ZSWIM4 regulates embryonic patterning and BMP signaling by promoting nuclear Smad1 degradation.

The dorsoventral gradient of BMP signaling plays an essential role in embryonic patterning. Zinc Finger SWIM-Type Containing 4 (zswim4) is expressed in the Spemann-Mangold organizer at the onset of Xenopus gastrulation and is then enriched in the developing neuroectoderm at the mid-gastrula stages. Knockdown or knockout of zswim4 causes ventralization. Overexpression of zswim4 decreases, whereas knockdown of zswim4 increases the expression levels of ventrolateral mesoderm marker genes. Mechanistically, ZSWIM4 attenuates the BMP signal by reducing the protein stability of SMAD1 in the nucleus. Stable isotope labeling by amino acids in cell culture (SILAC) identifies Elongin B (ELOB) and Elongin C (ELOC) as the interaction partners of ZSWIM4. Accordingly, ZSWIM4 forms a complex with the Cul2-RING ubiquitin ligase and ELOB and ELOC, promoting the ubiquitination and degradation of SMAD1 in the nucleus. Our study identifies a novel mechanism that restricts BMP signaling in the nucleus.

An Optimized Proteomics Approach Reveals Novel Alternative Proteins in Mouse Liver Development.

Alternative ORFs (AltORFs) are unannotated sequences in genome that encode novel peptides or proteins named alternative proteins (AltProts). Although ribosome profiling and bioinformatics predict a large number of AltProts, mass spectrometry as the only direct way of identification is hampered by the short lengths and relative low abundance of AltProts. There is an urgent need for improvement of mass spectrometry methodologies for AltProt identification. Here, we report an approach based on size-exclusion chromatography for simultaneous enrichment and fractionation of AltProts from complex proteome. This method greatly simplifies the variance of AltProts discovery by enriching small proteins smaller than 40 kDa. In a systematic comparison between 10 methods, the approach we reported enabled the discovery of more AltProts with overall higher intensities, with less cost of time and effort compared to other workflows. We applied this approach to identify 89 novel AltProts from mouse liver, 39 of which were differentially expressed between embryonic and adult mice. During embryonic development, the upregulated AltProts were mainly involved in biological pathways on RNA splicing and processing, whereas the AltProts involved in metabolisms were more active in adult livers. Our study not only provides an effective approach for identifying AltProts but also novel AltProts that are potentially important in developmental biology.

The Small Open Reading Frame-Encoded Peptides: Advances in Methodologies and Functional Studies.

Small open reading frames (sORFs) are an important class of genes with less than 100 codons. They were historically annotated as noncoding or even junk sequences. In recent years, accumulating evidence suggests that sORFs could encode a considerable number of polypeptides, many of which play important roles in both physiology and disease pathology. However, it has been technically challenging to directly detect sORF-encoded peptides (SEPs). Here, we discuss the latest advances in methodologies for identifying SEPs with mass spectrometry, as well as the progress on functional studies of SEPs.

Airway microecology in rifampicin-resistant and rifampicin-sensitive pulmonary tuberculosis patients.

