RNA-Seq is a widely used technology that allows an efficient genome-wide quantification of gene expressions for, for example, differential expression (DE) analysis. After a brief review of the main ...issues, methods and tools related to the DE analysis of RNA-Seq data, this article focuses on the impact of both the replicate number and library size in such analyses. While the main drawback of previous relevant studies is the lack of generality, we conducted both an analysis of a two-condition experiment (with eight biological replicates per condition) to compare the results with previous benchmark studies, and a meta-analysis of 17 experiments with up to 18 biological conditions, eight biological replicates and 100 million (M) reads per sample. As a global trend, we concluded that the replicate number has a larger impact than the library size on the power of the DE analysis, except for low-expressed genes, for which both parameters seem to have the same impact. Our study also provides new insights for practitioners aiming to enhance their experimental designs. For instance, by analyzing both the sensitivity and specificity of the DE analysis, we showed that the optimal threshold to control the false discovery rate (FDR) is approximately 2
, where r is the replicate number. Furthermore, we showed that the false positive rate (FPR) is rather well controlled by all three studied R packages:
, and
. We also analyzed the impact of both the replicate number and library size on gene ontology (GO) enrichment analysis. Interestingly, we concluded that increases in the replicate number and library size tend to enhance the sensitivity and specificity, respectively, of the GO analysis. Finally, we recommend to RNA-Seq practitioners the production of a pilot data set to strictly analyze the power of their experimental design, or the use of a public data set, which should be similar to the data set they will obtain. For individuals working on tomato research, on the basis of the meta-analysis, we recommend at least four biological replicates per condition and 20 M reads per sample to be almost sure of obtaining about 1000 DE genes if they exist.
Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations in SOX2 are ...the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted high-throughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation of ptch1 in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate that PTCH1 mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in either PTCH1 or SOX2.
Background: Early abnormalities in adipose tissue dietary fatty acid storage have been proposed as an important mechanism linking obesity to the development of insulin resistance and type 2 diabetes ...(T2D). Although exercise has been shown to improve peripheral insulin sensitivity, the mechanisms driving these changes and the impact on fatty acid fluxes are not fully understood. Here, we investigated the effects of a single high-intensity interval training (HIIT) session and 12-weeks (3×/week) of HIIT training on postprandial metabolism in women and men (45-75 years-old; BMI > 25 kg/m2) with prediabetes. Methods: Individuals participated in three postprandial metabolic protocols: before training, 18-24 h after an exercise session and after 12 weeks of training. Participants ingested a standard liquid meal (853 kcal; 33% fat, 18% protein, 49% carbohydrates) containing U-13C palmitate and the long-chain fatty acid PET analog 18F-FTHA. Whole-body PET scans were performed 3, 4, 5 and 6 h after consuming the meal to quantify organ-specific dietary fatty acid partitioning and postprandial fatty acid metabolism. Results: Preliminary results demonstrate that there were no significant differences between the three postprandial protocols for suda and 6-h AUG of insulin (n = 9). For circulating lipids, there were no significant difference between baseline and after 12 weeks of exercise in the 6-hour AUG of plasma18F- triglycerides (TG) (-1.0 ± 17.8 %ID/100 mL·6 h),18F- chylomicron-TG (-0.1 ± 13.8% ID/100 mL·6 h) and18F-VLDL-TG (-1.3 ± 4.5 %ID/100 mL·6 h) (n = 13). Evaluation of organ-specific dietary fatty acid partitioning is currently underway. Conclusions: Our full cohort (n = 48) will allow us to better characterize postprandial responses, including the biodistribution of dietary fatty acids, following a single session and after 36 sessions of HIIT in a prediabetic population.
Introduction
The loss of ovarian function and the accompanying decrease in estradiol during menopause has an impact on the metabolic activity of many tissues and organs, resulting in a reduction in ...resting energy expenditure. In rodents, ovariectomy or the absence of the estrogen receptor α suppresses brown adipose tissue (BAT) thermogenesis. It is therefore hypothesized that a reduction in BAT volume and thermogenesis may contribute to the reduction in resting energy expenditure in postmenopausal women.
