Intensive Treatment of Organic Wastewater by Three-Dimensional Electrode System within Mn-Loaded Steel Slag as Catalytic Particle Electrodes.

Developing a green, low-carbon, and circular economic system is the key to achieving carbon neutrality. This study investigated the organics removal efficiency in a three-dimensional electrode reactor (3DER) constructed from repurposed industrial solid waste, i.e., Mn-loaded steel slag, as the catalytic particle electrodes (CPE). The CPE, a micron-grade material consisting primarily of transition metals, including Fe and Mn, exhibited excellent electric conductivity, catalytic ability, and recyclability. High rhodamine B (RhB) removal efficiency in the 3DER was observed through a physical modelling experiment. The optimal operating condition was determined through a single-factor experiment in which 5.0 g·L-1 CPE and 3 mM peroxymonosulfate (PMS) were added to a 200 mL solution of 10 mM RhB under a current intensity of 0.5 A and a 1.5 to 2.0 cm distance between the 2D electrodes. When the initial pH value of the simulated solution was 3 to 9, the RhB removal rate exceeded 96% after 20 min reaction. In addition, the main reactive oxidation species in the 3DER were determined. The results illustrated that HO• and SO4•- both existed, but that the contribution of SO4•- to RhB removal was much lower than that of HO• in the 3DER. In summary, this research provides information on the potential of the 3DER for removing refractory organics from water.

Predictors and influence of postoperative moderate-to-severe pain of PACU in the patients with malignancy.

BACKGROUND: This study was identified the risk factors for and designed to investigate influence of postoperative moderate-to-severe pain of post anaesthesia care unit (PACU) in patients with malignancy. METHODS: A retrospective study was performed on 22,600 cancer patients with malignancy who underwent elective radical surgery in the new hospital of First Affiliated Hospital of Wenzhou Medical University, between January 2016 and June 2021. All patients were transferred to the PACU after tracheal extubation. Patients were divided into two groups according to a visual analogue scale (VAS) score of > 3: the no-moderate-severe-pain group and moderate-to-severe-pain group. Data pertaining to demographic, surgical, anaesthetic, and other factors were recorded. Lasso and logistic regression analysis was performed to explore the risk factors, then a nomogram was constructed to predict the moderate-severe-pain in the PACU. Validation was performed by using another 662 cancer patients in old hospital. The ROC curves and calibration curve were used to evaluate the accuracy and predictive ability of the nomogram. RESULTS: The incidence of postoperative moderate-to-severe pain of PACU in patients with malignancy was 1.42%. Gender, type of surgery, postoperative use of PCA, intraoperative adjuvant opioid agonists, NSAIDS, epidural analgesia, duration of anaesthesia, intraoperative massive haemorrhage, PACU vomiting were independent predictors for postoperative moderate-to-severe pain of PACU in the patients with malignancy. The area under the ROC curve of the predictive models in the primary and validation groups were 0.817 and 0.786, respectively. Moderate-to-severe pain in the PACU correlated with hypertension, hyperglycaemia, agitation, and hypoxemia (P < 0.05). CONCLUSIONS: The prediction model for postoperative moderate-to-severe pain of PACU in patients with malignancy has good predictive ability and high accuracy, which is helpful for PACU medical staff to identify and prevent postoperative moderate-to-severe pain in advance. TRIAL REGISTRATION: The study was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (No.KY2021-097) and registered in the Chictr.org.cn registration system on 06/12/2021 (ChiCTR2100054013).

Influence of endotracheal tube and laryngeal mask airway for general anesthesia on perioperative adverse events in patients undergoing laparoscopic hysterectomy: A propensity score-matched analysis.