BACKGROUND: Pulmonary tuberculosis is a chronic infectious disease of the respiratory system. It is still one of the leading causes of death from a single infectious disease, but it has been stuck in the study of a single pathogen. Recent studies have shown that many diseases are associated with disruption of the native microbiota. In this study we investigated the occurrence of tuberculosis and the correlation between drug resistance and respiratory flora. High-throughput 16 S rRNA gene sequencing was used to characterize the respiratory microbiota composition of 30 tuberculosis (TB) affected patients and compared with 30 healthy (H) controls. According to their Gene Xpert results, 30 pulmonary tuberculosis patients were divided into 12 persons in the drug-sensitive group (DS0) and 18 persons in the drug-resistant group (DR0). The microbial flora of the two were compared with the H group. RESULTS: The data generated by sequencing showed that Firmicutes, Proteus, Bacteroides, Actinomyces and Fusobacterium were the five main bacterial phyla detected, and they constituted more than 96% of the microbial community. The relative abundances of Fusobacterium, Haemophilus, Porphyromonas, Neisseria, TM7, Spirochetes, SR1, and Tenericutes in the TB group was lower than that of the H group, and Granulicatella was higher than the H group. The PcoA diagrams of the two groups had obvious clustering differences. The Alpha diversity of the TB group was lower than that of the H group, and the Beta diversity was higher than that of the H group (P < 0.05). The relative abundance of Streptococcus in the DS0 group was significantly higher than that in the DR0 group (P < 0.05). CONCLUSION: Pulmonary tuberculosis can cause disorders of the respiratory tract microbial flora, in which the relative abundance of Streptococcus was significantly different between rifampicin-sensitive and rifampicin-resistant patients.

SETD2 deficiency accelerates MDS-associated leukemogenesis via S100a9 in NHD13 mice and predicts poor prognosis in MDS.

SETD2, the histone H3 lysine 36 methyltransferase, previously identified by us, plays an important role in the pathogenesis of hematologic malignancies, but its role in myelodysplastic syndromes (MDSs) has been unclear. In this study, low expression of SETD2 correlated with shortened survival in patients with MDS, and the SETD2 levels in CD34+ bone marrow cells of those patients were increased by decitabine. We knocked out Setd2 in NUP98-HOXD13 (NHD13) transgenic mice, which phenocopies human MDS, and found that loss of Setd2 accelerated the transformation of MDS into acute myeloid leukemia (AML). Loss of Setd2 enhanced the ability of NHD13+ hematopoietic stem and progenitor cells (HSPCs) to self-renew, with increased symmetric self-renewal division and decreased differentiation and cell death. The growth of MDS-associated leukemia cells was inhibited though increasing the H3K36me3 level by using epigenetic modifying drugs. Furthermore, Setd2 deficiency upregulated hematopoietic stem cell signaling and downregulated myeloid differentiation pathways in the NHD13+ HSPCs. Our RNA-seq and chromatin immunoprecipitation-seq analysis indicated that S100a9, the S100 calcium-binding protein, is a target gene of Setd2 and that the addition of recombinant S100a9 weakens the effect of Setd2 deficiency in the NHD13+ HSPCs. In contrast, downregulation of S100a9 leads to decreases of its downstream targets, including Ikba and Jnk, which influence the self-renewal and differentiation of HSPCs. Therefore, our results demonstrated that SETD2 deficiency predicts poor prognosis in MDS and promotes the transformation of MDS into AML, which provides a potential therapeutic target for MDS-associated acute leukemia.

Different roles of E proteins in t(8;21) leukemia: E2-2 compromises the function of AETFC and negatively regulates leukemogenesis.

The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 20% of acute myeloid leukemia (AML) cases. In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AML1-ETO-containing transcription factor complex (AETFC), that contains multiple transcription (co)factors. Among these AETFC components, HEB and E2A, two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA-binding capacity to AETFC, and are essential for leukemogenesis. However, the third E protein, E2-2, is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the bHLH DNA-binding domain. RNA-seq and ChIP-seq analyses reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with dendritic cell differentiation and represses MYC target genes. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates leukemogenesis. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a heterogeneity of AETFC, which improves our understanding of the precise mechanism of leukemogenesis and assists development of diagnostic/therapeutic strategies.

D283 Med, a Cell Line Derived from Peritoneal Metastatic Medulloblastoma: A Good Choice for Missing Protein Discovery.