Objective
The primary aim was to investigate the impact of estrogen on BAT volume and its distribution across the various depots in pre‐ and postmenopausal women.
Methods
A cohort of 22 premenopausal (33 years, 95% CI: 29 to 37 years) and 22 postmenopausal women (63 years, 95% CI: 60 to 67 years) took part in a 3‐hr cold exposure using a liquid‐conditioned suit perfused with 18°C water. During cold exposure, an i.v. bolus of 11C‐acetate and 18FFDG were given sequentially, with each injection followed by a 30 min list‐mode dynamic PET acquisition to quantify BAT oxidative metabolism and glucose uptake, respectively. Finally, a whole‐body static PET acquisition was performed to quantify the whole‐body biodistribution of glucose and estimate BAT volume and the distribution of this BAT volume across the various depots. BAT volume was determined according to the following criteria: 18FFDG ≥ 2 SUVmean and a radiodensity between ‐10 and ‐250 HU.
Results
There was significant variability in BAT volume and its distribution in both pre‐ and postmenopausal women. Premenopausal women had greater total BAT volume (53 mL, 95% CI: 34 to 72 mL) compared to postmenopausal women (21 mL, 95% CI: 8 to 34 mL, P = 0.006), although both groups included individuals with undetectable BAT. In premenopausal women, 65% of BAT was localized in the supraclavicular depot (95% CI: 57 to 73%) and 9% in the paravertebral depot (95% CI: 5 to 13%), with the remaining depots (e.g. cervical, axillary, mediastinal, and perirenal) accounting for 25% (95% CI: 16 to 34%). In postmenopausal women, 45% of BAT was localized in the supraclavicular depot (95% CI: 24 to 66%) while the paravertebral depot represented 32% (95% CI: 12 to 52%) of total BAT. The remaining 23% (95% CI: 5 to 41 %) was distributed among the other depots. Thus, in postmenopausal women, there was a 20% reduction in the relative contribution of supraclavicular depots to the total volume and a 23% increase in the relative contribution of paravertebral depots to the total BAT volume compared to premenopausal women.
Conclusion
Here we show that total BAT volume is lower in postmenopausal women and results in a redistribution of BAT. However, there was tremendous variability in both BAT volume and distribution in both premenopausal and postmenopausal women. Further, it is difficult to distinguish the effects of estrogen from the effect of age or adiposity in the observed differences between our two cohorts.
Background
An inverse relationship between brown adipose tissue (BAT) volume and shivering intensity has previously been reported in humans. Considering its small volume in adult humans, this inverse ...relationship may be explained by the regional distribution of BAT. It has been postulated that the paraspinal depot may be critical to heating the spinal cord to maintain neural conductivity under cold stimulation. However, this local heating may also suppress the drive to shiver, a phenomenon previously shown in guinea pigs.
Objective
The research objective was to determine whether the presence of paraspinal BAT can modulate the intensity and pattern of shivering in lean, healthy, adult humans.
Hypothesis
We propose that paraspinal BAT thermogenesis can supress shivering intensity and modulate the shivering pattern.
Methodology
The present data includes 23 young women who completed a 3h mild cold exposure protocol, using a liquid‐conditioned suit perfused with water at 18°C. During cold exposure, participants remained supine in a PET/CT scanner. An i.v. bolus of 11C‐acetate and 18FFDG were given sequentially, with each injection followed by a 30 min list‐mode dynamic PET acquisition to quantify depot‐specific BAT oxidative metabolism and glucose uptake, respectively. Finally, a whole‐body static PET acquisition was performed to quantify the total BAT volume and distribution. Surface electromyography (sEMG) was used to characterize shivering activity in 8 different muscles. Shivering intensity and shivering pattern were determined using custom‐designed EMG algorithms. In brief, the two distinct shivering patterns (continuous vs burst shivering) are distinguished according to differences in frequency of occurrence (4‐8 Hz for continuous vs. 0.1‐0.2 Hz for bursts) and intensity 2‐5% maximal voluntary contraction (MVC) for continuous vs. 7‐15% MVC for bursts.
