Oleic acid-PPARγ-FABP4 loop fuels cholangiocarcinoma colonization in lymph node metastases microenvironment.

BACKGROUND AND AIMS: Lymph node metastasis is a significant risk factor for patients with cholangiocarcinoma, but the mechanisms underlying cholangiocarcinoma colonization in the lymph node microenvironment remain unclear. We aimed to determine whether metabolic reprogramming fueled the adaptation and remodeling of cholangiocarcinoma cells to the lymph node microenvironment. APPROACH AND RESULTS: Here, we applied single-cell RNA sequencing of primary tumor lesions and paired lymph node metastases from patients with cholangiocarcinoma and revealed significantly reduced intertumor heterogeneity and syntropic lipid metabolic reprogramming of cholangiocarcinoma after metastasis to lymph nodes, which was verified by pan-cancer single-cell RNA sequencing analysis, highlighting the essential role of lipid metabolism in tumor colonization in lymph nodes. Metabolomics and in vivo CRISPR/Cas9 screening identified PPARγ as a crucial regulator in fueling cholangiocarcinoma colonization in lymph nodes through the oleic acid-PPARγ-fatty acid-binding protein 4 positive feedback loop by upregulating fatty acid uptake and oxidation. Patient-derived organoids and animal models have demonstrated that blocking this loop impairs cholangiocarcinoma proliferation and colonization in the lymph node microenvironment and is superior to systemic inhibition of fatty acid oxidation. PPARγ-regulated fatty acid metabolic reprogramming in cholangiocarcinoma also contributes to the immune-suppressive niche in lymph node metastases by producing kynurenine and was found to be associated with tumor relapse, immune-suppressive lymph node microenvironment, and poor immune checkpoint blockade response. CONCLUSIONS: Our results reveal the role of the oleic acid-PPARγ-fatty acid-binding protein 4 loop in fueling cholangiocarcinoma colonization in lymph nodes and demonstrate that PPARγ-regulated lipid metabolic reprogramming is a promising therapeutic target for relieving cholangiocarcinoma lymph node metastasis burden and reducing further progression.

Global chromosome rearrangement induced by CRISPR-Cas9 reshapes the genome and transcriptome of human cells.

Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. Large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved global chromosome rearrangement (GCR) in human cells. The copy number variations of the GCR genomes showed typical patterns observed in tumor genomes. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by GCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival. Our study provided a new application of CRISPR-Cas9 and a practical approach for GCR in complex mammalian genomes.

The composition dynamics of transposable elements in human blastocysts.

Transposable elements (TEs) are mobile DNA sequences that can replicate themselves and play significant roles in embryo development and chromosomal structure remodeling. In this study, we investigated the variation of TEs in blastocysts with different parental genetic backgrounds. We analyzed the proportions of 1137 TEs subfamilies from six classes at the DNA level using Bowtie2 and PopoolationTE2 in 196 blastocysts with abnormal parental chromosomal diseases. Our findings revealed that the parental karyotype was the dominant factor influencing TEs frequencies. Out of the 1116 subfamilies, different frequencies were observed in blastocysts with varying parental karyotypes. The development stage of blastocysts was the second most crucial factor influencing TEs proportions. A total of 614 subfamilies exhibited different proportions at distinct blastocyst stages. Notably, subfamily members belonging to the Alu family showed a high proportion at stage 6, while those from the LINE class exhibited a high proportion at stage 3 and a low proportion at stage 6. Moreover, the proportions of some TEs subfamilies also varied depending on blastocyst karyotype, inner cell mass status, and outer trophectoderm status. We found that 48 subfamilies displayed different proportions between balanced and unbalanced blastocysts. Additionally, 19 subfamilies demonstrated varying proportions among different inner cell mass scores, and 43 subfamilies exhibited different proportions among outer trophectoderm scores. This study suggests that the composition of TEs subfamilies may be influenced by various factors and undergoes dynamic modulation during embryo development.

Current status and influencing factors of self-management in knee joint discomfort among middle-aged and elderly people: a cross-sectional study.

