PABPN1L assemble into “ring-like” aggregates in the cytoplasm of MII oocytes and is associated with female infertility†

2021 ◽  
Author(s):  
Ying Wang ◽  
Tianhao Feng ◽  
Mingcong Zhu ◽  
Xiaodan Shi ◽  
Zerui Wang ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mouse Model ◽  
Genetic Marker ◽  
Large Scale ◽  
Scale Up ◽  
Mrna Degradation ◽  
Female Infertility ◽  
Rna Seq ◽  
Maternal Mrna

Abstract Infertility affects 10% - 15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A resent study showed that PABPN1L recruited BTG4 to mRNA 3′-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated an PABPN1L-antibody and found “ring-like” PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L-EGFP proteins spontaneously formed“ring-like” aggregates in vitro. This phenomenon is similar with CCR4–NOT catalytic subunit, CNOT7, when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l  −/− and Pabpn1l  tm1a/tm1a) simulating the intron1-exon2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l−/− MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l-♀/+♂zygote. Both the Btg4 and Cnot61 genes are necessary for the deadenylation process and Pabpn1l  −/− resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4-♀/+♂ zygote and 84.2% genes stabilized in the Cnot6l-♀/+♂zygote were also stabilized in Pabpn1l-♀/+♂ zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4–NOT-mediated maternal mRNA degradation and PABPN1L variants on intron1-exon2 could be a genetic marker of female infertility. Summary sentence. “Ring-like” PABPN1L aggregates was found in the cytoplasm of MII oocytes and in vitro; intron1-exon2 abnormality of Pabpn1l leads female sterile in mice.

scholarly journals Maternal factor PABPN1L is essential for maternal mRNA degradation during maternal-to-zygotic transition

2020 ◽  
Author(s):  
Ying Wang ◽  
Tianhao Feng ◽  
Xiaodan Shi ◽  
Siyu Liu ◽  
Zerui Wang ◽  
...  
Keyword(s):  
Mouse Model ◽  
Large Scale ◽  
Mrna Degradation ◽  
Female Infertility ◽  
Control Group ◽  
Rna Seq ◽  
Cell Stage ◽  
Maternal Mrna ◽  
Maternal To Zygotic Transition ◽  
Group A

AbstractInfertility affects 10% - 15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. We constructed a mouse model (Pabpn1l -/-) simulating the splicing abnormality of human PABPN1L and found that the female was sterile and the male was fertile. The Pabpn1l -/- oocytes can be produced, ovulated and fertilized normally, but cannot develop beyond the 2-cell stage. Using RNA-Seq, we found a large-scale upregulation of RNA in Pabpn1l -/- MII oocytes. Of the 2401 transcripts upregulated in Pabpn1l-/- MII oocytes, 1523 transcripts (63.4%) were also upregulated in Btg4 -/- MII oocytes, while only 53 transcripts (2.2%) were upregulated in Ythdf2 -/- MII oocytes. We documented that transcripts in zygotes derived from Pabpn1l -/- oocytes have a longer poly(A) tail than the control group, a phenomenon similar to that in Btg4-/- mice. Surprisingly, the poly(A) tail of these mRNAs was significantly shorter in the Pabpn1l -/- MII oocytes than in the Pabpn1l +/+. These results suggest that PABPN1L is involved in BTG4-mediated maternal mRNA degradation, and may antagonize poly(A) tail shortening in oocytes independently of its involvement in maternal mRNA degradation. Thus, PABPN1L variants could be a genetic marker of female infertility.

Preparative chromatographic purification of cyclosporine metabolites

1993 ◽  
Vol 39 (3) ◽  
pp. 457-466 ◽  
Author(s):  
C A Brooks ◽  
S M Cramer ◽  
T G Rosano
Keyword(s):  
Animal Studies ◽  
Large Scale ◽  
Scale Up ◽  
Reversed Phase ◽  
Preparative Scale ◽  
Primary Metabolites ◽  
Rapid Processing ◽  
Adsorption Desorption ◽  
Elution Chromatography

