kaplan-Meier method. Patients with follow-up under 3 months were excluded from analysis. Survival in complete remission was calculated from the date of diagnosis to the date of first tumour recurrence in patients AWD or died of disease (DOD) at last follow-up. Patients in complete remission were censored at their last follow-up visit. Analyses were performed using the XLStat software . Multivariable analysis of survival in complete remission was done by using a stepwise Cox proportional hazards model that used forward covariate entry to the model. The proportions of patients either CR or AWD, or at stages I or II or III versus IV who had either a MCPyV negative or MCPyV positive PBMC sample were compared using the exact Fischer test. All Thirty nine patients with MCC attended the Dermatology Departments of Bichat and Cochin hospitals. Six patients without retrieved MCC material were excluded from the study. The remaining 33 patients included 14 males and 19 females (sex ratio  = 0.6). Their median age at diagnosis was 77 years (range 39–88). Four patients were immunocompromised, because of corticoid therapy for rheumatoid arthritis, hepatic transplantation, lymphopenia and recurring hairy cell leukaemia. Thirteen (39%) patients had a history of cancer other than MCC (non MCC skin cancer and/or non skin cancer) . Primary−3 to 3.103). Negative results observed in one FFPE section and one fresh frozen sample from primary MCC were confirmed using the previously described LT3 and VP1 PCR assays We analyzed 43 MCC samples consisting of 26 primary MCC  specimens), 14 skin metastasis (12 fresh-frozen and 2 FFPE specimens) and 3 fresh-frozen regional lymph node metastasis samples. Viral DNA was detected in 41/43 samples from 31/33 patients, with a median viral load quantified in 37 samples of 3 copies per cell (range 3.103 copies per sample (range 5–2.106). MCPyV DNA was also found in 22/38 (58%) urine samples from 18/28 (64%) MCC patients, with a median load of 6.102 copies/ml (range 100–4.105). In addition, MCPyV was amplified from 20/49 (41%) PBMC samples from 15/30 (50%) patients with a median load of 102 copies per ml whole blood (range 10–5.104). MCPyV DNA was detected in 1/6 FFPE non MCC cancer samples from 5 patients  nasal swabs from 21/21 patients, with a median load of 3.10patients . We wondP = 0.00083). Moreover, among AWD patients, MCPyV tended to be more frequently detected in patients with distant metastasis than at less advanced stages of the disease . In twenty eight patients with follow up greater than three months, the detection of MCPyV in PBMC was associated with poorer outcome, since patients with at least one positive sample had a median survival of 28 months (95% IC  = 19 to 36) whereas all patients with no detectable MCPyV survived after a median follow-up of 71 months (Kaplan Meier log-rank P = 0.003)  than in patients in complete remission (CR)  . All cliUsing the DIPS-PCR method, we demonstrated the integration of MCPyV DNA in six MCC cases. We first confirmed viral integration in metastatic tissue from two patients, and found virus-host genome junctions identical to those previously reported in their primary tumours We sequenced the whole second exon of LT gene in MCC and non MCC tissues. Fifty-two sequences from 26 patients displayed >99% homology with prototypes MCC350, MCC339, MKL-1 and TKS published sequences Fourteen MCC sequences  were obtained from 12 patients. In two patients, we verified that sequences from distinct metastasis exhibited 100% homology. Mutations which truncate LT were characterized in nine cases, consisting in stop mutations (5 cases), or deletions causing frameshifts which generate stop codons (3 cases) . In the Then, we analyzed 16 nasal and 8 urine sequences. All sequences were complete except three obtained from weak positive samples, probably reflecting sensitivity limits of the method used rather than truncations. Two sequential nasal sequences from the same case showed 100% homology. All full-length sequences were wild-type. None of the 9 non MCC samples from 7 patients who displayed a truncated LT in MCC harboured the tumour-specific molecular signature . These rPBMC sequences were obtained in 14 cases, including partial sequences obtained from 7 weak positive samples. Complete PBMC and MCC sequences from 4 patients were compared . In one Since the discovery by Feng et al who identified MCPyV in 6/8 MCC in vitro and in vivo in models of SV40 and MPyV-induced carcinogenesis Monoclonal integration of MCPyV is viewed as a key element in oncogenesis. We identified in metastatic lesions from two patients the same virus-host genome integration characteristics previously described in their primary tumours, sustaining the hypothesis that viral integration constitutes an early event in MCC oncogenesis The lifecycle of MCPyV in the host is unknown. Serological studies showed that infection is common in the general population and occurs before the third decade Lastly, our results show evidence of high rates of MCPyV DNA in MCC patients' PBMC, in contrast with low rates (0–8%) reported in the serum, whole blood or PBMC of non MCC subjects Altogether, our results provide new insights in the life cycle of MCPyV during MCC pathogenesis. The low number of cases studied might weaken the statistical power of our results. However, we suggest that quantitative and qualitative molecular analysis of MCPyV in tumour and non tumour sites of MCC patients may be a useful tool to characterize their disease stage and manage their follow-up. We are currently designing a prospective study to confirm these results in large series of patients.Table S1Primers and probes used for PCR, DIPS-PCR and sequencing.(0.03 MB XLS)Click here for additional data file.Table S2Non MCC cancer data of MCC patients. FFPE samples were tested for MCPyV DNA. Case number as in (0.02 MB XLS)Click here for additional data file.


