Lagovirus Non-structural Protein p23: A Putative Viroporin That Interacts With Heat Shock Proteins and Uses a Disulfide Bond for Dimerization

The exact function(s) of the lagovirus non-structural protein p23 is unknown as robust cell culture systems for the Rabbit haemorrhagic disease virus (RHDV) and other lagoviruses have not been established. Instead, a range of in vitro and in silico models have been used to study p23, revealing that p23 oligomerizes, accumulates in the cytoplasm, and possesses a conserved C-terminal region with two amphipathic helices. Furthermore, the positional homologs of p23 in other caliciviruses have been shown to possess viroporin activity. Here, we report on the mechanistic details of p23 oligomerization. Site-directed mutagenesis revealed the importance of an N-terminal cysteine for dimerization. Furthermore, we identified cellular interactors of p23 using stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics; heat shock proteins Hsp70 and 110 interact with p23 in transfected cells, suggesting that they ‘chaperone’ p23 proteins before their integration into cellular membranes. We investigated changes to the global transcriptome and proteome that occurred in infected rabbit liver tissue and observed changes to the misfolded protein response, calcium signaling, and the regulation of the endoplasmic reticulum (ER) network. Finally, flow cytometry studies indicate slightly elevated calcium concentrations in the cytoplasm of p23-transfected cells. Taken together, accumulating evidence suggests that p23 is a viroporin that might form calcium-conducting channels in the ER membranes

Auxin Response Factor2 (ARF2) and Its Regulated Homeodomain Gene HB33 Mediate Abscisic Acid Response in Arabidopsis

The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth

Genetics of early-life head circumference and genetic correlations with neurological, psychiatric and cognitive outcomes

This is the final version. Available from BMC via the DOI in this record. Availability of data and materials GWAS summary data will be deposited at the EGG website (https://egg-consortium.org/) at publication. Individual study data are available from the corresponding author on reasonable request.Abstract Background: Head circumference is associated with intelligence and tracks from childhood into adulthood. Methods: We performed a genome-wide association study meta-analysis and follow-up of head circumference in a total of 29,192 participants between 6 and 30 months of age. Results: Seven loci reached genome-wide signifcance in the combined discovery and replication analysis of which three loci near ARFGEF2, MYCL1, and TOP1, were novel. We observed positive genetic correlations for early-life head circumference with adult intracranial volume, years of schooling, childhood and adult intelligence, but not with adult psychiatric, neurological, or personality-related phenotypes. Conclusions: The results of this study indicate that the biological processes underlying early-life head circumference overlap largely with those of adult head circumference. The associations of early-life head circumference with cognitive outcomes across the life course are partly explained by genetics.Wellcome TrustSimons FoundationWellcome TrustMRC & WTUniversity of Southern DenmarkMax Planck core societ

Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice.

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury

Both presynaptic and postsynaptic components are present at neuromuscular junctions in 14 d regenerated muscle of Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> KOs.

<p>(A) Proportion of NMJs positive for synaptophysin and BGTX in saline- or CTX-injected TA of Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> KOs or grouped Myf5/Tam LCs. (B) Representative images showing localization of NMJ pre-synaptic (synaptophysin, red) and post-synaptic markers (BGTX, green) in Myf5/<i>Fktn</i> KO and Tam/<i>Fktn</i> KO and LC TAs injected with saline or CTX. Nuclei stained with DAPI (blue). Scale bar = 100μm. n = 7, LC; n = 4, Myf5 KO; n = 6 Tam KO.</p

Minor delay in progression of glycolytic type 2x fiber switching in TA of Myf5/<i>Fktn</i> KO mice normalizes by 8 weeks.

<p>Glycolytic intermediate-twitch 2x fibers in Myf5/<i>Fktn</i>-deficient (KO) and control (LC) (A) iliopsoas and (B) TA muscles. *, p<0.05; **, p<0.01; ***, p<0.001, two-way ANOVA with Bonferroni’s post-test. Whole tissue (C) iliopsoas and (D) TA maps of sections stained with antibody detecting all myosin isoforms except type 2x (red), with basement membrane perlecan or sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Unstained (negative) fibers were counted to measure type 2x. Scale bar = 100μm. n = 4 for all 2 and 4 wko measurements (except Ilio 4wko LC, n = 3); n = 5 per 8 wko group.</p

Frequencies of type 2a oxidative and type 2b glycolytic fast-twitch fibers are unchanged in iliopsoas and TA between Myf5/<i>Fktn</i> KO and LC mice.

<p>(A) Oxidative type 2a fibers in iliopsoas (top) and TA (bottom) of 2, 4, and 8 wko Myf5/<i>Fktn</i>-deficient (KO) and control (LC) mice. (B) Glycolytic type 2b fibers in iliopsoas (top) and TA (bottom) of 2, 4, and 8 wko Myf5/<i>Fktn-</i>deficient (KO) and control (LC) mice. *, p<0.05; two-way ANOVA with Bonferroni’s post-test. n = 4 for all 2 and 4 wko experimental groups (except Ilio 4 wko LC, n = 3); n = 5 per 8 wko group.</p

Slow oxidative fibers are decreased in Myf5/<i>Fktn</i> KO and Tam/<i>Fktn</i> LC mice following CTX injection.

<p>(A) Quantification of slow type 1 oxidative fibers in TA muscle of saline- or CTX-injected Myf5/<i>Fktn</i> and Tam/<i>Fktn</i> LC or KO mice. *, p<0.05; **, p<0.01; ***, p < .001; two-way ANOVA with Bonferroni’s post-test (all genotypes combined) depicted on figures; two-way ANOVA per strain (Myf5/<i>Fktn</i> or Tam/<i>Fktn</i>) are also reported. (B) Whole tissue maps of CTX-injected TA muscle stained with anti-myosin heavy chain type 1 antibody (red), with sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Scale bar = 100 μm. n = 5, Myf5 LC; n = 7, Myf5 KO and Tam LC; n = 8, Tam KO.</p

Frequency of type 1 oxidative fibers decreases with age in iliopsoas and TA.

<p>Oxidative type 1 fibers in (A) iliopsoas and (B) TA of 2, 4, and 8 wko Myf5/<i>Fktn</i>-deficient (KO) and control (LC) mice. *, p<0.05; **, p<0.01; ***, p<0.001, two-way ANOVA with Bonferroni’s post-test. Whole tissue (C) iliopsoas and (D) TA maps of sections stained with anti-myosin heavy chain type 1 antibody (red), with basement membrane perlecan or sarcolemmal αDG core protein (green) and nuclear (blue) counterstains. Scale bar = 100 μm. n = 4 for all 2 and 4 wko measurements (except Ilio LC, n = 3); n = 5 for all 8 wko measurements.</p