Background: To compare perioperative adverse events between general anesthesia with endotracheal tube (ETT) and general anesthesia with laryngeal mask airway (LMA) in patients undergoing laparoscopic hysterectomy. Materials and Methods: This was a large sample retrospective, propensity score-matched (PSM) study. We collected the data of 6739 female patients who underwent laparoscopic hysterectomy between January 2016 and June 2021 in our hospital, China. Patients were divided into two groups (ETT group and LMA group) according to different airway management modes. Data on all perioperative adverse events were collected. PSM analysis was performed to control confounding factors and differences in baseline values between the two groups. Finally, 4150 female patients were recruited after PSM. Results: The total number of patients taking intraoperative vasoactive drugs during surgery was higher in the ETT group than in the LMA group (P = 0.04). The LMA group had a higher incidence of vomiting (51 [2.46%]) and somnolence (165 [7.95]) in the postanesthesia care unit (PACU) than the ETT group (71 [3.42%] and 102 [4.92%], respectively) (P = 0.02 and P < 0.001). Hypothermia was significantly higher in the LMA group (183 [10.36%]) than in the ETT group (173 [8.34%]) in the PACU (P = 0.03). The number of patients with sore throat was significantly higher in the ETT group (434 [20.02%]) than in the LMA group (299 [14.41%]) in the ward (P < 0.001). Other variables such as hypoxemia, moderate to severe pain, abdominal distension, diarrhea, sleep disorders, wound bleeding, and skin itch were not significantly different between the two groups (P > 0.05). Conclusion: The ETT group had more incidences of vomiting, sore throat, and cough complications and needed more drug treatment than the LMA group. LMA is a better airway management mode and LMA general anesthesia can be safely used in patients undergoing laparoscopic nonemergency hysterectomy.

Predicting changes in molluscan spatial distributions in mangrove forests in response to sea level rise.

Mollusks are an important component of the mangrove ecosystem, and the vertical distributions of molluscan species in this ecosystem are primarily dictated by tidal inundation. Thus, sea level rise (SLR) may have profound effects on mangrove mollusk communities. Here, we used dynamic empirical models, based on measurements of surface elevation change, sediment accretion, and molluscan zonation patterns, to predict changes in molluscan spatial distributions in response to different sea level rise rates in the mangrove forests of Zhenzhu Bay (Guangxi, China). The change in surface elevation was 4.76-9.61 mm year-1 during the study period (2016-2020), and the magnitude of surface-elevation change decreased exponentially as original surface elevation increased. Based on our model results, we predicted that mangrove mollusks might successfully adapt to a low rate of SLR (2.00-4.57 mm year-1) by 2100, with mollusks moving seaward and those in the lower intertidal zones expanding into newly available zones. However, as SLR rate increased (4.57-8.14 mm year-1), our models predicted that surface elevations would decrease beginning in the high intertidal zones and gradually spread to the low intertidal zones. Finally, at high rates of SLR (8.14-16.00 mm year-1), surface elevations were predicted to decrease across the elevation gradient, with mollusks moving landward and species in higher intertidal zones blocked by landward barriers. Tidal inundation and the consequent increases in interspecific competition and predation pressure were predicted to threaten the survival of many molluscan groups in higher intertidal zones, especially arboreal and infaunal mollusks at the landward edge of the mangroves, resulting in a substantial reduction in the abundance of original species on the landward edge. Thus, future efforts to conserve mangrove floral and faunal diversity should prioritize species restricted to landward mangrove areas and protect potential species habitats.

Silencing miRNA-324-3p protects against cerebral ischemic injury via regulation of the GATA2/A1R axis.

Previous studies have suggested that miR-324-3p is related to the pathophysiology of cerebral ischemia, but the mechanism underlying this relationship is unclear. In this study, we found that miR-324-3p expression was decreased in patients with acute ischemic stroke and in in vitro and in vivo models of ischemic stroke. miR-324-3p agomir potentiated ischemic brain damage in rats subjected to middle cerebral artery occlusion, as indicated by increased infarct volumes and cell apoptosis rates and greater neurological deficits. In a PC12 cell oxygen-glucose deprivation/reoxygenation model, a miR-324-3p mimic decreased cell viability and expression of the anti-apoptotic protein BCL2 and increased expression of the pro-apoptotic protein BAX and rates of cell apoptosis, whereas treatment with a miR-324-3p inhibitor had the opposite effects. Silencing miR-324-3p increased adenosine A1 receptor (A1R) expression through regulation of GATA binding protein 2 (GATA2). These findings suggest that silencing miR-324-3p reduces ischemic brain damage via the GATA2/A1R axis.

Prognostic Value of Blood Urea Nitrogen to Serum Albumin Ratio in Intensive Care Unit Patients with Lung Cancer.

