Here, we aimed to establish an efficient protocol for maximizing generation of PDX1+/NKX6.1+ population, we manipulated in vitro culture conditions during differentiation by dissociating densely formed endodermal cells and re-plating them at different densities. These dissociated cells were subjected to an augmented duration of retinoid and fibroblast growth factor (FGF)10 signaling to induce higher PDX1 and NKX6.1 expression.In order to enhance the PDX1+/NKX6.1+ progenitors (~90%) in monolayer, higher than the previously published protocols, as well as upregulated key TFs controlling pancreatic development. The improved efficiency of pancreatic differentiation was complemented by an inhibited hepatic specification and an increased proliferation of NKX6.1+ cells. Interestingly, we were able to enrich a novel PDX1–/NKX6.1+ population by manipulating the re-plating density; these oriented themselves in three-dimensional clusters. Further differentiation validated the ability of our PDX1+/NKX6.1+ progenitors to generate NGN3+ endocrine progenitors.Our optimized protocol dramatically increased the expression of NKX6.1, leading to an increase in the proportion of PDX1+/NKX6.1+ PPs with an elevated self-replicating capacity, thereby aiding scalable production of functional β cells from hPSCs in vitro. Our innovative method also enriches a novel NKX6.1+/PDX1– population, with characteristics of proposed endocrine precursors, allowing further studies on deciphering routes to β-cell development.We provide a novel technique that facilitates appropriate cellular rearrangement in monolayer culture to yield a high proportion of PDX1The online version of this article (doi:10.1186/s13287-017-0759-z) contains supplementary material, which is available to authorized users. Diabetes is a globally widespread disease that exists in two major forms: type 1 diabetes (T1D) and type 2 diabetes (T2D). Both forms of this disease are characterized by loss of pancreatic β cells. T1D is characterized by autoimmune destruction of insulin-producing β cells of the pancreas, whereas in T2D pancreatic β-cell failure is a result of β-cell exhaustion after hypersecretion of insulin to overcome insulin resistance –9. These+/NKX6.1– cells differentiate into poly-hormonal or glucagon-secreting cells [Notably, differentiating hPSCs into pancreatic progenitors that co-express a panel of markers indispensable for inducing a β-cell fate is a key, decisive step for in vitro generation of β cells. Differentiation of the definitive endoderm (DE) into pancreatic progenitors is controlled by pancreatic and duodenal homeobox 1 (PDX1) TF which promotes pancreatic differentiation in concert with other TFs, such as NK6 homeobox transcription factor-related locus 1 (NKX6.1) . When alng cells . Therefong cells –9.http://viacyte.com/clinical/clinical-trials/). Nevertheless, to encounter the issue of scaling up the production of hPSC-derived pancreatic β cells, optimization of in vitro protocols that generate a high yield of the PDX1+/NKX6.1+ population and enhance their proliferative capacity is needed to accelerate their clinical use. While much focus has been assigned to determining the appropriate cytokine cocktail to mimic in vivo development [While the functionality and efficiency of in vitro generated β cells is widely debated , 14–16, elopment , 18, 19,elopment –26.+/NKX6.1+ pancreatic progenitors [+/NKX6.1+ pancreatic progenitors from hPSCs and maximizing their proliferative capacity. We show that dissociation and re-plating of endodermal cells at half their density in monolayer culture, followed by an extended duration of retinoid and fibroblast growth factor (FGF) signaling, specifically promotes NKX6.1 expression. Interestingly, our method also enriched a novel NKX6.1+ population, devoid of PDX1 expression, that may be a new source of pancreatic β cells.More recently, Nostro et al. provided the optimum temporal window and cytokine cocktail for higher induction of NKX6.1 in pancreatic progenitors in adherent culture . Anothergenitors , 28. HerThe H1 human embryonic stem cells (H1-hESCs) and IMR90-hiPSCs were obtained from WiCell Research Institute . Both cell lines were maintained in mTesR1 medium  on Matrigel-coated dishes . Cells were passaged when they reached above 70% confluency by detachment with ReLeSR  and resuspended in mTesR1 containing 10 μM Y-27632 . Cells were supplemented with fresh mTesR1 every day.3  and 1% penicillin/streptomycin as basal media and supplemented with 100 ng/ml hActivin A , 2 μM CHIR99021 , 0.25 mM vitamin C , and 10 μM Y-27632  for day 1 of differentiation (stage 1 day 1). For days 2–3, the basal media were supplemented with 100 ng/ml hActivin A, 0.25 mM vitamin C, and 5 ng/ml basic fibroblast growth factor . On day 4 (stage 2 day 1), the cells for each protocol were either dissociated using TrypLE and re-plated on fresh Matrigel (1:50) coated dishes or left adherent. Basal media as prepared in stage 1 were supplemented with 0.25 mM vitamin C, 50 ng/ml hFGF10 , 0.25 μM CHIR99021, and 50 ng/ml NOGGIN  for 2 days of stage 2 (days 4–5). For all experiments, except when stated otherwise, the dissociated cells were re-plated at densities within the range 2.5–3.5 × 105 cells/cm2, which was about half the endoderm density for the individual experiments. For Protocol 1  and non-dissociated (ND)), stage 3 treatment was performed for 2 days, while for Protocol 2  and non-dissociated (ND)) this was performed for 4 days. For stage 3 differentiation, DMEM  was supplemented with 1% penicillin/streptomycin, 1% vol/vol B27 supplement without vitamin A , 0.25 mM vitamin C, 50 ng/ml hFGF10, 50 ng/ml hNOGGIN, 2 μM retinoic acid , and 0.25 μM SANT-1  for the specified number of days for each protocol. At the end of stage 3, media were changed to DMEM supplemented with 1% vol/vol B27, 0.25 mM vitamin C, 50 ng/ml hFGF10, 50 ng/ml hNOGGIN, 100 ng/ml hEGF, and 10 mM nicotinamide  for 4 days of stage 4 treatment for each protocol  using two previously published protocols with slight modifications in media composition . hPSCs dcol Fig. . For genDifferentiated hPSCs derived using different protocols were washed twice with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 min. The cells were permeabilized for 15 min with 0.4% Triton X-100 in PBS (PBST), and blocked for at least 2 h with 6% BSA in PBST at room temperature. They were then incubated at 4 °C overnight with the following antibodies: mouse anti-SOX17 , rabbit anti-FOXA2 , rabbit anti-OCT4 , guinea pig anti-PDX1 , mouse anti-NKX6.1 , goat anti-SOX9 , mouse anti-NKX2.2 , sheep anti-NGN3 , rabbit anti-Chromogranin A , mouse anti-AFP , E-cadherin , and mouse anti-Ki67-647 conjugate . The cells were washed three times with tris-buffered saline with 0.3% Tween 20 (TBST) and then incubated with the following secondary antibodies: Alexa Fluor 488-labeled anti-guinea pig IgG, Alexa Fluor 568-labeled anti-mouse IgG, Alexa Fluor 488-labeled anti-rabbit IgG, or Alexa Fluor 568-labeled anti-goat IgG . Nuclei were counterstained with Hoechst 33342 . The plates were examined by inverted fluorescence microscopy (Olympus) and the images were processed using the Adobe Photoshop software. The antibody details are listed in Table RNA was extracted using the Qiagen Miniprep RNA extraction kit (Qiagen) and cDNA was synthesized using the superscript™ IV, first strand synthesis system kit . For real-time PCR, we used the SYBR Green-based detection system  and amplification was detected using Quant Studio 7 system  in triplicate for each protocol. Average Ct values for each protocol were normalized to average Ct for P1-ND and fold-change in gene expression for each protocol was determined with respect to P1-ND. Primer details are listed in Table Differentiated cells at specific stages were fixed with 70% ethanol and blocked overnight with 6% BSA in PBST. Cells were stained with the primary antibodies goat anti-PDX1 , goat anti-SOX9 , mouse anti-NKX6.1 , goat anti-NKX6.1 , and mouse anti-Ki67-647 conjugate  for 3–4 h at room temperature. The cells were incubated with Alexa-fluor secondary antibodies  for 40 min at room temperature. The results were analyzed using the BD Accuri C6 flow analyzer and the results were processed using FlowJo.To determine the proliferation of differentiated cells generated from the different protocols, the cells were incubated with BrdU  for 6 h in the differentiation media. DNA synthesis and cell cycle analyses were performed as previously described , 30. Bri+ endodermal cells that were further directed towards PDX1+/NKX6.1+ progenitors  layer. In order to maximize pancreatic specification, it is important to obtain a high proportion of DE. We induced DE differentiation following a previously published protocol with some modifications, as described in Fig. ors Fig. .+/NKX6.1+ pancreatic progenitors, first we reproduced the two protocols established by Nostro et al. [+/NKX6.1+)    . For each of these protocols, we compared the efficiency of pancreatic fate induction between the non-dissociated (P1-ND and P2-ND) and those dissociated (P1-D and P2-D)  maximized the co-localization of PDX1 and NKX6.1 in P2-D generated progenitors  Fig. . HoweverND) Fig. . We notiure Fig. . In orde 1) Fig. . The endD) Figs. . Using iors Fig. . Re-plators Fig. . These nors Fig. , and parors Fig. .Fig. 2Ge+ cells of P2-D compared to other protocols  resulted in the appearance of three-dimensional (3D) structures   Fig. . These r1+) Fig. .Fig. 4Ef–/NKX6.1+ structures co-expressed the endocrine marker NKX2.2, while all of them were negative for the master regulator of endocrine progenitors, NGN3 (manuscript submitted for publication). However, one unexpected observation was the absence of chromogranin A (CHGA) in this population. Our findings strongly suggest that they are a novel pancreatic progenitor population and are currently under characterization (manuscript submitted for publication). These findings indicate that re-plating the endodermal cells at a very low density on fresh Matrigel can generate a novel population expressing NKX6.1 in the absence of PDX1 (PDX1–/NKX6.1+), which may have the potential to generate functional pancreatic β cells.We have evaluated the potential of this population to be specified towards the endocrine lineage. Interestingly, we saw that some of these PDX1Even though the dissociated cells were re-plated in smaller numbers than the non-dissociated cells, we observed that the densities of the cells in P1-D and P2-D were high at the end of stage 4 suggesting an increase in proliferation of the dissociated cells. To investigate whether the improvement in the differentiation efficiency is associated with alterations in the cell proliferation profiles, we analyzed the DNA synthesis and cell cycle at different time points during the differentiation into pancreatic progenitors. To measure the DNA synthesis, the cells were exposed to BrdU incorporation for 6 h. At the end of stage 2 (day 5 of