Glutathione safeguards TET-dependent DNA demethylation and is critical for the acquisition of totipotency and pluripotency during preimplantation development.

During early development, both genome-wide epigenetic reprogramming and metabolic remodeling are hallmark changes of normal embryogenesis. However, little is known about their relationship and developmental functions during the preimplantation window, which is essential for the acquisition of totipotency and pluripotency. Herein, we reported that glutathione (GSH), a ubiquitous intracellular protective antioxidant that maintains mitochondrial function and redox homeostasis, plays a critical role in safeguarding postfertilization DNA demethylation and is essential for establishing developmental potential in preimplantation embryos. By profiling mitochondria-related transcriptome that coupled with different pluripotency, we found GSH is a potential marker that is tightly correlated with full pluripotency, and its beneficial effect on prompting developmental potential was functionally conformed using in vitro fertilized mouse and bovine embryos as the model. Mechanistic study based on preimplantation embryos and embryonic stem cells further revealed that GSH prompts the acquisition of totipotency and pluripotency by facilitating ten-eleven-translocation (TET)-dependent DNA demethylation, and ascorbic acid (AsA)-GSH cycle is implicated in the process. In addition, we also reported that GSH serves as an oviductal paracrine factor that supports development potential of preimplantation embryos. Thus, our results not only advance the current knowledge of functional links between epigenetic reprogramming and metabolic remodeling during preimplantation development but also provided a promising approach for improving current in vitro culture system for assisted reproductive technology.

Mitochondrial genome undergoes de novo DNA methylation that protects mtDNA against oxidative damage during the peri-implantation window.

Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, in vivo and in vitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

"Meiosis arrester" C-natriuretic peptide directly stimulates oocyte mtDNA accumulation and is implicated in aging-associated oocyte mtDNA loss.

C-natriuretic peptide (CNP) is the central regulator of oocyte meiosis progression, thus coordinating synchronization of oocyte nuclear-cytoplasmic maturation. However, whether CNP can independently regulate cytoplasmic maturation has been long overlooked. Mitochondrial DNA (mtDNA) accumulation is the hallmark event of cytoplasmic maturation, but the mechanism underlying oocyte mtDNA replication remains largely elusive. Herein, we report that CNP can directly stimulate oocyte mtDNA replication at GV stage, and deficiency of follicular CNP may contribute largely to lower mtDNA copy number in in vitro matured oocytes. The mechanistic study showed that cAMP-PKA-CREB1 signaling cascade underlies the regulatory role of CNP in stimulating mtDNA replication and upregulating related genes. Of interest, we also report that CNP-NPR2 signaling is inhibited in aging follicles, and this inhibition is implicated in lower mtDNA copy number in oocytes from aging females. Together, our study provides the first direct functional link between follicular CNP and oocyte mtDNA replication, and identifies its involvement in aging-associated mtDNA loss in oocytes. These findings, not only update the current knowledge of the functions of CNP in coordinating oocyte maturation but also present a promising strategy for improving in vitro fertilization outcomes of aging females.

The mRNA-destabilizing protein Tristetraprolin targets "meiosis arrester" Nppc mRNA in mammalian preovulatory follicles.

C-natriuretic peptide (CNP) and its receptor guanylyl cyclase, natriuretic peptide receptor 2 (NPR2), are key regulators of cyclic guanosine monophosphate (cGMP) homeostasis. The CNP-NPR2-cGMP signaling cascade plays an important role in the progression of oocyte meiosis, which is essential for fertility in female mammals. In preovulatory ovarian follicles, the luteinizing hormone (LH)-induced decrease in CNP and its encoding messenger RNA (mRNA) natriuretic peptide precursor C (Nppc) are a prerequisite for oocyte meiotic resumption. However, it has never been determined how LH decreases CNP/Nppc In the present study, we identified that tristetraprolin (TTP), also known as zinc finger protein 36 (ZFP36), a ubiquitously expressed mRNA-destabilizing protein, is the critical mechanism that underlies the LH-induced decrease in Nppc mRNA. Zfp36 mRNA was transiently up-regulated in mural granulosa cells (MGCs) in response to the LH surge. Loss- and gain-of-function analyses indicated that TTP is required for Nppc mRNA degradation in preovulatory MGCs by targeting the rare noncanonical AU-rich element harbored in the Nppc 3' UTR. Moreover, MGC-specific knockout of Zfp36, as well as lentivirus-mediated knockdown in vivo, impaired the LH/hCG-induced Nppc mRNA decline and oocyte meiotic resumption. Furthermore, we found that LH/hCG activates Zfp36/TTP expression through the EGFR-ERK1/2-dependent pathway. Our findings reveal a functional role of TTP-induced mRNA degradation, a global posttranscriptional regulation mechanism, in orchestrating the progression of oocyte meiosis. We also provided a mechanism for understanding CNP-dependent cGMP homeostasis in diverse cellular processes.

