A Functional Screen Provides Evidence for a Conserved, Regulatory, Juxtamembrane Phosphorylation Site in Guanylyl Cyclase A and B

Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ∼20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation – processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200–1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor 32PO4 co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders

How to realize a robust practical Majorana chain in a quantum dot-superconductor linear array

Semiconducting nanowires in proximity to superconductors are promising experimental systems for Majorana fermions, which may ultimately be used as building blocks for topological quantum computers. A serious challenge in the experimental realization of the Majorana fermions is the supression of topological superconductivity by disorder. We show that Majorana fermions protected by a robust topological gap can occur at the ends of a chain of quantum dots connected by s-wave superconductors. In the appropriate parameter regime, we establish that the quantum dot/superconductor system is equivalent to a 1D Kitaev chain, which can be tuned to be in a robust topological phase with Majorana end modes even in the case where the quantum dots and superconductors are both strongly disordered. Such a spin-orbit coupled quantum dot - s-wave superconductor array provides an ideal experimental platform for the observation of non-Abelian Majorana modes.Comment: 8 pages; 3 figures; version 2: Supplementary material updated to include more general proof for localized Majorana fermion

Attenuated Induction of the Unfolded Protein Response in Adult Human Primary Astrocytes in Response to Recurrent Low Glucose

This is the final version. Available on open access from Frontiers Media via the DOI in this record. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.AIMS/HYPOTHESIS: Recurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure. METHODS: Human primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq, respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform. RESULTS: 24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following low glucose (LG) exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures. CONCLUSIONS/INTERPRETATION: These data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be a consequence of a successful metabolic adaptation, as previously reported, that better preserves intracellular energy levels and a reduced necessity for the UPR.Diabetes UKJDRF postdoctoral fellowshipMedical Research Council (MRC)Wellcome TrustBiotechnology & Biological Sciences Research Council (BBSRC)Novo Nordisk UK Research FoundationMary Kinross Charitable TrustEuropean Foundation for the Study of Diabetes/Novo Nordisk Programm

Parental recommendations and exercise attitudes in congenital hearts.

BACKGROUND: Children and young people with CHD benefit from regular physical activity. Parents are reported as facilitators and barriers to their children's physical activity. The aim of this study was to explore parental factors, child factors, and their clinical experience on physical activity participation in young people with CHD. METHODS: An online questionnaire was co-developed with parents (n = 3) who have children with CHD. The survey was then distributed in the United Kingdom by social media and CHD networks, between October 2021 and February 2022. Data were analysed using mixed methods. RESULTS: Eighty-three parents/guardians responded (94% mothers). Young people with CHD were 7.3 ± 5.0 years old (range 0-20 years; 53% female) and 84% performed activity. Parental participation in activity (X2(1) = 6.9, P < 0.05) and perceiving activity as important for their child were positively associated with activity (Fisher's Exact, P < 0.05). Some parents (∼15%) were unsure of the safety of activity, and most (∼70%) were unsure where to access further information about activity. Fifty-two parents (72%) had never received activity advice in clinic, and of the 20 who received advice, 10 said it was inconsistent. Qualitative analysis produced the theme "Knowledge is power and comfort." Parents described not knowing what activity was appropriate or the impact of it on their child. CONCLUSION: Parental participation and attitudes towards activity potentially influence their child's activity. A large proportion of young people performed activity despite a lack and inconsistency of activity advice offered by CHD clinics. Young people with CHD would benefit from activity advice with their families in clinics

Temperature dependence on the mass susceptibility and mass magnetization of superparamagnetic Mn–Zn–ferrite nanoparticles as contrast agents for magnetic imaging of oil and gas reservoirs

The mass susceptibility (χmass) and mass magnetization (Mmass) were determined for a series of ternary manganese and zinc ferrite nanoparticles (Mn–Zn ferrite NPs, MnxZn1−xFe2O4) with different Mn:Zn ratios (0.08 ≤ x ≤ 4.67), prepared by the thermal decomposition reaction of the appropriate metal acetylacetonate complexes, and for the binary homologs (MxFe3−xO4, where M = Mn or Zn). Alteration of the Mn:Zn ratio in Mn–Zn ferrite NPs does not significantly affect the particle size. At room temperature and low applied field strength the mass susceptibility increases sharply as the Mn:Zn ratio increases, but above a ratio of 0.4 further increase in the amount of manganese results in the mass susceptibility decreasing slightly, reaching a plateau above Mn:Zn ≈ 2. The compositional dependence of the mass magnetization shows less of a variation at room temperature and high applied fields. The temperature dependence of the mass magnetization of Mn–Zn ferrite NPs is significantly less for Mn-rich compositions making them more suitable for downhole imaging at higher temperatures (>100 °C). For non-shale reservoirs, replacement of nMag by Mn-rich Mn–Zn ferrites will allow for significant signal-to-noise enhancement of 6.5× over NP magnetite

Opportunities and priorities for breast surgical research

The Breast Cancer Campaign Gap analysis (2013) established breast cancer research priorities without specific focus on surgical research nor the role of surgeons. The majority of breast cancer patients encounter a surgeon at diagnosis or during treatment, thus surgical involvement in design and delivery of high-quality research to improve patient care is critical. This review aims to identify opportunities and priorities for breast surgical research to complement the previous gap analysis

Tissue Proteome of 2-Hydroxyacyl-CoA Lyase Deficient Mice Reveals Peroxisome Proliferation and Activation of ω-Oxidation

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of β-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1−/− mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1−/− liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1−/− mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1−/− mice in better understanding disease mechanism in fatty acid α-oxidation disorders