differentiation), flow cytometry analysis for BrdU and DNA content showed an increase in BrdU incorporation in dissociated cells in comparison with those of non-dissociated cells Fig. . Further+/NKX6.1+ population generated from our optimized P2-D protocol using the proliferation marker Ki67. Our results showed that the percentage of cells co-expressing NKX6.1 and Ki67 was higher in P2-D (~89%) in comparison to that of P1-ND (~64%), indicating that our optimized P2-D protocol elevates the proliferative capacity of NKX6.1+ cells , we co-stained them with Ki67 and PDX1. Our results showed that the 3D structures (PDX1–/NKX6.1+ cells) exhibited highly proliferative activity in comparison with other surrounding PDX1+ progenitor cells .We next gauged the proliferative activity of the PDX1lls Fig. . Furtherlls Fig. . To assells Fig. . As expells Fig. . Taken tIn order to further characterize the efficiency of our optimized protocol, we investigated the effect of dissociation on hepatic specification since pancreatic and hepatic progenitors commonly bud from the DE layer. mRNA levels of key hepatic markers such as alpha-fetoprotein (AFP) and ALBUMIN were remarkably decreased in dissociated progenitors (P1-D and P2-D) compared with the non-dissociated protocols (P1-ND and P2-ND) Fig. . Further+/NKX6.1+ cells generated using our protocol (P2-D), we further differentiated the PDX1+/NKX6.1+ progenitors generated from P2-D into stage 5 to generate endocrine progenitors in adherent monolayer culture  density within the novel PDX1–/NKX6.1+ population is in accord with a recent study reporting that NKX6.1+ cells can be produced at low-density culture in the presence of ROCK-NM II inhibitor [The significance of cell density and cell-cell contact in promoting pancreatic differentiation has been previously reported, specifically in regulating endocrine differentiation . A previnduction . Of notenhibitor .–/NKX6.1+ population re-oriented themselves in well-defined, compact, 3D structures. Formation of 3D cell clusters has been shown to contribute to islet function and maturation [+/NKX6.1+ and PDX1–/NKX6.1+ pancreatic progenitors, both of which are proposed to be β-cell precursors.Interestingly, we found that the novel PDX1turation , 33. Expturation , 35. Addturation , stronglNonetheless, re-plating the dissociated endodermal cells on a fresh Matrigel matrix may also have beneficial effects on further differentiation into pancreatic progenitors. The crucial role played by the ECM in directing stem cells towards a specific fate has been previously reported , 38. Mat+/NKX6.1– cells  induced the highest NKX6.1 expression following dissociation of endoderm, which was higher than those treated for only 2 days. This is in contrast to the recent study that showed that NKX6.1 is predominantly induced by a shorter stage 3 treatment, and its expression is lowest in extended stage 3 (4 days) induced progenitors . It is no et al. ) and inso et al. , with thRetinoid and FGF signaling are activators for PDX1 in the pancreas ; therefo+ pancreatic precursors develop at the expense of hepatic progenitors [Both pancreatic and hepatic progenitors originate from DE cells during development and it has been reported that PDX1genitors , 41. In genitors , 18, 42.genitors . This fi+/SOX9+ progenitors [+ cells to enhance the efficiency of obtaining mono-hormonal endocrine cells. An increased fraction of dissociated cells entering S phase facilitated the generation of ~90% NKX6.1+/Ki67+ proliferative progenitors at the end of stage 4 in our optimized protocol. Moreover, few studies have reported the essential role of cell proliferation regulators in enhancing pancreatic differentiation [+/NKX6.1+ proliferative pancreatic progenitors could facilitate their scalable production for transplantation therapy.Expansion of pancreatic progenitors is a necessity for scaling up their production for clinical use. Since pancreatic progenitors are multipotent cells , 41 it igenitors , it is cntiation , 46. The+/NKX6.1+ cells generated by our optimized protocol were able to generate NGN3+/NKX6.1+ endocrine progenitors. The induction of NGN3 expression is essential for pancreatic progenitors to be directed toward the endocrine fate [PDX1ine fate , 41. Speine fate . Our endAlthough the generation of mono-hormonal β cells in vitro has been reported –9, the d+/NKX6.1+ and studying pathways regulating β-cell development and functionality. Indeed, our novel method for maximizing PDX1+/NKX6.1+ progenitors from hPSCs in monolayer culture could serve as a source of highly proliferative pancreatic progenitors, providing a platform for future scalable production of functional β cells in vitro.In conclusion, we demonstrated that manipulating the cell seeding density, cell-cell contact, and cues from the extracellular matrix are essential elements in improving pancreatic differentiation efficiency and proliferation, thereby providing a simplified differentiation method for generating pancreatic progenitors in vitro under adherent culture conditions. Our analyses indicate that pancreatic progenitors produced by our protocol (P2-D) efficiently express key markers necessary for generating functional β cells. Our further work will focus on optimizing endocrine differentiation conditions using P2-D-derived PDX1Additional file 1: Figure S1.+ progenitors derived using P2-D. Scale bars = 100 μm. (JPG 3473 kb)Expression of E-cadherin in pancreatic progenitors generated from different protocols. Double immunofluorescence staining for PDX1 and E-cadherin in pancreatic progenitors generated using different protocols. Note the highest induction of E-cadherin, an epithelial marker, in PDX1Additional file 2: Figure S2.+/SOX9+ in progenitors derived using P2-D. Scale bars = 100 μm. (JPG 2484 kb)Co-expression of NKX6.1 and SOX9 in pancreatic progenitors generated from different protocols. Double immunofluorescence staining for NKX6.1and SOX9 in pancreatic progenitors generated using different protocols. Note the highest induction of NKX6.1


In the nuclei of most mammalian cells, pericentric heterochromatin is characterized by DNA methylation, histone modifications such as H3K9me3 and H4K20me3, and specific binding proteins like heterochromatin-binding protein 1 isoforms (HP1 isoforms). Maintenance of this specialized chromatin structure is of great importance for genome integrity and for the controlled repression of the repetitive elements within the pericentric DNA sequence. Here we have studied histone modifications at pericentric heterochromatin during primordial germ cell (PGC) development using different fixation conditions and fluorescent immunohistochemical and immunocytochemical protocols.We observed that pericentric heterochromatin marks, such as H3K9me3, H4K20me3, and HP1 isoforms, were retained on pericentric heterochromatin throughout PGC development. However, the observed immunostaining patterns varied, depending on the fixation method, explaining previous findings of a general loss of pericentric heterochromatic features in PGCs. Also, in contrast to the general clustering of multiple pericentric regions and associated centromeres in DAPI-dense regions in somatic cells, the pericentric regions of PGCs were more frequently organized as individual entities. We also observed a transient enrichment of the chromatin remodeler ATRX in pericentric regions in embryonic day 11.5 (E11.5) PGCs. At this stage, a similar and low level of major satellite repeat RNA transcription was detected in both PGCs and somatic cells.These results indicate that in pericentric heterochromatin of mouse PGCs, only minor reductions in levels of some chromatin-associated proteins occur, in association with a transient increase in ATRX, between E11.5 and E13.5. These pericentric heterochromatin regions more frequently contain only a single centromere in PGCs compared to the surrounding soma, indicating a difference in overall organization, but there is no de-repression of major satellite transcription.The online version of this article (doi:10.1186/s13072-017-0119-3) contains supplementary material, which is available to authorized users. X in female cells). Facultative heterochromatin is frequently marked by specific histone modifications such as H2AK119Ub and H3K27me3, mediated by the polycomb repressor complexes (PRC) 1 and 2, respectively. Constitutive heterochromatin forms at specific regions of the genome, which are characterized by arrays of tandem DNA repeats: at the centromeres (minor satellite repeats), telomeres (telomeric repeats), and pericentric regions (major satellite repeats). Here we focus on the pericentric heterochromatin. A known hallmark of this chromatin type is the lack of histone modifications that generally mark active chromatin, such as histone acetylation. Conversely, there is an accumulation of repressive histone marks such as H3K9me3 and H4K20me3 [Chromatin is composed of DNA, histones, and other tightly associated proteins. Modifications of the DNA and of histones directly or indirectly control the regulation of DNA-related processes like transcription. Globally, the chromatin in a nucleus can be functionally divided into active and accessible euchromatin and inactive and condensed heterochromatin. Heterochromatin exists in two forms: facultative and constitutive heterochromatin. Facultative heterochromatin is a flexible form of heterochromatin and can be found in various chromosomal regions, when gene-coding regions need to be repressed. Its size varies from gene clusters to an entire chromosome  also undergo genome-wide epigenetic reprogramming, and this occurs between E8.0 and E13.5. It includes changes in histone modifications , reactivation of the inactive  embryos , 24.Initiation of imprint erasure in PGCs takes place between E10.5 and E11.5 , 26, andWe first reanalysed the reported dynamics of H3K9me3  in PGCs Specific histone modifications recruit certain proteins. Members of the heterochromatin protein 1 (HP1) protein family bind H3K9me3 and mark constitutive heterochromatin. In mammals, there are three different HP1 isoforms: HP1α, HP1β, and HP1γ also known as CBX5, CBX1, and CBX3, respectively. We examined the localization of these three isoforms during PGC development. In the regular fixation protocol and using paraffin sections, HP1α immunostaining marked pericentric heterochromatin in E10.5 PGCs. Already at this stage, the signal for HP1α appeared lower in PGCs compared to surrounding somatic cells. Thereafter, HP1α was undetectable in developing germ cells  regions Fig. a. This oOct4 promoter. We isolated RNA and analysed mRNA expression in the FACs-sorted cell fractions, heart tissue, and NIH3T3 cells , due to the advanced developmental stage of the embryo. Embryos were kept in ice-cold PBS at all times, before any further processing.After embryo isolation from the uteri, embryo regions containing the developing germ cells were dissected from E10.5 to E11.5 embryos. Gonads were isolated from the E13.5 embryos, and the sex was determined by morphology. E10.5 and E11.5 gonadal regions were fixed in ice-cold 4% PFA for 2 h and 3 h, respectively, followed by consecutive washes in PBS. Gonads were fixed for 1.5 h in ice-cold 4% PFA. Tissues were then processed for OCT or paraffin embedding using standard histology procedures. Cryo- and paraffin sections were 10 and 5 μm, respectively.For the regular and extended fixation, sections were fixed for an additional 10 min at room temperature or for 30 min at 37 °C, respectively, followed by brief PBS washes. The fixation step was performed after the OCT or paraffin was removed from the sections.Embryo trunks containing the germ cells from E10.5, E11.5 and gonads from E13.5 embryos were dissected, pooled as indicated in figure legends, and incubated in 500 μl TrypLE™ Express (Thermo Fisher Scientific) for 6 min at 37 °C. Dissociation was followed by two washes with 5% FBS in PBS. Spreads of nuclei for immunocytochemistry were obtained as described by .Single plane images at optimal focus were acquired with a Zeiss LSM 700 microscope  with the same exposure time for each nucleus of the same stage. A homemade ImageJ macro was used to measure the mean fluorescence intensity of each pericentric heterochromatin marker used for immunofluorescence, in the whole nucleus (defined by the DAPI-positive area), or in pericentric heterochromatin regions, defined by the area that contained signal above the set background threshold for the corresponding marker, and corresponding to regions more strongly stained with DAPI.® Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Confocal imaging was performed on a Zeiss LSM700 microscope . In this study, the following primary antibodies were used: goat anti-OCT3/4 (N-19) by Santa Cruz (sc-8628) diluted 1:800 for sections and 1:50 for spread preparations, rabbit anti-OCT4 by Abcam (ab19857) diluted 1:250 for sections and 1:50 for spread preparations, rat anti-TRA98 by Abcam , rabbit anti-DDX4/MVH by Abcam , anti-rabbit H3K9me3 by Abcam , rabbit anti-H4K20me3 diluted 1:300 [Heat-mediated (900 W in a microwave for 20 min) epitope retrieval in citrate buffer pH = 6 was performed on paraffin sections. The following staining protocol was performed in all samples. Sections and nuclear spreads were blocked with 2% BSA, 5% donkey serum in PBS (blocking solution) for 30 min at room temperature, followed by primary antibody incubation, diluted in blocking solution, at 4 °C overnight in a humid chamber. The next day, slides were washed in PBS (3 × 5 min) and blocked with secondary antibodies, diluted in blocking buffer, for 1 h at room temperature, in a humid chamber. Slides were then washed in PBS (3 × 5 min) and mounted with ProLonged 1:300 , goat anDrying-down or nuclear spread preparations of germ cells section). Equal numbers of PGCs and somatic cells were isolated using the SORP-FACSAria II flow cytometer (BD). In more detail, 600 cells per cell population were sorted at 4 °C in 40ul lysis buffer  in a 96-well plate containing additionally 2U/μl RNAseOUT . Thereafter, the lysis buffer containing the cells was split into four tubes (two for +RT and two for –RT experiments) and cDNA reactions were performed as described below (RT-qPCR). For NIH-3T3 and heart tissue, RNA was isolated with TRIzol reagent . Thereafter, RNA was treated with Turbo DNAse  according to the manufacturer’s instructions.Female DBA2 mice were mated with OCT4-GOF18/GFP C57BL/6 males to produce F1 fetuses carrying the OCT4-GFP transgene . StagingFor quantitative RT-PCR (RT-qPCR), the lysis buffer  containing the cells was processed for cDNA according to the manufacturer’s instructions. cDNA from NIH-3T3 cells and heart tissue was made with Superscript III  according to the manufacturer’s instructions.® 480 SYBR Green I Master , primers to a final concentration of 0,2 μM and 1 μl of cDNA. The following primers were used: Oct4 Fw CCCCAATGCCGTGAAGTTG and Rv TCAGCAGCTTGGCAAACTGTT, major satellites Fw GGCGAGAAAACTGAAAATCACG and Rv AGGTCCTTCAGTGTGCATTTC [Atrx Fw GAGCTTGACGTGAAACGAAGAG and Rv TTGTTGCTGTTGCTGCTGAG, Actin Fw ACTATTGGCAACGAGCGGTTC and Rv AGAGGTCTTTACGGATGTCAACG.All samples were analysed in triplicate in a 15-μl final reaction volume using the BioRad CFX 384 Real-time System. Each reaction contained LightCyclerTGCATTTC , Atrx FwActin expression according to the 2-ΔCt method. For the major satellites, the Ct values were compared.After an initial hold at 94 °C for 4 min, reaction mixtures underwent 40 cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C. Gene expression levels were normalized over


Dogs have been proposed as spontaneous animal models of human breast cancer, based on clinicopathologic similarities between canine and human mammary carcinomas. We hypothesized that a better knowledge of the natural history and prognostic factors of canine invasive mammary carcinomas would favor the design of preclinical trials using dogs as models of breast cancer.The 2-year outcome of 350 female dogs with spontaneous invasive mammary carcinoma was studied. The investigated prognostic factors included age at diagnosis, pathologic tumor size, pathologic nodal stage, lymphovascular invasion, histological grade, and expression of Estrogen Receptor alpha (ERα), Progesterone Receptor, Ki-67, Human Epidermal Growth Factor Receptor 2, basal cytokeratins 5/6, and Epidermal Growth Factor Receptor. Multivariate survival analyses were performed using the Cox proportional hazards model.The overall survival after mastectomy was 11 months. Within 1 year post mastectomy, 41.5% of dogs (145/350) died from their mammary carcinoma. By multivariate analysis, the significant prognostic factors for overall survival included a pathologic tumor size larger than 20 mm [HR 1.47 ], a positive nodal stage , a histological grade III [HR 1.32 (1.02–1.69)], ERα negativity [HR 1.39 (1.01–1.89)], a high Ki-67 proliferation index [HR 1.32 (1.04–1.67)], and EGFR absence [HR 1.33 (1.04–1.69)].The short natural history of spontaneous canine invasive mammary carcinomas and high rate of cancer-related death allow for rapid termination of preclinical investigations. The prognostic factors of invasive mammary carcinomas are remarkably similar in dogs and humans, highlighting the similarities in cancer biology between both species. Breast cancer represents the most prevalent cancer and the leading cause of cancer death in women worldwide . DespiteAlthough their prevalence decreases in regions where early preventive ovariectomy is routinely performed, canine mammary carcinomas (CMCs) remain the most common canine cancer, with an estimated annual incidence of 182 per 100,000 female dogs . Recent Here, we hypothesized that (1) knowledge of the natural history of CMCs would emphasize the aggressive and short course of the disease, and could be useful for the design of preclinical therapeutic trials in dogs with CMC, as translational models of human breast cancer; (2) knowledge of the prognostic factors of CMCs would highlight the biological similarities between spontaneous CMCs and breast cancers.The aims of this study were thus to describe the natural history of invasive CMCs, i.e., cancer progression and mortality rates, in the largest cohort collected so far ; to describe invasive CMCs using human pathological criteria including immunohistochemical markers; and to validate these criteria as prognostic factors able to predict patients' outcome. In part 2 of this article, we evaluated the prognostic significance of the immunohistochemical classification of human breast cancer applied to dogs.This retrospective study included 350 female dogs with invasive mammary carcinoma, but free from other cancer, initially diagnosed in two laboratories of veterinary histopathology  between 2007 and 2010. The owners’ written consent and approval from the Oniris College of Veterinary Medicine local Animal Welfare Committee were obtained prior to inclusion.Dogs were eligible for inclusion when a histological diagnosis of invasive mammary carcinoma was established and confirmed by an absent layer of p63-positive myoepithelial cells  by immunohistochemistry (IHC) that differentiates invasive from in situ mammary carcinomas . All dogHistological examination was performed on 3-μm-thick hematoxylin–eosin-saffron (HES) stained sections. The 350 tumors were classified according to the human breast cancer classification adapted to dogs  , 47, andThe methods used for IHC were detailed previously . BrieflyERα, PR, and Ki-67 were assessed based on the number of positive nuclei among > 500 neoplastic cells , and considered positive at threshold ≥ 10% for ERα and PR , 49, CK5Negative controls were included in each IHC run, and consisted in replacing the primary antibody with normal mouse or rabbit serum . The positive controls were mostly internal . For HER2, the pathway HER2 4-in-1 control slides (Roche Diagnostics) were chosen to assess the quality of staining for each HER2 score .Four veterinary pathologists  and 1 medical pathologist (DL) examined the HES and IHC slides blindly . In case of discrepancy between evaluators, cases were collectively reviewed in order to achieve a consensual diagnosis, grade, and immunohistochemical scoring.® statistical software  was used. Continuous variables are expressed as median, [range], mean ± standard deviation. Correlations between categorical variables were analyzed using the Pearson Chi-square test. The Kaplan–Meier method and log-rank tests were used for univariate survival analyses, and Cox proportional hazards models for multivariate survival analyses, whose results are reported using the Hazard Ratio (HR), its confidence interval (95%-CI), and the p value of each covariate. For all statistical tests, a p value < 0.05 was considered significant.The MedCalcn = 78, 22.3%) outnumbered Poodles , German Shepherds , Brittany and Labrador Retrievers , and Yorkshire Terriers .The cohort comprises 350 female dogs with invasive CMC, including 253 (72.3%) intact and 97 (27.7%) spayed female dogs. The main characteristics of patients and CMCs are given in Table n = 49; 14.0%) slightly outnumbered multiparous  females. History of contraceptive use was reported in 20 (5.7%) dogs.In 235 dogs (67.1%), the invasive carcinoma was the first mammary lesion detected, whereas 115 (32.9%) dogs had a history of previous non-malignant mammary lesions. Parity was unknown in 269 dogs (76.9%), and nulliparous females  in 256 cases (73.1%), the thoracic mammary glands (M1–M2) in 50 (14.3%), both in 11 (3.1%), and location was unrecorded in 33 cases (9.4%). The most common surgical procedure was radical mastectomy (excision of the 5 mammary glands of the affected side) in 156 dogs (44.6%), followed by regional (M1–M3 or M3–M5) mastectomy in 112 cases (32.0%), and single mastectomy in 64 cases (18.3%). Information on the surgical procedure was missing in 18 dogs (5.1%).n = 227 dogs]; in the other cases, the pathologic tumor size could not be precisely