A proteomic atlas of ligand-receptor interactions at the ovine maternal-fetal interface reveals the role of histone lactylation in uterine remodeling.

Well-orchestrated maternal-fetal cross talk occurs via secreted ligands, interacting receptors, and coupled intracellular pathways between the conceptus and endometrium and is essential for successful embryo implantation. However, previous studies mostly focus on either the conceptus or the endometrium in isolation. The lack of integrated analysis impedes our understanding of early maternal-fetal cross talk. Herein, focusing on ligand-receptor complexes and coupled pathways at the maternal-fetal interface in sheep, we provide the first comprehensive proteomic map of ligand-receptor pathway cascades essential for embryo implantation. We demonstrate that these cascades are associated with cell adhesion and invasion, redox homeostasis, and the immune response. Candidate interactions and their physiological roles were further validated by functional experiments. We reveal the physical interaction of albumin and claudin 4 and their roles in facilitating embryo attachment to endometrium. We also demonstrate a novel function of enhanced conceptus glycolysis in remodeling uterine receptivity by inducing endometrial histone lactylation, a newly identified histone modification. Results from in vitro and in vivo models supported the essential role of lactate in inducing endometrial H3K18 lactylation and in regulating redox homeostasis and apoptotic balance to ensure successful implantation. By reconstructing a map of potential ligand-receptor pathway cascades at the maternal-fetal interface, our study presents new concepts for understanding molecular and cellular mechanisms that fine-tune conceptus-endometrium cross talk during implantation. This provides more direct and accurate insights for developing potential clinical intervention strategies to improve pregnancy outcomes following both natural and assisted conception.

Dynamic monitoring of TGW6 by selective autophagy during grain development in rice.

Crop yield must increase to achieve food security in the face of a growing population and environmental deterioration. Grain size is a prime breeding target for improving grain yield and quality in crop. Here, we report that autophagy emerges as an important regulatory pathway contributing to grain size and quality in rice. Mutations of rice Autophagy-related 9b (OsATG9b) or OsATG13a causes smaller grains and increase of chalkiness, whereas overexpression of either promotes grain size and quality. We also demonstrate that THOUSAND-GRAIN WEIGHT 6 (TGW6), a superior allele that regulates grain size and quality in the rice variety Kasalath, interacts with OsATG8 via the canonical Atg8-interacting motif (AIM), and then is recruited to the autophagosome for selective degradation. In consistent, alteration of either OsATG9b or OsATG13a expression results in reciprocal modulation of TGW6 abundance during grain growth. Genetic analyses confirmed that knockout of TGW6 in either osatg9b or osatg13a mutants can partially rescue their grain size defects, indicating that TGW6 is one of the substrates for autophagy to regulate grain development. We therefore propose a potential framework for autophagy in contributing to grain size and quality in crops.

Early seed development requires the A-type ATP-binding cassette protein ABCA10.

A-type ATP-binding cassette (ABCA) proteins transport lipids and lipid-based molecules in humans, and their malfunction is associated with various inherited diseases. Although plant genomes encode many ABCA transporters, their molecular and physiological functions remain largely unknown. Seeds are rapidly developing organs that rely on the biosynthesis and transport of large quantities of lipids to generate new membranes and storage lipids. In this study, we characterized the Arabidopsis (Arabidopsis thaliana) ABCA10 transporter, which is selectively expressed in female gametophytes and early developing seeds. By 3 d after flowering (DAF), seeds from the abca10 loss-of-function mutant exhibited a smaller chalazal endosperm than those of the wild-type. By 4 DAF, their endosperm nuclei occupied a smaller area than those of the wild-type. The endosperm nuclei of the mutants also failed to distribute evenly inside the seed coat and stayed aggregated instead, possibly due to inadequate expansion of abca10 endosperm. This endosperm defect might have retarded abca10 embryo development. At 7 DAF, a substantial portion of abca10 embryos remained at the globular or earlier developmental stages, whereas wild-type embryos were at the torpedo or later stages. ABCA10 is likely involved in lipid metabolism, as ABCA10 overexpression induced the overaccumulation of triacylglycerol but did not change the carbohydrate or protein contents in seeds. In agreement, ABCA10 localized to the endoplasmic reticulum (ER), the major site of lipid biosynthesis. Our results reveal that ABCA10 plays an essential role in early seed development, possibly by transporting substrates for lipid metabolism to the ER.

