Identification of FOXP3+ epithelial cells contributing to pancreatic proliferation and angiogenesis.

Forkhead box protein 3 (FOXP3), traditionally recognized as a specific transcription factor for regulatory T cells (Tregs), has also been identified in various tumor epithelial cells (named as cancer-FOXP3, c-FOXP3). However, the natural state and functional role of FOXP3 positive tumor epithelial cells remain unknown. Monoclonal cells expressing varying levels of c-FOXP3 were isolated from established PANC-1 cells using limited dilution. Whole transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) were conducted on these subsets, followed by in vitro and in vivo functional investigations. In addition, we identified c-FOXP3+E-cadherin- epithelial cells in human pancreatic cancer tissues after radical resection by immunofluorescence co-staining. We also investigated the connection between c-FOXP3+E-cadherin- epithelial cells and their clinicopathological features. Our study uncovered a distinct subset of c-FOXP3+ tumor epithelial cells characterized by reduced E-cadherin expression. C-FOXP3+E-cadherin- cells displayed significant proliferation potential and pro-angiogenic effect through the expression of chemokines, including C-X-C motif ligand 1 (CXCL1), C-X-C motif ligand 5 (CXCL5), and C-X-C motif ligand 8 (CXCL8). Notably, higher counts of c-FOXP3+E-Cadherin- cells correlated with poorer prognosis, lower tumor differentiation, lymph node metastasis, and vascular invasion in pancreatic ductal adenocarcinoma (PDAC). In conclusion, this work revealed the stable expression of FOXP3 in tumor epithelial cells, marking a distinct subset. C-FOXP3+E-cadherin- epithelial cells exhibit active proliferation and promote angiogenesis in a vascular endothelial growth factor A (VEGFA) independent manner. These findings provide novel insights into PDAC prognosis and therapeutic avenues.NEW & NOTEWORTHY In this study, we revealed a novel c-FOXP3+ tumor epithelial cell subset marked by diminished E-cadherin and stable FOXP3 expression. These subpopulations not only show robust proliferation and drive angiogenesis via CXCL1, CXCL5, and CXCL8, bypassing VEGFA pathways, but their heightened presence also correlates with adverse PDAC outcomes. By challenging traditional epithelial cell definitions and extending lymphocyte markers to these cells, our findings present innovative targets for PDAC treatment and enrich our understanding of cell biology.

Molecular characterization of the cytochrome P450 enzyme CYP18A1 in Henosepilachna vigintioctopunctata.

In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half became malformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvßFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.

The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNAPyl pairs in the same organism.

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNAPyl pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNAPyl pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNAPyl2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNAPyl2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNAPyl pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.

Single-atom iron boosts electrochemiluminescence for ultrasensitive carcinoembryonic antigen detection.

A simple and ultrasensitive sandwich-type electrochemiluminescence (ECL) immunosensor has been developed using porous three-dimensional gold nanoparticles (Au NPs) iron(Fe)-zinc(Zn) metal-organic frameworks (Au NPs-FeZn-MOFs@luminol) as high-efficiency ECL signal probes with Fe single-atom catalysts (SACs) (Fe-N-C SACs) as potentially advanced coreaction accelerators and dissolved oxygen as a coreaction agent to realize an H2O2-free amplification method for detecting carcinoembryonic antigen (CEA). The cathodic ECL of luminol, which was usually negligible, increased first. Because the Fe-N-C SACs exhibited an outstanding catalytic performance and a unique electronic structure, different reactive oxygen species (ROS) were generated via the oxygen reduction reaction. ROS oxidized the luminol anions to luminol anion radicals, preventing the time-consuming luminol electrochemical oxidation. Furthermore, the luminol anion radicals generated in situ reacted with ROS to produce potent cathodic ECL emissions. The immunosensor exhibited favorable analytical accuracy (detection range: 0.1 pg mL-1 - 80 ng mL-1), and its detection limit for serum samples was 0.031 pg mL-1 (S/N = 3). Consequently, the proposed strategy offers a new approach for early screening of CEA.

Real-time imaging of ipsilateral parathyroid glands by retrograde injection of methylene blue into the superior thyroid artery: a new intraoperative parathyroid protection method.