determined due to larger size and/or positive margins. In 236 dogs (67.4%), the pathologic nodal stage was pNX due to absence of lymph node sampling for histopathology. Nodal stage pN+ (with metastasis of any size) was confirmed in 75 cases (21.4%). Six dogs (1.7%) had evidence of distant metastasis (M1) at diagnosis.The mean pathologic tumor size was 18 ± 7 mm .All of the included cases correspond to invasive mammary carcinomas according to breast cancer classification. The predominant histological types were simple tubulopapillary , cutaneous ulceration in 50 cases (14.3%), abdominal or thoracic muscle infiltration in 65 cases (18.6%), peritumoral inflammation in 168 cases (48.0%), and central necrosis in 261 cases (74.6%).n = 203) did not express ERα at all. The mean PR index was 5.4 ± 14.8% (0–92.0%); 65.4% (n = 229) of CMCs did not express PR at all. At positive threshold 10%, 57 CMCs (16.3%) were ER + and 40 (11.4%) were PR+ ; 58.0% of cases . HER2 score 0 was predominant , followed by HER2 score 1 + : the strongest predictor of locoregional relapse was ERα positivity (HR 0.48), followed by the pathological nodal stage pN + (HR 1.92), the presence of lymphovascular invasion, and positive margins (HR = 1.55 for each).By univariate analysis, 11 parameters were significantly associated with the LRR risk Table , of whicThe risk of distant metastasis was 17% at 1 year and 24% at 2 years post diagnosis : the strongest was lymphovascular invasion (HR 2.66), followed by age at diagnosis (HR 2.16 for older dogs), multicentricity (HR 1.89), and the Ki-67 proliferation index (HR 1.0149).By univariate analysis, six parameters were significantly associated with DMFI Table , of whicThe median DFI was 34.4 months. Cancer progression  was recorded in 34% of dogs at 1 year post diagnosis, and 45% at 2 years.p < 0.0001): the pathologic nodal stage pN + (HR 1.92), ERα negativity (HR 1.69), a high proliferation index (HR 1.59), and positive margins (HR 1.54).By univariate analysis, 13 parameters were significantly associated with DFI Table , of whicDuring the follow-up period, 310 dogs (88.6%) died Fig. c. The mep < 0.0001). The strongest prognostic factors were the pathologic nodal stage (pN + : HR 1.89) and pathologic tumor size (pT ≥ 20 mm: HR 1.47), followed by the histological grade, ERα positivity, the Ki-67 index, and EGFR expression (HR 1.32–1.39).By univariate analysis, 16 parameters were significantly associated with OS Table , of whicThe median time to death attributable to cancer was 19.5 months [2 days–56 months] . The most significant predictors of cancer-related death were those that define the stage of invasive CMCs: the pathologic tumor size (pT ≥ 20 mm: HR 1.41), pathologic nodal stage (pN + : HR = 1.82), and the presence of distant metastases at diagnosis (M1: HR 2.61). Peritumoral inflammation (HR 1.54), ERα negativity (HR 1.56), and a high Ki-67 proliferation index (HR 1.67) were also associated with cancer-related death, independently of the stage of the carcinoma at diagnosis.By univariate analysis, 15 clinicopathologic parameters were significantly associated with cancer-related death Table , of whicDogs with invasive mammary carcinomas have been proposed as a useful resource for preclinical research in comparative oncology due to epidemioclinical, biological, and pathological similarities with human breast cancer –11. TherThe epidemiological characteristics of CMCs in this cohort, although in agreement with some previous reports , 33, areAnother particularity of this cohort is the absence of any HER2-positive CMC, as previously reported . HoweverThe natural history of invasive CMCs is much shorter in dogs (54% cancer-related death at 2 years post diagnosis in this study) than in human breast cancer , confirmThe results of the present study confirm that canine invasive mammary carcinomas have a short disease course, which is predictable with clinicopathologic criteria close to those of human oncology. In the second part of this article, we hypothesized that CMCs could be subdivided into luminal and triple-negative cases with different outcomes, as in human breast cancer.


Purpose: The present study aimed to compare four methods of estimating anaerobic energy production during supramaximal exercise.Methods: Twenty-one junior cross-country skiers competing at a national and/or international level were tested on a treadmill during uphill (7°) diagonal-stride (DS) roller-skiing. After a 4-minute warm-up, a 4 × 4-min continuous submaximal protocol was performed followed by a 600-m time trial (TT). For the maximal accumulated O2 deficit (MAOD) method the 2-speed regression relationship was used to estimate the 2 demand during the TT, either including  or excluding  a fixed Y-intercept for baseline 2. The gross efficiency (GE) method (method 3) involved calculating metabolic rate during the TT by dividing power output by submaximal GE, which was then converted to a 2 demand. An alternative method based on submaximal energy cost  was also used to estimate 2 demand during the TT.Results: The GE/EC remained constant across the submaximal stages and the supramaximal TT was performed in 185 ± 24 s. The GE and EC methods produced identical 2 demands and O2 deficits. The 2 demand was ~3% lower for the 4+Y method compared with the 4-Y and GE/EC methods, with corresponding O2 deficits of 56 ± 10, 62 ± 10, and 63 ± 10 mL·kg−1, respectively (P < 0.05 for 4+Y vs. 4-Y and GE/EC). The mean differences between the estimated O2 deficits were −6 ± 5 mL·kg−1 , −7 ± 1 mL·kg−1  and −1 ± 5 mL·kg−1 (4-Y vs. GE/EC), with respective typical errors of 5.3, 1.9, and 6.0%. The mean difference between the O2 deficit estimated with GE/EC based on the average of four submaximal stages compared with the last stage was 1 ± 2 mL·kg−1, with a typical error of 3.2%.Conclusions: These findings demonstrate a disagreement in the O2 deficits estimated using current methods. In addition, the findings suggest that a valid estimate of the O2 deficit may be possible using data from only one submaximal stage in combination with the GE/EC method. It was hypothesized that the GE/EC methods using one submaximal stage vs. four stages would result in similar values of the estimated O2 deficit.Despite clear computational differences between the GE and MAOD methods, only one direct comparison has examined the OEleven male and 10 female junior cross-country skiers  competing at a national and/or international level were recruited from two specialist ski schools. Testing was performed off-season, starting 2 weeks after the last ski competition, while the athletes were still completing ~8–9 h of endurance training and two gym-based training sessions per week. The skiers were instructed to abstain from alcohol for at least 24 h before testing and from caffeine on the day of the trial before testing. All athletes had experience of treadmill roller-skiing and the test protocols as part of their seasonal training and performance monitoring. The study was pre-approved by the Regional Ethical Review Board of Umeå University, Umeå, Sweden and all participants were fully informed of the nature of the study before providing written consent. Additional parental consent was obtained for those under 18 years.2 data, as well as to determine 2max. The 600-m TT was completed ~2.5 h after the pre-test. Participants arrived at the laboratory on the morning of testing in a fed and rested state, having completed only light training on the previous day. They were familiarized to the TT twice in non-experimental training sessions before the test day.Participants were tested twice on a treadmill under laboratory conditions on a single test day, employing the DS cross-country skiing sub-technique during all testing. The first test served as a pre-test to the uphill (7°) 600-m time trial (TT) and was used to obtain baseline and submaximal −1·s−1) or decreased (0.40 km·h−1·s−1) the speed if the athlete moved to the front or rear of the belt, respectively, maintaining a constant speed otherwise  and calibration of the flowmeter was performed at low, medium and high flow rates with a 3-L air syringe . Ambient conditions were monitored with an external apparatus  and the laboratory temperature was 18.0 ± 0.3°C during all testing. Heart rate (HR) was monitored continuously  during all testing and blood lactate concentration was measured from a fingertip sample collected 3 min after the 2 max test .All tests were performed on a motor-driven treadmill designed for roller-skiing . Height and body mass of the participants, as well as the mass of the roller-skis, were measured before testing . Pro-Ski C2 classical roller skis  with a rolling-resistance coefficient of 0.021 ± 0.001 (mean ± SD) were used. The rolling resistance of the skis was determined as described previously by Ainegren et al. . Before 2 measured continuously. The warm-up and first stage were completed at 5.2–7.0 km·h−1 and the treadmill speed was increased thereafter by either 0.8 or 1.0 km·h−1 per stage, up to final speeds of 7.6–10.0 km·h−1. A fixed value for baseline 2 of 5.1 mL·kg−1·min−1 was used as the Y-intercept in the linear speed-2 relationship, based on previous observations made by Medbø et al. . The highest 30-s moving average was used to calculate 2max, maximal ventilation rate, respiratory exchange ratio and maximal HR. The selected speeds during the submaximal test and 2max test were based on sex, ability of the participant and previous test results. After a 2.5-h break participants completed the 600-m TT, which commenced with a 15-min warm-up followed by a 1-min break, then the self-paced TT , a rating of perceived exertion (RPE) was recorded.Following a 4-min warm-up, participants performed 4 × 4-min continuous, submaximal stages at a fixed incline of 7° with ø et al.  using a tot) against gravity and to overcome rolling resistance:External power output (PO) was calculated as the sum of the power exerted to elevate the body mass and skiing equipment , respiratory exchange ratio (RER: 2/2) and gross energy expenditure (Egross), where Egross was calculated according to the equation introduced by Weir  of submaximal roller-skiing was calculated as:The MR was based on the The GE and net efficiency (NE) during the final 30 s of each stage in the submaximal tests were calculated using the following equations:BL is the baseline MR calculated from a fixed baseline 2 of 5.1 mL·kg−1·min−1  included in (4+Y) or excluded from (4-Y) the model. The Y-intercept in the 4+Y model was based on all five data points in the regression . The two regression equations were used to estimate the 2 demand (in mL·kg−1·min−1) at the individual average speed attained during the TT. In addition, a similar method was used where the linear relationship between 2 and speed was modified to a linear relationship between MR  and speed. The required metabolic rate (MRreq) during the supramaximal TT could then be estimated by using the regression equation and