BACKGROUND: This study aims to identify the current status and factors influencing self-management of knee discomfort in middle-aged and elderly people in China. METHODS: A stratified multistage cluster sampling method was used to select participants from communities in China from January 15 to May 31, 2020. A cross-sectional survey was conducted using the general information questionnaire and the Knee Joint Discomfort Self-management Scale. Univariate analysis and a generalized linear model were used to analyze the factors influencing self-management. RESULTS: The prevalence of knee discomfort was 77%. Moderate to severe discomfort accounted for 30.5%. The average item score of self-management in 9640 participants was 1.98 ± 0.76. The highest and lowest levels were: 'daily life management' and 'information management'. Gender, ethnicity, education level, economic source, chronic disease, knee pain in the past month, and the degree of self-reported knee discomfort were significant predictors of self-management. CONCLUSION: The self-management of knee discomfort in middle-aged and elderly people is poor, and the degree of discomfort is a significant predictor. Healthcare providers should consider socioeconomic demographic and clinical characteristics to help these individuals improve their self-management skills. Attention should also be given to improving their ability to access health information and making them aware of disease risks.

Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA-damaging chemotherapeutic agents.

The bromodomain-containing protein 7 (BRD7) is a tumour suppressor protein with critical roles in cell cycle transition and transcriptional regulation. Whether BRD7 is regulated by post-translational modifications remains poorly understood. Here, we find that chemotherapy-induced DNA damage leads to the rapid degradation of BRD7 in various cancer cell lines. PARP-1 binds and poly(ADP)ribosylates BRD7, which enhances its ubiquitination and degradation through the PAR-binding E3 ubiquitin ligase RNF146. Moreover, the PARP1 inhibitor Olaparib significantly enhances the sensitivity of BRD7-positive cancer cells to chemotherapeutic drugs, while it has little effect on cells with low BRD7 expression. Taken together, our findings show that PARP1 induces the degradation of BRD7 resulting in cancer cell resistance to DNA-damaging agents. BRD7 might thus serve as potential biomarker in clinical trial for the prediction of synergistic effects between chemotherapeutic drugs and PARP inhibitors.

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. RESULTS: We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3'-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. CONCLUSIONS: Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study.

The wide distribution and horizontal transfers of beta satellite DNA in eukaryotes.

Beta satellite DNA (satDNA), also known as Sau3A sequences, are repetitive DNA sequences reported in human and primate genomes. It is previously thought that beta satDNAs originated in old world monkeys and bursted in great apes. In this study, we searched 7821 genome assemblies of 3767 eukaryotic species and found that beta satDNAs are widely distributed across eukaryotes. The four major branches of eukaryotes, animals, fungi, plants and Harosa/SAR, all have multiple clades containing beta satDNAs. These results were also confirmed by searching whole genome sequencing data (SRA) and PCR assay. Beta satDNA sequences were found in all the primate clades, as well as in Dermoptera and Scandentia, indicating that the beta satDNAs in primates might originate in the common ancestor of Primatomorpha or Euarchonta. In contrast, the widely patchy distribution of beta satDNAs across eukaryotes presents a typical scenario of multiple horizontal transfers.

Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy.

Plant type [2Fe-2S] ferredoxins function primarily as electron transfer proteins in photosynthesis. Studying the unfolding-folding of ferredoxins in vitro is challenging, because the unfolding of ferredoxin is often irreversible due to the loss or disintegration of the iron-sulfur cluster. Additionally, the in vivo folding of holo-ferredoxin requires ferredoxin biogenesis proteins. Here, we employed atomic force microscopy-based single-molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and refolding of a plant type [2Fe-2S] ferredoxin from cyanobacteria Anabaena. Our results indicate that upon stretching, ferredoxin unfolds in a three-state mechanism. The first step is the unfolding of the protein sequence that is outside and not sequestered by the [2Fe-2S] center, and the second one relates to the force-induced rupture of the [2Fe-2S] metal center and subsequent unraveling of the protein structure shielded by the [2Fe-2S] center. During repeated stretching and relaxation of a single polyprotein, we observed that the completely unfolded ferredoxin can refold to its native holo-form with a fully reconstituted [2Fe-2S] center. These results demonstrate that the unfolding-refolding of individual ferredoxin is reversible at the single-molecule level, enabling new avenues of studying both folding-unfolding mechanisms, as well as the reactivity of the metal center of metalloproteins in vitro.

Serial number tagging reveals a prominent sequence preference of retrotransposon integration.