Prostate cancer is a leading cause of male cancer specific mortality. When cure by radical prostatectomy is not possible the next line of prostate cancer treatment is androgen deprivation. However prolonged androgen deprivation often results in relapse and androgen-independent prostate cancer that is inevitably fatal despite optimal chemotherapy. The Hedgehog signalling pathway has recently been implicated in prostate cancer development and metastasis. EGFR or ErbB2 expression has been also correlated with androgen independence, shorter survival and metastasis.We determined that the Hedgehog and ErbB signalling pathways are active in circulating tumour cells isolated from androgen-independent prostate cancer patients and in the androgen-independent prostate cancer cell line LNCaP C4-2B. As a basis for synergistic chemotherapy protocols combinations of the Hedgehog specific inhibitor cyclopamine and the ErbB signalling inhibitors gefitinib or lapatinib were tested in this study. Androgen-independent prostate cancer cell growth was inhibited by a SMO inhibitor (cyclopamine) which blocks Hedgehog signalling and by ErbB inhibitors (gefitinib and lapatinib). The isobologram and combination index method of Chou and Talalay was used to evaluate drug interactions. Synergistic antiproliferation effects were observed when the Hedgehog and ErbB inhibitors were combined.Androgen-independent prostate cancer cell proliferation was associated with activity of the Hedgehog and ErbB signalling pathways. Cyclopamine, gefitinib or lapatinib treatment significantly decreased the proliferation of androgen-independent prostate cancer cells. The Hedgehog pathway therefore represents a promising new therapeutic target in androgen-independent prostate cancer. Synergistic effects were observed when Hedgehog and ErbB inhibitors were used together. This study may have clinical implications for improving the treatment of advanced prostate cancer. Prostate cancer is a leading cause of male cancer related deaths  and autoAIPC that arises as a consequence of androgen deprivation therapy may be due to increased activity of the androgen receptor (AR) or cell signalling pathways . Growth The Hedgehog pathway has also recently been implicated in prostate cancer development and metastasis . PatchedTo clarify the role of ErbB and Hedgehog signalling in AIPC we determined that these pathways are active in both circulating tumour cells (CTC) isolated from patients with androgen-independent prostate cancer and in the androgen-independent prostate cancer cell line LNCaP C4-2B. The specific hedgehog pathway inhibitor cyclopamine and the ErbB pathway inhibitors gefitinib or lapatinib significantly decreased the proliferation of androgen-independent prostate cancer cells. A synergistic effect of Hedgehog and ErbB inhibitors on prostate cancer cell growth was also observed, consistent with both Hedgehog and ErbB signalling contributing to the proliferation of androgen-independent prostate cancer cells. The Hedgehog pathway therefore represents a promising new therapeutic target in androgen-independent prostate cancer.p < 0.005), PTCH (p < 0.005) and DD3PCA3   and are therefore androgen-independent, . The use of CTC is advantageous as biopsies are rarely performed on patients at this cancer stage and obtaining CTC is a low risk minimally invasive technique compared to biopsy. Immunofluorescence analysis showed that each prostate cancer patient sample contained more than 5 nucleated, EpCAM positive CTC, which has been associated with a poor prognosis in breast and prostate cancer . No CTC ) Figure , consistin vivo  assay is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form dark blue formazan crystals which are largely impermeable to cell membranes, thus resulting in its accumulation within viable cells. The colour can then be quantified using a simple colorimetric assay. Cells were seeded onto 96 well plates at 2–4 × 1050 doses were determined by exposing cells to various concentrations of the drugs 10-7–10-3M for 72 hours. The medium with drug was aspirated and the MTT assay described above was performed. The IC50 was defined as the concentration of drug at which there was a 50% less growth when compared to control cells. Each experiment was performed in triplicate.Androgen-independent LNCaP C4-2B cells growing in androgen free medium were harvested by trypsinisation, washed and seeded at 30,000 per well in 96 well plates and left to attach overnight. The IC50 across a range of activities and viability was evaluated using the MTT assay at each dosage. Data from cell viability assay were expressed as the fraction of cells inhibited by drug treatments compared with untreated cells. Interaction between pairs of drugs was determined using the Calcusyn computed isolobogram and combination index (CI). The isobologram is a graphical representation of the interaction between two drugs and is formed by plotting the individual drug doses required to achieve a single agent effect on their respective x and y axes, a line connecting the two points is drawn and the concentrations of the two drugs used in combination to achieve the same effect are plotted on the isobologram. Combination data points that fall on the line represent an additive interaction, whereas points above or below represent antagonism or synergy respectively. The CI analysis is similar to the isobologram provides qualitative information on the drug interaction and a numerical CI value is calculated based on the following equation: CI = (D)1/(Dx)1 + (D)2/(Dx)2 + (D)1(D)2/(Dx)1(Dx)2, where (D)1 and (D)2 are the doses of drug 1 and drug 2 that have x% effect when used in combination, and (Dx)1 and (Dx)2 are the doses of drug 1 and drug 2 that have the same x% effect when used alone. The CI indicates synergism when <0.9, antagonism when >1.1 and additivity when 0.9–1.1. The Calcusyn software also calculates the median effect dose (Dm) of each combination, shape of the dose-effect curve (m) and linear correlation coefficient of the median effect plot (r) indicating conformity of date.The isobologram and combination index (CI) were calculated according to the Chou and Talalay median effect principal  using CaThe author(s) declare that they have no competing interests.GS: Experiments . DMP: Experimental design, synergy calculations, manuscript preparation. All authors have read and approved the final manuscript.


The intensity and degree of heterogeneous epithelial marker expression were evaluated immunohistochemically in 29 mucosal biopsy specimens from 7 ulcerative colitis (UC) patients with dysplasia. Biopsy specimens from UC patients with mild (n = 7) or severe (n = 6) inflammation and from histologically normal samples (n = 7) served as controls. HLA-DR showed heterogeneous epithelial expression in all lesions with high grade dysplasia and in 6 of 8 with low grade dysplasia. SC was heterogeneous stained in 17 of 21 lesions with high grade dysplasia and in all but two lesions with low grade dysplasia. In histologically normal mucosa, SC was homogeneously expressed and epithelial DR was virtually absent. In mildly inflamed UC lesions, SC exhibited patchy distribution in only one sample and DR in two, whereas both SC and DR showed a slight degree of heterogeneous expression in all lesions with severe inflammation. Moreover, the overall intensity of SC staining tended to decrease with increasing degree of inflammation, whereas the opposite was seen for DR. Decreased SC and increased DR expression thus seemed to be related to intensified inflammatory activity, whereas heterogeneous expression of these markers was significantly more related to dysplasia.