converted to a 2 demand by using the following equation:For the MAOD method, the linear relationship between treadmill speed at 7° and time is the time to complete the 600-m TT. Assuming a 100% carbohydrate utilization during supramaximal exercise , using the energetic equivalent for 1 L of consumed O2 according to Weir  by the pre-determined GE according to the criteria described previously by Andersson et al. (r2 < 0.50). If velocity dependency was observed (r2 ≥ 0.50) the equation of the linear regression was used for prediction of the supramaximal GE. In addition, the individual relationship between speed and GE was only considered as being speed dependent if the relative effect of speed on GE was >1.1%  together with an r2 ≥ 0.50. An analogous method to the GE method is the use of submaximal EC (see Equation 4) to estimate the supramaximal MRreq using the following equation  and thedist is the 600-m TT distance. For both the GE and EC methods the MRreq during the TT was converted to a 2 demand (in mL·kg−1·min−1) according to Equation (7). For all methods described, the total accumulated O2 deficit (in mL) was given by subtracting the accumulated VO2 from the accumulated VO2 demand during the TT.where TT2 demands and anaerobic capacities determined from the two MAOD methods  and the GE/EC method. The assumption of sphericity was tested using Mauchly's test. Partial eta squared , Y-intercepts and mean slopes based on the two different linear regressions  were compared with a paired t-test. In addition, the relative and absolute typical error for the comparisons were computed by taking the SD for the pair-wise mean differences  divided by the square root of two.The Statistical Package for the Social Sciences  was used to carry out statistical analyses and the level of significance was set at α ≤ 0.05. Data were checked for normality by visual inspection of Q-Q plots and histograms together with the Shapiro-Wilks analysis and are presented as mean ± standard deviation (SD), except in the case of HR and RPE, where data are presented as median and interquartile range (IQR). One-way repeated measures ANOVA tests with a Bonferroni α correction were used to analyze the 2max of 61.2 ± 7.1 mL·kg−1·min−1 , a maximal ventilation rate of 157.3 ± 33.4 L·min−1, respiratory exchange ratio of 1.17 ± 0.05 and a maximal HR of 202 (IQR = 197–206) beats·min−1. The RPE on completing the 2max test was 19 (IQR = 19–20) and the blood lactate concentration after 3 min was 11.0 ± 2.1 mmol·L−1.The skiers completed the incremental test to exhaustion in 6.5 ± 1.2 min and reached a P < 0.05). The mean ± SD of 2 during the four stages, together with the extrapolated 2 demand during the TT and the associated estimate using the GE model, are displayed in Figure 2 (mL·kg−1·min−1) and speed (km·h−1) for the 4+Y and 4-Y methods demonstrated correlation coefficients of r = 1.00 ± 0.00 and 0.99 ± 0.01, SEE of 0.8 ± 0.4 and 0.7 ± 0.5 mL·kg−1·min−1, Y-intercepts of 4.9 ± 0.1 and 1.4 ± 2.8 mL·kg−1·min−1, and mean slopes of 5.2 ± 0.2 and 5.6 ± 0.3 mL·kg−1·min−1 per km·h−1, respectively .The cardiorespiratory variables, two various concepts of efficiency , together with relative EC at each of the four submaximal speeds, are shown in Table −1 and an average PO of 330 ± 78 W . The maximal HR during the TT was 196 (IQR = 192–200) beats·min−1 with an RPE value reported immediately after the TT of 19 (IQR = 18-19). Based on the estimated 2 demands during the TT, the TT speed/power output corresponded to exercise intensities of 112 ± 5, 116 ± 5, and 116 ± 5% of 2max for the 4+Y, 4-Y, and GE methods, respectively. Applying the EC method resulted in identical estimated O2 deficit values as for the GE method. The 2 demands and O2 deficits calculated using the three methods  are shown in Table The participants completed the TT in 185 ± 24 s, with an average speed of 11.9 ± 1.6 km·h2 deficit estimated with the 4+Y vs. 4-Y method was −6.3 ± 4.9 mL·kg−1, with the 4+Y vs. GE/EC method was −7.2 ± 1.2 mL·kg−1 and with the 4-Y vs. GE/EC method was −1.0 ± 5.3 mL·kg−1, with respective typical errors of 5.3% (3.5 mL·kg−1), 1.9% (0.8 mL·kg−1), and 6.0% (3.8 mL·kg−1).The 95% limits of agreement and scatter plots for the comparisons between the three models are presented in Figure 2 deficit estimated with GE/EC based on the average of four submaximal stages compared with the last submaximal stage was 1.1 ± 2.1 mL·kg−1 . The comparisons between the two MAOD methods (4+Y and 4-Y) using linear extrapolation of the 2-speed relationship (4+Y and 4-Y [2]) and the MR-speed relationship (4+Y and 4-Y [MR]) are presented in Figures 2 deficit estimated with the 4+Y [2] and 4+Y [MR] method was 0.9 ± 0.6 mL·kg−1, and between the 4-Y [2] and 4-Y [MR] method was −2.1 ± 1.0 mL·kg−1, with respective typical errors of 0.7% (0.4 mL·kg−1) and 1.1% (0.7 mL·kg−1).The mean difference between the O1 Figure , with a 2 demand during a 600-m DS roller-skiing TT was 3% lower when a fixed value for baseline 2 was included in the MAOD method  as compared to no inclusion of baseline 2  and the GE/EC methods (methods 3 and 4). The higher Y-intercept in the 4+Y vs. 4-Y method resulted in an 8% lower slope of the regression line. Although the estimated values of O2 deficit between the four methods were highly correlated (r = 0.86–0.99), the limits of agreements ranged from 5 to 21 mL·kg−1 and typical errors ranged from 1.9 to 6.0%, indicating that the different methods should not be used interchangeably. Moreover, since GE/EC was independent of speed, the O2 deficits estimated with the GE/EC methods using one submaximal stage vs. four stages were highly related (r = 0.98) and highly similar (bias of 1 mL·kg−1), as hypothesized.The main findings of the current study were that the estimated supramaximal 2 deficit during isolated knee-extension exercise . This was based on previous findings showing no differences in the estimated 2 demand when using continuous vs. discontinuous protocols  resulted in a lower 2 demand compared with the other methods (2–4). The modeled Y-intercept resulting from the 4-Y method in the current study was 1.4 mL/kg/min, which is considerably lower than the modeled Y-intercept of 4.9 mL/kg/min for the 4+Y method involving a fixed value for baseline 2 of 5.1 mL/kg/min, based on previous suggestions by Medbø et al.  up to high submaximal speeds  resulted in a delta efficiency that was 2.5 percentage points higher than for the 4-Y method, which explains the differences in the estimated 2 demands between the two methods. Although the typical error for the differences in O2 deficits estimated with the 4+Y vs. GE/EC was low (1.9%), and results were highly correlated (r = 0.99), a high systematic bias was observed between the methods with significantly lower O2 deficit values (7.2 mL·kg−1) for the 4+Y method. In contrast to the present findings, Noordhof et al. , which relate to different exercise modalities (cycling vs. DS roller-skiing) and the wider range of submaximal stages (30–90% of 2max) employed in the study by Noordhof et al. . This is supported by previous findings of Green and Dawson . Therefore, the findings of the current study suggest that only one submaximal stage may be needed  when using the GE and/or EC methods to estimate O2 deficit during supramaximal exercise, provided GE/EC is found to be independent of speed. The constant GE/EC observed in the current study for DS roller-skiing at relatively high submaximal exercise intensities , is similar to previous observations of well-trained athletes during cycle ergometry (Ransom et al., The GE method is commonly used for estimating anaerobic work and/or power during supramaximal exercise (Noordhof et al., n et al. . Moreove2 demand and hence O2 deficit. Therefore, future studies should, in combination with 2 measurements, include continuous blood lactate sampling (Björklund et al., 2 demand through the use of energetic equivalents for changes in blood lactate concentration (di Prampero and Ferretti, 2-speed relationship during submaximal exercise. Although a continuous submaximal protocol, as used in the current study, is more time-efficient than discontinuous protocols conducted over several days (Medbø et al., 2 slow component, with the possibility for overestimating the supramaximal 2 demand (Noordhof et al., 2 slow component (Jones et al., 2 deficit will be underestimated, which constitutes a potential limitation of the present study as well as studies using the conventional GE and MAOD methods (Noordhof et al., 2 demand and hence O2 deficit, as proposed by Noordhof et al. (At present, there is no gold standard method for estimating anaerobic capacity during whole-body exercise (Noordhof et al., f et al. .2 deficit calculation minimally when using the GE method.In the current study, external power output was based solely on power causing propulsion similar to previous studies (Sandbakk et al., 2-speed relationship suggested by Medbø et al. (2 deficit estimates were highly correlated. However, the level of bias and relatively high typical errors suggest that the different methods should not be used interchangeably, except the GE and EC methods, which produced identical results and may therefore be used interchangeably. Since GE/EC was found to be independent of speed during DS roller-skiing on a treadmill in highly trained athletes, the current findings indicate that only one submaximal stage may be needed for estimating anaerobic energy production. This could be included in a warm-up before a maximal test, for example, so may from both methodological and practical perspectives be preferable to the traditional MAOD method for estimating anaerobic capacity. However, this novel proposition requires further investigation and the one-stage method requires validation across a variety of exercise modes before it can be recommended as standard practice.In summary, the primary aim of the current study was to compare four different methods of estimating anaerobic energy production during supramaximal uphill DS roller-skiing. Secondary intentions were to introduce new methodological concepts and to compare these with currently existing methods. Although the MAOD method has been suggested as having the potential to indirectly quantify anaerobic energy production (Bangsbo et al., ø et al.  is that EA and KM designed the study and KM collected data. EA performed the data and statistical analyses. EA and KM interpreted the results, wrote the draft and revised the manuscript. EA and KM approve the final version to be published and agree to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.The 5’terminal oligopyrimidine (5’TOP) motif is a DOI:http://dx.doi.org/10.7554/eLife.24146.001 This moppp) cap .The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway plays a prominent role in the control of TOP mRNA translation . In respbona fide trans-acting factor that represses TOP mRNA translation downstream of mTORC1 remained enigmatic.Genome-wide ribosome