Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 µg of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation through Noncanonical Ubiquitination

The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging

Minicircle HBV cccDNA with a Gaussia luciferase reporter for investigating HBV cccDNA biology and developing cccDNA-targeting drugs

Chronic Hepatitis B Virus (HBV) infection is generally not curable with current anti-viral drugs. Virus rebounds after stopping treatment from the stable HBV covalently-closed-circular DNA (cccDNA). The development of drugs that directly target cccDNA is hampered by the lack of robust HBV cccDNA models. We report here a novel HBV cccDNA technology that will meet the need. We engineered a minicircle HBV cccDNA with a Gaussia Luciferase reporter (mcHBV-GLuc cccDNA), which serves as a surrogate to measure cccDNA activity. The mcHBV-GLuc cccDNA was easily produced in bacteria, and it formed minichromosomes as HBV cccDNA episome DNA does when it was transfected into human hepatocytes. Compared to non-HBV minicircle plasmids, mcHBV-GLuc cccDNA showed persistent HBV-GLuc activity and HBx-dependent gene expression. Importantly, the mcHBV-GLuc cccDNA showed resistance to interferons (IFN) treatment, indicating its unique similarity to HBV cccDNA that is usually resistant to long-term IFN treatment in chronic HBV patients. Most importantly, GLuc illuminates cccDNA as a surrogate of cccDNA activity, providing a very sensitive and quick method to detect trace amount of cccDNA. The mcHBV-GLuc cccDNA model is independent of HBV infection, and will be valuable for investigating HBV cccDNA biology and for developing cccDNA-targeting drugs

The Ubiquitin Ligase ASB4 Promotes Trophoblast Differentiation through the Degradation of ID2

Vascularization of the placenta is a critical developmental process that ensures fetal viability. Although the vascular health of the placenta affects both maternal and fetal well being, relatively little is known about the early stages of placental vascular development. The ubiquitin ligase Ankyrin repeat, SOCS box-containing 4 (ASB4) promotes embryonic stem cell differentiation to vascular lineages and is highly expressed early in placental development. The transcriptional regulator Inhibitor of DNA binding 2 (ID2) negatively regulates vascular differentiation during development and is a target of many ubiquitin ligases. Due to their overlapping spatiotemporal expression pattern in the placenta and contrasting effects on vascular differentiation, we investigated whether ASB4 regulates ID2 through its ligase activity in the placenta and whether this activity mediates vascular differentiation. In mouse placentas, ASB4 expression is restricted to a subset of cells that express both stem cell and endothelial markers. Placentas that lack Asb4 display immature vascular patterning and retain expression of placental progenitor markers, including ID2 expression. Using JAR placental cells, we determined that ASB4 ubiquitinates and represses ID2 expression in a proteasome-dependent fashion. Expression of ASB4 in JAR cells and primary isolated trophoblast stem cells promotes the expression of differentiation markers. In functional endothelial co-culture assays, JAR cells ectopically expressing ASB4 increased endothelial cell turnover and stabilized endothelial tube formation, both of which are hallmarks of vascular differentiation within the placenta. Co-transfection of a degradation-resistant Id2 mutant with Asb4 inhibits both differentiation and functional responses. Lastly, deletion of Asb4 in mice induces a pathology that phenocopies human pre-eclampsia, including hypertension and proteinuria in late-stage pregnant females. These results indicate that ASB4 mediates vascular differentiation in the placenta via its degradation of ID2

CHIP Is a U-box-dependent E3 Ubiquitin Ligase: IDENTIFICATION OF Hsc70 AS A TARGET FOR UBIQUITYLATION

Proper folding of proteins (either newly synthesized or damaged in response to a stressful event) occurs in a highly regulated fashion. Cytosolic chaperones such as Hsc/Hsp70 are assisted by cofactors that modulate the folding machinery in a positive or negative manner. CHIP (carboxyl terminus of Hsc70-interacting protein) is such a cofactor that interacts with Hsc70 and, in general, attenuates its most well characterized functions. In addition, CHIP accelerates ubiquitin-dependent degradation of chaperone substrates. Using an in vitro ubiquitylation assay with recombinant proteins, we demonstrate that CHIP possesses intrinsic E3 ubiquitin ligase activity and promotes ubiquitylation. This activity is dependent on the carboxyl-terminal U-box. CHIP interacts functionally and physically with the stress-responsive ubiquitin-conjugating enzyme family UBCH5. Surprisingly, a major target of the ubiquitin ligase activity of CHIP is Hsc70 itself. CHIP ubiquitylates Hsc70, primarily with short, noncanonical multiubiquitin chains but has no appreciable effect on steady-state levels or half-life of this protein. This effect may have heretofore unanticipated consequences with regard to the chaperoning activities of Hsc70 or its ability to deliver substrates to the proteasome. These studies demonstrate that CHIP is a bona fide ubiquitin ligase and indicate that U-box-containing proteins may comprise a new family of E3s

Regulation of ankyrin repeat and suppressor of cytokine signalling box protein 4 expression in the immortalized murine endothelial cell lines MS1 and SVR: a role for tumour necrosis factor alpha and oxygen: REGULATION OF ASB4 EXPRESSION IN ENDOTHELIAL CELL LINES

