Loss-of-Function Mutations in WDR73 Are Responsible for Microcephaly and Steroid-Resistant Nephrotic Syndrome: Galloway-Mowat Syndrome

Galloway-Mowat syndrome is a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. Through a combination of autozygosity mapping and whole-exome sequencing, we identified WDR73 as a gene in which mutations cause Galloway-Mowat syndrome in two unrelated families. WDR73 encodes a WD40-repeat-containing protein of unknown function. Here, we show that WDR73 was present in the brain and kidney and was located diffusely in the cytoplasm during interphase but relocalized to spindle poles and astral microtubules during mitosis. Fibroblasts from one affected child and WDR73-depleted podocytes displayed abnormal nuclear morphology, low cell viability, and alterations of the microtubule network. These data suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular subset of individuals presenting with late-onset nephrotic syndrome, postnatal microcephaly, severe intellectual disability, and homogenous brain MRI features. WDR73 is another example of a gene involved in a disease affecting both the kidney glomerulus and the CNS.     

Mammalian expression of full-length bovine aggrecan and link protein: formation of recombinant proteoglycan aggregates and analysis of proteolytic cleavage by ADAMTS-4 and MMP-13

Aggrecan, a large chondroitin sulfate (CS) and keratan sulfate (KS) proteoglycan, has not previously been expressed as a full-length recombinant molecule. To facilitate structure/function analysis, we have characterized recombinant bovine aggrecan (rbAgg) and link protein expressed in COS-7 cells. We demonstrate that C-terminally truncated rbAgg was not secreted. Gel filtration chromatography of rbAgg and isolated glycosaminoglycan (GAG) chains, and their susceptibility to chondroitinase ABC digestion indicate that the GAG chains are predominantly CS, which likely occupy fewer serine residues than native aggrecan. To confirm functionality, we determined that rbAgg bound hyaluronan and recombinant link protein to form proteoglycan aggregates. In addition, cleavage of rbAgg by ADAMTS-4 revealed that the p68 form of ADAMTS-4 preferentially cleaves within the CS-2 domain, whereas the p40 form only effectively cleaves within the interglobular domain (IGD). MMP-13 cleaved rbAgg within the IGD, but cleaved more rapidly at a site within the CS domains, suggesting a role in C-terminal processing of aggrecan. Our results demonstrate that recombinant aggrecan can be used for in vitro analyses of matrix protease-dependent degradation of aggrecan in the IGD and CS domains, and both recombinant aggrecan and link protein can be used to study the assembly of proteoglycan aggregates with hyaluronan.     

A possible role for insulin-like growth factor-binding protein-3 autocrine/paracrine loops in controlling hepatocellular carcinoma cell proliferation.

Hepatocellular carcinoma (HCC) is a common malignancy, but treatment outcomes have generally remained poor. Specific factors important for the pathogenesis of HCC are incompletely understood. Insulin-like growth factors (IGFs) are potent autocrine and paracrine mitogens for liver cancer cell proliferation, and their bioactivity is reduced by IGF-binding protein 3 (IGFBP-3). In the present study, we report that IGFBP-3 protein levels were either undetectable (28.5%) or low (71.5%) in human HCC samples examined compared with matched non-neoplastic liver tissue by Western blotting. IGFBP-3 was localized to nontumor liver cells by immunohistochemistry with greater immunointensity than neoplastic liver cells. Levels of type I receptor (IGF-IR) were found to be low in approximately 39% of human HCC samples examined compared with matched nontumor tissues. IGF-II was overexpressed in 32%, whereas IGF-I expression was decreased in 100% of HCC samples. In vitro studies revealed that IGF-I and IGF-II induced HepG2 cell proliferation in a dose-dependent manner. Treatment of HepG2 cells with either human recombinant IGFBP-3 (hrIGFBP-3) or IGF-II antibody led to a significant reduction in cell proliferation. Cotreating these cells with hrIGFBP-3 significantly attenuated the mitogenic activity of IGF-I. IGF-I-induced phosphorylation of IGF-IR beta subunit, IRS-1, mitogen-activated protein kinase, Elk-1, and Akt-1 as well as phosphatidylinositol 3'-kinase activity was significantly attenuated when hepG2 cells were pretreated with hrIGFBP-3. Our data indicate that loss of autocrine/paracrine IGFBP-3 loops may lead to HCC tumor growth and suggest that modulating production of the IGFs, IGFBP-3, and IGF-IR may represent a novel approach in the treatment of HCC.     