BACKGROUND: Postoperative hypoparathyroidism caused by parathyroid injury is a problem faced by thyroid surgeons. The current technologies for parathyroid imaging all have some defects. METHODS: Patients with differentiated thyroid carcinoma (DTC) who underwent unilateral thyroidectomy plus ipsilateral central lymph node dissection were recruited. We dissected the main trunk of the superior thyroid artery entering the thyroid gland and placed the venous indwelling tube into the artery. The sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) were calculated. RESULTS: A total of 132 patients enrolled in this single-arm clinical trial, 105 of them completed retrograde catheterization via the superior artery. The sensitivity was 69.23 and 83.33% respectively. The specificity was 72.91 and 64.89%. The accuracy was 72.91 and 64.89%. The PPV was 85.71 and 81.08%. The NPV was 22.58 and 45.45%. There were no patients with allergic reactions to the methylene blue, or methylene blue toxicity. CONCLUSIONS: Retrograde injection of methylene blue via the superior thyroid artery is an effective and safe method to visualize parathyroid glands. This method can accurately locate the target organ by ultraselecting the blood vessel and injecting the contrast agent while avoiding background contamination and reducing the amount of contrast agent. TRIAL REGISTRATION: Clinical trial registration numbers and date of registration: ChiCTR2300077263、02/11/2023.

New Set of Isobaric Labeling Reagents for Quantitative 16Plex Proteomics.

Peptide labeling by isobaric tags is a powerful approach for the relative quantitative analysis of proteomes in multiple groups. There has been a revolution in the innovation of new isobaric reagents; however, great effort is being made to expand simultaneous labeling groups to identify more labeled peptides and reduce reporter ion signal suppression. We redesigned the original chemical structure of the deuterium isobaric amine-reactive tag developed in our laboratory. We optimized the synthetic pathway to create a new set of 16-plex isobaric tags (IBT-16plex). The novel reagent enabled almost complete labeling of peptides within 90 min, with all labeling reporter ions exhibiting comparable MS/MS signals. Compared to a typical 16plex reagent, TMTpro-16plex, the peptides and proteins identified by IBT-16plex in trypsinized HeLa cells were significantly increased by 14.8 and 8.6%, respectively. Moreover, differences in peptide abundance within 10-fold among multiple groups were barely suppressed in IBT-16plex, whereas the dynamic range in TMTpro-16plex-labeled groups was smaller. After quantitative examination of MCF7 cell proteins, IBT-16plex was confirmed as feasible and useful for evaluating protein responses of glucose-starved MCF7 cells to a glucose-rich medium.

Prediction and Validation of Mouse Meiosis-Essential Genes Based on Spermatogenesis Proteome Dynamics.

The molecular mechanism associated with mammalian meiosis has yet to be fully explored, and one of the main reasons for this lack of exploration is that some meiosis-essential genes are still unknown. The profiling of gene expression during spermatogenesis has been performed in previous studies, yet few studies have aimed to find new functional genes. Since there is a huge gap between the number of genes that are able to be quantified and the number of genes that can be characterized by phenotype screening in one assay, an efficient method to rank quantified genes according to phenotypic relevance is of great importance. We proposed to rank genes by the probability of their function in mammalian meiosis based on global protein abundance using machine learning. Here, nine types of germ cells focusing on continual substages of meiosis prophase I were isolated, and the corresponding proteomes were quantified by high-resolution MS. By combining meiotic labels annotated from the mouse genomics informatics mouse knockout database and the spermatogenesis proteomics dataset, a supervised machine learning package, FuncProFinder (https://github.com/sjq111/FuncProFinder), was developed to rank meiosis-essential candidates. Of the candidates whose functions were unannotated, four of 10 genes with the top prediction scores, Zcwpw1, Tesmin, 1700102P08Rik, and Kctd19, were validated as meiosis-essential genes by knockout mouse models. Therefore, mammalian meiosis-essential genes could be efficiently predicted based on the protein abundance dataset, which provides a paradigm for other functional gene mining from a related abundance dataset.

Online Video Anomaly Detection.

With the popularity of video surveillance technology, people are paying more and more attention to how to detect abnormal states or events in videos in time. Therefore, real-time, automatic and accurate detection of abnormal events has become the main goal of video-based surveillance systems. To achieve this goal, many researchers have conducted in-depth research on online video anomaly detection. This paper presents the background of the research in this field and briefly explains the research methods of offline video anomaly detection. Then, we sort out and classify the research methods of online video anomaly detection and expound on the basic ideas and characteristics of each method. In addition, we summarize the datasets commonly used in online video anomaly detection and compare and analyze the performance of the current mainstream algorithms according to the evaluation criteria of each dataset. Finally, we summarize the future trends in the field of online video anomaly detection.