Since the Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2010, many techniques have been adopted for the discovery of missing proteins (MPs). Because of these efforts, only 1481 MPs remained as of July 2020; however, by relying only on technique optimization, researchers have reached a bottleneck in MP discovery. Protein expression is tissue- or cell-type-dependent. The tissues of the human testis and brain have been reported to harbor a large number of tissue-specific genes and proteins; however, few studies have been performed on human brain tissue or cells to identify MPs. Herein a metastatic cell line derived from brain cancer, D283 Med, was used to search for MPs. With a traditional and simple shotgun workflow to separate the peptides into 20 fractions, 12 MPs containing at least two unique non-nested peptides (amino acid length ≥9) were identified in this cell line with a protein false discovery rate of <1%. Following the same experimental protocol, only one MP was found in a nonmetastatic brain cancer cell line, U-118 MG. Furthermore, 12 MPs were verified as having two non-nested unique peptides by matching them with corresponding chemically synthesized peptides through parallel reaction monitoring. These results clearly demonstrate that the appropriate selection of experimental materials, either tissues or cell lines, is imperative for MP discovery. The data obtained in this study are available via ProteomeXchange (PXD021482) and PeptideAtlas (PASS01627).

Exploration of Missing Proteins by a Combination Approach to Enrich the Low-Abundance Hydrophobic Proteins from Four Cancer Cell Lines.

The mission of the Chromosome-Centric Human Proteome Project (C-HPP) to discover missing proteins (MPs) has become increasingly difficult due to the remaining low-abundance, high-hydrophobicity, or low-molecular-weight MPs. We have reported two approaches to resolve these identification problems for the low-abundance and high-hydrophobicity MPs, respectively. In this study, to improve the identification of low-abundance MPs with high hydrophobicity, we combined two approaches and obtained MPs from several different cancer cell lines. Their membrane fractions were isolated by ultracentrifugation, and the low-abundance proteins were enriched at the protein level with the ProteoMiner kit. After that, the peptides from the enriched proteins were separated by high concentrations of organic solvents according to their hydrophobicity as the first dimension of separation at the peptide level, and the second and third dimensions of separation involved a high pH reversed-phase and an acid reversed-phase column, respectively. In total, 16 MPs (at least two non-nested unique peptides with ≥9 amino acids) with 61 unique peptides were identified from four human cancer cell lines, including 2, 8, 2, and 7 MPs from HeLa, HCT116, SNU-1, and HepG2 cells, respectively. Furthermore, all MPs were verified with two non-nested unique peptides through parallel reaction monitoring (PRM) by matching the peptides with their chemically synthesized peptides. Interestingly, two additional MPs were verified from the same cell line by PRM assay, although the two non-nested unique peptides with ≥9 amino acids for each MP were identified from different MS injections or cell lines by data-dependent acquisition (DDA). Thus, a total of 18 MPs were dug out in this study. The data are available via ProteomeXchange (PXD014058) and PeptideAtlas (PASS01388).

A Reliable Prognosis Approach for Degradation Evaluation of Rolling Bearing Using MCLSTM.

Prognostics and health management technology (PHM), a measure to ensure the reliability and safety of the operation of industrial machinery, has attracted attention and application adequately. However, how to use the monitored information to evaluate the degradation of rolling bearings is a significant issue for its predictive maintenance and autonomic logistics. This work presents a reliable health prognosis approach to estimate the health indicator (HI) and remaining useful life (RUL) of rolling bearings. Firstly, to accurately capture the degradation process, a novel health index (HI) is constructed based on correlation kurtosis for different iteration periods and a Gaussian process latency variable model (GPLVM). Then, a multiple convolutional long short-term memory (MCLSTM) network is proposed to predict HI values and RUL values. Finally, we perform experimental datasets of rolling bearings, demonstrating that the presented method surpasses other state-of-the-art prognosis approaches. The results also confirm the feasibility of the presented method in industrial machinery.

Alternative Strategy To Explore Missing Proteins with Low Molecular Weight.