BACKGROUND: We aimed to evaluate the prognostic ability of blood urea nitrogen (BUN) to serum albumin ratio (BAR) to predict in-hospital mortality in patients with lung cancer in the intensive care unit (ICU). METHODS: Medical Information Mart for Intensive Care IV (MIMIC-IV v1.0) database was used to identify patients who were diagnosed with lung cancer. The primary outcome was in-hospital mortality. Multivariate COX regression was used to investigate the association between BAR and in-hospital mortality and propensity score matching (PSM) and inverse probability of treatment weighting (IPTW) were also used to ensure the robustness of our findings. eICU-CRD database (validation cohort) was also applied to validate our findings. RESULTS: The optimal cut-off value for BAR was 6.8mg/g. Among 1202 patients who were diagnosed with lung cancer, 287 high-BAR group (≥6.8mg/g) patients and 287 low-BAR group (<6.8mg/g) patients, who had similar propensity scores were included in this study. After matching, the high-BAR group had significantly higher in-hospital mortality (hazard ratio, HR, 2.24, 95% confidence index, 95% CI, 1.57-3.19, P<0.001) even after adjustment for confounding factors. Moreover, the performance of BAR was superior to that of BUN and serum albumin alone and could add net benefit in predicting in-hospital mortality. Those results were further confirmed in the validation cohort. CONCLUSION: As an easily accessible and cost-effective parameter, BAR could serve as a good prognostic predictor for lung cancer patients in ICU.

Description and Comparison of Acute Pain Characteristics After Laparoscope-Assisted Vaginal Hysterectomy, Laparoscopic Myomectomy and Laparoscopic Adnexectomy.

PURPOSE: To determine the characteristics of the acute pain after laparoscopic-assisted vaginal hysterectomy (LAVH), laparoscopic myomectomy (LM), and laparoscopic adnexectomy (LA) and compare them with each other. PATIENTS AND METHODS: Patients undergoing LAVH, LM, and LA under general anaesthesia at the First Affiliated Hospital of Wenzhou Medical University between December 2017 and December 2019 were selected. Their data were collected before, during, and after the surgery. We evaluated the degrees of pain in each group of patients and compared them. RESULTS: There were differences in the baseline characteristics of the patients in the LAVH, LM, and LA groups. The severity and incidence of postoperative pain were higher in the LAVH group than in the LM and LA groups, followed by the LM and LA groups. Compared with the LA group, the postoperative pain in the LAVH and LM groups was more complicated. The LA group had the lowest incidence of two or more types of moderate to severe pain. The LAVH and LM groups mainly had visceral pain and low back pain, and the LA group mainly had incisional pain. Shoulder pain had the lowest incidence in the three groups. CONCLUSION: There were different postoperative pain characteristics after the LAVH, LM, and LA, and we should clinically adjust analgesia programs for different gynaecological laparoscopic surgeries.

Elevated retrotransposon activity and genomic instability in primed pluripotent stem cells.

BACKGROUND: Naïve and primed pluripotent stem cells (PSCs) represent two different pluripotent states. Primed PSCs following in vitro culture exhibit lower developmental potency as evidenced by failure in germline chimera assays, unlike mouse naïve PSCs. However, the molecular mechanisms underlying the lower developmental competency of primed PSCs remain elusive. RESULTS: We examine the regulation of telomere maintenance, retrotransposon activity, and genomic stability of primed PSCs and compare them with naïve PSCs. Surprisingly, primed PSCs only minimally maintain telomeres and show fragile telomeres, associated with declined DNA recombination and repair activity, in contrast to naïve PSCs that robustly elongate telomeres. Also, we identify LINE1 family integrant L1Md_T as naïve-specific retrotransposon and ERVK family integrant IAPEz to define primed PSCs, and their transcription is differentially regulated by heterochromatic histones and Dnmt3b. Notably, genomic instability of primed PSCs is increased, in association with aberrant retrotransposon activity. CONCLUSIONS: Our data suggest that fragile telomere, retrotransposon-associated genomic instability, and declined DNA recombination repair, together with reduced function of cell cycle and mitochondria, increased apoptosis, and differentiation properties may link to compromised developmental potency of primed PSCs, noticeably distinguishable from naïve PSCs.

Tn5 Transposase Applied in Genomics Research.

