Structural and Functional Characterization of Hyper-Phosphorylated GRK5 Protein Expressed From E. coli

G protein-coupled receptor (GPCR) kinases (GRKs) are proteins in the cell responsible for regulating GPCRs located on the cell membrane. GRKs regulate active GPCRs by phosphorylating them at certain sites which causes them to stop normal signaling on the membrane. This ultimately affects how the cell responds to its environment. GRK5 is a kinase of particular interest due to its involvement in the pathology of diseases such as cardiac failure, cancers, and diabetes. Understanding the structure and function of GRK5 is essential for discovering ways to manipulate its behavior with these diseases, but not much is known about how GRK5 interacts with GPCRs. Although past studies used mammalian and insect cells to produce GRK5, this study aims to use E. coli cells to discover more about GRK5’s structure and function. Previous studies revealed E. coli produce a hyper-phosphorylated version of the GRK5 protein. We attempted to crystalize this GRK5 produced from E. coli to reveal its conformation in a phosphorylated state that we hypothesize to be similar to its form when bound to GPCRs. We also tested the functionality of this GRK5 to reveal the effects of phosphorylation. We genetically edited the GRK5 gene in multiple E. coli samples to create GRK5 with less phosphorylation sites and tested activity levels by measuring the phosphorylation of GPCRs mediated by each GRK5 variant. Successfully creating an E. coli system for structural and functional analysis of GRK5 would help reduce time and costs for GRK5 research, and it could speed up the full understanding of the interactions between GRK5 and GPCRs

A sense of unfairness reduces charitable giving to a third -party:Evidence from behavioral and electrophysiological data

Unfairness commonly impacts human economic decision-making. However, whether inequity aversion impairs pro-social decisions and the corresponding neural processes, is poorly understood. Here, we conducted two experiments to investigate whether human gifting behavior and brain activity are affected by inequity aversion. In experiment 1, participants played as a responder in a joint donation game in which they were asked to decide whether or not to accept a donation proposal made by the proposer. In experiment 2, participants played a donation game similar to experiment 1, but the charity projects were classified as high-deservingness and low-deservingness projects. The results in both of two experiments showed that the participants were more likely to reject an unfair donation proposal and the late positivity potential (LPP)/P300 elicited by fair offers was more positive than moderately unfair and highly unfair offers regardless of charity deservingness. Moreover, after principal component analysis, the differences in P300 amplitude between fair and highly unfair conditions were positively correlated with the acceptance rates in experiment 2. Taken together, our study revealed that late positivity (LPP/P300) reflected the evaluation of fairness of proposals, and could predict subsequent pro-social decisions. This study is the first to demonstrate that inequity aversion reduces pro-social motivation to help innocent third party

An assessment of land energy balance over East Asia from multiple lines of evidence and the roles of the Tibet Plateau, aerosols, and clouds

With high emissions of aerosols and the known world's “Third Pole” of the Tibet Plateau (TP) in East Asia, knowledge on the energy budget over this region has been widely concerned. This study first attempts to estimate the present-day land energy balance over East Asia by combining surface and satellite observations as well as the atmospheric reanalysis and Coupled Model Intercomparison Project Phase 6 (CMIP6) simulations. Compared to the global land budget, a substantially larger fraction of atmospheric shortwave radiation of 5.2 % is reflected, highly associated with the higher aerosol loadings and more clouds over East Asian land. While a slightly smaller fraction of atmospheric shortwave absorption of 0.6 % is unexpectedly estimated, possibly related to the lower water vapor content effects due to the thinner air over the TP to overcompensate for the aerosol and cloud effects over East Asian land. The weaker greenhouse effect and fewer low clouds due to the TP are very likely the causes of the smaller fraction of East Asian land surface downward longwave radiation. Hence, high aerosol loadings, clouds, and the TP over East Asia play vital roles in the shortwave budgets, while the TP is responsible for the longwave budgets during this regional energy budget assessment. The further obtained cloud radiative effects suggest that the presence of clouds results in a larger cooling effect on the climate system over East Asian land than that over the globe. This study provides a perspective to understand fully the roles of potential factors in influencing the different energy budget assessments over regions.</p