Abstract Polar and primary metabolites of cyclosporin A (CsA) have successfully been isolated by a novel separation protocol. An efficient, easy-to-scale-up chromatographic adsorption/desorption operation recovers polar and primary CsA metabolite pools from large volumes of urine; purified CsA metabolites are subsequently obtained by high-resolution preparative elution chromatography of the semipurified metabolite pools. Separations performed on a semipreparative scale [with a 250 x 9.4 mm (i.d.) reversed-phase HPLC column] yielded microgram quantities of CsA metabolites at > 97% purity, as determined by fast atom bombardment mass spectrometry. These separations also yielded two previously unreported CsA metabolites, similar to AM1A but with an additional hydroxylation. The yield of metabolites was increased to several milligrams by performing the separations with a preparative-scale [250 x 21.2 mm (i.d.)] reversed-phase column. The production rate of purified primary CsA metabolites was greatly increased by performing the separation with the preparative-scale column under conditions of severe mass overloading. In a single chromatographic run, we successfully isolated 11.0 and 5.0 mg of AM1 and AM1c, respectively, at a purity of > 97%. As expected, this increase in the yield of purified metabolites was accompanied by a decrease in the overall recovery. This separation scheme enables the rapid processing of large volumes of urine for isolation of the milligram quantities of CsA metabolites necessary to assess their biological activity. The procedure is applicable to small- or large-scale metabolite isolation and provides a ready source of purified metabolites for in vitro and whole-animal studies.

scholarly journals Autophagy Influences Maternal mRNA Degradation and Apoptosis in Porcine Parthenotes Developing In Vitro

2012 ◽  
Vol 58 (5) ◽  
pp. 576-584 ◽  
Author(s):  
Yong-Nan XU ◽  
Xing-Hui SHEN ◽  
Seung-Eun LEE ◽  
Jung-Suk KWON ◽  
Deuk-Joong KIM ◽  
...  
Keyword(s):  
Mrna Degradation ◽  
Maternal Mrna

scholarly journals A protein microarray-based in vitro transglutaminase assay platform for epitope mapping and peptide discovery

2021 ◽  
Author(s):  
Chen Liu
Keyword(s):  
Large Scale ◽  
Matrix Protein ◽  
Tissue Transglutaminase ◽  
Protein Microarray ◽  
Scale Up ◽  
Alpha Synuclein ◽  
Extracellular Matrix Protein ◽  
Dependent Manner ◽  
Scanning Method

Transglutaminases (TGs) are a family of crosslinking enzymes catalyzing the formation of intra- and inter-molecular glutamine-lysine isopeptide bonds in a calcium dependent manner. Activation of transglutaminases is pathogenically associated with severe human diseases including neurodegenerations, cardiovascular diseases, and autoimmune diseases. Although continuous efforts determining the enzymes substrate preference have been witnessed, a high-throughput assay platform with the omic efficiency is still missing for the global identification of substrate-specific TG modification sites. In this study we report a protein microarray-based in vitro TG assay platform for rapid and large-scale (up to 30000 reactions per chip) determination of the glutamine (Q)-bearing TG modification motifs. With this platform we identified the Q16 in superoxide dismutase 1 and Q109 in alpha-synuclein as the modification sites for tissue transglutaminase (TG2), the most ubiquitous member of the enzyme family. Of particular interest, we found a close match between the modification motif and published vaccine epitope sequence in alpha-synuclein. Our data collectively suggest the glutamine and its follow-up five residues on the C terminal compose a minimal determinant motif for TG2 modification. To screen for site-specific interfering peptides and assist gene editing-based protein engineering, we developed an onchip amino-acid scanning method for the optimization of TG2 modification motifs. Using this approach we optimized the TG2 modification motif QQIV in the extracellular matrix protein fibronectin and obtained 14 variants with significantly higher TG2 reactivity that might serve as the competitive inhibitor peptides and 1 with lower reactivity. We further confirmed the efficacy of this approach using 12-mer peptides, the longest ones that could be synthesized on the chip. Taken together, our synthetic assay platform will be able to not only deliver a precise epitope blueprint for personalized immunotherapy and vaccination but also provide proof-of-concept and directional studies for TG-based peptide discovery and protein design.

scholarly journals C-STEM: ENGINEERING NICHE-LIKE MICRO-COMPARTMENTS FOR OPTIMAL AND SCALE-INDEPENDENT EXPANSION OF HUMAN PLURIPOTENT STEM CELLS IN BIOREACTORS

2021 ◽  
Author(s):  
Philippe J.R. Cohen ◽  
Elisa Luquet ◽  
Justine Pletenka ◽  
Andrea Leonard ◽  
Elise Warter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Scale Up ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Human Pluripotent Stem Cells ◽  
Cell Therapies ◽  
Cellular Source