Fisetin Delays Postovulatory Oocyte Aging by Regulating Oxidative Stress and Mitochondrial Function through Sirt1 Pathway.

The quality of oocytes determines the development potential of an embryo and is dependent on their timely fertilization after ovulation. Postovulatory oocyte aging is an inevitable factor during some assisted reproduction technology procedures, which results in poor fertilization rates and impairs embryo development. We found that fisetin, a bioactive flavonol contained in fruits and vegetables, delayed postovulatory oocyte aging in mice. Fisetin improved the development of aged oocytes after fertilization and inhibited the Sirt1 reduction in aged oocytes. Fisetin increased the GSH level and Sod2 transcription level to inhibit ROS accumulation in aged oocytes. Meanwhile, fisetin attenuated aging-induced spindle abnormalities, mitochondrial dysfunction, and apoptosis. At the molecular level, fisetin decreased aging-induced aberrant expression of H3K9me3. In addition, fisetin increased the expression levels of the mitochondrial transcription factor Tfam and the mitochondrial genes Co2 and Atp8 by upregulating Sirt1 in aged oocytes. Finally, inhibition of Sirt1 reversed the anti-aging effects of fisetin. Taken together, fisetin delayed postovulatory oocyte aging by upregulating Sirt1.

Endoplasmic Reticulum Stress and Mitochondrial Stress in Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a widespread and harmful disease closely linked to mitochondrial and endoplasmic reticulum stress (ERS). Globally, severe drug-induced hepatitis, cirrhosis, and liver cancer are the primary causes of liver-related morbidity and mortality. A hallmark of DILI is ERS and changes in mitochondrial morphology and function, which increase the production of reactive oxygen species (ROS) in a vicious cycle of mutually reinforcing stress responses. Several pathways are maladapted to maintain homeostasis during DILI. Here, we discuss the processes of liver injury caused by several types of drugs that induce hepatocyte stress, focusing primarily on DILI by ERS and mitochondrial stress. Importantly, both ERS and mitochondrial stress are mediated by the overproduction of ROS, destruction of Ca2+ homeostasis, and unfolded protein response (UPR). Additionally, we review new pathways and potential pharmacological targets for DILI to highlight new possibilities for DILI treatment and mitigation.

Effects of overexpression of histone H3K9me3 demethylase on development of porcine cloned embryos.

The abnormal modification of histone is an important factor restricting development of porcine cloned embryos. Overexpression of histone H3K9me3 demethylase KDM4 family can effectively improve the developmental efficiency of cloned embryos. In order to explore the effects of overexpression of H3K9me3 demethylase on the development of porcine cloned embryos, KDM4A mRNA and KDM4D mRNA were injected respectively into porcine cloned embryos at the 1-cell stage and 2-cell stage to detect the blastocyst rate; 2-cell stage cloned embryos injected with KDM4A mRNA and embryo injection water (the control group) at the 1-cell stage were collected to detect the expression level of H3K9me3, and 4-cell stage cloned embryos were collected for single cell transcriptome sequencing, then the sequencing data was analyzed with KEGG and GO. The results showed that the blastocyst rate of porcine cloned embryos injected with KDM4A mRNA at 1-cell stage was significantly higher than that of the control group (25.32 ± 0.74% vs 14.78 ± 0.87%), while cloned embryos injected with KDM4D mRNA had a similar blastocyst rate with cloned embryos in control group (16.27 ± 0.77% vs 14.78 ± 0.87%). Porcine cloned embryos injected with KDM4A mRNA and KDM4D mRNA at 2-cell stage had a similar blastocyst rate with cloned embryos in control group (32.18 ± 1.67%, 30.04 ± 0.91% vs 31.22 ± 1.40%). The expression level of H3K9me3 in cloned embryos injected with KDM4A mRNA at 1-cell stage was lower than that in control group. There were 133 differentially expressed genes detected by transcriptome sequencing, including 52 up-regulated genes and 81 down-regulated genes. Pathways enriched by GO analyses were mainly related to protein localization. Pathways enriched by KEGG analyses were related to cellular senescence and acute myeloid leukemia. These results suggest that overexpression of histone H3K9me3 demethylase KDM4A can significantly improve the developmental efficiency of porcine cloned embryos.