Synthesis of Surface-Functionalized Molybdenum Disulfide Nanomaterials for Efficient Adsorption and Deep Profiling of the Human Plasma Proteome by Data-Independent Acquisition.

Blood is one of the most important clinical samples for protein biomarker discovery, as it provides rich physiological and pathological information and is easy to obtain with low invasiveness. However, the discovery of protein biomarkers in the blood by mass spectrometry (MS)-based proteomic strategies has been shown to be highly challenging due to the particularly large concentration range of proteins and the strong interference by the high-abundant proteins in the blood. Therefore, developing sensitive methods for low-abundant biomarker protein identification is a key issue that has received great attention. Here, we report the synthesis and characterization of surface-functionalized magnetic molybdenum disulfide (MoS2) for the large-scale adsorption of low-abundant plasma proteins and deep profiling by MS. MoS2 nanomaterials resulted in the coverage of more than 3400 proteins (including a single-peptide hit) in a single LC-MS analysis without peptide prefractionation using pooled plasma samples, which were five times more than those obtained by the direct analysis of the plasma proteome. A detection limit in the low ng L-1 range was obtained, which is rare compared with previous reports.

A Potential Research Target for Scleral Remodeling: Effect of MiR-29a on Scleral Fibroblasts.

INTRODUCTION: The purpose of this study was to determine whether miR-29a regulates cell survival and apoptosis and the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), MMP-2, and collagen I in scleral fibroblasts. METHODS: We transfected scleral fibroblasts with the miR-29a mimic and inhibitor. The effects of miR-29a on cell proliferation and apoptosis were determined using the CCK-8 assay and flow cytometry, respectively. Quantitative polymerase chain reaction (qPCR) was used to determine whether miR-29a regulates the mRNA levels of PTEN, MMP-2, and collagen I. The protein expression of PTEN, MMP-2, and collagen I was also assessed by western blot analysis. RESULTS: The results of CCK-8 showed that, at 0, 24, 48, and 72 h after transfection, the relative optical density values in the mimic group were 0.233 ± 0.005, 0.380 ± 0.008, 0.650 ± 0.040, and 0.906 ± 0.032, and in the inhibitor group were 0.272 ± 0.011, 0.393 ± 0.029, 0.597 ± 0.059, and 0.950 ± 0.101, respectively. The flow cytometry results showed that the apoptosis rates of each group were as follows: the mimic group (0.043 ± 0.007), the NC group (0.040 ± 0.006), the inhibitor group (0.032 ± 0.003), the inhibitor NC group (0.027 ± 0.010), the lipofectamine group (0.027 ± 0.005), and the blank group (0.031 ± 0.009). The qPCR results indicated that in the mimic group, PTEN (0.795 ± 0.182, p = 0.2783), MMP-2 (0.621 ± 0.105, p = 0.0033), and COL1A1 (0.271 ± 0.100, p = 0.0002) expression decreased, whereas in the inhibitor group, PTEN (1.211 ± 0.100, p = 0.2614), MMP-2 (1.161 ± 0.053, p = 0.1190), and COL1A1 (1.7040 ± 0.093, p = 0.0003) increased. Western blot analysis showed that in the mimic group, the expression of PTEN (0.392 ± 0.039, p < 0.0001), MMP-2 (0.577 ± 0.017, p < 0.0001), and COL1A1 (0.072 ± 0.006, p < 0.0001) protein decreased, whereas in the inhibitor group, PTEN (1.043 ± 0.042, p = 0.9413), MMP-2 (1.397 ± 0.075, p = 0.0002), and COL1A1 (1.935 ± 0.081, p < 0.0001) expression increased. CONCLUSION: MiR-29a inhibits the expression of PTEN, MMP-2, and collagen I on scleral fibroblasts, which may provide a basis studies in sclera.

