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Abstract Background Tong-Xin-Luo (TXL) – a mixture of herbal extracts, has been used in Chinese medicine with established therapeutic efficacy in patients with coronary artery disease. Methods We investigated the protective role of TXL extracts on endothelial cells injured by a known risk factor – palmitic acid (PA), which is elevated in metabolic syndrome and associated with cardiovascular complications. Human aortic endothelial cells (HAECs) were preconditioned with TXL extracts before exposed to PA for 24 hours. Results We found that PA (0.5 mM) exposure induced 73% apoptosis in endothelial cells. However, when HAECs were preconditioned with ethanol extracted TXL (100 μg/ml), PA induced only 7% of the endothelial cells into apoptosis. Using antibody-based protein microarray, we found that TXL attenuated PA-induced activation of p38-MAPK stress pathway. To investigate the mechanisms involved in TXL's protective effects, we found that TXL reduced PA-induced intracellular oxidative stress. Through AMPK pathway, TXL restored the intracellular antioxidant system, which was depressed by the PA treatment, with an increased expression of thioredoxin and a decreased expression of the thioredoxin interacting protein. Conclusion In summary, our study demonstrates that TXL protects endothelial cells from PA-induced injury. This protection is likely mediated by boosting intracellular antioxidant capacity through AMPK pathway, which may account for the therapeutic efficacy in TXL-mediated cardiovascular protection.

Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells., (© 2022. The Author(s).)

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Peña-Pérez, Kharazi, Frengen, Krstic, Bouderlique, Hauenstein, He, Somuncular, Li Wang, Dahlberg, Gustafsson, Johansson, Walfridsson, Kadri, Woll, Kierczak, Qian, Westerberg, Luc and Månsson.)

Ras-Erk MAPK signaling controls many of the principal pathways involved in metazoan cell motility, drives metastasis of multiple cancer types and is targeted in chemotherapy. However, its putative roles in immune cell functions or in infections have remained elusive. Here, using primary dendritic cells (DCs) in an infection model with the protozoan Toxoplasma gondii , we show that two pathways activated by infection converge on Ras-Erk MAPK signaling to promote migration of parasitized DCs. We report that signaling through the receptor tyrosine kinase Met (also known as HGF receptor) contributes to T. gondii -induced DC hypermotility. Furthermore, voltage-gated Ca 2+ channel (VGCC, subtype Ca V 1.3) signaling impacted the migratory activation of DCs via calmodulin-calmodulin kinase II. We show that convergent VGCC signaling and Met signaling activate the GTPase Ras to drive Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively) phosphorylation and hypermotility of T. gondii -infected DCs. The data provide a molecular basis for the hypermigratory mesenchymal-to-amoeboid transition (MAT) of parasitized DCs. This emerging concept suggests that parasitized DCs acquire metastasis-like migratory properties that promote infection-related dissemination., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

HPV16 late L1 mRNAs encode a short central exon that is located between HPV16 3'-splice site SA3358 and HPV16 5'-splice site SD3632. While SA3358 is used to produce both HPV16 early mRNAs encoding the E6 and E7 oncogenes, and late mRNAs encoding E4, L1 and L2, SD3632 is used exclusively to produce late L1 mRNA. We have previously identified an 8-nucleotide regulatory RNA element that is required for inclusion of the exon between SA3358 and SD3632 to produce L1 mRNAs at the expense of mRNAs polyadenylated at the HPV16 early polyadenylation signal pAE. Here we show that this HPV16 8-nucleotide splicing enhancer interacts with hnRNP G. Binding of hnRNP G to this element prevents inclusion of the exon between SA3358 and SD3632 on the HPV16 late L1 mRNAs. We concluded that hnRNP G has a splicing inhibitory role and that hnRNP G can control HPV16 mRNA splicing.

Liver transplantation has been established as a curative therapy for acute and chronic liver failure, as well as liver-based inherited metabolic diseases. Because of the complexity of organ transplantation and the worldwide shortage of donor organs, hepatocyte transplantation is being developed as a bridging therapy until donor organs become available, or for amelioration of inherited liver-based diseases. The Gunn rat is a molecular and metabolic model of Crigler-Najjar syndrome type 1, which is characterized by lifelong unconjugated hyperbilirubinemia due to the lack of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-mediated bilirubin glucuronidation. Gunn rats are convenient for evaluating the effect of hepatocyte transplantation or gene therapy, because the extent of UGT1A1 replacement can be assessed by serial determination of serum bilirubin levels, and excretion of bilirubin glucuronides in bile provide definitive evidence of the function of the transplanted hepatocytes or the effect of gene therapy. The core techniques involved in hepatocyte transplantation in Gunn rats are discussed in this chapter.