Knockdown of Notch1 inhibits nasopharyngeal carcinoma cell growth and metastasis via downregulation of CCL2, CXCL16, and uPA

Notch pathway is a highly conserved cell signaling system that plays very important roles in controlling multiple cell differentiation processes during embryonic and adult life. Multiple lines of evidence support the oncogenic role of Notch signaling in several human solid cancers; however, the pleiotropic effects and molecular mechanisms of Notch signaling inhibition on nasopharyngeal carcinoma (NPC) remain unclear. In this study, we evaluated Notch1 expression in NPC cell lines (CNE1, CNE2, SUNE1, HONE1, and HK1) by real-time quantitative PCR and Western blot analysis, and we found that CNE1 and CNE2 cells expressed a higher level of Notch1 compared with HONE1, SUNE1, and HK1 cells. Then Notch1 expression was specifically knocked down in CNE1 and CNE2 cells by Notch1 short hairpin RNA (shRNA). In Notch1 knockdown cells, cell proliferation, migration, and invasion were significantly inhibited. The epithelial-mesenchymal transition of tumor cells was reversed in Notch1-shRNA-transfected cells, accompanied by epithelioid-like morphology changes, increased protein levels of E-cadherin, and decreased expression of vimentin. In addition, knockdown of Notch1 markedly inhibited the expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and chemokines C-C motif chemokine ligand 2 and C-X-C motif chemokine ligand 16, indicating that these factors are downstream targets of Notch1. Furthermore, deleting uPA expression had similar effects as Notch1. Finally, knockdown of Notch1 significantly diminished CNE1 cell growth in a murine model concomitant with inhibition of cell proliferation and induction of apoptosis. These results suggest that Notch1 may become a novel therapeutic target for the clinical treatment of NPC.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151248/1/mc23082_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151248/2/mc23082.pd

Real-projective-plane hybrid-order topological insulator realized in phononic crystals

The manifold of the fundamental domain of the Brillouin zone is always considered to be a torus. However, under the synthetic gauge field, the Brillouin manifold can be modified by the projective symmetries, resulting in unprecedented topological properties. Here, we realize a real-projective-plane hybrid-order topological insulator in a phononic crystal by introducing the Z_2 gauge field. Such insulator hosts two momentum-space non-symmorphic reflection symmetries, which change the Brillouin manifold from a torus to a real projective plane. These symmetries can simultaneously lead to Klein-bottle and quadrupole topologies in different bulk gaps. The non-symmorphic reflection symmetries on Brillouin real projective plane, edge states of Klein-bottle insulator, and corner states of quadrupole insulator are observed. These results evidence the hybrid-order topology on Brillouin manifold beyond the torus, and enrich the topological physics.Comment: 4 figure

Identification of compound heterozygous variants in the noncoding RNU4ATAC gene in a Chinese family with two successive foetuses with severe microcephaly

Background: Whole-exome sequencing (WES) over the last few years has been increasingly employed for clinical diagnosis. However, one caveat with its use is that it inevitably fails to detect disease-causative variants that occur within noncoding RNA genes. Our experience in identifying pathogenic variants in the noncoding RNU4ATAC gene, in a Chinese family where two successive foetuses had been affected by severe microcephaly, is a case in point. These foetuses exhibited remarkably similar phenotypes in terms of their microcephaly and brain abnormalities; however, the paucity of other characteristic phenotypic features had made a precise diagnosis impossible. Given that no external causative factors had been reported/identified during the pregnancies, we sought a genetic cause for the phenotype in the proband, the second affected foetus. Results: A search for chromosomal abnormalities and pathogenic copy number variants proved negative. WES was also negative. These initial failures prompted us to consider the potential role of RNU4ATAC, a noncoding gene implicated in microcephalic osteodysplastic primordial dwarfism type-1 (MOPD1), a severe autosomal recessive disease characterised by dwarfism, severe microcephaly and neurological abnormalities. Subsequent targeted sequencing of RNU4ATAC resulted in the identification of compound heterozygous variants, one being the most frequently reported MOPD1-causative mutation (51G>A), whereas the other was a novel 29T>A variant. Four distinct lines of evidence (allele frequency in normal populations, evolutionary conservation of the affected nucleotide, occurrence within a known mutational hotspot for MOPD1-causative variants and predicted effect on RNA secondary structure) allowed us to conclude that 29T>A is a new causative variant for MOPD1. Conclusions: Our findings highlight the limitations of WES in failing to detect variants within noncoding RNA genes and provide support for a role for whole-genome sequencing as a first-tier genetic test in paediatric medicine. Additionally, the identification of a novel RNU4ATAC variant within the mutational hotspot for MOPD1-causative variants further strengthens the critical role of the 5′ stem-loop structure of U4atac in health and disease. Finally, this analysis enabled us to provide prenatal diagnosis and genetic counselling for the mother’s third pregnancy, the first report of its kind in the context of inherited RNU4ATAC variants