profiling studies demonstrate that TOP mRNAs are the class of transcripts that are most sensitive to translation suppression by mTOR inhibitors . TOP mRNRecently, we and others  identifi7Gppp) moiety and the invariant first cytidine of TOP mRNAs. Biochemical analyses reveal that LARP1 selectively prevents the binding of eIF4E to the m7Gppp cap to block the assembly of the eIF4F complex on TOP mRNAs. These important findings highlight a previously unrecognized dynamic interplay between LARP1 and eIF4F in the control of TOP mRNA translation and reconcile earlier, seemingly contradictory models of TOP mRNA translation control.Most importantly, our crystallographic data revealed an unexpected, but seminal role for the DM15 region of LARP1 in specialized cap-binding of TOP mRNAs. We show that the DM15 region of LARP1 specifically binds the 7-methylguanosine 5’−5’ triphosphate . The sequence and length choice was informed by the dimensions of the identified RNA binding site in the structure of apo DM15 and the results of nuclease protection assays performed on a complex of DM15 with the first 42 nucleotides of the RPS6 mRNA  of human LARP1 bound to an RNA oligonucleotide spanning a segment of the 5’TOP motif of ribosomal protein S6 (RPS6) mRNA. We selected nucleotides 4–11 of the 42-nucleotide TOP sequence of RPS6 for co-crystallization experiments  of the RNA bound to the protein in the neighboring unit cell . G8 sits7GTP) to 1.8 Å resolution. m7GTP binds the same pocket as G8* in the DM15-RNA co-crystal  a binding pocket complementary to the size and shape of a guanosine that (2) specifically recognizes its Watson-Crick face, and (3) sits upstream of the 5’ terminal cytidine of the bound TOP RNA led us to hypothesize that DM15 binds the cap of TOP mRNAs. To test this, we determined the co-crystal structure of DM15 bound to the cap analog 7-methylguanosine triphosphate  participate in the recognition of the invariant cytidine in the first position of TOP mRNAs (C1) and the guanosine (G8*) 5’ to it . All of 7GTP between two conserved aromatic amino acids, Y883 and Y922. In addition, E886 hydrogen bonds with the Watson-Crick face of the guanosine moiety, using acidic side chains for this recognition in a mechanism reminiscent to that of other cap-binding proteins . Capping the TOP 42-mer RPS6 RNA substrate increases the affinity 360-fold ; 21 nM o7GTP and m7GpppC is increasingly stabilizing to the DM15 fold , 124-fold (2.6 μM), and greater than ~950 fold , respectively . CappingM15 fold ; notablynslation . Interesnslation . Higher-nslation , indicatposition .7GpppG  has been linked to the control of TOP mRNA translation . Moreove7Gppp cap structure (Y883) (To investigate the mechanism by which LARP1 hinders the assembly of the eIF4F complex on TOP mRNAs we generated mutations in two key amino acids (R840 and Y883) that play pivotal roles in TOP mRNA binding (R840 and Y883) and in recognizing the me (Y883) . We compe (Y883) . This fie (Y883) , indicate (Y883) , these d7GpppC motif, a unique feature of TOP mRNAs that encode all the core protein components of the ribosome and the translation apparatus. Cap-binding proteins display a variety of mRNA regulatory functions  cells and plated on LB supplemented with kanamycin. Cells from one ~90% confluent plate were transferred to 500 mL ZY auto-induction media for 3 hr at 37°C and then flasks were transferred overnight to 18°C (The DM15 construct (aa 796–946) from LARP1a (BC001460.2) was expressed, purified, concentrated, and stored as previously reported . Point m to 18°C . Cells w6-MBP-eIF4E was TEV-cleaved overnight in Q/SP start buffer . Cleaved eIF4E was separated from contaminating MBP and TEV by tandem anion and cation exchange chromatography.Cells expressing eIF4E were resuspended in lysis buffer  and homogenized. Lysed cells were clarified by centrifugation and the soluble fraction was incubated with Ni-NTA nickel resin , cat. no. 88221). Resin was washed three times with wash buffer  and eluted in elution buffer . His7GTP , cat. no. M6133) was resuspended at a concentration of 4 mM in the same buffer. DM15 and m7GTP were incubated at a 1:1.3 ratio (850 µM DM15: 1100 µM m7GTP) in 50 mM HEPES, pH 7.0, 50 mM NaCl, 2 mM DTT at room temperature for 30 min. Crystals were grown at room temperature by sitting drop vapor diffusion using a 1:1 ratio of protein to mother liquor solution of 0.1 M HEPES, pH 7.5%, 10% PEG 6000. Crystals were stabilized in the same buffer +5% ethylene glycol and frozen in liquid nitrogen.DM15 was concentrated to 30 mg/mL (1620 µM) for crystallization in DM15 crystallization buffer . mapo DM15 structure (PDBID: 5C0V) as the search model  using a FR-E rotating anode generator and an R-AXIS HTC IP. Data were indexed, merged, and scaled using HKL-2000 . Initialch model . Iteratich model . Ligand ch model . A simul2, 50 mM HEPES, pH 7.0, 25% PEG MME 550 (Hampton Research Natrix HR2-116 #31). Crystals were looped directly from the drop and frozen in liquid nitrogen.DM15 was concentrated to 20 mg/mL for crystallization in 50 mM HEPES, pH 7.0, 2 mM DTT. RPS6 RNA oligonucleotide was synthesized by IDT (5’ CUUUUCCG 3’) and was resuspended in 20 mM sodium cacodylate , pH 6.5. DM15 and RNA were incubated at a 1:1.1 ratio of DM15:RNA with a final concentration of 10 mg/mL DM15 in the presence of 4X binding buffer . This usually resulted in precipitation, so 4 M NaCl was added 1 µL at a time and mixed until the solution cleared . Crystals were grown by vapor diffusion at room temperature by sitting drop using 1:1 ratio of protein-RNA complex to mother liquor solution of 5 mM MgClapo DM15 structure (PDBID: 5C0V) as the search model  was diluted at a concentration of 4 mM in the same buffer. DM15 and m7GpppC were incubated at a 1:1.3 ratio (850 µM DM15: 1100 µM m7GpppC) in 50 mM HEPES, pH 7.0, 100 mM NaCl, 2 mM DTT at room temperature for 30 min. Protein/RNA complex will precipitate quickly at 4°C, so all reagents must be kept at room temperature. Crystals were grown at room temperature by sitting drop vapor diffusion using a 1:1 ratio of protein to mother liquor solution of 0.08 M Magnesium acetate tetrahydrate, 0.05 M Sodium cacodylate trihydrate pH 6.5, 30% w/v PEG 4000 (#25 Index-116). Crystals were looped directly from the drop and frozen in liquid nitrogen.DM15 was concentrated to 26 mg/mL (1405 µM) for crystallization in DM15 crystallization buffer . mX-ray diffraction data were collected using the home source at the Department of Structural Biology (University of Pittsburgh) using a FR-E rotating anode generator and an R-AXIS HTC IP. Data were indexed, merged, and scaled using HKL-2000 . InitialAll figures containing refined models were generated with the PyMOL Molecular Graphics System ).32P]-ATP. To generate capped RNAs, the 5’ triphosphate required for the capping reaction was regenerated in TOP RNAs by incubation of 50 nM RNA with T4 PNK in PNK buffer A (ThermoFisher Scientific cat. no. EK0032) with 3 mM ATP for 20 min at 37°C followed by addition of 5 units of nucleoside monophosphate kinase . RNA was purified by phenol-chloroform extraction, MicroSpin G-25 desalting columns , cat. no. 27-5325-01), and ethanol precipitation. RNAs with a 5’ triphosphate were subsequently capped and radiolabeled using vaccinia capping enzyme , cat. no. M2080S) and [α-32P]-GTP or GTP according to the manufacturer’s protocol.RNAs were in vitro transcribed and purified as described previously . For stuEMSAs were performed and imaged as reported previously using the same amount of RNA (≤200 pM) regardless of labeling efficiency . All RNA7GpppG-RPS6 for 30 min on ice in 8 µL reactions. 2 µL of 5X cold competitor RNA was titrated for final reaction conditions of 20 mM Tris, pH 8, 150 mM NaCl, 10% glycerol, 1 mM DTT, 0.5 µg BSA, 0.5 µg tRNA. Competitions were incubated for an additional 30 min on ice before loading and analyzing on native gels using the same methods as the EMSAs.DM15 was prepared as 5X protein stocks in protein dilution buffer  and prebound to mDM15 or eIF4E 5X protein dilutions were prepared in protein dilution buffer and pre-bound to RNA substrates for 30 min on ice in 8 µL reactions. 2 µL of 5X competitor protein was titrated for final reaction conditions of 20 mM Tris, pH 8, 150 mM NaCl, 10% glycerol, 1 mM DTT, 0.5 µg BSA, 0.5 µg tRNA. Competitions were incubated for an additional 30 min on ice before and analyzing on native gels using the same methods as the EMSAs.The RNA oligonucleotides used for crystallization and biochemical assays were as follows:RPS6 8mer ): 5’ CUUUUCCG 3’RPS6 42mer (Sigma-Aldrich):5’ CCUCUUUUCCGUGGCGCCUCGGAGGCGUUCAGCUGCUUCAAG 3’.G-RPS6 was T7-transcribed from a DNA template to generate the RNA sequence:5’ GCCUCUUUUCCGUGGCGCCUCGGAGGCGUUCAGCUGCUUCAAG 3’.RPL32 was T7-transcribed from a DNA template designed with a self-cleaving 5’ hammerhead ribozyme to generate a 5’ C, resulting in the final RNA sequence:5’ CUCUCUUCCUCGGCGCUGCCUACGGAGGUGGCAGCCAUCUCC 3’.7GTP (1 mM) in 1X EMSA binding buffer and 1X SYPRO orange , cat. no. s5692) in 50 µL reactions at room temperature for 20 min. Protein unfolding was measured by monitoring fluorescence of SYPRO orange at 570 nm during a temperature ramp from (25–95°C). Fluorescence data were analyzed using R-studio and the Boltzmann model to calculate the melting temperature. Three biological replicates were performed for each experiment and averaged. The ∆Tm was determined to be the difference between the Tm of the protein alone and Tm of the protein in the presence of the ligand.100 µM protein was incubated with a saturating amount of GTP, ATP or mSequences were aligned with BLAST  and freqTM and lot no. 59521234), used in 2.HEK293T cells  C-terminally myc- and FLAG-tagged. Typically, 4 to 8 μg of plasmid DNA were used for transfecting HEK293T cells in a 15 cm dish. Cells were transfected with lipofectamine 2000 reagent , cat. no. 11668–019) as per manufacturer’s instructions. Plasmids were expressed for 24 hr prior to lysis in CHAPS extraction buffer containing 40 mM HEPES (pH 7.5 at room temperature), 0.3 (w/v) CHAPS zwitterionic detergent, 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 50 mM sodium fluoride (Ser/Thr phosphatase inhibitor), 1.5 mM sodium orthovanadate (Tyr phosphatase inhibitor), 1 mM DTT, complete EDTA-free protease inhibitors mixture tablets . Briefly, cells were washed once with 10 mL ice-cold phosphate buffer saline followed by 3 mL of CHAPS lysis buffer. Cells were incubated with lysis buffer for 45 min and then scraped and collected in a microfuge tube. Lysates were pre-cleared by centrifugation at 21,000 x g for 1 min at 4°C. Supernatant was collected and 1 mL lysate was used for immunoprecipitation with 10 μL of eIF4G antibody , cat. no. 2469) or 5 μL of FLAG antibody . Lysates were incubated with antibody for 1 hr at 4°C mixing end-over-end. Twenty microliters of bed-volume of protein G-conjugated magnetic dynabeads , cat. no. 10004D) were added to the lysate/antibody mixture and incubated for an additional 30 min at 4°C with mixing end-over-end. Beads were collected with a magnetic rack and washed 3 times with 1 vol (1 mL) of CHAPS extraction buffer described above. RNA was then extracted with RNAzol RT solution . 