Successful Pregnancy after Improving Insulin Resistance with the Glucagon-Like Peptide-1 Analogue in a Woman with Polycystic Ovary Syndrome: A Case Report and Review of the Literature.

The polycystic ovary syndrome (PCOS) is a common cause of anovulatory infertility. It is diagnosed by the presence of hyperandrogenemia, insulin resistance (IR), obesity and other endocrine or metabolic disorders. Exenatide (EX) is a kind of glucagon-like peptide, which is a new option for patients with diabetes mellitus. We present a patient with infertility for PCOS. She was overweight and her medical history included IR, right-sided ovarian mucinous cystadenomas, and left-sided teratoma. Although she had been treated with ovarian surgery, clomiphene citrate and gonadotropins, weight loss and metformin, which have been effective for dominant follicle development, she still failed to conceive. Then EX was initiated to intervene for 2 months. EX treatment was successful to improve IR; after that the infertile woman with PCOS became pregnant. EX improves IR and reproduction capacity in PCOS patients, reducing insulin level and ameliorating endocrine disorders, thereby improving ovarian function, promoting follicle development, and providing new avenues for the treatment of infertility with PCOS.

Single-embryo transcriptomic atlas of oxygen response reveals the critical role of HIF-1α in prompting embryonic zygotic genome activation.

Adaptive response to physiological oxygen levels (physO2; 5% O2) enables embryonic survival in a low-oxygen developmental environment. However, the mechanism underlying the role of physO2 in supporting preimplantation development, remains elusive. Here, we systematically studied oxygen responses of hallmark events in preimplantation development. Focusing on impeded transcriptional upregulation under atmospheric oxygen levels (atmosO2; 20% O2) during the 2-cell stage, we functionally identified a novel role of HIF-1α in promoting major zygotic genome activation by serving as an oxygen-sensitive transcription factor. Moreover, during blastocyst formation, atmosO2 impeded H3K4me3 and H3K27me3 deposition by deregulating histone-lysine methyltransferases, thus impairing X-chromosome inactivation in blastocysts. In addition, we found atmosO2 impedes metabolic shift to glycolysis before blastocyst formation, thus resulting a low-level histone lactylation deposition. Notably, we also reported an increased sex-dimorphic oxygen response of embryos upon preimplantation development. Together, focusing on genetic and epigenetic events that are essential for embryonic survival and development, the present study advances current knowledge of embryonic adaptive responses to physO2, and provides novel insight into mechanism underlying irreversibly impaired developmental potential due to a short-term atmosO2 exposure.

Computational protocol for the identification of X-linked genes contributing to X chromosome upregulation from RNA-sequencing datasets.

The X chromosome/autosome ratio has been widely used to profile XCU at the chromosomal level. However, this approach overlooks features of inside genes. Here, we present a computational protocol for the identification of X-linked genes contributing to X chromosome upregulation from RNA-sequencing datasets. We describe steps for selecting data, preparing software, processing data, and data analysis. This protocol quantifies the contribution value and contribution increment of each X-linked gene to XCU. For complete details on the use and execution of this protocol, please refer to Lyu et al. (2022).1.

Response to Lentini and Reinius.

Lyu et al. respond to the letter from Lentini and Reinius.

Intrafollicular Retinoic Acid Signaling Is Important for Luteinizing Hormone-Induced Oocyte Meiotic Resumption.

It has been clear that retinoic acid (RA), the most active vitamin A (VA) derivative, plays a central role in governing oocyte meiosis initiation. However, it has not been functionally determined if RA participates in luteinizing hormone (LH)-induced resumption from long-lasting oocyte meiotic arrest, which is essential for haploid oocyte formation. In the present study, using well-established in vivo and in vitro models, we identified that intrafollicular RA signaling is important for normal oocyte meiotic resumption. A mechanistic study indicated that mural granulosa cells (MGCs) are the indispensable follicular compartment for RA-prompted meiotic resumption. Moreover, retinoic acid receptor (RAR) is essential for mediating RA signaling to regulate meiotic resumption. Furthermore, we found zinc finger protein 36 (ZFP36) is the transcriptional target of RAR. Both RA signaling and epidermal growth factor (EGF) signaling were activated in MGCs in response to LH surge, and two intrafollicular signalings cooperate to induce rapid Zfp36 upregulation and Nppc mRNA decrease, which is critical to LH-induced meiotic resumption. These findings extend our understanding of the role of RA in oocyte meiosis: RA not only governs meiotic initiation but also regulates LH-induced meiotic resumption. We also emphasize the importance of LH-induced metabolic changes in MGCs in this process.