Transposable elements (TE) have both negative and positive impact on the biology of their host. As a result, a balance is struck between the host and the TE that relies on directing integration to specific genome territories. The extraordinary capacity of DNA sequencing can create ultra dense maps of integration that are being used to study the mechanisms that position integration. Unfortunately, the great increase in the numbers of insertion sites detected comes with the cost of not knowing which positions are rare targets and which sustain high numbers of insertions. To address this problem we developed the serial number system, a TE tagging method that measures the frequency of integration at single nucleotide positions. We sequenced 1 million insertions of retrotransposon Tf1 in the genome of Schizosaccharomyces pombe and obtained the first profile of integration with frequencies for each individual position. Integration levels at individual nucleotides varied over two orders of magnitude and revealed that sequence recognition plays a key role in positioning integration. The serial number system is a general method that can be applied to determine precise integration maps for retroviruses and gene therapy vectors.

Ion presence during thermal processing modulates the performance of rice albumin/anthocyanin binary system.

Thermal processing with salt ions is widely used for the production of food products (such as whole grain food) containing protein and anthocyanin. To date, it is largely unexplored how salt ion presence during thermal processing regulates the practical performance of protein/anthocyanin binary system. Here, rice albumin (RA) and black rice anthocyanins (BRA) were used to prepare RA/BRA composite systems as a function of temperature (60-100 °C) and NaCl concentration (10-40 mM) or CaCl2 concentration (20 mM). It was revealed that the spontaneous complexing reaction between RA and BRA was driven by hydrophobic interactions and hydrogen bonds and becomes easier and more favorable at a higher temperature (≤90 °C), excessive temperature (100 °C), however, may result in the degradation of BRA. Moreover, the salt ion presence during thermal processing may bind with RA and BRA, respectively, which could restrict the interaction between BRA and RA. Additionally, the inclusion of Na+ or Ca2+ at 20 mM endowed the binary system with strengthened DPPH radical scavenging capacity (0.95 for Na+ and 0.99 for Ca2+). Notably, Ca2+ performed a greater impact on the stability of the system than Na+.

Incorporating edible oil during cooking tailors the microstructure and quality features of brown rice following heat moisture treatment.

While brown rice (BR) has numerous nutritional properties, the consumption potential of which is seriously restricted since the poor cooking quality and undesirable flavor. Here, edible oils (pork lard and corn oil, 1-5 wt%) were incorporated during the cooking of BR following heat moisture treatment. Incorporating corn oil rather than lard significantly ameliorated the texture properties (e.g. hardness, cohesiveness, and chewiness) and sensory properties of cooked BR. Both lard- and corn oil-incorporated cooked BR showed obvious structural changes accompanied by the formation of amylose-lipid complexes during cooking. It was confirmed that the incorporation of lard and corn oil allowed a higher degree of short-range molecular order, more V-type starch crystallites, and elevated nano-structural arrangements. Additionally, a decreased hardness (from 559.04 g to 424.18 g and 385.91 g, respectively) and enriched resistant starch (RS) were also observed, the highest RS content (15.95 % and 16.32 %, respectively) was observed when 1 wt% of lard and corn oil were incorporated.

Preparing gelatin-containing polycaprolactone / polylactic acid nanofibrous membranes for periodontal tissue regeneration using side-by-side electrospinning technology.

Electrospinning technology has recently attracted increased attention in the biomedical field, and preparing various cellulose nanofibril membranes for periodontal tissue regeneration has unique advantages. However, the characteristics of using a single material tend to make it challenging to satisfy the requirements for a periodontal barrier film, and the production of composite fibrous membranes frequently impacts the quality of the final fiber membrane due to the influence of miscibility between different materials. In this study, nanofibrous membranes composed of polylactic acid (PLA) and polycaprolactone (PCL) fibers were fabricated using side-by-side electrospinning. Different concentrations of gelatin were added to the fiber membranes to improve their hydrophilic properties. The morphological structure of the different films as well as their composition, wettability and mechanical characteristics were examined. The results show that PCL/PLA dual-fibrous composite membranes with an appropriate amount of gelatin ensures sufficient mechanical strength while obtaining improved hydrophilic properties. The viability of L929 fibroblasts was evaluated using CCK-8 assays, and cell adhesion on the scaffolds was confirmed by scanning electron microscopy and by immunofluorescence assays. The results demonstrated that none of the fibrous membranes were toxic to cells and the addition of gelatin improved cell adhesion to those membranes. Based on our findings, adding 30% gelatin to the membrane may be the most appropriate content for periodontal tissue regeneration, considering the scaffold's mechanical qualities, hydrophilic properties and biocompatibility. In addition, the PCL-gelatin/PLA-gelatin dual-fibrous membranes prepared using side-by-side electrospinning technology have potential applications for tissue engineering.