The development of high-throughput sequencing (next-generation sequencing technology (NGS)) and the continuous increase in experimental throughput require the upstream sample processing steps of NGS to be as simple as possible to improve the efficiency of the entire NGS process. The transposition system has fast "cut and paste" and "copy and paste" functions, and has been innovatively applied to the NGS field. For example, the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) uses high-throughput sequencing to detect chromatin regions accessible by Tn5 transposase. Linear Amplification via Transposon Insertion (LIANTI) uses Tn5 transposase for linear amplification, haploid typing, and structural variation detection. Not only is it efficient and simple, it effectively shortens the time for NGS sample library construction, realizes large-scale and rapid sequencing, improves sequencing resolution, and can be flexibly modified for more technological innovation.

Expanded potential: the key to synthetic embryo?

How does an embryo acquire totipotency and develop into an adult is a fundamental scientific question. Stem cells derived from pre-implantation embryos or reprogrammed from somatic cells with totipotency features have been established. They have enriched molecular features, including transcription, epigenetic modification, chromatin structure and metabolism, similar to early embryos from 2 cell (2C) to morula. Functionally, they display a broader developmental potential to differentiate into cell types in the embryonic and extraembryonic tissues. The expanded developmental potential was further demonstrated by inducing these stem cells into embryo-like structures alone or aggregating with other embryo-derived stem cells. The synthetic embryo-like structures not only facilitate the dissection of key events in early embryonic development, but also serve as a model for investigating pregnancy related complications.

Genome-wide investigation and transcriptional analysis of cytosine-5 DNA methyltransferase and DNA demethylase gene families in tea plant (Camellia sinensis) under abiotic stress and withering processing.

DNA methylation is a highly conserved epigenetic modification involved in many biological processes, including growth and development, stress response, and secondary metabolism. In the plant kingdom, cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes have been identified in some plant species. However, to the best of our knowledge, no investigator has focused on the identification and analysis of C5-MTase and dMTase genes in tea plants (Camellia sinensis) based on genome-wide levels. In this study, eight CsC5-MTases and four dMTases were identified in tea plants. These CsC5-MTase genes were divided into four subfamilies, including CsMET, CsCMT, CsDRM and CsDNMT2. The CsdMTase genes can be classified into CsROS, CsDME and CsDML. Based on conserved domain analysis of these genes, the gene loss and duplication events occurred during the evolution of CsC5-MTase and CsdMTase. Furthermore, multiple cis-acting elements were observed in the CsC5-MTase and CsdMTase, including light responsiveness, phytohormone responsiveness, stress responsiveness, and plant growth and development-related elements. Then, we investigated the transcript abundance of CsC5-MTase and CsdMTase under abiotic stress (cold and drought) and withering processing (white tea and oolong tea). Notably, most CsC5-MTases, except for CsCMT1 and CsCMT2, were significantly downregulated under abiotic stress, while the transcript abundance of all four CsdMTase genes was significantly induced. Similarly, the same transcript abundance of CsC5-MTase and CsdMTase was found during withering processing of white tea and oolong tea, respectively. In total, our findings will provide a basis for the roles of CsC5-MTase and CsdMTase in response to abiotic stress and the potential functions of these two gene families in affecting tea flavor during tea withering processing.

Functional Oocytes Derived from Granulosa Cells.

The generation of genomically stable and functional oocytes has great potential for preserving fertility and restoring ovarian function. It remains elusive whether functional oocytes can be generated from adult female somatic cells through reprogramming to germline-competent pluripotent stem cells (gPSCs) by chemical treatment alone. Here, we show that somatic granulosa cells isolated from adult mouse ovaries can be robustly induced to generate gPSCs by a purely chemical approach, with additional Rock inhibition and critical reprogramming facilitated by crotonic sodium or acid. These gPSCs acquired high germline competency and could consistently be directed to differentiate into primordial-germ-cell-like cells and form functional oocytes that produce fertile mice. Moreover, gPSCs promoted by crotonylation and the derived germ cells exhibited longer telomeres and high genomic stability like PGCs in vivo, providing additional evidence supporting the safety and effectiveness of chemical induction, which is particularly important for germ cells in genetic inheritance.

Genome-wide investigation of superoxide dismutase (SOD) gene family and their regulatory miRNAs reveal the involvement in abiotic stress and hormone response in tea plant (Camellia sinensis).

