An essential signaling function of cytoplasmic NELFB is independent of RNA polymerase II pausing.

The four-subunit negative elongation factor (NELF) complex mediates RNA polymerase II (Pol II) pausing at promoter-proximal regions. Ablation of individual NELF subunits destabilizes the NELF complex and causes cell lethality, leading to the prevailing concept that NELF-mediated Pol II pausing is essential for cell proliferation. Using separation-of-function mutations, we show here that NELFB function in cell proliferation can be uncoupled from that in Pol II pausing. NELFB mutants sequestered in the cytoplasm and deprived of NELF nuclear function still support cell proliferation and part of the NELFB-dependent transcriptome. Mechanistically, cytoplasmic NELFB physically and functionally interacts with prosurvival signaling kinases, most notably phosphatidylinositol-3-kinase/AKT. Ectopic expression of membrane-tethered phosphatidylinositol-3-kinase/AKT partially bypasses the role of NELFB in cell proliferation, but not Pol II occupancy. Together, these data expand the current understanding of the physiological impact of Pol II pausing and underscore the multiplicity of the biological functions of individual NELF subunits.

Pickering emulsions prepared using zein-sugarcane leaves polyphenol covalent crosslinking nanoparticles via ultrasonication: Capacities in storage stability, lipid oxidation, in vitro digestion and safety evaluation.

This study firstly used sugarcane leaf polyphenols (SGLp) to modify zein to form covalent nanoparticles (SGLpZ) and used SGLpZ as an emulsifier to stabilize pickering emulsions (SZP) via ultrasonic method. The results showed that the addition of SGLp could alter the physicochemical properties of zein, including improving increasing the hydrophilicity of zein and the antioxidant properties of zein (three basic antioxidant activities test in vitro). SGLpZ could be able to form a dense film on the surface of the pickering emulsions which inhibited lipid oxidation as the concentration of SGLp increased at 4 ℃ for 20 days, thus stabilizing pickering emulsions (SZP). Further assessment of storage stability of pickering emulsions stabilized by SGLp was evaluated via measuring the free fatty acids (FFA) release in vitro gastrointestinal digestion. The results showed that the FFA release of SZP decreased from 20.61 ± 0.10% to 16.14 ± 0.69%. In addition, SGLp gave SZP a yellow color, which inspired that SZP could be used in the food industry to make yellow-colored functional foods. Finally, the safety of SZP initially assessed by in-vitro hemocompatibility and cytotoxicity (MTT) assays. In conclusion, our fingdings were beneficial for the further design and development of SGLp in food fields and enabled the development a new type in functional protein-plant polyphenols food pickering emulsions.

BRCA1 deficiency in mature CD8+ T lymphocytes impairs antitumor immunity.

Women with BRCA1 germline mutations have approximately an 80% lifetime chance of developing breast cancer. While the tumor suppressor function of BRCA1 in breast epithelium has been studied extensively, it is not clear whether BRCA1 deficiency in non-breast somatic cells also contribute to tumorigenesis. Here, we report that mouse Brca1 knockout (KO) in mature T lymphocytes compromises host antitumor immune response to transplanted syngeneic mouse mammary tumors. T cell adoptive transfer further corroborates CD8+ T cell-intrinsic impact of Brca1 KO on antitumor adaptive immunity. T cell-specific Brca1 KO mice exhibit fewer total CD8+, more exhausted, reduced cytotoxic, and reduced memory tumor-infiltrating T cell populations. Consistent with the preclinical data, cancer-free BRCA1 mutation-carrying women display lower abundance of circulating CD8+ lymphocytes than the age-matched control group. Thus, our findings support the notion that BRCA1 deficiency in adaptive immunity could contribute to BRCA1-related tumorigenesis. We also suggest that prophylactic boosting of adaptive immunity may reduce cancer incidence among at-risk women.

RNA polymerase II pausing factor NELF in CD8+ T cells promotes antitumor immunity.