Identifying more missing proteins (MPs) is an important mission of C-HPP. With the number of identified MPs being attenuated year by year (2,949 to 2,129 MPs from 2016 to 2019), we have realized that the difficulty of exploring the remaining MPs is a challenge in technique. Herein, we propose a comprehensive strategy to effectively enrich, separate, and identify proteins with low molecular weights, aiming at the discovery of MPs. Basically, a protein extract from human placenta was passed through a C18 SPE column, and the bound proteins that were eluted were further separated with an SDS-PAGE gel or a 50 kDa cutoff filter. The separated proteins were subjected to trypsin digestion, and the MS/MS signals were searched against data sets with two different digestion modes (full-trypsin and semitrypsin). The strategy was adopted, resulting in the identification of 4 MPs with 8 unique peptides (≥2 non-nested unique peptides with ≥9 amino acids). Importantly, the identification of 6 out of 8 of the unique peptides derived from the MPs was further supported by parallel reaction monitoring, which confirmed the identification of 3 MPs from human placenta tissues (Q6NT89: TMF-regulated nuclear protein 1; A0A183: late cornified envelope protein 6A; and Q6UWQ7: insulin growth factor-like family member 2, mapped to chromosomes 1, 1, and 19, respectively). The three proteins ranged in length from 80 aa to 227 aa. The study not only establishes a feasible strategy for analyzing proteins with low molecular weights but also fills a small part of a large gap in the list of MPs. The data obtained in this study are available via ProteomeXchange (PXD014083) and PeptideAtlas (PASS01389).

Improvement of Peptide Separation for Exploring the Missing Proteins Localized on Membranes.

Following an enormous effort by the global scientific community coordinated by HUPO's Human Proteome Project, the number of proteins without high-quality MS or other evidence (colloquially termed missing proteins) has substantially decreased; however, some highly hydrophobic MPs remain on the list. We believe that efficient peptide separation is an approach that can be used to improve the identification of these hydrophobic MPs. We propose that peptides prepared from the membrane fractions of human cell lines and placental tissue can be well separated from hydrophilic peptides in organic solvents at high concentrations due to the precipitation of hydrophilic peptides with lower solubility. Using a combination strategy of peptide separation in 98% acetonitrile prior to traditional 2D reverse-phase liquid chromatography, more hydrophobic peptides were detected in the supernatants of the organic solvent extractions than were found in the pellets. When this strategy was adopted, 30 MPs (≥2 non-nested unique peptides with ≥9 amino acids) with 114 unique peptides were identified at protein false discovery rate (FDR) < 1%, including 7, 12, and 13 MPs obtained from membrane preparations derived from K562, HeLa cells, and human placenta, respectively. Of the 30 MPs identified in this study, 19 were categorized as membrane proteins or extracellular matrix proteins. Furthermore, 20 were verified to possess two non-nested unique peptides through parallel reaction monitoring with the corresponding chemically synthesized peptides. The use of organic solvents at high concentrations was shown to be an efficient way to improve the exploration of hydrophobic MPs. The data obtained in this study are available via ProteomeXchange (PXD010630) and PeptideAtlas (PASS01218).

[Effects of miR-145 on the migration and invasion of prostate cancer PC3 cells by targeting DAB2].

It was reported that miR-145, which is significantly decreased in a variety of tumor cells, is associated with cell proliferation and metastasis. In this study, using bioinformatics analysis and in vitro assays, we identified DAB2 (Disabled homolog 2) as a downstream target gene of miR-145 during tumor metastasis process. DAB2 has been characterized as an important tumor suppressor, and is usually expressed at low levels in tumor cells. However, in this study, the relative high-level expression of DAB2 gene was observed in the highly invasive prostate cancer PC3 cells. Moreover, enforced expression of miR-145 could significantly down-regulate the expression level of DAB2 in PC3 cells, and inhibit the migration and invasion of PC3 cells. Notably, dysfunction of PC3 cells induced by miR-145 overexpression can be rescued by co-overexpression of DAB2. These results demonstrate that miR-145 regulates the migration and invasion of highly invasive prostate cancer cells through targeting DAB2 gene.