Superoxide dismutases (SODs), as a family of metalloenzymes related to the removal of reactive oxygen species (ROS), have not previously been investigated at genome-wide level in tea plant. In this study, 10 CsSOD genes were identified in tea plant genome, including 7 Cu/Zn-SODs (CSDs), 2 Fe-SODs (FSDs) and one Mn-SOD (MSD), and phylogenetically classified in three subgroups, respectively. Physico-chemical characteristic, conserved motifs and potential protein interaction analyses about CsSOD proteins were carried out. Exon-intron structures and codon usage bias about CsSOD genes were also examined. Exon-intron structures analysis revealed that different CsSOD genes contained various number of introns. On the basis of the prediction of regulatory miRNAs of CsSODs, a modification 5' RNA ligase-mediated (RLM)-RACE was performed and validated that csn-miR398a-3p-1 directly cleaves CsCSD4. By prediction of cis-acting elements, the expression patterns of 10 CsSOD genes and their regulatory miRNAs were detected under cold, drought, exogenous methyl jasmonate (MeJA) and gibberellin (GA3) treatments. The results showed that most of CsSODs except for CsFSD2 were induced under cold stress and CsCSDs may play primary roles under drought stress; exogenous GA3 and MeJA could also stimulated/inhibited distinct CsSODs at different stages. In addition, we found that csn-miR398a-3p-1 negatively regulated the expression of CsCSD4 may be a crucial regulatory mechanism under cold stress. This study provides a certain basis for the studies about stress resistance in tea plants, even provide insight into comprehending the classification, evolution, diverse functions and influencing factors of expression patterns for CsSOD genes.

Telomere dysfunction impairs epidermal stem cell specification and differentiation by disrupting BMP/pSmad/P63 signaling.

Telomere shortening is associated with aging and age-associated diseases. Additionally, telomere dysfunction resulting from telomerase gene mutation can lead to premature aging, such as apparent skin atrophy and hair loss. However, the molecular signaling linking telomere dysfunction to skin atrophy remains elusive. Here we show that dysfunctional telomere disrupts BMP/pSmad/P63 signaling, impairing epidermal stem cell specification and differentiation of skin and hair follicles. We find that telomere shortening mediated by Terc loss up-regulates Follistatin (Fst), inhibiting pSmad signaling and down-regulating P63 and epidermal keratins in an ESC differentiation model as well as in adult development of telomere-shortened mice. Mechanistically, short telomeres disrupt PRC2/H3K27me3-mediated repression of Fst. Our findings reveal that skin atrophy due to telomere dysfunction is caused by a previously unappreciated link with Fst and BMP signaling that could be explored in the development of therapies.

De novo transcriptome and phytochemical analyses reveal differentially expressed genes and characteristic secondary metabolites in the original oolong tea (Camellia sinensis) cultivar 'Tieguanyin' compared with cultivar 'Benshan'.

BACKGROUND: The two original plants of the oolong tea cultivar ('Tieguanyin') are "Wei shuo" 'Tieguanyin'-TGY (Wei) and "Wang shuo" 'Tieguanyin'-TGY (Wang). Another cultivar, 'Benshan' (BS), is similar to TGY in its aroma, taste, and genetic make-up, but it lacks the "Yin Rhyme" flavor. We aimed to identify differences in biochemical characteristics and gene expression among these tea plants. RESULTS: The results of spectrophotometric, high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) analyses revealed that TGY (Wei) and TGY (Wang) had deeper purple-colored leaves and higher contents of anthocyanin, catechins, caffeine, and limonene compared with BS. Analyses of transcriptome data revealed 12,420 differentially expressed genes (DEGs) among the cultivars. According to a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the flavonoid, caffeine, and limonene metabolic pathways were highly enriched. The transcript levels of the genes involved in these three metabolic pathways were not significantly different between TGY (Wei) and TGY (Wang), except for two unigenes encoding IMPDH and SAMS, which are involved in caffeine metabolism. The comparison of TGY vs. BS revealed eight up-regulated genes (PAL, C4H, CHS, F3'H, F3H, DFR, ANS, and ANR) and two down-regulated genes (FLS and CCR) in flavonoid metabolism, four up-regulated genes (AMPD, IMPDH, SAMS, and 5'-Nase) and one down-regulated XDH gene in caffeine metabolism; and two down-regulated genes (ALDH and HIBADH) in limonene degradation. In addition, the expression levels of the transcription factor (TF) PAP1 were significantly higher in TGY than in BS. Therefore, high accumulation of flavonoids, caffeine, and limonene metabolites and the expression patterns of their related genes in TGY might be beneficial for the formation of the "Yin Rhyme" flavor. CONCLUSIONS: Transcriptomic, HPLC, and GC-MS analyses of TGY (Wei), TGY (Wang), and BS indicated that the expression levels of genes related to secondary metabolism and high contents of catechins, anthocyanin, caffeine, and limonene may contribute to the formation of the "Yin Rhyme" flavor in TGY. These findings provide new insights into the relationship between the accumulation of secondary metabolites and sensory quality, and the molecular mechanisms underlying the formation of the unique flavor "Yin Rhyme" in TGY.