Surveillance for HIV transmitted drug resistance (TDR) is performed using HIV genotype results from individual specimens. Pyrosequencing, through its massive parallel sequencing ability, can analyze large numbers of specimens simultaneously. Instead of using pyrosequencing conventionally, to sequence a population of viruses within an individual, we interrogated a single combined pool of surveillance specimens to demonstrate that it is possible to determine TDR rates in HIV protease from a population of individuals.The protease region from 96 treatment naïve, HIV+ serum specimens was genotyped using standard Sanger sequencing method. The 462 bp protease amplicons from these specimens were pooled in equimolar concentrations and re-sequenced using the GS FLX Titanium system. The nucleotide (NT) and amino acid (AA) differences from the reference sequence, along with TDR mutations, detected by each method were compared. In the protease sequence, there were 212 nucleotide and 81 AA differences found using conventional sequencing and 345 nucleotide and 168 AA differences using pyrosequencing. All nucleotide and amino acid polymorphisms found at frequencies ≥5% in pyrosequencing were detected using both methods with the rates of variation highly correlated. Using Sanger sequencing, two TDR mutations, M46L and I84V, were each detected as mixtures at a frequency of 1.04% (1/96). These same TDR mutations were detected by pyrosequencing with a prevalence of 0.29% and 0.34% respectively. Phylogenetic analysis established that the detected low frequency mutations arose from the same single specimens that were found to contain TDR mutations by Sanger sequencing. Multiple clinical protease DR mutations present at higher frequencies were concordantly identified using both methods.We show that pyrosequencing pooled surveillance specimens can cost-competitively detect protease TDR mutations when compared with conventional methods. With few modifications, the method described here can be used to determine population rates of TDR in both protease and reverse transcriptase. Furthermore, this pooled pyrosequencing technique may be generalizable to other infectious agents where a survey of DR rates is required. Surveillance of transmitted HIV drug resistance (TDR) is an essential public health component of a comprehensive HIV strategy. Information obtained from TDR surveillance facilitates individual drug selection, where there are many available combinations, and informs drug selection for national treatment programs in resource-limited settingsPyrosequencing provides massive parallel sequencing that can be used to produce complete genome coverage from a conserved sequence or create an array of reads from mixed sequencespol sequences (http://www.bioafrica.net/virus-genotype/html/subtyping.html).Ninety-six anonymized, remnant serum specimens from an IRB approved national HIV TDR surveillance program were used in the analysis. Previous work had shown that the specimen subtypes were 90 subtype B; 4 subtype C, and one each of A1 and CRF02_AG. HIV-1 subtypes were determined using REGA HIV-1 Automated Subtyping Tool based upon 5′-TGAARGAITGYACTGARAGRCAG GCTAAT-3′ and POR 5′-CCTCATTYTTGCATAYTTYCCTGTT-3′ with nested primers PIF 5′- YTCAGARCAGRCCRGARCCAACAGC-3′ and PIR 5′-GGYTCTTGRTAAATTTGRTATGTCCA-3′. All reactions were carried out using standard conditions with annealing temperatures of 53°C. PCR amplicons were then purified and diluted to 15 ng/µl for DNA sequencing using ABI Prism BigDye 3.1 Cycle Sequencing System  following manufacturer's instructions. The sequencing PCR primers included both nested PCR primers and two additional primers PS1 5′-CTGGTGTYTCATTRTTKRTACTAGGT-3′ and PS2 5′-TTYTGGGARGTYCARY TAGGRATACC-3′.HIV-1 nucleic acid was extracted from 200 µl of serum using the Nuclisens EasyMag system  following manufacturer's instructions. HIV-1 protease (PR) and reverse transcriptase (RT), up to codon 236, were bidirectional sequenced with an in-house protocol. Briefly, viral RNA was reverse transcribed and amplified according to the manufacturer's directions using the QIAGEN one-step RT-PCR kit . RT-primers were POF 5′-TCCCTCARATCACTCTTTGG-3′ and PIR-p 5′-GGRTTTTYAGGCCCAATTTT-3′ with an annealing temperature of 59°C. Individual PCR products were purified using Millipore's Amicon Microcon-PCR Centrifugal filter devices , pooled in equimolar amount with Beckman Biomex FX system equipped with a DTX880 Multimode Detector , and sequenced using the GS FLX Titanium pyrosequencing kit (Roche Applied Science. USA). Pyrosequencing of the 96 specimens was conducted using 1/16 the capacity of a full PicoTiterPlate. Overall the reaction produced 42,928 sequence reads. Further analysis was only performed on the 19,106 (44.5%) reads that were: passed by the quality control software; of sufficient read length to cover the amplicon and; successfully mapped to the HXB-2 reference sequence. Based on the approximation of the expected pyrosequencing read length (400∼500 bp) and our template size (462 bp), the mapping criteria was 60% overlapping and 75% matching to the reference.Briefly, a 462 nucleotide fragment containing the entire PR gene was produced by amplifying RT-PCR products using PIF-p The frequency of differences from HIV-1 HXB2 (Accession number: K03455) was calculated for all pyrosequencing reads. The polymorphism and DR rates obtained from the pooled pyrosequencing reads were then compared with the polymorphism and DR rates calculated from the Sanger sequencing reads. Initial comparisons were done for all detected polymorphisms including those present at an extremely low levels. Analysis was repeated on those polymorphisms that were present at frequencies ≥0.2%. This threshold was chosen to be comparable with the sensitivity of Sanger sequencing based on the following assumptions: under ideal circumstances bulk, Sanger sequencing can detect a 20% mixture; the pool of specimens will consist of 100 specimens; thus, a 20% mixture in a single specimen will be present in the 96-member pyrosequencing pool at 0.2%.Conventional sequences were assembled and edited in Seqscape v2.5  with sequence variations identified by aligning the sequences with HXB-2 (Accession number: K03455). All polymorphisms/mutations were confirmed through agreement of bidirectional sequencing results. The 19,106 valid pyrosequencing reads were aligned with HXB-2 and the frequency and distribution of nucleotide and AA changes determined using an in-house Perl script. TDR mutations were identified using a surveillance drug resistance mutation (SDRM) list Phylogenetic analysis was performed on the group of pyrosequencing reads, containing the TDR mutation of interest, in a background of the 96 sequences obtained from Sanger sequencing. Trees were constructed using Neighbor-Joining approach using the K-2-P model with 100 bootstrap replicates as implemented in MEGA 4.0Using conventional Sanger sequencing-based genotypic DR test satisfactory results were obtained from all 96 specimens. Among all of the genotypes, a total of 212 nucleotide (NT) and 81 amino acid (AA) differences from HXB-2 were identified in the PR region. The 19,106 satisfactory pyrosequencing reads (average length: 437) represented an average redundancy of 17,911 for each nucleotide locus. Statistically, each base