Angiopoietin-like protein 8 (ANGPTL8)/betatrophin, a newly identified protein, is primarily expressed in the liver and regulates the glucose metabolic transition during fasting and re-feeding in mice with or without insulin resistance. These findings strongly suggest that ANGPTL8/betatrophin could be a novel glucose-lowering candidate medicine for type 2 diabetes. However, the molecular mechanisms by which ANGPTL8/betatrophin regulates glucose metabolism are poorly understood in human. Two sub-clones of HepG2 cells, ANGPTL8/betatrophin knockouts and ANGPTL8/betatrophin over-expressors, were established using TALENs (transcription activator-like effector nucleases) and through stable transfection, respectively. Over-expression of ANGPTL8/betatrophin enhanced the insulin-stimulated activation of the Akt-GSK3β or Akt-FoxO1 pathway, no matter whether the cells were present with insulin resistance or not. In contrast, knockout of ANGPTL8/betatrophin did not affect the Akt-GSK3β or Akt-FoxO1 pathway unless the HepG2 cells were preset with insulin resistance. Our results suggest that ANGPTL8/betatrophin might play an important role in glucose metabolism in the context of insulin resistance., (Copyright © 2016. Published by Elsevier Inc.)

AF1Q, a mixed lineage leukemia gene fusion partner, is identified as a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetic and adult myelodysplastic syndrome. AF1Q is highly regulated during hematopoietic progenitor differentiation and development but its regulatory mechanism has not been defined clearly. In the present study, we used pharmacological and genetic approaches to influence chaperone-mediated autophagy (CMA) and explored the degradation mechanism of AF1Q. Pharmacological inhibitors of lysosomal degradation, such as chloroquine, increased AF1Q levels, whereas activators of CMA, including 6-aminonicotinamide and nutrient starvation, decreased AF1Q levels. AF1Q interacts with HSPA8 and LAMP-2A, which are core components of the CMA machinery. Knockdown of HSPA8 or LAMP-2A increased AF1Q protein levels, whereas overexpression showed the opposite effect. Using an amino acid deletion AF1Q mutation plasmid, we identified that AF1Q had a KFERQ-like motif which was recognized by HSPA8 for CMA-dependent proteolysis. In conclusion, we demonstrate for the first time that AF1Q can be degraded in lysosomes by CMA., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: BioGlue-a surgical adhesive composed of bovine albumin and glutaraldehyde-is commonly used in cardiovascular operations. The objectives of this study were to determine whether BioGlue injures nerves and cardiac conduction tissues, and whether a water-soluble gel barrier protects against such injury., Materials and Methods: In 18 pigs, diaphragmatic excursion during direct phrenic nerve stimulation was measured at baseline and at 3 and 30 min after nerve exposure to albumin (n = 3), glutaraldehyde (n = 3), BioGlue (n = 6), or water-soluble gel followed by BioGlue (n = 6). Additionally, BioGlue was applied to the cavoatrial junction overlying the sinoatrial node (SAN), either alone (n = 12) or after application of gel (n = 6)., Results: Mean diaphragmatic excursions in the BioGlue and glutaraldehyde groups were lower at 3 min and 30 min than in the albumin group (P < 0.05). Mean excursions in the gel group were similar to those of the albumin group (P = 0.9). Five BioGlue pigs (83%) and one gel pig (17%) had diaphragmatic paralysis by 30 min (P < 0.05 and P = 0.3 versus albumin, respectively). Coagulation necrosis extended into the myocardium at the cavoatrial junction in all 12 BioGlue pigs but only two gel pigs (33%, P < 0.01). Two BioGlue pigs (17%), but no gel pigs, had focal SAN degeneration and persistent bradycardia (P < 0.01)., Conclusions: BioGlue causes acute nerve injury and myocardial necrosis that can lead to SAN damage. A water-soluble gel barrier is protective.

Objective: We investigated the histological and mechanical changes in addition to viable cellular recovery in cryopreserved blood vessels., Materials and Methods: Rabbit carotids were cryopreserved in a cryoprotective medium containing 1.5 M of 1,2-propanediol (PD) and then were thawed slowly in an ice bag that had been precooled in liquid nitrogen. Fresh carotids were used as the control. The fresh and freeze-thawed arteries were cultured for the growth of vascular smooth muscle cells (VSMCs). The freeze-thawed arterial tissues were perfused in vitro for 6, 12, or 24 h, respectively, to assess the integrity of carotid walls and the mechanical properties., Results: The results showed that it took almost the same time (24 approximately 36 h) for the VSMCs of the PD-cryopreserved arteries to regenerate as those from the fresh arteries. Their growing speeds also were similar. On the contrary, Me2SO-cryopreserved (1.5 M) arteries were unable to regenerate VSMCs in culture. After freeze-thawing, the mechanical properties decreased significantly (P < 0.003 for elastic modulus and P < 0.001 for fracture strength). After in vitro perfusion of the freeze-thawed carotid arteries, all of the survived endothelial cells fell off, and some of the VSMCs denaturalized or necrosed. The internal elastic fibers and collagen showed various degrees of cracking. The mechanical properties were decreased (P < 0.05)., Conclusion: Our findings demonstrate that the PD-containing cryoprotective medium can preserve regenerative capacity of VSMCs, which makes it a useful technique for viable VSMC recovery. However, the freeze-thawing process and the in vitro perfusion caused serious disruption in the arterial mechanical properties, rendering the cryopreserved blood vessels less useful for vessel reconstruction.