Results
Total BAT volume was estimated at 53 mL (95% CI: 34 to 72 mL) with paraspinal BAT volume accounting for 9% (95% CI: 5 to 13%) of total BAT. Mean shivering intensity under this cold stimulus was 3.0 % MVC (95% CI: 2.0 to 3.8 %). Shivering bursts occurred at a frequency of 2.8 bursts/min (95% CI: 2.3 to 3.2 bursts/min), eliciting a shivering intensity of 9.6 % MVC (95% CI: 5.9 to 13.3 % MVC), while the more continuous low‐intensity shivering was maintained at 3.0 % MVC (95% CI: 1.6 to 4.4% MVC). Pearson correlations revealed no associations between total BAT volume, shivering intensity or shivering pattern. Similarly, paraspinal BAT volume was not associated with any shivering outcomes.
Conclusion
The role of BAT in humans is still not clearly elucidated. Here we showed that in premenopausal women, neither total BAT volume nor paraspinal BAT are associated with shivering intensity or shivering pattern. Further studies are required to determine whether BAT plays a more regionalized role, unique to each depot, or more globally on whole‐body energy metabolism.
In rodents, loss of estradiol (E
) reduces brown adipose tissue (BAT) metabolic activity. Whether E
impacts BAT activity in women is not known. BAT oxidative metabolism was measured in premenopausal ...(
= 27; 35 ± 9 yr; body mass index = 26.0 ± 5.3 kg/m
) and postmenopausal (
= 25; 51 ± 8 yr; body mass index = 28.0 ± 5.0 kg/m
) women at room temperature and during acute cold exposure using
Cacetate with positron emission tomography coupled with computed tomograph. BAT glucose uptake was also measured during acute cold exposure using 2-deoxy-2-
Ffluoro-d-glucose. To isolate the effects of ovarian hormones from biological aging, measurements were repeated in a subset of premenopausal women (
= 8; 40 ± 4 yr; BMI = 28.0 ± 7.2 kg/m
) after 6 mo of gonadotropin-releasing hormone agonist therapy to suppress ovarian hormones. At room temperature, there was no difference in BAT oxidative metabolism between premenopausal (0.56 ± 0.31 min
) and postmenopausal women (0.63 ± 0.28 min
). During cold exposure, BAT oxidative metabolism (1.28 ± 0.85 vs. 0.91 ± 0.63 min
,
= 0.03) and net BAT glucose uptake (84.4 ± 82.5 vs. 29.7 ± 31.4 nmol·g
·min
,
< 0.01) were higher in premenopausal than postmenopausal women. In premenopausal women who underwent gonadotropin-releasing hormone agonist, cold-stimulated BAT oxidative metabolism was reduced to a similar level (from 1.36 ± 0.66 min
to 0.91 ± 0.41 min
) to that observed in postmenopausal women (0.91 ± 0.63 min
). These results provide the first evidence in humans that reproductive hormones are associated with BAT oxidative metabolism and suggest that BAT may be a target to attenuate age-related reduction in energy expenditure and maintain metabolic health in postmenopausal women.
In rodents, loss of estrogen reduces brown adipose tissue (BAT) activity. Whether this is true in humans is not known. We found that BAT oxidative metabolism and glucose uptake were lower in postmenopausal compared to premenopausal women. In premenopausal women who underwent ovarian suppression to reduce circulating estrogen, BAT oxidative metabolism was reduced to postmenopausal levels. Thus the loss of ovarian function in women leads to a reduction in BAT metabolic activity independent of age.