Progesterone Activates the Histone Lactylation-Hif1α-glycolysis Feedback Loop to Promote Decidualization.

Decidualization is a progesterone-dependent cellular differentiation process that is essential for establishing pregnancy. Robust activation of glycolysis and lactate synthesis during decidualization is remarkable, but their developmental functions remain largely unknown. Herein, we identify that endometrial lactate production plays a critical role in establishing local histone lactylation, a newly identified histone modification, and is important for ensuring normal decidualization. Enhanced endometrial glycolysis is the hallmark metabolic change and is tightly coupled with H4K12la during decidualization. Inhibition of histone lactylation impaired decidualization, in either physiological conception or in vivo and in vitro induced decidualization models. Mechanistic study based on CUT&Tag and ATAC-seq revealed that a transcriptional factor hypoxia-inducible factor 1 α (Hif1α) is the critical regulatory target of H4K12la, and in turn forms an H4K12la-Hif1α-glycolysis feedback loop to drive decidualization. Moreover, we demonstrate that the loop is directly activated by progesterone during decidualization. Our study not only advances the current knowledge of the role of lactate in regulating uterine function, but also establishes a novel functional link among the major endocrine factors, endometrial metabolic change, and epigenetic modification during endometrial remodeling. These findings present valuable clues to develop clinical intervention strategies to improve pregnancy outcomes following both natural conception and assisted reproduction.

Three-dimensional visualization of the vascular bundle in a branched bamboo node.

Bamboo is a natural vascular bundle (VB) reinforced composite material used in more than 10 fields such as construction and furniture. The nodes in bamboo are crucial to its mechanical properties, but understanding of its performance is limited by lack of knowledge about the three-dimensional (3D) structure of the node. This work aimed to non-destructively identify the multi-dimensional characteristics of the VB in a bamboo branched node (BN) using X-ray microtomography (µCT). The VB was segmented from the BN using deep learning combined with the Watershed algorithm. The 3D model reconstruction and characterization of the VB were also conducted. It was found that the structure of VBs showed significant changes along the height of the BN. The VBs formed a complex 3D structure, VBs of the culm are connected with those of the branch, and the connectivity of the conducting tissue and fibers was 88.91% and 99.95%, respectively. The conducting tissue and the fibers had similar shapes but varying thicknesses, which enabled VBs to perform both water transport and mechanical support functions. The volumes fraction of parenchyma, fibers, and conducting tissue in the BN were 61.3%, 35.3%, and 3.4%, respectively, but the tissue proportion of the different heights of the BN varied from each other. The nodal ridge was a mechanical weak point of the BN, with a maximum fibers proportion of 43.8%. This study contributes to understanding the relationship of VBs between the branch and the culm. It provides a structural perspective for understanding the mechanical properties of BN and a theoretical basis for optimizing bamboo utilization efficiency.

Multifaceted roles of zinc finger proteins in regulating various agronomic traits in rice.

Rice is an important cereal crop, which provides staple food for more than half of the world's population. To meet the demand of the ever-growing population in the next few decades, an extra increase in rice yield is an urgent need. Given that various agronomic traits contribute to the yield of rice, deciphering the key regulators involved in multiple agronomic trait formation is particularly important. As a superfamily of transcription factors, zinc finger proteins participate in regulating multiple genes in almost every stage of rice growth and development. Therefore, understanding zinc finger proteins underlying regulatory network would provide insights into the regulation of agronomic traits in rice. To this end, we intend to summarize the current advances in zinc finger proteins, with emphasis on C2H2 and CCCH proteins, and then discuss their potential in improving rice yield.

ABCG11 modulates cytokinin responses in Arabidopsis thaliana.