T cell factor 1 (TCF1) is required for memory and stem-like CD8+ T cell functions. How TCF1 partners with other transcription factors to regulate transcription remains unclear. Here we show that negative elongation factor (NELF), an RNA polymerase II (Pol II) pausing factor, cooperates with TCF1 in T cell responses to cancer. Deletion of mouse Nelfb, which encodes the NELFB subunit, in mature T lymphocytes impairs immune responses to both primary tumor challenge and tumor antigen-mediated vaccination. Nelfb deletion causes more exhausted and reduced memory T cell populations, whereas its ectopic expression boosts antitumor immunity and efficacy of chimeric antigen receptor T-cell immunotherapy. Mechanistically, NELF is associated with TCF1 and recruited preferentially to the enhancers and promoters of TCF1 target genes. Nelfb ablation reduces Pol II pausing and chromatin accessibility at these TCF1-associated loci. Our findings thus suggest an important and rate-limiting function of NELF in anti-tumor immunity.

Label-free Au NRs-based SERS coupled with chemometrics for rapid quantitative detection of thiabendazole residues in citrus.

Citrus is a highly consumed fruit worldwide. However, the excessive use of thiabendazole (TBZ) pesticides during citrus cultivation poses a health risk to people. Hence, a rapid and quantitative method has been established for TBZ determination in citrus by coupling gold nanorods (Au NRs) based surface enhanced Raman scattering (SERS) coupling chemometrics. The results show that support vector machine (SVM) can distinguish TBZ residues of different orders of magnitude with 99.1667% accuracy and that genetic algorithm-partial least squares (GA-PLS) had the best performance in the quantitative prediction of TBZ residues (Rp2 = 0.9737, RMSEP = 0.1179 and RPD = 5.85) in citrus. The limit of detection (LOD) was 0.33 µg mL-1. Furthermore, the proposed method was validated by a standard HPLC method using t-test with no significant difference. Therefore, the proposed Au NRs-based SERS technique can be used for the rapid quantitative analysis of TBZ residues in citrus.

"Without the need for a second visit" initiative improves patient satisfaction with updated services of outpatient clinics in China.

BACKGROUND: To implement the "without the need for a second visit" (WNASV) initiative in our hospital by optimizing the outpatient clinic services via an upgraded information system, in order to increase the quality of outpatient medical services and improve patients' satisfaction. METHODS: An Internet-based care delivery approach was developed and applied to improve the delivery of health care services, simplify the treatment process, and reduce patient waiting time. The patient waiting time and consultation time in the outpatient clinics of our hospital during the peak service intervals and the proportions of various payment methods for outpatient services during the period from May 2017 to September 2019 were retrospectively analyzed. Also, the patients' satisfaction with the outpatient process was surveyed. RESULTS: The waiting time for consultation was shortened from 32.25 min to 28.42 min; the consultation time was shortened from 6.52 min to 3.15 min; and the waiting time for payment decreased from 7.40 min to 4.31 min. The proportion of payment via a counter was reduced from 86.80 to 21.79%, the proportion of self-service payment increased from 9.99 to 16.05%, and the proportion of payment during a consultation increased from 3.21 to 61.91%. The scores of the patients' satisfaction with the outpatient services increased from an average of 89.10 points in 2017 to an average of 90.26 points in 2019. CONCLUSION: The continuous improvement of the service process markedly increases the efficiency of the outpatient services, and effectively improves patient's satisfaction with the outpatient process, this initiative thus deserves further application.

Sonic hedgehog signaling regulates the mammalian cardiac regenerative response.

Certain organisms, including zebrafish, are capable of complete cardiac regeneration in response to injury. This response has also been observed in newborn mice, although in this case, the regenerative capacity is lost at approximately one week of age. The mechanisms regulating this short temporal window of cardiac regeneration in mice are not well understood. Here, we show that sonic hedgehog (Shh) signaling modulates the neonatal mouse regenerative response. In particular, we demonstrate that following apical resection of the heart on postnatal day 1, mice activate Shh ligand expression and downstream signaling. This response is largely absent when surgery is performed on non-regenerative, postnatal day 7 pups. Furthermore, an enhanced cardiac regeneration response was detected in ptch heterozygous mice which have a genetically-based constitutive increase in Shh signaling. We further show that Shh ligand is produced in the myocardium by non-myocytes and appears to regulate cardiomyocyte proliferation, as well as the recruitment of monocytes/macrophages to the regenerating area. Finally, we demonstrate that a small molecule activator of Shh signaling promotes heart regeneration, whereas an inhibitor of Shh signaling impairs the regenerative response. Together, these results implicate Shh signaling as a regulator of mammalian heart regeneration and suggest that modulating this pathway may lead to new potential therapies for cardiovascular diseases.