[Regulation of ischemic stroke by circadian rhythm and intervention by traditional Chinese medicine].

Circadian rhythm is an internal regulatory mechanism formed in organisms in response to the circadian periodicity in the environment, which modulates the pathophysiological events, occurrence and development of diseases, and the response to treatment in mammals. It significantly influences the susceptibility, injury, and recovery of ischemic stroke, and the response to therapy. Accumulating evidence indicates that circadian rhythms not only regulate the important physiological factors of ischemic stroke events, such as blood pressure and coagulation-fibrinolysis system, but also participate in the immuno-inflammatory reaction mediated by glial cells and peripheral immune cells after ischemic injury and the regulation of neurovascular unit(NVU). This article aims to link molecular, cellular, and physiological pathways in circadian biology to the clinical consequences of ischemic stroke and to illustrate the impact of circadian rhythms on ischemic stroke pathogenesis, the regulation of NVU, and the immuno-inflammatory responses. The regulation of circadian rhythm by traditional Chinese medicine is reviewed, and the research progress of traditional Chinese medicine intervention in circadian rhythm is summarized to provide a reasonable and valuable reference for the follow-up traditional Chinese medicine research and molecular mechanism research of circadian rhythm.

Dynamic Responses of Chromosome-Binding Protein Complexes to Meiotic Prophase I of Mouse Spermatocyte.

Meiotic prophase I (MPI) is the most important event in mammalian meiosis. The status of the chromosome-binding proteins (CBPs) and the corresponding complexes and their functions in MPI have not yet been well scrutinized. Quantitative proteomics focused on MPI-related CBPs was accomplished, in which mouse primary spermatocytes in four different subphases of MPI were collected, and chromosome-enriched proteins were extracted and quantitatively identified. According to a stringent criterion, 1136 CBPs in the MPI subphases were quantified. Looking at the dynamic patterns of CBP abundance in response to MPI progression, the patterns were broadly divided into two groups: high abundance in leptotene and zygotene or that in pachytene and diplotene. Furthermore, 152 such CBPs were regarded as 26 CBP complexes with strict filtration, in which some of these complexes were perceived to be MPI-dependent for the first time. These complexes basically belonged to four functional categories, while their dynamic abundance changes following MPI appeared; the functions of DNA replication decreased; and transcription and synapsis were activated in zygotene, pachytene, and diplotene; in contrast to the traditional prediction, condensin activity weakened in pachytene and diplotene. Profiling of protein complexes thus offered convincing evidence of the importance of CBP complexes in MPI.

Global Quantification of Glutathione S-Transferases in Human Serum Using LC-MS/MS Coupled with Affinity Enrichment.

The members of the glutathione S-transferase (GST) superfamily often exhibit functional overlap and can compensate for each other. Their concentrations in serum are considered as disease biomarkers. A global and quantitative evaluation of serum GSTs is therefore urgent, but there is a lack of efficient approaches due to technological limitations. GSH magnetic beads were examined for their affinity to enrich GSTs in serum, and the enriched GSTs were quantitatively targeted using a Q Exactive HF-X mass spectrometer in parallel reaction monitoring (PRM) mode. To optimize the quantification of GST peptides, sample types, trypsin digestion, and serum loading were carefully assessed; a biosynthetic method was employed to generate isotope-labeled GST peptides, and instrumental parameters were systematically optimized. A total of 134 clinical sera were collected for GST quantification from healthy donors and patients with four liver diseases. Using the new approach, GSTs in healthy sera were profiled: 14 GST peptides were quantified, and the abundance of five GST families was ranked GSTM > GSTP > GSTA > MGST1 > GSTT1, ranging from 0.1 to 4 pmol/L. Furthermore, combining the abundance of multiple GST peptides could effectively distinguish different types of liver diseases. Quantification of serum GSTs through targeted proteomics, therefore, has apparent clinical potential for disease diagnosis.

Dissecting the molecular mechanism of russeting in sand pear (Pyrus pyrifolia Nakai) by metabolomics, transcriptomics, and proteomics.