During vascular development, endothelial cells are exposed to a variety of rapidly changing factors, including fluctuating oxygen levels. We have previously shown that Ankyrin Repeat and SOCS Box Protein 4 (ASB4) is the most highly differentially expressed gene in the vascular lineage during early differentiation and is expressed in the embryonic vasculature at a time when oxygen tension is rising due to the onset of placental blood flow. In order to further our understanding of the regulation of ASB4 expression in endothelial cells, we tested the effect of various stressors for their ability to alter ASB4 expression in the immortalized murine endothelial cell lines MS1 and SVR. ASB4 expression is decreased during hypoxic insult and shear stress whereas it is increased in response to TNF-α. Further investigation indicated that Nuclear Factor kappa B (NF-κB) is the responsible transcription factor involved in the TNF-α-induced upregulation of ASB4, placing ASB4 downstream of NF-κB in the TNF-α signaling cascade and identifying it as a potential regulator for TNF-α’s numerous functions associated with inflammation, angiogenesis and apoptosis

BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions

Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects

Adherence to diabetes risk reduction diet and the risk of head and neck cancer: a prospective study of 101,755 American adults

BackgroundAdherence to the diabetes risk reduction diet (DRRD) may potentially reduce the risk of developing head and neck cancer (HNC) as the diet includes fruits and limits red and processed meats, known risk factors for HNC. However, there is currently no epidemiological research to investigate this potential association.MethodsThe present study utilized data on demographics, lifestyles, medications, and diets of participants from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial to explore the potential association between adherence to DRRD and the risk of HNC. We used a DRRD score to evaluate adherence to the dietary pattern and employed Cox regression analysis to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for HNC risk. Several subgroup analyses were carried out to identify potential effect modifiers, and multiple sensitivity analyses were performed to evaluate the stability of the correlation. The nine components of the DRRD was assessed separately for its association with the risk of HNC.ResultsDuring a mean follow up of 8.84 years, 279 cases of HNC were observed. DDRD score was found to be inversely associated with the risk of HNC (HR Q4 vs. Q1: 0.582; 95% CI: 0.396, 0.856; p = 0.005 for trend) in a linear dose–response manner (p = 0.211 for non-linearity). Subgroup analysis indicated this inverse correlation was more pronounced among participants who had never smoked (HRQ4 vs. Q1: 0.193; 95% CI: 0.073, 0.511; p < 0.001 for trend) compared to current or former smokers (p = 0.044 for interaction). The primary association of DDRD and HNC risk remained robust after several sensitivity analyses. Regarding the individual components of DRRD, an inverse association was also observed between the risk of HNC and increased intake of cereal fiber and whole fruit (all p < 0.05 for trend).ConclusionOur findings provide evidence that following the DRRD pattern may reduce the risk of NHC, especially for non-smokers

PRDM6 is enriched in vascular precursors during development and inhibits endothelial cell proliferation, survival, and differentiation

The mechanisms that regulate the differentiation program of multipotential stem cells remain poorly understood. In order to define the cues that delineate endothelial commitment from precursors, we screened for candidate regulatory genes in differentiating mouse embryoid bodies. We found that the PR/SET domain protein, PRDM6, is enriched in flk1(+) hematovascular precursor cells using a microarray-based approach. As determined by 5′ RACE, full length PRDM6 protein contains a PR domain and four Krüpple-like zinc fingers. In situ hybridization in mouse embryos demonstrates staining of the primitive streak, allantois, heart, outflow tract, para-aortic splanchnopleura (P-Sp)/aorto-gonadal-mesonephric (AGM) region and yolk sac, all sites known to be enriched in vascular precursor cells. PRDM6 is also detected in embryonic and adult-derived endothelial cell lines. PRDM6 is co-localized with histone H4 and methylates H4-K20 (but not H3) in vitro and in vivo, which is consistent with the known participation of PR domains in histone methyltransferase activity. Overexpression of PRDM6 in mouse embryonic endothelial cells induces apoptosis by activating caspase-3 and inducing G1 arrest. PRDM6 inhibits cell proliferation as determined by BrdU incorporation in endothelial cells, but not in rat aortic smooth muscle cells. Overexpression of PRDM6 also results in reduced tube formation in cultured endothelial cells grown in Matrigel. Taken together, our data indicate that PRDM6 is expressed by vascular precursors, has differential effects in endothelial cells and smooth muscle cells, and may play a role in vascular precursor differentiation and survival by modulating local chromatin-remodeling activity within hematovascular subpopulations during development

Pre‐symptomatic transmission of novel coronavirus in community settings

We used contact tracing to document how COVID‐19 was transmitted across 5 generations involving 10 cases, starting with an individual who became ill on January 27. We calculated the incubation period of the cases as the interval between infection and development of symptoms. The median incubation period was 6.0 days (interquartile range, 3.5‐9.5 days). The last two generations were infected in public places, 3 and 4 days prior to the onset of illness in their infectors. Both had certain underlying conditions and comorbidity. Further identification of how individuals transmit prior to being symptomatic will have important consequences.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163478/2/irv12773.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163478/1/irv12773_am.pd