position of every constituent specimen in the pooled mixture was oversampled 187 times. Analysis of all of the reads demonstrated an aggregate of 1173 NT and 995 AA differences from HXB2. Re-analysis of the data, identifying only those variations present at a level of ≥0.2 revealed 345 NT and 168 AA differences from the reference. The numbers of sequence variations detected and categorized according to the frequency of detection by pyrosequencing are shown in The concordance rates between methods, for detecting NT and AA changes, were proportional to their abundances in the viral population Figure 1Comparison of the measured frequency of variants by pyrosequencing, or the calculated frequency of variants by Sanger sequencing, showed that both methods produced comparable results across a range of frequencies Table 1.Thirteen protease TDR mutations were detected by pyrosequencing at frequencies ranging from 0.20% to 0.64% Table 2.Except for K20R which was detected by pyrosequencing only, 17 clinical, minor protease DR mutations were detected by both methods with extremely high concordance across a broad range of rates Figure 5Successful TDR surveillance programs typically acquire sequencing results from large numbers of antiretroviral naïve subjects and produce an estimate of the percentage of drug resistance based upon the aggregate results. The percentage resistance to protease inhibitors is not described in the context of the individual but instead is attributed to the population under study. Thus far, pyrosequencing of HIV has been used to explore HIV DR in a population of viruses within an individual The findings presented here are strongly supportive of analyzing pooled specimens for the determination of the prevalence of HIV TDR in HIV PR. The concordance of DRM detection when the results from PR sequencing were analyzed is astonishing. At a mutation level detected by pyrosequencing of greater than 5%, there is a 100% concordance of the prevalence of specific NT or AA changes. The overall correlation between all protease DR mutations between methods is similarly impressive. In situations where the mutations were present at frequencies greater than 1%, phylogenetic analysis demonstrated that there were a discrete number of clusters that corresponded to the number of Sanger sequences with that mutation. This finding further demonstrates that the results from pooled pyrosequencing reflect the bulk sequence content of the constituent specimens.Determining the protease TDR prevalence using dendograms derived from the pyrosequencing reads presented an interesting challenge. Pyrosequencing reads of both the SDR mutations, M46L and I84V, identified by Sanger sequencing, were found be tightly clustered and almost exclusively associated with the mutant Sanger sequences. These mutations were detected as 0.29% and 0.34% of the total number of reads consistent with their presence as mixtures in the bulk Sanger sequence. The dendograms for the M46L and I84V mutations detected by pyrosequencing were unique among those SDRM found at frequencies of less than 1%. With further validation, this pattern of clustering may allow one to reproducibly identify those mutations that would also be observed as mixtures in Sanger sequences. We also observed cases where there were only a few mutant pyrosequencing reads but they were related, and they also clustered with a specific Sanger sequence. We believe that the observed mutant reads reflect minority variants undetectable by bulk sequencing methodology. Finally, there were the occasional mutant pyrosequencing reads that were not clearly associated with any single sequences and these may represent PCR artifact only. Further studies will confirm the predictive value of phylogenetic patterns of pyrosequencing reads but one implication is that thresholds for calling mixtures in TDR surveillance need to be standardized.The possibility of falsely elevating the rate of TDR by separately counting TDR mutations arising from a single specimen was addressed in this study. After reviewing 803 pyrosequencing reads with TDR mutations in PR, we found 38 reads that contained more than one drug resistance mutation (DRM) in this region. The reads with more than one mutation were not associated with any one single DRM and occurred in less than 5% of the mutant reads. Thus, the absolute contribution of a multiply drug resistant specimen to the final results is negligible.pol, will require some means of associating individual pyrosequencing reads. In fact in this study, the observation of high rates of non-SDRM protease mutations is entirely consistent with single specimens harbouring multiple DRMs. Methods have been described for constructing haplotypes from pyrosequencing reads using computational methodsFuture studies that would allow for reporting on linked mutations that exist in one coding region, or linked mutations in different coding regions in To further evaluate the application potential of pyrosequencing in HIV DR surveillance, we calculated and compared the material and labor costs for Sanger sequencing and pyrosequencing  and S3. Although pyrosequencing pooled specimens is a promising approach for TDR surveillance, there are limitations to extrapolating the results from our study. The high sensitivity and data throughput of pyrosequencing result in high number of NT variants being detected, many of which exist at low frequencies. The phylogenetic analysis is helpful in resolving this issue but further improvements will be obtained by using the multiplex identifier approach. Second, in order to accurately reflect the true TDR prevalence in the viral population, the PCR amplicons from each component specimen need to be pooled at equimolar concentrations with extreme precision. Any bias introduced at this step can have a significant effect on reported TDR prevalence. Third, although high numbers of sequence reads are generated for each pyrosequencing run, the quality and usability of these reads vary. Stringent sequence alignment criteria are essential to screen out unreliable shorter reads and those containing false insertion/deletion mutations.pol gene making it possible to use this approach for TDR surveillance for mutations in both protease and reverse transcriptase. The significance of minor sequence variants, which exist within individuals in surveillance cohort but below the detection threshold of conventional techniques, remains to be explored. Applications for this technique may be found in other disciplines where surveillance of mutations within a population is required.In summary, we have demonstrated that pooled pyrosequencing of the HIV protease identifies protease TDR mutations with comparable costs to conventional Sanger sequencing. Pyrosequencing results were highly correlated with those produced by conventional Sanger sequencing of individual specimens. Advances in labeling of pyrosequencing specimens will facilitate linkage TDR mutations across the Table S1Comparison of material costs for DR testing of 96 specimens.(0.05 MB DOC)Click here for additional data file.Table S2Comparison of labour cost for DR testing of 96 specimens.(0.05 MB DOC)Click here for additional data file.Table S3Comparison of total cost for DR testing of 96 specimens.(0.03 MB DOC)Click here for additional data file.