Brown coloration and a rough appearance as russet and semi-russet (partial russet) are features unique to the popular Asian sand pear (Pyrus pyrifolia Nakai). The degree of russeting is different between different genotypes. Russeting is sensitive to water fluctuations, where excessive rainwater can trigger/stimulate its development. However, the molecular mechanism of russeting is currently unclear. Here, we employed multi-omics, i.e., metabolomics, transcriptomics, and proteomics, and analyzed the effect of different sand pear genotypes and artificial rainfall on russeting of pear fruits. This led to the identification of 79, 64, and 29 differentially produced/expressed metabolites, transcripts, and proteins that are involved in the biosynthesis of suberin, phenylpropane, cutin, and waxes. Further analysis of these differentially expressed genes and their encoded proteins revealed that four of them exhibited high expression at both transcript and protein levels. Transient expression of one such gene, PbHHT1 (accession number 103966555), which encodes ω-hydroxypalmitate-O-feruloyl transferase, in young green non-russet fruits triggered premature suberization in the russeting pear genotypes. This coincided with increased production of 16-feruloyloxypalmitic acid, a conjugated compound between phenols and esters during the polymerization for suberin formation. Collectively, our data from the combined three omics demonstrate that russeting in sand pear is a complex process involving the biosynthesis and transport of suberin and many other secondary metabolites.

Effect of ALA-PDT on inhibition of oral precancerous cell growth and its related mechanisms.

BACKROUND: Early treatment of oral precancerous lesions is considered as a key strategy for in oral carcinogenesis prevention. Increasing evidence has suggested that the transforming growth factor beta (TGF-ß) signaling pathway is tightly involved in the process of oral-carcinogenesis. In this study, we investigated the inhibition effect and potential mechanism of 5-aminolaevulinic acid photodynamic therapy (ALA-PDT) in human oral precancerous cells via TGF-ß pathway. MATERIALS AND METHODS: Here, the dysplastic oral keratinocyte (DOK) cells were incubated with ALA concentration of 1 mM/mL for 4 h and then irradiated with a Helium-Neon (He-Ne) ion laser at 633 nm (200 mW/cm2). The control cells were cultured in Dulbecco's modified Eagle's medium (DMEM) medium. We analyzed the differentially expressed genes and correlated pathways in oral precancerous cells following ALA-PDT using Affymetrix microarrays. TGF-ß pathway was analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. Bioinformatics analysis was performed to evaluate the expression of TGF-ß1 in human oral cancer samples and adjacent normal samples. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), flow cytometry, 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and wound healing assay were used to assess the effects of ALA-PDT plus TGF-ß receptor inhibitor (LY2109761) in DOK cells. RESULTS: The TGF-ß signaling could exert in suppressive effects on DOK cells after ALA-PDT. The cell proliferation and migration rate of DOK cells was significantly reduced and apoptosis and ROS generation induced more effectively by ALA-PDT combined with LY2109761. Furthermore, cell cycle analysis revealed that the combined treatment resulted in G0/G1 phase arrest. CONCLUSIONS: ALA-PDT suppresses the growth of oral precancerous cells by regulating the TGF-ß signaling pathway, and its suppressive effect was enhanced using LY2109761. These results indicate that it could be a promising alternative treatment against oral precancerous lesions.

Genome-Wide Analysis of Genes Involved in the GA Signal Transduction Pathway in 'duli' Pear (Pyrus betulifolia Bunge).

Gibberellic acid (GA) is an important phytohormone that regulates every aspect of plant growth and development. While elements involved in GA signaling have been identified and, hence, their functions have been well studied in model plants, such as Arabidopsis and rice, very little is known in pear. We, therefore, analyzed the genes related to GA signaling from the recently sequenced genome of the wildtype 'duli' pear (Pyrus betulifolia Bunge), a widely used rootstock for grafting in pear cultivation in China due to its vigorous growth and resistance to abiotic and biotic stress. In total, 15 genes were identified, including five GA receptors PbGID1s (GA-INSENSTIVE DWARF 1), six GA negative regulators, PbDELLAs, and four GA positive regulators, PbSLYs. Exogenous application of GA could promote the expression of PbGID1s but inhibit that of PbDELLAs and PbSLYs in tissue culture 'duli' pear seedlings. The expression profiles of these genes in field-grown trees under normal growth conditions, as well as in tissue-cultured seedlings treated with auxin (IAA), GA, paclobutrazol (PAC), abscisic acid (ABA), and sodium chloride (NaCl), were also studied, providing further evidence of the involvement of these genes in GA signaling in 'duli' pear plants. The preliminary results obtained in this report lay a good foundation for future research into GA signaling pathways in pear. Importantly, the identification and preliminary functional verification of these genes could guide molecular breeding in order to obtain the highly desired dwarf pear rootstocks for high-density plantation to aid easy orchard management and high yielding of pear fruits.