Transcriptome and Phytochemical Analyses Provide New Insights Into Long Non-Coding RNAs Modulating Characteristic Secondary Metabolites of Oolong Tea (Camellia sinensis) in Solar-Withering.

Oolong tea is a popular and semi-fermented beverage. During the processing of tea leaves, withering is the first indispensable process for improving flavor. However, the roles of long non-coding RNAs (lncRNAs) and the characteristic secondary metabolites during the withering of oolong tea leaves remain unknown. In this study, phytochemical analyses indicated that total polyphenols, flavonoids, catechins, epigallocatechin (EGC), catechin gallate (CG), gallocatechin gallate (GCG), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) were all less abundant in the solar-withered leaves (SW) than in the fresh leaves (FL) and indoor-withered leaves (IW). In contrast, terpenoid, jasmonic acid (JA), and methyl jasmonate (MeJA) contents were higher in the SW than in the FL and IW. By analyzing the transcriptome data, we detected 32,036 lncRNAs. On the basis of the Kyoto Encyclopedia of Genes and Genomes analysis, the flavonoid metabolic pathway, the terpenoid metabolic pathway, and the JA/MeJA biosynthesis and signal transduction pathway were enriched pathways. Additionally, 63 differentially expressed lncRNAs (DE-lncRNAs) and 23 target genes were identified related to the three pathways. A comparison of the expression profiles of the DE-lncRNAs and their target genes between the SW and IW revealed four up-regulated genes (FLS, CCR, CAD, and HCT), seven up-regulated lncRNAs, four down-regulated genes (4CL, CHI, F3H, and F3'H), and three down-regulated lncRNAs related to flavonoid metabolism; nine up-regulated genes (DXS, CMK, HDS, HDR, AACT, MVK, PMK, GGPPS, and TPS), three up-regulated lncRNAs, and six down-regulated lncRNAs related to terpenoid metabolism; as well as six up-regulated genes (LOX, AOS, AOC, OPR, ACX, and MFP2), four up-regulated lncRNAs, and three down-regulated lncRNAs related to JA/MeJA biosynthesis and signal transduction. These results suggested that the expression of DE-lncRNAs and their targets involved in the three pathways may be related to the low abundance of the total polyphenols, flavonoids, and catechins (EGC, CG, GCG, ECG, and EGCG) and the high abundance of terpenoids in the SW. Moreover, solar irradiation, high JA and MeJA contents, and the endogenous target mimic (eTM)-related regulatory mechanism in the SW were also crucial for increasing the terpenoid levels. These findings provide new insights into the greater contribution of solar-withering to the high-quality flavor of oolong tea compared with the effects of indoor-withering.

ICBP90 mediates Notch signaling to facilitate human hepatocellular carcinoma growth.