1 mL of RNAzol was added to the beads/antibody/protein/RNA mixture, followed by the addition of 1 mL of CHAPS lysis buffer. Samples were vortexed for 15 s and incubated at room temperature for 15 min, and then centrifuged at maximal speed on a table-top centrifuge for 15 min at 4°C. The aqueous phase was collected (900 μL) and ethanol precipitated. The total RNA pellet was air-dried and resuspended in 100 μL RNAse-free water  for inputs and in 10 μL RNAse-free water for immunoprecipitates.RNA immunoprecipitation was carried out as follows: HEK293T cells were seeded at approximately 15 million cells per plate on 15 cm plates and propagated in DMEM  supplemented with 10% (v/v) fetal bovine serum  and 1% (v/v) penicillin/streptomycin in a humidified incubator at 37°C and 5% (v/v) COHercules, CA), cat. no. 172–5038) as per manufacturer’s protocol with modifications. Briefly, 4 μL of 5X advanced reaction mix were added to 1 μL advanced reverse transcriptase and 10 μL of RNA template supplemented with RNAse-free water to a final volume of 20 μL. The reaction mixture was incubated at 46°C for 1 hr followed by 95°C for 1 min. The cDNA reaction was then diluted 1000X for eIF4G IPs and 5000X for (LARP1 IPs) in RNAse free water prior to analysis by digital droplet PCR (ddPCR). Each ddPCR reaction was carried out by adding 10 μL QX200 ddPCR EvaGreen Supermix , 0.2 μL of each primer (forward and reverse) at a stock concentration of 10 μM, 8 μL of diluted cDNA, and 1.6 μL RNAse-free water to a final volume of 20 μL reaction. The reaction mixtures were transferred to DG8 Cartridges for the QX100/QX200 Droplet Generator  and 70 μL Droplet Generation Oil for EvaGreen were added . Samples were processed on the droplet generator and then transferred to 96-well ddPCR plates followed by sealing with aluminum foil. Thermal cycling was run using the following conditions: 95°C for 5 min, 95°C for 30 s, ramp down 2°C/s until it temperature reaches 62°C, then ramp up temperature to 95°C; repeat this cycle 45 times. Samples were then cooled to 4°C for 5 min, heated up to 95°C 5 min, and lastly held at 12°C indefinitely. Samples were analyzed on Bio-Rad QX200 droplet plate reader.Reverse-transcription reactions were carried out using the iScript advanced cDNA synthesis kit  and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Alan G Hinnebusch (Reviewer #3).Thank you for submitting your article "La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:7G cap with that of the 5'-TOP sequences, particularly a conserved 5'-terminal C residue. Consistent with these data, ectopic expression of wild type, but not mutant, LARP1 reduces co-IP of two native TOP mRNAs with eIF4G, but not the non-TOP actin mRNA.This is an elegant study that reveals a new and unanticipated mechanism by which LARP1 regulates translation of TOP mRNAs by competing with translation initiation factors for binding to the mRNA CAP. Efficient translation of these mRNAs in nutrient-replete cells depends on mTORC1. LARP1 has been implicated recently as a target of mTORC1 and was previously shown by this group to bind the 5'TOP motif on TOP mRNAs via the DM15 region. In the present manuscript, the authors discover that DM15 also has cap-binding activity by noting fortuitous interactions in their crystal structure of the human LARP1 DM15 domain bound to an RNA fragment. In the crystal, a guanine nucleotide from one LARP-RNA complex bound to a site in a neighboring LARP1 protein that the authors cleverly surmised might mimic the guanine cap of the remaining RNA fragment. This hypothesis is supported by a crystal structure of LARP1 bound to an mRNA CAP analogue, in vitro binding studies demonstrating a preference for capped TOP mRNA and competition studies showing that LARP1 DM15 competes for mRNA binding to the initiation factor, eIF4E. LARP1 is thus a novel cap-binding protein that couples recognition of the mThe three reviewers were enthusiastic about this exciting addition to our understanding of the mechanism by which mTORC1, which regulates LARP1 activity, enhances TOP mRNA translation and thereby stimulates growth. By revealing a previously unknown activity of LARP1, the work represents a conceptual advance in understanding how translation of components of the protein synthesis machinery is regulated.Essential revisions:7GpppC cap. The authors should also cite and discuss results from Fonseca et al., 2015, JBC that support their new model, including the finding that LARP1 overexpression led to a shift of mRNAs encoding ribosomal proteins towards lighter fractions in polysome density gradients, while knocking down LARP1 caused the re-distribution of TOP mRNAs from light to heavy polysomal fractions.The manuscript is somewhat tersely written and would thus benefit overall from more detailed explanations of the authors' logic and relation of their results to prior work. The Introduction needs more details about exactly what has already been shown for LARP1 in terms of inhibiting the translation of TOP mRNAs and mediating the stimulatory effects of mTORC1 on TOP mRNA translation in extracts or cells. The authors should also do a better job of explaining to the general readership how the interactions in the crystal lattice led them to speculate that they were mimicking binding of a capped RNA and how the contacts they observe co-specify binding to the m Essential revisions:7GpppC cap. The authors should also cite and discuss results from Fonseca et al., 2015, JBC that support their new model, including the finding that LARP1 overexpression led to a shift of mRNAs encoding ribosomal proteins towards lighter fractions in polysome density gradients, while knocking down LARP1 caused the re-distribution of TOP mRNAs from light to heavy polysomal fractions.The manuscript is somewhat tersely written and would thus benefit overall from more detailed explanations of the authors' logic and relation of their results to prior work. The Introduction needs more details about exactly what has already been shown for LARP1 in terms of inhibiting the translation of TOP mRNAs and mediating the stimulatory effects of mTORC1 on TOP mRNA translation in extracts or cells. The authors should also do a better job of explaining to the general readership how the interactions in the crystal lattice led them to speculate that they were mimicking binding of a capped RNA and how the contacts they observe co-specify binding to the m7GpppC, which has been included in We have significantly lengthened the manuscript. We include a detailed explanation of the logic used to deduce that LARP1 is a cap-binding protein. We have also expanded the Introduction to more adequately address the contributions of Fonseca et al., 2015 to set the stage for understanding LARP1 in TOP mRNA translation regulation. Finally, we have determined the crystal structure of DM15 bound to m


Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples’ genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing. These fIn this study, we report a new method that simplifies and accelerates nanopore-based short-length DNA library preparation and sequencing, and apply this method to test a panel of normal and aneuploid genomic DNA samples. We were able to accurately screen for aneuploidy using pure genomic DNA in 5 multiplexed samples in under 3 hr, including the time required for library preparation and sequencing. Using this method, the MinION nanopore sequencer can be used for a wide range of applications requiring rapid short-read DNA sequencing.Normal male (NA12877), normal female (NA12878), Monosomy X (NG08006), trisomy 21 (NG05397) and trisomy 18, 15s+ (NG08016) genomic DNA (gDNA) from the Coriell Institute were used for development and testing of this protocol. The study was approved by the Institutional Review Board of Albert Einstein College of Medicine and the Institutional Review Board of Columbia University Medical Center and complied with Coriell Institute NIGMS Human Genetic Cell Repository and NIA Aging Cell Culture Repository policies.Table S1)  were added to each ligation reaction and incubated at 25° for 10 min (http://imagej.nih.gov/ij/) densitometry analysis with 2 technical replicates  and 2 pmol of a 34 bp adapter with a 3′-AGCAAT and 5′ blunt-end (ME-6bp) were used (r 10 min . Each liplicates .Table S1). 4 uL 200 nM MP1, 4 µL 200 nM ME-A adapters, and 2 µL 2 mM Tris-Cl, pH 8 were subjected to each ligation condition as described above (The efficiency of the TA ligation was estimated by the same method as 6-bp sticky-end ligation but using a different pair of adapters that had 3′ T/A overhangs (MP1-T and ME-A) (ed above .http://imagej.nih.gov/ij/) (Although the 12 native barcoding (NB) adapters in a 1D native barcoding kit  are designed to be supplied at 670 nM , the actual concentration of each NB adapters in a kit can vary from 2-13 ng/µL. Thus, the DNA content of each NB adapter was quantified using the Qubit dsDNA HS assay . To determine the right amount of NB adapter needed in a 1-step ligation reaction, a titration ligation experiment was performed. 6.5 ng, 9.8 ng, or 13 ng NB adapters were added to a 21.2 µL ligation reaction mixture containing 0.2 pmole dA-tailed 434bp, 1.6 pmole MP1-6bp, 10 µL Blunt/TA ligase master mix, and 1.2 µL enhancer mix  in 2 mM Tris-HCl. Each ligation reaction mixture was incubated at 25° for 10 min, and purified using a DNA Clean & Concentrator-5 column as described above. The purified ligation products were analyzed using 3% agarose gel electrophoresis and ImageJ densitometry analysis (gov/ij/) .pCR-Blunt vector  using Q5 High-Fidelity DNA Polymerase  (Table S1) as reported previously  in a clean 8-well 0.2 mL PCR strip and incubated for 5min at 20° for end-repair reaction, and then 5 min at 65° for dA-tailing reaction. 3 conditions were tested to optimize end-repair/dA-tailing: 1) ∼0.5 pmol DNA (∼162.5 ng) in 60uL total reaction mixture; 2) ∼0.5 pmol DNA in 30 µL total reaction mixture with addition of 0.9 µL 100 mM dATP before the 65° incubation; 3) ∼0.5 pmol DNA control fragment in 30 µL reaction mixture with addition of 0.9 µL 100 mM dATP and 1µL Bst 2.0 WarmStart DNA polymerase  before the 65° incubation. Each reaction mixture was then purified using a DNA Clean & Concentrator-5 column  following the manufacturer’s protocol as described above.Table S1). The adapter was diluted to 0.4 μM in MinION adapter buffer , and ligated to the dA-tailed control fragment in a 10:1 ratio as previously reported (http://imagej.nih.gov/ij/) (The efficiency of end-repair/dA-tailing of each of the 3 conditions was assessed using a 57-bp asymmetric adapter with a 3′T overhang (MP1-T) (gov/ij/) .Table S1) and 100 ng of a 50 bp ladder  were mixed in Buffer EB  to make a 60 µL reaction mixture.10% PEG 8000 (PEG) wash buffer  was prepared following the protocol in the Oxford Nanopore MAP003 kit. 9%, 8.5%, and 8% PEG buffers were also prepared to determine which concentration would most efficiently remove excessive adapters through AMPure XP bead purification . PEG wash buffers were stored at 4°. 