Identification of Important Factors Causing Developmental Arrest in Cloned Pig Embryos by Embryo Biopsy Combined with Microproteomics.

The technique of pig cloning holds great promise for the livestock industry, life science, and biomedicine. However, the prenatal death rate of cloned pig embryos is extremely high, resulting in a very low cloning efficiency. This limits the development and application of pig cloning. In this study, we utilized embryo biopsy combined with microproteomics to identify potential factors causing the developmental arrest in cloned pig embryos. We verified the roles of two potential regulators, PDCD6 and PLK1, in cloned pig embryo development. We found that siRNA-mediated knockdown of PDCD6 reduced mRNA and protein expression levels of the pro-apoptotic gene, CASP3, in cloned pig embryos. PDCD6 knockdown also increased the cleavage rate and blastocyst rate of cloned porcine embryos. Overexpression of PLK1 via mRNA microinjection also improved the cleavage rate of cloned pig embryos. This study provided a new strategy to identify key factors responsible for the developmental defects in cloned pig embryos. It also helped establish new methods to improve pig cloning efficiency, specifically by correcting the expression pattern of PDCD6 and PLK1 in cloned pig embryos.

Supplementation of SDF1 during Pig Oocyte In Vitro Maturation Improves Subsequent Embryo Development.

The quality of in vitro matured oocytes is inferior to that of in vivo matured oocytes, which translates to low developmental capacity of embryos derived from in vitro matured oocytes. The developmental potential of in vitro matured oocytes is usually impaired due to oxidative stress. Stromal cell-derived factor-l (SDF1) can reduce oxidative stress and inhibit apoptosis. The aim of this study was to investigate the effects of SDF1 supplementation during pig oocyte in vitro maturation (IVM) on subsequent embryo development, and to explore the acting mechanisms of SDF1 in pig oocytes. We found that the IVM medium containing 20 ng/mL SDF1 improved the maturation rate of pig oocytes, as well as the cleavage rate and blastocyst rate of embryos generated by somatic cell nuclear transfer, in vitro fertilization, and parthenogenesis. Supplementation of 20 ng/mL SDF1 during IVM decreased the ROS level, increased the mitochondrial membrane potential, and altered the expression of apoptosis-related genes in the pig oocytes. The porcine oocyte transcriptomic data showed that SDF1 addition during IVM altered the expression of genes enriched in the purine metabolism and TNF signaling pathways. SDF1 supplementation during pig oocyte IVM also upregulated the mRNA and protein levels of YY1 and TET1, two critical factors for oocyte development. In conclusion, supplementation of SDF1 during pig oocyte IVM reduces oxidative stress, changes expression of genes involved in regulating apoptosis and oocyte growth, and enhances the ability of in vitro matured pig oocytes to support subsequent embryo development. Our findings provide a theoretical basis and a new method for improving the developmental potential of pig in vitro matured oocytes.

Discovery of Missing Proteins from an Aneuploidy Cell Line Using a Proteogenomic Approach.

With the steadfast development of proteomic technology, the number of missing proteins (MPs) has been continuously shrinking, with approximately 1470 MPs that have not been explored yet. Due to this phenomenon, the discovery of MPs has been increasingly more difficult and elusive. In order to face this challenge, we have hypothesized that a stable aneuploid cell line with increased chromosomes serves as a useful material for assisting MP exploration. Ker-CT cell line with trisomy at chromosome 5 and 20 was selected for this purpose. With a combination strategy of RNA-Seq and LC-MS/MS, a total of 22 178 transcripts and 8846 proteins were identified in Ker-CT. Although the transcripts corresponding to 15 and 15 MP genes located at chromosome 5 and 20 were detected, none of the MPs were found in Ker-CT. Surprisingly, 3 MPs containing at least two unique non-nest peptides of length ≥9 amino acids were identified in Ker-CT, whose genes are located on chromosome 3 and 10, respectively. Furthermore, the 3 MPs were verified using the method of parallel reaction monitoring (PRM). These results suggest that the abnormal status of chromosomes may not only impact the expression of the corresponding genes in trisomy chromosomes, but also influence that of other chromosomes, which benefits MP discovery. The data obtained in this study are available via ProteomeXchange (PXD028647) and PeptideAtlas (PASS01700), respectively.