The alarming rise in the obesity epidemic and growing concern for the pathologic consequences of the metabolic syndrome warrant great need for development of obesity-related pharmacotherapeutics. The search for such therapeutics is severely limited by the slow throughput of animal models of obesity. Amenable to placement into a 96 well plate, zebrafish larvae have emerged as one of the highest throughput vertebrate model organisms for performing small molecule screens. A method for visually identifying non-toxic molecular effectors of fat metabolism using a live transparent vertebrate was developed. Given that increased levels of nicotinamide adenine dinucleotide (NAD) via deletion of CD38 have been shown to prevent high fat diet induced obesity in mice in a SIRT-1 dependent fashion we explored the possibility of directly applying NAD to zebrafish.Zebrafish larvae were incubated with daily refreshing of nile red containing media starting from a developmental stage of equivalent fat content among siblings  and continuing with daily refreshing until 7 dpf.C. elegans support that future GPR142 antagonists may be effective non-toxic anti-obesity therapeutics.PPAR activators, beta-adrenergic agonists, SIRT-1 activators, and nicotinic acid treatment all caused predicted changes in fat, cholesterol, and gene expression consistent with a high degree of evolutionary conservation of fat metabolism signal transduction extending from man to zebrafish larvae. All changes in fat content were visually quantifiable in a relative fashion using live zebrafish larvae nile red fluorescence microscopy. Resveratrol treatment caused the greatest and most consistent loss of fat content. The resveratrol tetramer Vaticanol B caused loss of fat equivalent in potency to resveratrol alone. Significantly, the direct administration of NAD decreased fat content in zebrafish. Results from knockdown of a zebrafish G-PCR ortholog previously determined to decrease fat content in Owing to the apparently high level of evolutionary conservation of signal transduction pathways regulating lipid metabolism, the zebrafish can be useful for identifying non-toxic small molecules or pharmacological target gene products for developing molecular therapeutics for treating clinical obesity. Our results support the promising potential in applying NAD or resveratrol where the underlying target protein likely involves Sirtuin family member proteins. Furthermore data supports future studies focused on determining whether there is a high concentration window for resveratrol that is effective and non-toxic in high fat obesity murine models. Obesity has been increasing in every state in the nation  and has Several monogenic obesity genes have been cloned in mice including leptin, leptin receptor, carboxypeptidase, melanocortin-4 receptor, and the orexigenic agouti protein (for a review see ). MutatiDanio rerio is currently the simplest model organism complete with the full complement of vertebrate organs that can be used in forward genetic screens. ENU mutagenesis screens have been performed to isolate mutant zebrafish with defects in their temperature maintenance in an attempt to identify genes potentially involved in regulating lipid metabolism , which is GPR142a. A morpholino with sequence CGGGCGTCGTGCCATTGTGCCAGTC was used to target the GPR142a mRNA. As a negative control, three unrelated morpholinos were injected targeting the ortholog to B3 (TATACGAAAATGAGCGACCGTGTTG) and heat shock protein. From 3 to 7 dpf zebrafish larvae were incubated with daily refreshing of nile red before relative quantification by fluorescence microscopy.At least 30 embryos were injected with 2 nL of 0.3 mM (0.6 pmol) anti-sense morpholino per protocols for each of three separate experiments as described previously . Morphols screen . The C. Early development of the zebrafish proceeds quickly to insure greater chance for survival in nature. By just 3 dpf the protruding mouth has been formed. Thus oral delivery of small molecules can be taken into consideration by this stage of development. At 3 dpf zebrafish siblings generally have equivalent amounts of lipid content, thus providing a good baseline for quantifying subsequent changes in lipid metabolism. Volume measurements reveal that over 50% of the volume of the yolk sac is depleted for development from 4 to 7 dpf  and CD36 are classic PPARγ target genes in mammals [2 overnight caused a greater than 10 fold increase in the amount of FABP4 and CD36 transcripts in zebrafish similar to what has been observed in mammals , which similarly activates PPARγ [2 and nicotinic acid), SIRT-1 (resveratrol), beta-adrenergic receptor , or GPR109a (nicotinic acid but not nicotinamide) pharmacology as related to fat metabolism  or SIRT-1 . To determine whether we could visually detect increases in fat we used specific activators of PPARγ  while the other leads to decreases in fat (C. elegans F56B6.5). Knockdown of the zebrafish ortholog to C. elegans F56B6.5 resulted in developmental defects thus suggesting that complete antagonism of this GPCR may be toxic. Knockdown of H09F14.1 however, did cause detectable decreases in fat  and indirect (nicotinic acid) PPARγ agonists caused increased FABP4 and CD36 gene expression in 7 dpf zebrafish larvae  failed to detect changes in zebrafish larvae fat content due to water insolubility (unpublished observations). For testing molecules with poor solubility in zebrafish it is necessary to inject them. This makes the zebrafish assay not as high throughput, but it does work and an examination of the logP of octanol:water partitioning can generally predict whether injection will be necessary [While changes in fat content are visually quantifiable under these ecessary .Of all the molecules tested here, we see the most consistently dramatic effects in reducing nile red detectable fat after treatment with 100 μM resveratrol Fig. . ResveraNewly discovered pathways are now being targeted for therapeutic clinical obesity drug development. Knockdown of zebrafish GPR142a causes a significant decrease in detectable fat without causing overt developmental defects Fig. , thus suWhile it was commonly believed that hormone sensitive lipase is the rate-limiting enzyme in lipolysis, the recent discovery of triglyceride lipases revealed a more complex picture . HormoneOur experiments reveal for the first time that the direct addition of NAD causes a decrease in total fat content Fig. . These rThe authors declare that they have no competing interests.WTP developed the original conceptual framework for the study, performed most of the experiments, and prepared the manuscript. KSJ performed initial experiments developing the zebrafish-based assay. LAW measured cholesterol. RJJ measured total phospholipids and helped prepare the manuscript. APA and HLR performed quantitative real-time PCR. All authors read and approved the final version.