The Notch signaling pathway plays a key role in cell proliferation and development that is closely related to an inverted CCAAT box binding protein (ICBP90), but little is known about whether there is a correlation between Notch signaling and ICBP90. The aim of the current study was to elucidate this. MTT assay and flow cytometry were used to determine the proliferation, cell cycle and apoptosis of HepG2 or Hepa1-6 cells treated by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a specific inhibitor of the Notch pathway. RT-PCR, Western Blot and in situ immunofluorescence staining were employed to examine expression of ICBP90 in the cells. DAPT caused inhibition of the activation of the Notch signaling pathway, followed by preventing the cells at the G0/G1 phases to enter S and G2/M phases. ICBP90 and Hes-1 proteins were highly expressed in the untreated cells. The reduced levels of Notch intracellular domain (NICD) protein were observed in the DAPT-treated cells, thereby bringing about the down-regulation of ICBP90 with the increment of the DAPT dose. Consistent with this, knockdown of the Hes-1 gene, which encodes a critical transcriptional factor in the Notch pathway, also led to the attenuation of ICBP90. On the contrary, Jagged-1, a Notch ligand, facilitated ICBP90 production. Adriamycin could result in the reduction of ICBP90, which was not accompanied with the alteration of Hes-1. ICBP90 was almost fully distributed within the nuclei, but Hes-1 was visible within both the cytoplasm and nuclei. Our novel findings strongly indicate that inactivation of the Notch signaling pathway impedes hepatocellular carcinoma progress via reduction of ICBP90.

Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells.

Feeder cells like mouse embryonic fibroblasts (MEFs) have been widely applied for culture of pluripotent stem cells, but their roles remain elusive. Noticeably, ESCs cultured on the feeders display transcriptional heterogeneity. We investigated roles of feeder cells by examining the telomere maintenance. Here we show that telomere is longer in mESCs cultured with than without the feeders. mESC cultures without MEF feeders exhibit telomere loss, chromosomal fusion, and aneuploidy with increasing passages. Notably, feeders facilitate heterogeneous transcription of 2-cell genes including Zscan4 and telomere elongation. Moreover, feeders produce Fstl1 that together with BMP4 periodically activate Zscan4. Interestingly, Zscan4 is repressed in mESCs cultured in 2i (inhibitors of Mek and Gsk3ß signaling) media, associated with shorter telomeres and increased chromosome instability. These data suggest the important role of feeders in maintaining telomeres for long-term stable self-renewal and developmental pluripotency of mESCs.

Dynamics of Telomere Rejuvenation during Chemical Induction to Pluripotent Stem Cells.

Chemically induced pluripotent stem cells (CiPSCs) may provide an alternative and attractive source for stem cell-based therapy. Sufficient telomere lengths are critical for unlimited self-renewal and genomic stability of pluripotent stem cells. Dynamics and mechanisms of telomere reprogramming of CiPSCs remain elusive. We show that CiPSCs acquire telomere lengthening with increasing passages after clonal formation. Both telomerase activity and recombination-based mechanisms are involved in the telomere elongation. Telomere lengths strongly indicate the degree of reprogramming, pluripotency, and differentiation capacity of CiPSCs. Nevertheless, telomere damage and shortening occur at a late stage of lengthy induction, limiting CiPSC formation. We find that histone crotonylation induced by crotonic acid can activate two-cell genes, including Zscan4; maintain telomeres; and promote CiPSC generation. Crotonylation decreases the abundance of heterochromatic H3K9me3 and HP1α at subtelomeres and Zscan4 loci. Taken together, telomere rejuvenation links to reprogramming and pluripotency of CiPSCs. Crotonylation facilitates telomere maintenance and enhances chemically induced reprogramming to pluripotency.

DNA repair and replication links to pluripotency and differentiation capacity of pig iPS cells.

Pigs are proposed to be suitable large animal models for test of the efficacy and safety of induced pluripotent stem cells (iPSCs) for stem cell therapy, but authentic pig ES/iPS cell lines with germline competence are rarely produced. The pathways or signaling underlying the defective competent pig iPSCs remain poorly understood. By improving induction conditions using various small chemicals, we generated pig iPSCs that exhibited high pluripotency and differentiation capacity that can contribute to chimeras. However, their potency was reduced with increasing passages by teratoma formation test, and correlated with declined expression levels of Rex1, an important marker for naïve state. By RNA-sequencing analysis, genes related to WNT signaling were upregulated and MAPK signaling and TGFß pathways downregulated in pig iPSCs compared to fibroblasts, but they were abnormally expressed during passages. Notably, pathways involving in DNA repair and replication were upregulated at early passage, but downregulated in iPSCs during prolonged passage in cluster with fibroblasts. Our data suggests that reduced DNA repair and replication capacity links to the instability of pig iPSCs. Targeting these pathways may facilitate generation of truly pluripotent pig iPSCs, with implication in translational studies.