Human pluripotent stem cells (hPSCs) have emerged as the most promising cellular source for cell therapies. To overcome scale up limitations of classical 2D culture systems, suspension cultures have been developed to meet the need of large-scale culture in regenerative medicine. Despite constant improvements, current protocols relying on the generation of micro-carriers or cell aggregates only achieve moderate amplification performance. Here, guided by reports showing that hPSCs can self-organize in vitro into cysts reminiscent of the epiblast stage in embryo development, we developed a physio-mimetic approach for hPSC culture. We engineered stem cell niche microenvironments inside microfluidics-assisted core-shell microcapsules. We demonstrate that lumenized three-dimensional colonies maximize viability and expansion rates while maintaining pluripotency. By optimizing capsule size and culture conditions, we scale-up this method to industrial scale stirred tank bioreactors and achieve an unprecedented hPSC amplification rate of 282-fold in 6.5 days.

scholarly journals Downregulation of SETD2-H3K36me3 Tumor Suppression Axis Promotes MLL Leukemia through Activation of DOT1L-H3K79me2 Axis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 435-435
Author(s):  
Jiachen Bu ◽  
Aili Chen ◽  
Yunzhu Dong ◽  
Fuhong He ◽  
Xiaomei Yan ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Mouse Model ◽  
Histone H3 ◽  
The Body ◽  
Single Mutation ◽  
Rna Seq ◽  
Expression Levels ◽  
Gene Dysregulation ◽  
The Impact

Abstract SET domain containing 2 (SETD2) gene encodes the sole methyltransferase that is specific for histone H3 lysine 36 trimethylation (H3K36me3) in mammals. Previously we identified somatic SETD2 loss-of-function (LOF) mutations in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) patients. Interestingly, SETD2 mutations were identified in over 20% of leukemia patients with MLL gene rearrangement. Using an Mll-Af9 (MA9) knock-in AML mouse model, we found that downregulation of Setd2 by shRNA or genetic knock-out/knock-in strategies significantly accelerated disease development. However, the contribution of SETD2-H3K36me3 axis downregulation to MLL leukemia progression has not yet been fully understood. In MLL leukemia, high expression levels of MLL targets, driven by aberrant histone H3 lysine 79 dimethylation (H3K79me2), have been reported. Both H3K36me3 and H3K79me2 are enriched within the body of transcriptionally active genes and are associated with phosphorylated RNA Polymerase II. Whether H3K36me3 also contributes to gene dysregulation in MLL leukemia, whether SETD2-H3K36me3 downregulation has impacts on DOT1L-H3K79me2 axis, and how it promotes MLL leukemia progression are unclear. More importantly, SETD2 mutations were enriched in relapsed B-cell ALL patients. SETD2 LOF mutation could cause chemotherapy resistance or relapse after bone marrow transplantation. Thus, mechanistic-driven novel therapies are urgently needed for leukemia with SETD2 mutation. Firstly, to understand SETD2-H3K36me3 axis on gene regulation in MLL leukemia, we purified c-Kit+ hematopoietic stem/progenitor cells (HSPCs) fromMA9 and normal C57/BL/6 wild-type (WT) adult mice. Using immuno-blotting, we found increased H3K36me3 and H3K79me2 in the HSPCs from MA9 compared to those from WT mice. To further study the global histone modification and gene regulation, we performed ChIP-seq and RNA-seq analyses. Genome-wide increase of H3K36me3 and H3K79me2 were confirmed in the HSPCs from MA9 mice, and both modifications were positively correlated with gene expression. Our results indicate that not only increased H3K79me2, but also increased H3K36me3 are related to gene dysregulation in MLL leukemogenesis. Next, to explore the impact of SETD2-H3K36me3 loss on DOT1L-H3K79me2 axis and leukemia progression, we performed KD of Setd2 gene. Setd2 KD caused increased self-renewal and proliferation abilities of both WT and MA9 HSPCs. However, Setd2 KD-WT HSPCs could be replated only 3-4 times in vitro, suggesting that the SETD2 single mutation is not sufficient for leukemic transformation. In contrast, Setd2 KD significantly enhanced in vitro replating ability and in vivo leukemia development of MA9 HSPCs. Immuno-blotting and ChIP-seq results revealed the dramatic loss of H3K36me3 in Setd2 KD cells. Surprisingly, global H3K79me2 was further increased in Setd2 KD cells. As upregulation of MLL targets is the main driver for MLL leukemogenesis, we measured the expression levels of known MLL targets. However, no significant change was found either in the RNA-seq or qPCR validation. Moreover, up-regulated genes with higher H3K79me2 in their 5' gene bodies were not enriched in the classical known MLL targets but a different group of AML related genes including Arg, Erg and Bcl2l1. This partially explains the corporative activity between SETD2 LOF and MLL fusions. Thirdly, due to the further increase of H3K79me2 in Setd2 KD cells, we tested epigenetic inhibitors for DOT1L-H3K79me2 axis in MLL leukemia with SETD2 LOF mutant models. DOT1L inhibitor EPZ-5676 induced differentiation and cell death of Setd2 KD MA9 cellsor MA9/Setd2 LOF mutant cells with significantly lower concentration (450nM) compared to the case of MA9 cells (1000nM), indicating that DOT1L-H3K79me2 axis could be the tumor vulnerability of this chemo-resistant type of leukemia. In conclusion, using MA9 genetic knock-in mouse model and modulating SETD2-H3K36me3 axis, we found that, 1) not only H3K79me2, but also H3K36me3 were aberrantly modified in MLL leukemia which related to gene dysregulation, 2) Downregulation of SETD2-H3K36me3 axis could further upregulate DOT1L-H3K79me2 axis, leading to activation of a new set of AML related genes which could contributes to quick leukemia onset, 3) EPZ-5676 could be an effective therapeutic option for MLL leukemia patients with SETD2 mutations by targeting its tumor vulnerability. Disclosures No relevant conflicts of interest to declare.