Lentiviral vectors are under intense scrutiny as unique candidate viral vector vaccines against tumor and aggressive pathogens because of their ability to initiate potent and durable specific immune responses. Strategies that alleviate safety concerns will facilitate the clinical developments involving lentiviral vectors. In this respect, the development of integration deficient lentiviral vectors circumvents the safety concerns relative to insertional mutagenesis and might pave the way for clinical applications in which gene transfer is targeted to non-dividing cells. We thus evaluated the potential use of nonintegrative lentiviral vectors as vaccination tools since the main targeted cell in vaccination procedures is the non-dividing dendritic cell (DC). In this study, we demonstrated that a single administration of nonintegrative vectors encoding a secreted form of the envelope of a virulent strain of West Nile Virus (WNV) induces a robust B cell response. Remarkably, nonintegrative lentiviral vectors fully protected mice from a challenge with a lethal dose of WNV and a single immunization was sufficient to induce early and long-lasting protective immunity. Thus, nonintegrative lentiviral vectors might represent a safe and efficacious vaccination platform for the development of prophylactic vaccines against infectious agents. Prevention of infectious diseases through vaccine development is one of the greatest achievements of modern medicine. Nonetheless, considerable challenges remain for the development of new vaccines combining efficiency with enhanced safety profiles. Recently, human immunodeficiency virus (HIV)-1–derived lentiviral vectors have emerged as very promising vaccination tools. These vectors elicit both specific cytotoxic and strong humoral immune responses in several animal models Although lentiviral vector-based vaccines have been shown to elicit unparallel specific and protective immunity against tumor and infectious agents, their transition as vaccinal vectors from preclinical evaluation to clinical development is hindered by their status of integration-competent viral vectors and potential problems linked to the non-specific integration of the transgene in the genome of the transduced cells. However, several arguments point out that lentiviral vectors possess a much safer profile than other retroviral vectors, especially when they are applied to the field of vaccination. Firstly, in a vaccination scenario based on direct injection of Ag-encoding lentiviral vectors, transduced cells that express the relevant Ag become targets of the elicited immune response and are eliminated within a few weeks from the vaccinated organism in vitro different subpopulations of DC. We next investigated whether immunization with nonintegrative lentiviral vector particles was efficient to stimulate a specific and protective humoral immune response against West Nile Virus (WNV), a flavivirus responsible for the largest recognized epidemic of neuroinvasive human disease in North America.In this study, we asked whether integration-deficient lentiviral vectors could initiate a specific and protective immune response. We hypothesized that the induction of Ag specific immune responses in vaccine strategies with lentiviral vectors should not require vector integration, since targeted cells are non-dividing DC. To test this hypothesis, we firstly evaluated the ability of these vectors to transduce We show that integration-deficient lentiviral particles are not only efficient in transducing DC but also that a single immunization with these vectors coding for the secreted form of the WNV envelope can prime humoral Ag-specific responses and confers complete protection against lethal challenge.To test the hypothesis that integration deficient lentiviral vectors could be efficient tools to deliver Ags to non-dividing antigen-presenting cells such as DC, we initially evaluated their transduction efficiency in growth-arrested cells. For this purpose, HeLa cells treated with aphidicolin, a specific inhibitor of cell cycle, were exposed to increasing doses of nonintegrative or integrative lentiviral vectors encoding eGFP and transduction efficiency was determined by flow cytometry. It is important to note that, as previously described As shown in + B220−) and plasmacytoid (pDC) (CD11c+ B220+) and both these DC subtypes are able to stimulate Ag-specific immune responses de novo transcription of the transgene rather than from pseudotransduction ofencapsidated GFP proteins.We next studied the ability of integration-deficient lentiviral vectors to transduce DC. Murine DC are categorized into conventional (cDC)  which possesses neutralizing epitopes and we have demonstrated that these vectors could stimulate a massive production of anti-WNV antibodies WNV ranging from 1 to 100 ng of p24 antigen per mouse (corresponding to 1×105 to 1×107 transduction units). As a control, mice were inoculated with 100 ng of nonintegrative EWNV vector particles inactivated by heating to abrogate their transduction capacities. Three weeks after immunization, pooled sera collected from each group of immunized mice were tested for the presence of total anti-WNV antibodies. As expected, immunizations with 100 ng of heat-inactivated lentiviral vectors (HI 100) were not followed by the production of antibodies . We also included in this challenge experiment a group of mice immunized with 100 ng of integrative EWNV lentiviral vectors as a positive control of protection and another group of mice inoculated with D-PBS as a negative control. As expected, all mice that received D-PBS died within 12 days post-challenge  and were challenged 2-months post-immunization. As shown in WNV vector particles administrated and the degree of protection, with a full protection achieved at a dose of 100 ng of vaccine particles injected. In contrast, a 100% protection was achieved after a single injection of integrative EWNV vector particles regardless the dose. Thus, long-lasting immunity against WNV infection can be achieved with a nonintegrative lentiviral vector-based vaccine.As demonstrated earlier, immunization of mice with integrative lentiviral vectors coding for EWe have demonstrated for the first time that safe nonintegrative lentiviral vectors are not only effective at transducing different subpopulations of DC but also that a single immunization with these vectors was sufficient to