High-throughput sequencing of retrotransposon integration provides a saturated profile of target activity in Schizosaccharomyces pombe.

The biological impact of transposons on the physiology of the host depends greatly on the frequency and position of integration. Previous studies of Tf1, a long terminal repeat retrotransposon in Schizosaccharomyces pombe, showed that integration occurs at the promoters of RNA polymerase II (Pol II) transcribed genes. To determine whether specific promoters are preferred targets of integration, we sequenced large numbers of insertions using high-throughput pyrosequencing. In four independent experiments we identified a total of 73,125 independent integration events. These data provided strong support for the conclusion that Pol II promoters are the targets of Tf1 integration. The size and number of the integration experiments resulted in reproducible measures of integration for each intergenic region and ORF in the S. pombe genome. The reproducibility of the integration activity from experiment to experiment demonstrates that we have saturated the full set of insertion sites that are actively targeted by Tf1. We found Tf1 integration was highly biased in favor of a specific set of Pol II promoters. The overwhelming majority (76%) of the insertions were distributed in intergenic sequences that contained 31% of the promoters of S. pombe. Interestingly, there was no correlation between the amount of integration at these promoters and their level of transcription. Instead, we found Tf1 had a strong preference for promoters that are induced by conditions of stress. This targeting of stress response genes coupled with the ability of Tf1 to regulate the expression of adjacent genes suggests Tf1 may improve the survival of S. pombe when cells are exposed to environmental stress.

Effect of luteal-phase GnRH agonist on frozen-thawed embryo transfer during artificial cycles: a randomised clinical pilot study.

Purpose: This randomised clinical pilot study evaluated the effect of the mid-luteal additional single dose of gonadotropin-releasing hormone agonist (GnRH-a) on the clinical outcome of the females subjected to artificial cycle frozen-thawed embryo transfer (AC-FET). Methods: A total of 129 females were randomised into two groups (70 in the control group and 59 in the intervention group). Both groups received standard luteal support. The intervention group was given an extra dose of 0.1 mg GnRH-a in the luteal phase. The live birth rate served as the primary endpoint. The secondary endpoints were the positivity of pregnancy tests, the clinical pregnancy rate, the miscarriage rate, the implantation rate, and the multiple pregnancy rate. Results: There were more positive pregnancy tests, clinical pregnancies, live births, and twinning pregnancies, and fewer miscarriages observed in the intervention arm compared to the controls, though no statistical significance was concluded. No difference was found in the number of macrosomia in the two groups. There was no congenital abnormality newborn. Conclusion: Overall, the difference of 12.1 percentage points in the live births rate (40.7% vs 28.6%) between the two groups, however, is statistically insignificant. the improvement of the pregnancy outcome supports the non-inferiority of GnRH-a added during the luteal phase in AC-FET. Larger-scale clinical trials are required to further establish the positive benefits.

The local integration preference of the Tf1 retrotransposon in Schizosaccharomyces pombe.

Transposons are mobile DNAs that can move to different locations in host genomes. The integration site selection of transposons is critical for both themselves and host cells. Studies on the integration of retrotransposons and retroviruses have focused more on the global preference than on the local preference. The local preferences of retrotransposons are usually weak and of large diversity. Here, we analyzed hundreds of thousands of independent integration events of the Tf1 retrotransposon in Schizosaccharomyces pombe. The consensus sequence at the Tf1 integration sites shows a palindromic pattern, which can be divided into four sections, each of them contains one or more CGnTA units with a period of 10 base pairs, indicating interaction with subunits of the integrase oligomer in the pre-integration complex. Moreover, the analysis on the nucleosome occupancy flanking Tf1 target sites shows that Tf1 integration favors regions with one entire nucleosome depletion.

Comprehensive Evaluation of the Tendency of Vertical Collusion in Construction Bidding Based on Deep Neural Network.

To effectively diagnose and monitor the vertical collusion in construction project bidding, this paper developed a comprehensive evaluation model with deep neural network and transfer learning. By this model, the collusion characteristics of bidders, tenderers, and bid evaluation experts were mined from limited data set hidden and collusion tendency was evaluated. Firstly, 18 evaluation indicators were established from literature review, court file summarization, typical case analysis, and expert consultation. Then, a comprehensive evaluation model was developed with the deep neural network and transfer learning. Finally, the model was trained and tested with the collected data set. The test results showed that the developed model achieved 87.3% identification accuracy in collusion tendency evaluation of different subjects.