scholarly journals Stormbow: A Cloud-Based Tool for Reads Mapping and Expression Quantification in Large-Scale RNA-Seq Studies

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Shanrong Zhao ◽  
Kurt Prenger ◽  
Lance Smith
Keyword(s):  
Data Analysis ◽  
Large Scale ◽  
Scale Up ◽  
Local Environment ◽  
Transcriptome Profiling ◽  
Cost Effective ◽  
Rna Seq ◽  
Practical Challenge ◽  
Amazon Web Services ◽  
Computational Resources

RNA-Seq is becoming a promising replacement to microarrays in transcriptome profiling and differential gene expression study. Technical improvements have decreased sequencing costs and, as a result, the size and number of RNA-Seq datasets have increased rapidly. However, the increasing volume of data from large-scale RNA-Seq studies poses a practical challenge for data analysis in a local environment. To meet this challenge, we developed Stormbow, a cloud-based software package, to process large volumes of RNA-Seq data in parallel. The performance of Stormbow has been tested by practically applying it to analyse 178 RNA-Seq samples in the cloud. In our test, it took 6 to 8 hours to process an RNA-Seq sample with 100 million reads, and the average cost was $3.50 per sample. Utilizing Amazon Web Services as the infrastructure for Stormbow allows us to easily scale up to handle large datasets with on-demand computational resources. Stormbow is a scalable, cost effective, and open-source based tool for large-scale RNA-Seq data analysis. Stormbow can be freely downloaded and can be used out of box to process Illumina RNA-Seq datasets.

scholarly journals Predicting heterogeneity in clone-specific therapeutic vulnerabilities using single-cell transcriptomic signatures

2020 ◽  
Author(s):  
Chayaporn Suphavilai ◽  
Shumei Chia ◽  
Ankur Sharma ◽  
Lorna Tu ◽  
Rafael Peres Da Silva ◽  
...  
Keyword(s):  
Single Cell ◽  
Recommender System ◽  
In Silico ◽  
Large Scale ◽  
Drug Response ◽  
Response Prediction ◽  
Scale Analysis ◽  
Rna Seq ◽  
Large Scale Analysis

SummaryWhile understanding heterogeneity in molecular signatures across patients underpins precision oncology, there is increasing appreciation for taking intra-tumor heterogeneity into account. Single-cell RNA-seq (scRNA-seq) technologies have facilitated investigations into the role of intra-tumor transcriptomic heterogeneity (ITTH) in tumor biology and evolution, but their application to in silico models of drug response has not been explored. Based on large-scale analysis of cancer omics datasets, we highlight the utility of ITTH for predicting clinical outcomes. We then show that heterogeneous gene expression signatures obtained from scRNA-seq data can be accurately analyzed (80%) in a recommender system framework (CaDRReS-Sc) for in silico drug response prediction. Patient-derived cell lines capturing transcriptomic heterogeneity from primary and metastatic tumors were used as in vitro proxies for validating monotherapy predictions (Pearson r>0.6), as well as optimal drug combinations to target different subclonal populations (>10% improvement). Applying CaDRReS-Sc to the increasing number of publicly available tumor scRNA-seq datasets can serve as an in silico screen for further in vitro and in vivo drug repurposing studies.Graphical abstractHighlightsLarge-scale analysis to establish the impact of transcriptomic heterogeneity within tumors on clinical outcomesCalibrated recommender system for drug response prediction based on single-cell RNA-seq data (CaDRReS-Sc)Prediction of drug response in patient-derived cell lines with transcriptomic heterogeneityIn silico identification of drug combinations that work based on clonal vulnerabilities