afford complete protective immunity against a lethal viral challenge in mice. Transduction efficiency of both plasmacytoid and conventional DC with nonintegrative viral particles was dose dependent and led to the same levels of transduction than those obtained with integrative particles. Although transduction of both growth-arrested cells and DC with nonintegrative lentiviral vectors led to high levels of expression of the transgene, the levels were approximatively 2 fold lower than that achieved with integrative lentiviral vectors.4. However, dose-response experiments revealed that the minimal dose required for the induction of a B cell response was lower with TRIP.I particles compared to the TRIP.NI particles. One possible explanation for this result could be related to the ability of integrative lentiviral vectors to generate a more sustained production of Ag in transduced DC since, theoretically, high expression levels of the Ag in the DC could favor a more sustained presentation of antigenic peptides and may thus explain why low doses of integrative vector particles were sufficient to elicit a specific immune response. An alternative or complementary explanation is that VSV-G pseudotyped particles have a large cellular tropism and thus, may transduce at the site of injection other cell types than DC, including dividing cells. This could result in a more sustained expression of the Ag in vaccination experiments with integrative vector particles. Which cell types are transduced after in vivo injections of lentiviral vector particles and to what extend they are involved in the magnitude of the immune response elicited by integrative and nonintegrative lentiviral vectors is the subject of ongoing research.Immunizations of mice with a single dose of 100 ng of nonintegrative lentiviral vectors coding for a secreted form of the envelope of WNV induced an equivalent level of specific WNV antibodies to that obtained with the same dose of integrative vectors with a mean titer reaching 8×10WNV particles was sufficient to elicit a very early, long-lasting and fully protective immunity against a challenge with a lethal dose of WNV. Kinetic challenge experiments on vaccinated mice revealed that nonintegrative vector-based vaccines conferred a protective immunity as early as one week after a single injection of particles. Although the exact mechanisms involved in this early protection are not fully understood, we have detected specific WNV antibodies one week after immunizations with nonintegrative and integrative vector particles (data not shown). We have previously shown that this early wave of antibodies were exclusively composed of specific IgM, which have been shown to completely protect mice against WNV infection An important result of our study was the demonstration that vaccination with a single dose of 100 ng of nonintegrative EUntil now, potential applications of lentiviral vectors in the field of vaccinology were mainly restricted to therapeutic interventions against fatal diseases such as advanced cancers Even if further works are needed to explore the efficacy of nonintegrative lentiviral vectors especially in the field of specific cellular immune responses, we envision that nonintegrative- based vaccine strategy could represent a very promising platform for the development of vaccines with fewer safety concerns and potent protection immunity.Aedes pseudoscutellaris AP61 cell monolayers. Purification in sucrose gradients and virus titration on AP61 cells by focus immunodetection assay using anti-WNV hyperimmune mouse ascitic fluid were performed as previously described Hela cells, Human 293T cells and African green monkey kidney Vero cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10%  or 5% (Vero cells) heat-inactivated fetal calf serum (FCS), penicillin, streptomycin and Glutamax (GIBCO). WNV strain IS-98-ST1 (GenBank accession number AF 481 864) WNV and TRIP GFP vector plasmids were constructed as previously described The TRIP E6 cells/ml in culture medium consisting of RPMI with 10% FCS, L-glutamine, penicillin, streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, and 50 µM 2-mercaptoethanol supplemented with 100 ng/ml of recombinant mouse FLT3 ligand (R&D Systems).Bone marrow cells were isolated by flushing femurs and tibiae with RPMI supplemented with 10% FCS. Cells were then passed through a 45-µm cell strainer, centrifuged and resuspended in IO Test 3 lysing solution (Beckman Coulter) and incubated at 4°C for 5 min to lyze red blood cells. The cells were centrifuged and cultured for 8 days at 1×10For transduction experiments on non-dividing cells, Hela cells were seeded in 48 wells plates at 40,000 cells/well in the presence of 8 µM of aphidicolin (Sigma). Cells were transduced 24 hours after the aphidicolin block, which was replenished in the medium at the time of transduction. At 2 days post-transduction, cells were harvested and eGFP expression was analyzed by flow cytometry. For DC transduction experiments, FLT3L-generated-bone marrow-derived DC (FL-DC) were transduced at day 6 of the differentiation. At 2 days post-transduction, FL-DC were harvested and resuspended in PBS with 2% FCS and 0.01% sodium azide (staining buffer). Cells were stained with an APC-conjugated anti-CD11c antibody (BD biosciences) and a PerCP-conjugated anti-B220 antibody (BD biosciences), washed twice and analyzed by flow cytometry on a FACSCalibur .WNV vector particles in 0.1 ml Dulbecco's phosphate-buffered saline  supplemented with buffered 0.2% bovine serum albumin . WNV challenge was performed by i.p. inoculation of neurovirulent WNV strain IS-98-ST1 (i.p. LD 50 = 10 focus-forming units (FFU)) as previously described All animal experiments were conducted in accordance with the guidelines of the Office Laboratory of Animal Care at the Pasteur Institute. Six-week-old C57/Bl6 mice were intraperitoneally (i.p.) inoculated with varying doses of TRIP.EMice were bled via the periorbital route and serum samples were heat-inactivated 30 min at 56°C. Anti-WNV antibodies were detected by ELISA, by use of microtitration plates coated with sucrose-purified WNV IS-98-ST1. Peroxydase goat anti-mouse immunoglobulin (H+L) (Jackson Immuno Research) was used at a 1:4,000 dilution as secondary antibodies. The endpoint titer was calculated as the reciprocal of the last dilution eliciting twice the optical density of sera from nonimmunized mice.