The Arabidopsis ABC transporter ABCG11 transports lipidic precursors of surface coating polymers at the plasma membrane of epidermal cells. Mutants in ABCG11 exhibit severe developmental defects, suggesting that ABCG11 might also participate in phytohormone-mediated development. Here, we report that ABCG11 is involved in cytokinin-mediated development. The roots of abcg11 mutant seedlings failed to respond to cytokinins and accumulated more cytokinins than wild-type roots. When grown under short-day conditions, abcg11 exhibited longer roots and shorter hypocotyls compared to wild type, similar to abcg14, a knockout mutant in a cytokinin transporter. Treatment with exogenous trans-zeatin, which inhibits primary root elongation in the wild type, enhanced abcg11 primary root elongation. It also increased the expression of cytokinin-responsive Arabidopsis response regulator (ARR) genes, and the signal of the TCS::GFP reporter in abcg11 roots compared to wild-type roots, suggesting that cytokinin signaling was enhanced in abcg11 roots. When we treated only the roots of abcg11 with trans-zeatin, their shoots showed lower ARR induction than the wild type. The abcg14 abcg11 double mutant did not have additional root phenotypes compared to abcg11. Together, these results suggest that ABCG11 is necessary for normal cytokinin-mediated root development, likely because it contributes to cytokinin transport, either directly or indirectly.

A small proportion of X-linked genes contribute to X chromosome upregulation in early embryos via BRD4-mediated transcriptional activation.

Females have two X chromosomes and males have only one in most mammals. X chromosome inactivation (XCI) occurs in females to equalize X-dosage between sexes. Besides, mammals also balance the dosage between X chromosomes and autosomes via X chromosome upregulation (XCU) to fine-tune X-linked expression and thus maintain genomic homeostasis. Despite some studies highlighting the importance of XCU in somatic cells, little is known about how XCU is achieved and its developmental role during early embryogenesis. Herein, using mouse preimplantation embryos as the model, we reported that XCU initially occurs upon major zygotic genome activation and co-regulates X-linked expression in cooperation with imprinted XCI during preimplantation development. An in-depth analysis further indicated, unexpectedly, only a small proportion of, but not X chromosome-wide, X-linked genes contribute greatly to XCU. Furthermore, we identified that bromodomain containing 4 (BRD4) plays a key role in the transcription activation of XCU during preimplantation development. BRD4 deficiency or inhibition caused an impaired XCU, thus leading to reduced developmental potential and mitochondrial dysfunctions of blastocysts. Our finding was also supported by the tight association of BRD4 dysregulation and XCU disruption in the pathology of cholangiocarcinoma. Thus, our results not only advanced the current knowledge of X-dosage compensation and provided a mechanism for understanding XCU initiation but also presented an important clue for understanding the developmental and pathological role of XCU.

A New Strategy for High-Efficient Tandem Enrichment and Simultaneous Profiling of N-Glycopeptides and Phosphopeptides in Lung Cancer Tissue.

N-glycosylation and phosphorylation, two common posttranslational modifications, play important roles in various biological processes and are extensively studied for biomarker and drug target screening. Because of their low abundance, enrichment of N-glycopeptides and phosphopeptides prior to LC-MS/MS analysis is essential. However, simultaneous characterization of these two types of posttranslational modifications in complex biological samples is still challenging, especially for tiny amount of samples obtained in tissue biopsy. Here, we introduced a new strategy for the highly efficient tandem enrichment of N-glycopeptides and phosphopeptides using HILIC and TiO2 microparticles. The N-glycopeptides and phosphosites obtained by tandem enrichment were 21%-377% and 22%-263% higher than those obtained by enriching the two PTM peptides separately, respectively, using 160-20 µg tryptic digested peptides as the starting material. Under the optimized conditions, 2798 N-glycopeptides from 434 N-glycoproteins and 5130 phosphosites from 1986 phosphoproteins were confidently identified from three technical replicates of HeLa cells by mass spectrometry analysis. Application of this tandem enrichment strategy in a lung cancer study led to simultaneous characterization of the two PTM peptides and discovery of hundreds of differentially expressed N-glycosylated and phosphorylated proteins between cancer and normal tissues, demonstrating the high sensitivity of this strategy for investigation of dysregulated PTMs using very limited clinical samples.