2 pmole (∼260 ng) of a 204 bp DNA fragment  and purified according to the manufacturer’s protocol on a 96-well magnet plate . However, in the wash steps, the 10% PEG wash buffer described above was substituted for the 70% ethanol used in the manufacturer’s protocol. After two washes, the beads were resuspended in 20 µL Buffer EB  and incubated at 37° for 5 min. The resuspended beads were allowed to pellet on the magnet plate, and the eluate was carefully transferred to a 1.5 mL low retention tube . The AMPure XP bead purified products were analyzed with 2% agarose gel electrophoresis .To determine the optimal PEG concentration in PEG wash buffer to remove extra adapters, AMPure XP bead purification was performed using a 0.62:1 beads: sample ratio and washed in each PEG wash buffer described above .To test the efficiency of adapter removal for multiple reactions, 8 reaction mixtures were purified by 0.62X AMPure XP bead purification and washed in 8% PEG buffer as described above. The efficiency was not altered (data not shown).At the end of library preparation, each 1D barcoding sequencing adapter (BAM)  must be annealed to tethering oligonucleotides (tethers), which carry a hydrophobic group on their 5′end, included in the elution buffer (ELB) . This process is called tethering in the manufacturer’s protocol. Tethering in ELB buffer can assist the barcode sequencing adapters (BAM) to reach the nanopores faster. Motor proteins are pre-attached to BAM adapters. When a BAM adapter reaches a nanopore, the motor protein can unzip dsDNA into ssDNA and drive the resulting DNA strand through the nanopore at a fixed speed. The ideal tethering condition was determined by mixing the BAM adapter with ELB buffer in 1:1, 1:2, 1:3, and 1:4 ratios and incubated at 37° for 3 min. The BAM adapter was completely tethered at a 1:2 BAM: ELB ratio . TetheriEach optimized step was combined to form our rapid multiplex MinION sequencing library preparation. ∼500 ng gDNA was fragmented with a Covaris microTUBE using the manufacturer’s default 500-bp setting. ∼0.5 pmole of fragmented gDNA (165 ng ∼500-bp DNA) was subjected to a 30 µL end-repair/dA-tailing reaction using the NEBNext Ultra II End Repair/dA-Tailing Module  as detailed above with addition of 3 mM dATP after 5min incubation at 20°, followed by 5 min incubation at 65°. A Select-a-Size DNA Clean & Concentrator  was used to purify each end-repair/dA-tail reaction following the manufacturer’s protocol. 156 µL size selection mix  was added to each end-repair/dA-tailing reaction tube and gently mixed by pipetting. The size-selected DNA was then eluted in 12 µL 2 mM Tris-Cl, pH 8.A 1D native barcoding kit  and a 1D Ligation Sequencing Kit  were used for library preparation. As the native barcoding (NB) adapters in each kit  can vary in concentration, the DNA content was quantified using the Qubit dsDNA HS assay . 6-10 ng NB adapter were used for each adapter ligation reaction.In each 1-step ligation reaction, 15 µL ligation components , 15 µL Blunt/TA ligase master mix, and 3.6 µL enhancer mix were mixed thoroughly. The mixture was then incubated at 25° for 10 min. Two or more ligation reactions can be performed for 1 sample using 1 barcode to increase the final library concentration. In run 1, 1 NB adapter was used to barcode 1 sample and 4 ligation reactions were prepared for this sample; In run 2 and run 3, 5 NB adapters were used to barcode 5 sample and 2 ligation reactions were used for each sample . 26.4 µL12 µL of the library was loaded into a MinION MIN106 flow cell for sequencing following manufacturer’s protocol. MINKnow v1.3.30 software and run protocol NC_48Hr_sequencing_Run_FLO-MIN106_SQK-LSK108_plus_Basecaller were used to control and monitor the sequencing in real time. To maximize the data output for rapid MinION sequencing, the data acquisition was restarted after a 1 h run, and stopped when sufficient data were generated. A 30 min sequencing run generated enough reads for 1 sample; a 1-3 h sequencing run generated enough reads for 5 barcoded samples. The average number of real-time pores used for strand sequencing was 114-145 as monitored on the MINKnow software and the total number of pores generated sequences was >500 (data not shown).http://icebert.github.io/pblat/) using the parameters stated in Table S2 (Table S2). A new alignment software, minimap2, was also evaluated (Table S2)  p(x > 1.5 λ) = 0.0008; the type II error for false negatively detecting a 50% changes pβ(x’ < 1.5 λ) = 0.0021 and the type II error for false negatively detecting a 30% changes pβ(x’ < 1.3 λ) = 0.04 as estimated by ppois function in R. UA reads aligned to each chromosome were summarized and analyzed using the modified Z-score method to identify normal diploid and aneuploid chromosomes as reported previously (Table S3)  from each sample were used for aneuploidy detection analysis. Given 9,000 UA reads, >41 UA reads were assigned to chrY in a normal male sample. Data analysis and statistical analysis were performed in R version 3.4.0  and normal female (NA12878) from 3 sequencing runs in this study are available on European Genome-phenome Archive (EGA) with the accession number EGAS00001002650. The bed files of all each individual containing no personally identifying genetic information (PIGI) are available upon request.In this study we developed a rapid and practical protocol for preparing and sequencing a 1D multiplex genomic DNA short-read sequencing library using a nanopore sequencer. This was achieved by systematically and quantitatively evaluating and optimizing each module in the library preparation workflow. This provided robust reaction conditions in each module, and reduced required library preparation time from 2-4 h to ∼45 min and sequencing time from 1-4 h to <30 min when compared to the original protocol   DNA Table S1) with a 6bp overhang and a 34 bp adapter (ME-6bp) (Table S1) were mixed with the complementary 6-bp overhang in a 1:1 ratio and subjected to a range of ligation conditions (Table S1) was mixed with a 3′T overhang and a 20 bp adapter (ME-A) (Table S1) was mixed with the complementary 3′A overhang in a 1:1 ratio and both mixtures were subjected to the ligation conditions described above . In a 20 µL TA ligation reaction, addition of 6–12% volume of enhancer mix further reduced the percentage of unligated MP1 from 12.5 to 6.8–9.3%. Addition of 6–12% volume (1.2 to 2.4 µL) of enhancer mix further reduced the percentage of unligated MP1-6bp from 4 to ≤ 3% , and subjecting them to the optimized ligation method determined above. Using the same ligation conditions, TA ligation efficiency was affected by the proportion of fragments that had successfully been end-repaired and dA-tailed. The manufacturer’s Ultra II End-RepairR/dA-Tailing Module protocol did not provide comparable dA-tailing efficiency to that reported in our previous study  combines end repair and dA-tailing in one reaction. We evaluated the efficiency of this product by mixing dA-tailed DNA samples with 10-fold diluted MP1 adapter . The size selection and purification can be performed in ∼7 min.The second purification was performed after the ligation reaction. This purification step is gentle to protein and efficient in removing excess adapters. AMPure XP beads with PEG-based wash buffer were used to protect the motor protein attached to the BAM sequencing adapter. We first used 0.62-fold equivalent AMPure XP beads to achieve size selection for fragments <400 bp after the purification, but a noticeable amount of ∼200-bp adapter (BAM+NB) was retained after the bead purification. 1.2 to 2 pmol NB and BAM adapters were ligated to 0.2 pmol DNA fragments in a ligation reaction. To determine the optimal AMPure XP bead purification condition the purification efficiency was evaluated by mixing 100 ng 50 bp ladder and 2 pmol 204bp DNA fragment, and subjected to different purification conditions , 2E. oneTo concentrate the oligonucleotide to be sequenced at the bottom of the sequencing flow cell where the nanopores are located, the BAM adapter needs to be tethered to an oligonucleotide in the ELB buffer that attached to a hydrophobic group. Using Dynabeads MyOne Streptavidin C1 bead-based 2D library purification , the library was eluted from the beads in ELB buffer at 37° for 10 min  wei and. This doFigure S1). The mean Q-score is ∼9-10 . The platform generated ∼70K raw reads per hour. 55–80% reads could be assigned to a unique barcode, and 92–95% reads with a barcode could be aligned to a unique genomic location . Analyses can be performed in parallel in 30min given sufficient computational resources. A new nanopore sequence aligner, minimap2, can be a potential substitute in to perform data analysis in 10min with less computational resources at the cost of 5–6% loss in UA reads for downstream analysis (Table S2).This method does have limitations. During development, we evaluated the time needed using pure genomic DNA. In actual lab or clinical applications, time used to obtain sufficient DNA needs to be added. It takes ∼1 h to extract gDNA from body fluids and cell lines, and 2-3 h to extract gDNA from tissues, and longer for difficult samples such as fixed tissue and slides. Time and computational resources are also needed for data analysis. In the current pipeline, ∼30 min is needed to analyze each sample -based methods . ULCS-bae.g., 69, XXX). A Karyotype or SNP-based approaches is more reliable in polyploidy detection at this point ), preimplantation genetic screening (PGS) of embryos after in-vitro fertilization (IVF), and evaluation of miscarriage tissue (We then successfully used this method for aneuploidy testing in genomic DNA samples. Using this method, up to five samples can be multiplexed to produce sufficient sequencing data for aneuploidy detection in 1-3h on a single flow cell, including library preparation time. It also takes additional 1-3h for gDNA extraction from human tissues or body fluids, and 30min for computation analysis in the development phase. Faster computation analysis can be achieved by allocating more computational resources and performing analysis on a SSD drive, or using newly developed software. Fetal aneuploidy testing is routinely performed as an essential component of prenatal testing (We reported a robust rapid multiplex short-read MinION sequencing library protocol for ultra-fast aneuploidy detection for single and multiple samples. It shows promise for translation to clinical applications with a low assay cost and the fastest turnover time to date.www.g3journal.org/lookup/suppl/doi:10.1534/g3.118.200087/-/DC1.Supplemental Material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.