Catalytic activity of Setd2 is essential for embryonic development in mice: establishment of a mouse model harboring patient-derived Setd2 mutation.

SETD2 is the only enzyme responsible for transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3). Mutations in SETD2 cause human diseases including cancer and developmental defects. In mice, Setd2 is essential for embryonic vascular remodeling. Given that many epigenetic modifiers have recently been found to possess noncatalytic functions, it is unknown whether the major function(s) of Setd2 is dependent on its catalytic activity or not. Here, we established a site-specific knockin mouse model harboring a cancer patient-derived catalytically dead Setd2 (Setd2-CD). We found that the essentiality of Setd2 in mouse development is dependent on its methyltransferase activity, as the Setd2CD/CD and Setd2-/- mice showed similar embryonic lethal phenotypes and largely comparable gene expression patterns. However, compared with Setd2-/-, the Setd2CD/CD mice showed less severe defects in allantois development, and single-cell RNA-seq analysis revealed differentially regulated allantois-specific 5' Hoxa cluster genes in these two models. Collectively, this study clarifies the importance of Setd2 catalytic activity in mouse development and provides a new model for comparative study of previously unrecognized Setd2 functions.

Threonine dehydrogenase regulates neutrophil homeostasis but not H3K4me3 levels in zebrafish.

l-threonine dehydrogenase (Tdh) is an enzyme that links threonine metabolism to epigenetic modifications and mitochondria biogenesis. In vitro studies show that it is critical for the regulation of trimethylation of histone H3 lysine 4 (H3K4me3) levels and cell fate determination of mouse embryonic stem cells (mESCs). However, whether Tdh regulates a developmental process in vivo and, if it does, whether it also primarily regulates H3K4me3 levels in this process as it does in mESCs, remains elusive. Here, we revealed that, in zebrafish hematopoiesis, tdh is preferentially expressed in neutrophils. Knockout of tdh causes a decrease in neutrophil number and slightly suppresses their acute injury-induced migration, but, unlike the mESCs, the level of H3K4me3 is not evidently reduced in neutrophils sorted from the kidney marrow of adult tdh-null zebrafish. These phenotypes are dependent on the enzymatic activity of Tdh. Importantly, a soluble supplement of nutrients that are able to fuel the acetyl-CoA pool, such as pyruvate, glucose and branched-chain amino acids, is sufficient to rescue the reduction in neutrophils caused by tdh deletion. In summary, our study presents evidence for the functional requirement of Tdh-mediated threonine metabolism in a developmental process in vivo. It also provides an animal model for investigating the nutritional regulation of myelopoiesis and immune response, as well as a useful tool for high-throughput drug/nutrition screening.

Construction of multiple shRNAs expression vector that inhibits FUT1 gene expression and production of the transgenic SCNT embryos in vitro.

Enterotoxigenic Escherichia coli F18 is a major pathogen that causes postweaning diarrhoea and edema disease in piglets. The alpha(1,2)-fucosyltransferase (FUT1) gene has been identified as an ideal candidate gene for controlling the expression of the receptor for ECF18 bacteria. Therefore, the use of RNA interference (RNAi) to study the function of the FUT1 gene and to produce FUT1 knockdown transgenic pig would be highly beneficial. We developed an effective strategy for the expression of multiple small hairpin RNA simultaneously using multiple RNA polymerase III (hU6, hH1, mU6 and h7SK) promoters in a single vector to knockdown the FUT1 gene. Stable FUT1 knockdown transgenic fibroblast lines were generated by transfecting porcine fetal fibroblasts with the constructed vectors. Real-time RT-PCR indicated that the mRNA level of FUT1 in the transgenic fibroblast lines was significantly lower than that in the control, as much as 29 %. Finally, we successfully obtained transgenic SCNT porcine embryos. Overall, the results demonstrated that this vector-based RNAi expression system is an efficient approach to knockdown FUT1 gene expression in porcine fetal fibroblast cells, which could thereby provide donor cells for somatic cell nuclear cloning and the potential production of a marker-free transgenic pig resistant to F18 related diseases. Furthermore, it also provides strong evidence that this approach could be useful both in the production of transgenic livestock resistant to disease, and in the development of effective strategies for the suppression of gene expression in clinical gene therapy.