The ERAD Inhibitor Eeyarestatin I Is a Bifunctional Compound with a Membrane-Binding Domain and a p97/VCP Inhibitory Group

Protein homeostasis in the endoplasmic reticulum (ER) has recently emerged as a therapeutic target for cancer treatment. Disruption of ER homeostasis results in ER stress, which is a major cause of cell death in cells exposed to the proteasome inhibitor Bortezomib, an anti-cancer drug approved for treatment of multiple myeloma and Mantle cell lymphoma. We recently reported that the ERAD inhibitor Eeyarestatin I (EerI) also disturbs ER homeostasis and has anti-cancer activities resembling that of Bortezomib.Here we developed in vitro binding and cell-based functional assays to demonstrate that a nitrofuran-containing (NFC) group in EerI is the functional domain responsible for the cytotoxicity. Using both SPR and pull down assays, we show that EerI directly binds the p97 ATPase, an essential component of the ERAD machinery, via the NFC domain. An aromatic domain in EerI, although not required for p97 interaction, can localize EerI to the ER membrane, which improves its target specificity. Substitution of the aromatic module with another benzene-containing domain that maintains membrane localization generates a structurally distinct compound that nonetheless has similar biologic activities as EerI.Our findings reveal a class of bifunctional chemical agents that can preferentially inhibit membrane-bound p97 to disrupt ER homeostasis and to induce tumor cell death. These results also suggest that the AAA ATPase p97 may be a potential drug target for cancer therapeutics

Case Report: A novel de novo variant of COL1A1 in fetal genetic osteogenesis imperfecta

ObjectiveOsteogenesis imperfecta (OI) is a rare genetic disorder. Clinical severity is heterogeneous. The purpose of this study was to investigate the genetic characteristics of a fetus with OI by whole exome sequencing (WES) and identify the cause of the disease.MethodsIn this study, a fetus with osteogenic dysplasia was referred to our hospital. DNA was extracted from the aborted fetal tissue and peripheral blood of the parents. To identify the pathogenic genes, we conducted the trio-WES using DNA. A de novo variant in the COL1A1 gene is suspected to be the cause of the OI phenotype. We used Sanger sequencing for validation and various bioinformatics methods (such as SIFT, PolyPhen2, Mutation Taster, conservative analysis, SWISS Model, glycosylation site prediction, and I-Mutant 2.0) for analysis.ResultsBoth WES and Sanger sequencing identified a novel de novo variant of COL1A1 (c. 1309G&gt;A, p. Gly437Ser) in a fetus with OI. Bioinformatic analysis showed that the affected residue, p. Gly437, was highly conserved in multiple species and predicted that the variant was deleterious and may have an impact on protein function. This variant is present in highly conserved glycine residues of Gly-X-Y sequence repeats of the triple helical region of the collagen type I α chain, which may be the cause of OI.ConclusionThis study revealed that the c.1309G&gt;A (p. Gly437Ser) variant in the COL1A1 gene may be the genetic cause of fetal OI in this case. The discovery of this variant enriched the variation spectrum of OI. WES improves the accurate diagnosis of fetal OI, and doctors can provide patients with appropriate genetic counseling