Key proteins and pathways that regulate lifespan.

Here, we review three sets of key proteins and their corresponding downstream pathways that have been linked to extending lifespan and promoting health span in a wide range of organisms. In particular, we review the biology of the sirtuin family of proteins, the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway, and the mechanistic target of rapamycin (mTOR). Using insights derived from simple model organisms, mice, and humans we discuss how these proteins and pathways may potentially alter the rate of aging. We further describe how knowledge of these pathways may lead to the rational design of small molecules that modulate aging and hence alter the propensity for a host of age-related diseases.

The impact of aging on cardiac extracellular matrix.

Age-related changes in cardiac homeostasis can be observed at the cellular, extracellular, and tissue levels. Progressive cardiomyocyte hypertrophy, inflammation, and the gradual development of cardiac fibrosis are hallmarks of cardiac aging. In the absence of a secondary insult such as hypertension, these changes are subtle and result in slight to moderate impaired myocardial function, particularly diastolic function. While collagen deposition and cross-linking increase during aging, extracellular matrix (ECM) degradation capacity also increases due to increased expression of matrix metalloproteinases (MMPs). Of the MMPs elevated with cardiac aging, MMP-9 has been extensively evaluated and its roles are reviewed here. In addition to proteolytic activity on ECM components, MMPs oversee cell signaling during the aging process by modulating cytokine, chemokine, growth factor, hormone, and angiogenic factor expression and activity. In association with elevated MMP-9, macrophage numbers increase in an age-dependent manner to regulate the ECM and angiogenic responses. Understanding the complexity of the molecular interactions between MMPs and the ECM in the context of aging may provide novel diagnostic indicators for the early detection of age-related fibrosis and cardiac dysfunction.

Antiproliferation of berberine is mediated by epigenetic modification of constitutive androstane receptor (CAR) metabolic pathway in hepatoma cells.

Constitutive androstane receptor (CAR) regulates hepatic xenobiotic and energy metabolism, as well as promotes cell growth and hepatocarcinogenesis. Berberine is an ancient multipotent alkaloid drug which derived from Coptis chinensis plants. Here we report that berberine is able to be cellular uptake and accessible to chromatin in human hepatoma HepG2 cells. Berberine induces more apoptosis, cell cycle arrest, but less ROS production in CAR overexpressed mCAR-HepG2 cells. Moreover, berberine inhibits expressions of CAR and its target genes CYP2B6 and CYP3A4. Furthermore, berberine enhances DNA methylation level in whole genome but reduces that in promoter regions CpG sites of CYP2B6 and CYP3A4 genes under the presence of CAR condition. These results indicated that the antiproliferation of berberine might be mediated by the unique epigenetic modifying mechanism of CAR metabolic pathway, suggesting that berberine is a promising candidate in anticancer adjuvant chemotherapy, due to its distinct pharmacological properties in clinic.

Assessment of cardiac function in mice lacking the mitochondrial calcium uniporter.

Mitochondrial calcium is thought to play an important role in the regulation of cardiac bioenergetics and function. The entry of calcium into the mitochondrial matrix requires that the divalent cation pass through the inner mitochondrial membrane via a specialized pore known as the mitochondrial calcium uniporter (MCU). Here, we use mice deficient of MCU expression to rigorously assess the role of mitochondrial calcium in cardiac function. Mitochondria isolated from MCU(-/-) mice have reduced matrix calcium levels, impaired calcium uptake and a defect in calcium-stimulated respiration. Nonetheless, we find that the absence of MCU expression does not affect basal cardiac function at either 12 or 20months of age. Moreover, the physiological response of MCU(-/-) mice to isoproterenol challenge or transverse aortic constriction appears similar to control mice. Thus, while mitochondria derived from MCU(-/-) mice have markedly impaired mitochondrial calcium handling, the hearts of these animals surprisingly appear to function relatively normally under basal conditions and during stress.

Translational Initiation at a Non-AUG Start Codon for Human and Mouse Negative Elongation Factor-B.