Histone H3 lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodeling.

HYPB is a human histone H3 lysine 36 (H3K36)-specific methyltransferase and acts as the ortholog of yeast Set2. This study explored the physiological function of mammalian HYPB using knockout mice. Homozygous disruption of Hypb impaired H3K36 trimethylation but not mono- or dimethylation, and resulted in embryonic lethality at E10.5-E11.5. Severe vascular defects were observed in the Hypb(-/-) embryo, yolk sac, and placenta. The abnormally dilated capillaries in mutant embryos and yolk sacs could not be remodeled into large blood vessels or intricate networks, and the aberrantly rounded mesodermal cells exhibited weakened interaction with endothelial cells. The embryonic vessels failed to invade the labyrinthine layer of placenta, which impaired the embryonic-maternal vascular connection. These defects could not be rescued by wild-type tetraploid blastocysts, excluding the possibility that they were caused by the extraembryonic tissues. Consistent with these phenotypes, gene expression profiling in wild-type and Hypb(-/-) yolk sacs revealed that the Hypb disruption altered the expression of some genes involved in vascular remodeling. At the cellular level, Hypb(-/-) embryonic stem cell-derived embryonic bodies, as well as in vitro-cultured human endothelial cells with siRNA-mediated suppression of HYPB, showed obvious defects in cell migration and invasion during vessel formation, suggesting an intrinsic role of Hypb in vascular development. Taken together, these results indicate that Hypb is required for embryonic vascular remodeling and provide a tool to study the function of H3K36 methylation in vasculogenesis/angiogenesis.

A systematic survey of LU domain-containing proteins reveals a novel human gene, LY6A, which encodes the candidate ortholog of mouse Ly-6A/Sca-1 and is aberrantly expressed in pituitary tumors.

The Ly-6 and uPAR (LU) domain-containing proteins represent a large family of cell-surface markers. In particular, mouse Ly-6A/Sca-1 is a widely used marker for various stem cells; however, its human ortholog is missing. In this study, based on a systematic survey and comparative genomic study of mouse and human LU domain-containing proteins, we identified a previously unannotated human gene encoding the candidate ortholog of mouse Ly-6A/Sca-1. This gene, hereby named LY6A, reversely overlaps with a lncRNA gene in the majority of exonic sequences. We found that LY6A is aberrantly expressed in pituitary tumors, but not in normal pituitary tissues, and may contribute to tumorigenesis. Similar to mouse Ly-6A/Sca-1, human LY6A is also upregulated by interferon, suggesting a conserved transcriptional regulatory mechanism between humans and mice. We cloned the full-length LY6A cDNA, whose encoded protein sequence, domain architecture, and exon-intron structures are all well conserved with mouse Ly-6A/Sca-1. Ectopic expression of the LY6A protein in cells demonstrates that it acts the same as mouse Ly-6A/Sca-1 in their processing and glycosylphosphatidylinositol anchoring to the cell membrane. Collectively, these studies unveil a novel human gene encoding a candidate biomarker and provide an interesting model gene for studying gene regulatory and evolutionary mechanisms.

In vitro evaluation of a mammary gland specific expression vector encoding recombinant human lysozyme for development of transgenic dairy goat embryos.