Over the past thirty years several reports of the pairing or association of non-homologous centromeres during meiotic prophase have appeared in the literature. Recently, the homology-independent pairwise association of centromeres, termed centromere coupling, was also reported in budding yeast. It seems paradoxical that centromeres would pair with non-homologous partners during a process intended to align homologous chromosomes, yet the conservation of this phenomenon across a wide range of species suggests it may play an important role in meiosis.To better define the role of this phenomenon in budding yeast, experiments were preformed to place centromere coupling within the context of landmark meiotic events. Soon after the initiation of the meiotic program, centromeres were found to re-organize from a single cluster into non-homologous couples. Centromere coupling is detected as soon as chromosome replication is finished and persists while the recombination protein Dmc1 is loaded onto the chromosomes, suggesting that centromere coupling persists through the time of double strand break formation. In the absence of the synaptonemal complex component, Zip1, centromere coupling was undetectable, at all times examined, confirming the essential role of this protein on this process. Finally, the timely release of centromere coupling depends on the recombination-initiating enzyme, Spo11, suggesting a connection between events in homologous pairing/recombination and the regulation of centromere coupling.Based on our results we propose a role for centromere coupling in blocking interactions between homologous centromeres as recombination initiation is taking place. In order to correctly segregate from one another at meiosis I, homologous chromosomes need first to become associated. In most organisms the selection of the segregation partners is based on sequence homology and recombination along the chromosome arms, and not the centromeric regions, as manipulation of homologous chromosomes so that they have non-homologous centromeres has little deleterious effect on their segregation behavior Drosophila and humans are associated with elevated levels of chromosome segregation errors in meiosis In many organisms, including budding yeast, crossing-over is repressed near the centromeres Over thirty years ago experiments in onion meiocytes revealed that the centromeres became organized in non-homologous pairs or small groups, prior to the period of homologous chromosome synapsis Why, in many organisms, do centromeres become coupled with non-homologous partners when meiosis has evolved a series of elaborate mechanisms to ensure the pairing of homologous chromosomes? To help address this question we performed time course experiments to place the period of centromere coupling within the context of other meiotic landmark events, an approach that has not been taken by previous studies. The results favor a new model for the function of centromere coupling.To characterize centromere coupling we compared the kinetics of the formation of non-homologous and homologous centromere pairs in budding yeast cells harvested at intervals from cultures induced to enter meiosis. The kinetochore protein Mtw1 was epitope-tagged, allowing us to score the number of kinetochore foci in spread nuclei. Diploid yeast cells have sixteen homologous chromosome pairs. If the centromeres of the chromosomes are arranged in pairs, each nucleus will exhibit sixteen Mtw1 foci. As described previously I were GFP-tagged at their centromeres (CEN1) CEN1's will be juxtaposed; yielding one single or two closely paired fluorescent dots that co-localize with a single Mtw1 focus. Conversely, centromere coupling has been shown to occur largely between non-homologous partners CEN1 GFP-tags will usually be associated with different Mtw1 foci. In early time points, in chromosome spreads with 12–20 Mtw1 foci, the two copies of CEN1 (GFP) were nearly always associated with different Mtw1 foci . To distinguish between these possibilities both copies of chromosome tw1 foci . Homologtw1 foci . In samptw1 foci . This extw1 foci . Throughtw1 foci . In our tw1 foci . Thus, wtw1 foci . In sometw1 foci . The incCentromere coupling has been shown to require the SC protein Zip1 CEN1's associated with separate Mtw1 foci) exhibited linear SC . Thus, the timely release from centromere coupling depends on the presence of Spo11p.Centromere coupling was originally noted in valuated , spo11Δ.zip1 mutant cells in late meiotic prophase do not exhibit centromere coupling, suggesting that Zip1 is necessary either for the initiation of centromere coupling or its persistence zip1Δ). A requirement for Zip1 in initiation of coupling would predict that coupling would not be observed at any time point, whereas a role in maintenance could result in the observation of centromere coupling at early time points that does not persist until late meiotic prophase. We did not detect centromere coupling in zip1Δ cells at any time point evaluated. Instead, concomitant with the exit from centromere clustering we observed the appearance of chromosome spreads with dispersed centromeres (more than 20 Mtw1 foci) (zip1Δ and 3C). Some of the dispersed spreads we observe in zip1Δ mutants reflect a role for Zip1 in pairing the centromeres of homologous chromosomes in late prophase ZIP1, homologs become aligned, but because Zip1 is necessary for tight pairing of homologous centromeres in late meiotic prophase, their centromeres are often slightly separated and thus visualized as side-by-side Mtw1 foci zip1Δ strain the “dispersed” spreads from the four and five hour time points often feature side-by-side CEN1 GFP dots associated with separate but adjacent Mtw1 foci  ; spo11Δ zip1Δ double mutants transition from the clustered stage to a dispersed organization of centromeres.Previous work (single time point experiments) revealed that  = 5 hr) . The modDetermining the placement of centromere coupling among the landmark events of early meiosis might provide clues to the function of non-homologous centromere associations. Thus we compared the timing of centromere coupling to pre-meiotic DNA replication and the early stages of homologous meiotic recombination.Flow cytometry was used to monitor DNA replication in time course experiments. The emergence of 4n cells coincided with the release of the centromere clustering and the emergence of cells exhibiting centromere coupling . The perFormally, the experiments here do not reveal whether centromeres within the cluster are already organized in couples, though the apparent requirement for Zip1 for coupling and the absence of Zip1 from cells in the clustering stage make this model less appealing. The data do demonstrate that when replication has finished clusters are giving way to dispersed couples. To compare the relative timing of centromere coupling and the early steps of homologous recombination, we asked whether cells involved in coupling have their chromosomes decorated with Dmc1 . Dmc1 loCEN1's rarely precedes initiation of homologous recombination. It makes intuitive sense that the alignment of homologous chromosomes would be led by a homology-based recombination process rather than by elements (centromeres) that interact in a homology-independent fashion. When Dmc1 staining was evaluated on cells undergoing coupling, 71% were found to exhibit clear positive staining . Correct integration was confirmed genetically. The PCYC1-lacI-GFP cassette was inserted as part of pAFS152, a gift from Aaron Straight. Strain genotypes are reported in PCR-based methods were used to create complete deletions of ORFs and epitope-tags Meiotic nuclear spreads were prepared according to FACS analysis and determination of the proportion of 4n cells were performed according to published protocols Table S1Yeast strains used in this study.(0.07 MB DOC)Click here for additional data file.