Negative elongation factor (NELF), a four-subunit protein complex in metazoan, plays an important role in regulating promoter-proximal pausing of RNA polymerase II (RNAPII). Genetic studies demonstrate that the B subunit of mouse NELF (NELF-B) is critical for embryonic development and homeostasis in adult tissue. We report here that both human and mouse NELF-B proteins are translated from a non-AUG codon upstream of the annotated AUG. This non-AUG codon sequence is conserved in mammalian NELF-B but not NELF-B orthologs of lower metazoan. The full-length and a truncated NELF-B that starts at the first AUG codon both interact with the other three NELF subunits. Furthermore, these two forms of NELF-B have a similar impact on the transcriptomics and proliferation of mouse embryonic fibroblasts. These results strongly suggest that additional amino acid sequence upstream of the annotated AUG is dispensable for the essential NELF function in supporting cell growth in vitro. The majority of mouse adult tissues surveyed express the full-length NELF-B protein, and some contain a truncated NELF-B protein with the same apparent size as the AUG-initiated version. This result raises the distinct possibility that translational initiation of mouse NELF-B is regulated in a tissue-dependent manner.

Negative elongation factor controls energy homeostasis in cardiomyocytes.

Negative elongation factor (NELF) is known to enforce promoter-proximal pausing of RNA polymerase II (Pol II), a pervasive phenomenon observed across multicellular genomes. However, the physiological impact of NELF on tissue homeostasis remains unclear. Here, we show that whole-body conditional deletion of the B subunit of NELF (NELF-B) in adult mice results in cardiomyopathy and impaired response to cardiac stress. Tissue-specific knockout of NELF-B confirms its cell-autonomous function in cardiomyocytes. NELF directly supports transcription of those genes encoding rate-limiting enzymes in fatty acid oxidation (FAO) and the tricarboxylic acid (TCA) cycle. NELF also shares extensively transcriptional target genes with peroxisome proliferator-activated receptor α (PPARα), a master regulator of energy metabolism in the myocardium. Mechanistically, NELF helps stabilize the transcription initiation complex at the metabolism-related genes. Our findings strongly indicate that NELF is part of the PPARα-mediated transcription regulatory network that maintains metabolic homeostasis in cardiomyocytes.

Genetic and genomic analyses of RNA polymerase II-pausing factor in regulation of mammalian transcription and cell growth.

Many mammalian genes are occupied by paused RNA polymerase II (pol II) in the promoter-proximal region on both sides of the transcription start site. However, the impact of pol II pausing on gene expression and cell biology is not fully understood. In this study, we used a Cre-Lox system to conditionally knock out the b subunit of mouse negative elongation factor (Nelf-b), a key pol II-pausing factor, in mouse embryonic fibroblasts. We found that Nelf-b was associated with the promoter-proximal region of the majority of expressed genes, yet genetic ablation of Nelf-b only affected the steady-state mRNA levels of a small percentage of the Nelf-b-associated genes. Interestingly, Nelf-b deletion also increased levels of transcription start site upstream transcripts at multiple negative elongation factor-associated genes. The direct target genes of Nelf-b were highly enriched with those involved in the control of cell growth and cell death. Correspondingly, Nelf-b knock-out mouse embryonic fibroblasts exhibited slower progression from quiescence to proliferation, as well as in a cycling cell population. Furthermore, Nelf-b deletion also resulted in increased apoptosis. Thus, the genetic and genomic studies provide new physiological and molecular insight into Nelf-mediated pol II pausing.

Ubiquitin chain elongation enzyme Ufd2 regulates a subset of Doa10 substrates.

Ufd2 is the founding member of E4 enzymes that are specifically involved in ubiquitin chain elongation but whose roles in proteolysis remain scarce. Here, using a genome-wide screen, we identified one cellular target of yeast Ufd2 as the membrane protein Pex29. The ubiquitin chains assembled on Pex29 in vivo by Ufd2 mainly contain Lys-48 linkages. We found that the ubiquitin-protein E3 ligase for overexpressed Pex29 is Doa10, which is known to be involved in protein quality control. Interestingly, not all Doa10 substrates are regulated by Ufd2, suggesting that E4 involvement is not specific to a particular E3, but may depend on the spatial arrangement of the E3-substrate interaction. Cells lacking UFD2 elicit an unfolded protein response, expanding the physiological function of Ufd2. Our results lead to novel insights into the biological role of Ufd2 and further underscore the significance of Ufd2 in proteolysis.