Due to organ shortage, clinicians are prone to consider alternative type of organ donors among them donors deceased after circulatory death (DCD). However, especially using these organs which are ...more prone to graft dysfunction, there is a need to better understand mechanistic events ocuring during ischemia phase and leading to ischemia/reperfusion injuries (IRI). The aim of this study is to provide a dynamic transcriptomic analysis of preclinical porcine model kidneys subjected to ischemic stress mimicking DCD donor. We compared cortex and corticomedullary junction (CMJ) tissues from porcine kidneys submitted to 60 min warm ischemia (WI) followed by 0, 6 or 24 hours of cold storage in University of Wisconsin solution versus control non-ischemic kidneys (n = 5 per group). 29 cortex genes and 113 CMJ genes were significantly up or down-regulated after WI versus healthy kidneys, and up to 400 genes were regulated after WI followed by 6 or 24 hours of cold storage (p < 0.05). Functionnal enrichment analysis (home selected gene kinetic classification, Gene-ontology-biological processes and Gene-ontology-molecular-function) revealed relevant genes implication during WI and cold storage. We uncovered targets which we will further validate as biomarkers and new therapeutic targets to optimize graft kidney quality before transplantation and improve whole transplantation outcome.
Microorganisms constitute a reservoir of enzymes involved in environmental carbon cycling and degradation of plant polysaccharides through their production of a vast variety of Glycoside Hydrolases ...(GH). The CAZyChip was developed to allow a rapid characterization at transcriptomic level of these GHs and to identify enzymes acting on hydrolysis of polysaccharides or glycans.
This DNA biochip contains the signature of 55,220 bacterial GHs available in the CAZy database. Probes were designed using two softwares, and microarrays were directly synthesized using the in situ ink-jet technology. CAZyChip specificity and reproducibility was validated by hybridization of known GHs RNA extracted from recombinant E. coli strains, which were previously identified by a functional metagenomic approach. The GHs arsenal was also studied in bioprocess conditions using rumen derived microbiota.
The CAZyChip appears to be a user friendly tool for profiling the expression of a large variety of GHs. It can be used to study temporal variations of functional diversity, thereby facilitating the identification of new efficient candidates for enzymatic conversions from various ecosystems.
Introduction: Due to organ shortage, clinicians are prone to consider alternative type of organ donors among them donors deceased after circulatory death (DCD). However, especially using these organs ...which are more prone to graft dysfunction, there is a need to better understand mechanistic events occurring during ischemia phase and leading to ischemia/reperfusion injuries (IRI). The aim of this study is to provide a dynamic transcriptomic analysis of preclinical porcine model kidneys subjected to ischemic stress mimicking DCD donor.Methods: We compared cortex and corticomedullary junction (CMJ) tissues from porcine kidneys submitting to 60 min warm ischemia (WI) followed by 0, 6 or 24 h of cold storage in University of Wisconsin solution versus control non‐ischemic kidneys (n = 5 per group).Results: 29 cortex genes and 113 CMJ genes were significantly up or down‐regulated after WI versus healthy kidneys, and up to 400 genes were regulated after WI and more 6 or 24 h of cold storage (p < 0.05). Home selected gene kinetic classification, Gene‐ontology‐biological processes and Gene‐ontology‐molecular‐function functional enrichment analysis revealed relevant genes implication during WI and cold storage.Conclusion: We uncovered targets which we will further validate as biomarkers and new therapeutic targets to optimize graft kidney quality before transplantation and improve whole transplantation outcome.
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•We obtain homogeneous DNA transfer in μ-contact printing by dynamic inking process.•The speed of the triple line is found critical in the quality of dynamic inking process.•DNA ...inking time in μ-contact printing (μCP) is not key to inking quality.•DNA consumption is reduced 10-folds.•This method paves the way to μCP as an industrial solution for biochips fabrication.
Microcontact printing (μCP) is used as a patterning technique to produce simple, rapid and cost-effective DNA microarrays. The accuracy of the final transferred pattern drastically depends on the inking step. The usual way to ink a PDMS stamp by droplet deposition of labeled biomolecules using a pipette, results in irregular transfer of the biomolecules on the chip surface and leads to poor and irreproducible fluorescent signals. These drawbacks are likely due to irregular ‘coating’ of the biomolecules on the PDMS stamp. In this work, a novel approach for inking PDMS with DNA molecules is presented. It is based on the continuous displacement of the meniscus formed by the inking solution over the surface of the stamp. When compared with the conventional technique, this dynamic PDMS inking method proved to be very reproducible for producing regular prints/spots on a functionalized glass slide, and this method could be easily extrapolated at an industrial scale.
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