Identification of an integrase-independent pathway of retrotransposition.

Retroviruses and long terminal repeat retrotransposons rely on integrase (IN) to insert their complementary DNA (cDNA) into the genome of host cells. Nevertheless, in the absence of IN, retroelements can retain notable levels of insertion activity. We have characterized the IN-independent pathway of Tf1 and found that insertion sites had homology to the primers of reverse transcription, which form single-stranded DNAs at the termini of the cDNA. In the absence of IN activity, a similar bias was observed with HIV-1. Our studies showed that the Tf1 insertions result from single-strand annealing, a noncanonical form of homologous recombination mediated by Rad52. By expanding our analysis of insertions to include repeat sequences, we found most formed tandem elements by inserting at preexisting copies of a related transposable element. Unexpectedly, we found that wild-type Tf1 uses the IN-independent pathway as an alternative mode of insertion.

Interleukin 13 participates in terminal differentiation of esophageal squamous cell carcinoma cells.

Background: In China, esophageal squamous cell carcinoma (ESCC) accounts for more than 90% of all esophageal cancer cases. Interleukin 13 (IL-13) was widely reported to play a key role in tumor progression. Our previous study reported that IL-13 was a favorable predictive marker for the overall survival of esophageal squamous cell carcinoma (ESCC) patients, but how IL-13 contributes to ESCC progression remains unknown. This study aims to explore the role of IL-13 and its underlying downstream molecular mechanisms in ESCC progression. Methods: Tissue microarrays including 262 primary ESCC tumor tissues were collected and analyzed. The expression of IL-13 in ESCC tumor tissue was detected with immunohistochemistry staining (IHC). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to qualify the expressions of KRT13, KRT4 and 15-lipoxygenase-1 (15-LOX-1) in cultured ESCC cell lines with recombinant IL-13 treatment. Results: IL-13 was expressed in the esophageal epithelium cells and ESCC tumor cells. High IL-13 expression in ESCC tumor cells predicted a good prognosis for patients. Recombinant human IL-13 raised KRT13 and 15-LOX-1 mRNA levels, but lowered KRT4 mRNA level 15-LOX-1 in ESCC cells in vitro. Conclusions: In summary, our study suggests that IL-13 might improve the prognosis of ESCC by promoting the terminal differentiation of ESCC cells. This may offer potential new therapeutic target for early treatment of ESCC.

Starch-based materials encapsulating food ingredients: Recent advances in fabrication methods and applications.

Encapsulation systems have gained significant interest in designing innovative foods, as they allow for the protection and delivery of food ingredients that have health benefits but are unstable during processing, storage and in the upper gastrointestinal tract. Starch is widely available, cheap, biodegradable, edible, and easy to be modified, thus highly suitable for the development of encapsulants. Much efforts have been made to fabricate various types of porous starch and starch particles using different techniques (e.g. enzymatic hydrolysis, aggregation, emulsification, electrohydrodynamic process, supercritical fluid process, and post-processing drying). Such starch-based systems can load, protect, and deliver various food ingredients (e.g. fatty acids, phenolic compounds, carotenoids, flavors, essential oils, irons, vitamins, probiotics, bacteriocins, co-enzymes, and caffeine), exhibiting great potentials in developing foods with tailored flavor, nutrition, sensory properties, and shelf-life. This review surveys recent advances in different aspects of starch-based encapsulation systems including their forms, manufacturing techniques, and applications in foods.

The Integration Preference of Sleeping Beauty at Non-TA Site Is Related to the Transposon End Sequences.

Recently, we proved that Sleeping Beauty (SB) transposon integrates into non-TA sites at a lower frequency. Here, we performed a further study on the non-TA integration of SB and showed that (1) SB can integrate into non-TA sites in HEK293T cells as well as in mouse cell lines; (2) Both the hyperactive transposase SB100X and the traditional SB11 catalyze integrations at non-TA sites; (3) The consensus sequence of the non-TA target sites only occurs at the opposite side of the sequenced junction between the transposon end and the genomic sequences, indicating that the integrations at non-TA sites are mainly aberrant integrations; and (4) The consensus sequence of the non-TA target sites is corresponding to the transposon end sequence. The consensus sequences changed following the changes of the transposon ends. This result indicated that the interaction between the SB transposon end and genomic DNA (gDNA) may be involved in the target site selection of the SB integrations at non-TA sites.