295 EFFECT OF TREHALOSE DURING IN VITRO MATURATION OF PIG OOCYTES ON OOCYTE MATURATION AND EMBRYONIC DEVELOPMENT AFTER PARTHENOGENESIS

2015 ◽  
Vol 27 (1) ◽  
pp. 236
Author(s):  
Y. Jeon ◽  
B. Baasanjav ◽  
Y. I. Jeong ◽  
Y. W. Jeong ◽  
Y. W. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Large Scale ◽  
Treated Group ◽  
Developmental Competence ◽  
Control Group ◽  
Scale Production ◽  
Developmental Potential

Autophagy is a critical process for the maintenance of cellular homeostasis and mammalian early embryogenesis. Autophagy can be regulated by various chemical inducers. However, there are few reports about effect of autophagy inducer in vitro maturation (IVM) of porcine oocyte. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Immature oocytes were treated with various concentrations (0, 25, 50, and 100 mM) of trehalose in M-199 (Invitrogen, Carlsbad, CA) supplemented with 10 ng mL–1 of epidermal growth factor (EGF; Sigma-Aldrich Corp.), 1 ug mL–1 of insulin (Sigma-Aldrich Corp.), 4 IU mL–1 of pregnant mare serum gonadotropin (PMSG; Intervet, Boxmeer, Holland), 4 IU mL–1 of human chorionic gonadotropin (hCG; Intervet), and 10% (vol/vol) porcine follicular fluid (pFF) for 10 h, and transferred to another IVM medium without trehalose. Osmolality of each groups (0, 25, 50, and 100 mM trehalose) was in the 290 to 295, 310 to 315, 330 to 335, and 375 to 380 osmol range, respectively. After 44 h of IVM, trehalose treatment during IVM did not improve nuclear maturation rates of oocytes in any group (90.7, 92.1, 92.7, and 90.1%, respectively). The developmental competence of oocytes matured with different trehalose concentrations was evaluated after PA. There were no significant differences in cleavage rates. However, blastocyst (BL) formation was different. Oocytes treated with 25 mM of trehalose during IVM had a significantly higher (P < 0.05) BL formation rate (64.2%) after PA compared with the control (52.0%). The BL quality was also improved in the 25 mM trehalose-treated group. Early BL rate significantly reduced in the 25 mM trehalose-treated group as compared to control (19.6 v. 29.9%, P < 0.05). By contrast, expanded BL rate significantly increased in the 25 mM trehalose-treated group than of control (27.7 v. 11.0%, P < 0.05). Total cell numbers of BL were significantly higher (P < 0.05) in the 25 mM trehalose-treated group compared to those in the control group (52.2 v. 36.8). However, BL rate and quality of oocytes treated with 50 and 100 mM trehalose were similar with control group. In conclusion, these results indicate that 25 mM trehalose during IVM improved the developmental potential of porcine embryos. Trehalose will be useful for large-scale production of BL with good quality in porcine in vitro production.This work was supported by a grant from the Next-Generation Bio Green 21 Program (No. PJ009563032014), Rural Development Administration, Republic of Korea.

A Hybrid Bioprinting Approach for Scale-Up Tissue Fabrication

2014 ◽  
Vol 136 (6) ◽  
Author(s):  
Yin Yu ◽  
Yahui Zhang ◽  
Ibrahim T. Ozbolat
Keyword(s):  
Tissue Engineering ◽  
Self Assembly ◽  
Large Scale ◽  
Vascular System ◽  
Scale Up ◽  
Organ Printing ◽  
Promising Application ◽  
Organ Models

Tissue engineering has been focused on the fabrication of vascularized 3D tissue for decades. Most recently, bioprinting, especially tissue and organ printing, has shown great potential to enable automated robotic-based fabrication of 3D vascularized tissues and organs that are readily available for in vitro studies or in vivo transplantation. Studies have demonstrated the feasibility of the tissue printing process through bioprinting of scaffold-free cellular constructs that are able to undergo self-assembly for tissue formation; however, they are still limited in size and thickness due to the lack of a vascular network. In this paper, we present a framework concept for bioprinting 3D large-scale tissues with a perfusable vascular system in vitro to preserve cell viability and tissue maturation. With the help of a customized Multi-Arm Bioprinter (MABP), we lay out a hybrid bioprinting system to fabricate scale-up tissues and organ models and demonstrated envision its promising application for in vitro tissue engineering and its potential for therapeutic purposes with our proof of concept study.

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