A vector expressing human lysozyme (pBC1-hLYZ-GFP-Neo) was evaluated for gene and protein expression following liposome-mediated transformation of C-127 mouse mammary cancer cells. Cultures of G418-resistant clones were harvested 24-72 h after induction with prolactin, insulin and hydrocortisone. Target gene expression was analyzed by RT-PCR and Western blot and recombinant human lysozyme (rhLYZ) bacteriostatic activity was also evaluated. The hLYZ gene was correctly transcribed and translated in C-127 cells and hLYZ inhibited gram-positive bacterial growth, indicating the potential of this expression vector for development of a mammary gland bioreactor in goats. Guanzhong dairy goat skin fibroblasts transfected with pBC1-hLYZ-GFP-Neo were used to construct a goat embryo transgenically expressing rhLYZ by somatic nuclear transplantation with a blastocyst rate of 9.0 ± 2.8 %. These data establish the basis for cultivation of mastitis-resistant hLYZ transgenic goats.

Reproductive Performance of Zi-Goose Promoted by Red Color Illumination.

The color of light affects the reproductive performance of poultry, but it is not clear what efficient illumination strategy could be adopted to improve the reproductive performance of Zi-goose. Red light can increase the average weekly egg production rate, egg production, and qualified production. It can increase the serum GnRH level and decrease the serum PRL, MT, and T4 levels. In our study, red light for 12 h increased the average weekly laying rate, average qualified egg production, and hatching rate of Zi-goose eggs, and increased the serum levels of FSH, LH, P4, E2, MT, T3, and T4. Blue light at 14 h improved the average weekly egg production rate, average egg production, and average qualified egg production, and reduce serum PRL and MT levels to ensure the improvement of reproductive performance of goose. A total of 705,714 overlapping group sequences, 471,145 transcript sequences, and 268,609 single gene sequences were obtained from 18 sequencing samples, with a total length of 323.04, 668.53, and 247.88 M, respectively. About 176,416 unigenes were annotated successfully in six databases, accounting for 65.68% of the total unigenes obtained. 2,106, 2,142, and 8,892 unigenes were identified in the hypothalamus, pituitary gland, and ovary of the birds respectively, with different expressions of light regulation. The hypothalamus, ovary, and pituitary were involved in 279, 327, and 275 KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathways in response to light, respectively. Through further significance analysis and differential discovery rate control, a total of five metabolic pathways were obtained which were closely related to the reproductive hormones of goose. Ten candidate genes related to the reproductive performance of goslings were selected according to the identification results of differentially expressed genes of goslings under red light and white light conditions and the genes involved in metabolic pathways significantly related to the reproductive hormones of goslings. The expression levels of GnRh-1 in the hypothalamus, GnRH-R, FSH ß and LH ß in the pituitary gland, and FSH-R and LH-R candidate genes in the ovary were higher under the 12 h red light treatment than white light. However, the expression levels of VIP, PRL, and PRL-R candidate genes in the hypothalamus, pituitary and ovary were lower under 12 h red light than under 12 h white light.

Profiling the Bisecting N-acetylglucosamine Modification in Amniotic Membrane via Mass Spectrometry.

Bisecting N-acetylglucosamine (GlcNAc), a GlcNAc linked to the core ß-mannose residue via a ß1,4 linkage, is a special type of N-glycosylation that has been reported to be involved in various biological processes, such as cell adhesion and fetal development. This N-glycan structure is abundant in human trophoblasts, which is postulated to be resistant to natural killer cell-mediated cytotoxicity, enabling a mother to nourish a fetus without rejection. In this study, we hypothesized that the human amniotic membrane, which serves as the last barrier for the fetus, may also express bisected-type glycans. To test this hypothesis, glycomic analysis of the human amniotic membrane was performed, and bisected N-glycans were detected. Furthermore, our proteomic data, which have been previously employed to explore human missing proteins, were analyzed and the presence of bisecting GlcNAc-modified peptides was confirmed. A total of 41 glycoproteins with 43 glycopeptides were found to possess a bisecting GlcNAc, and 25 of these glycoproteins were reported to exhibit this type of modification for the first time. These results provide insights into the potential roles of bisecting GlcNAc modification in the human amniotic membrane, and can be beneficial to functional studies on glycoproteins with bisecting GlcNAc modifications and functional studies on immune suppression in human placenta.