Morphology heterogeneity within a Campylobacter jejuni helical population: the use of calcofluor white to generate rod‐shaped C. jejuni 81‐176 clones and the genetic determinants responsible for differences in morphology within 11168 strains

Campylobacter jejuni helical shape is important for colonization and host interactions with straight mutants having altered biological properties. Passage on calcofluor white (CFW) resulted in C. jejuni 81‐176 isolates with morphology changes: either a straight morphology from frameshift mutations and single nucleotide polymorphisms in peptidoglycan hydrolase genes pgp1 or pgp2 or a reduction in curvature due a frameshift mutation in cjj81176_1105, a putative peptidoglycan endopeptidase. Shape defects were restored by complementation. Whole genome sequencing of CFW‐passaged strains showed no specific changes correlating to CFW exposure. The cjj81176_1279 (recR; recombinational DNA repair) and cjj81176_1449 (unknown function) genes were highly variable in all 81‐176 strains sequenced. A frameshift mutation in pgp1 of our laboratory isolate of the straight genome sequenced variant of 11168 (11168‐GS) was also identified. The PG muropeptide profile of 11168‐GS was identical to that of Δpgp1 in the original minimally passaged 11168 strain (11168‐O). Introduction of wild type pgp1 into 11168‐GS did not restore helical morphology. The recR gene was also highly variable in 11168 strains. Microbial cell‐to‐cell heterogeneity is proposed as a mechanism of ensuring bacterial survival in sub‐optimal conditions. In certain environments, changes in C. jejuni morphology due to genetic heterogeneity may promote C. jejuni survival.     

Genome-wide association mapping and agronomic impact of cowpea root architecture

Key message

Genetic analysis of data produced by novel root phenotyping tools was used to establish relationships between cowpea root traits and performance indicators as well between root traits and Striga tolerance.

Abstract

Selection and breeding for better root phenotypes can improve acquisition of soil resources and hence crop production in marginal environments. We hypothesized that biologically relevant variation is measurable in cowpea root architecture. This study implemented manual phenotyping (shovelomics) and automated image phenotyping (DIRT) on a 189-entry diversity panel of cowpea to reveal biologically important variation and genome regions affecting root architecture phenes. Significant variation in root phenes was found and relatively high heritabilities were detected for root traits assessed manually (0.4 for nodulation and 0.8 for number of larger laterals) as well as repeatability traits phenotyped via DIRT (0.5 for a measure of root width and 0.3 for a measure of root tips). Genome-wide association study identified 11 significant quantitative trait loci (QTL) from manually scored root architecture traits and 21 QTL from root architecture traits phenotyped by DIRT image analysis. Subsequent comparisons of results from this root study with other field studies revealed QTL co-localizations between root traits and performance indicators including seed weight per plant, pod number, and Striga (Striga gesnerioides) tolerance. The data suggest selection for root phenotypes could be employed by breeding programs to improve production in multiple constraint environments.

     

Woody structure facilitates invasion of woody plants by providing perches for birds

Woody encroachment threatens prairie ecosystems globally, and thus understanding the mechanisms that facilitate woody encroachment is of critical importance. Coastal tallgrass prairies along the Gulf Coast of the US are currently threatened by the spread of several species of woody plants. We studied a coastal tallgrass prairie in Texas, USA, to determine if existing woody structure increased the supply of seeds from woody plants via dispersal by birds. Specifically, we determined if (i) more seedlings of an invasive tree (Tridacia sebifera) are present surrounding a native woody plant (Myrica cerifera); (ii) wooden perches increase the quantity of seeds dispersed to a grassland; and (iii) perches alter the composition of the seed rain seasonally in prairie habitats with differing amounts of native and invasive woody vegetation, both underneath and away from artificial wooden perches. More T. sebifera seedlings were found within M. cerifera patches than in graminoid‐dominated areas. Although perches did not affect the total number of seeds, perches changed the composition of seed rain to be less dominated by grasses and forbs. Specifically, 20–30 times as many seeds of two invasive species of woody plants were found underneath perches independent of background vegetation, especially during months when seed rain was highest. These results suggest that existing woody structure in a grassland can promote further woody encroachment by enhancing seed dispersal by birds. This finding argues for management to reduce woody plant abundance before exotic plants set seeds and argues against the use of artificial perches as a restoration technique in grasslands threatened by woody species.     

Melittin-loaded Iron Oxide Nanoparticles Prevent Intracranial Arterial Dolichoectasia Development through Inhibition of Macrophage-mediated Inflammation

Rationale: In intracranial arterial dolichoectasia (IADE) development, the feedback loop between inflammatory cytokines and macrophages involves TNF-α and NF-κB signaling pathways and leads to subsequent MMP-9 activation and extracellular matrix (ECM) degeneration. In this proof-of-concept study, melittin-loaded L-arginine-coated iron oxide nanoparticle (MeLioN) was proposed as the protective measure of IADE formation for this macrophage-mediated inflammation and ECM degeneration.Methods: IADE was created in 8-week-old C57BL/6J male mice by inducing hypertension and elastase injection into a basal cistern. Melittin was loaded on the surface of ION as a core-shell structure (hydrodynamic size, 202.4 nm; polydispersity index, 0.158). Treatment of MeLioN (2.5 mg/kg, five doses) started after the IADE induction, and the brain was harvested in the third week. In the healthy control, disease control, and MeLioN-treated group, the morphologic changes of the cerebral arterial wall were measured by diameter, thickness, and ECM composition. The expression level of MMP-9, CD68, MCP-1, TNF-α, and NF-κB was assessed from immunohistochemistry, polymerase chain reaction, and Western blot assay.Results: MeLioN prevented morphologic changes of cerebral arterial wall related to IADE formation by restoring ECM alterations and suppressing MMP-9 expression. MeLioN inhibited MCP-1 expression and reduced CD68-positive macrophage recruitments into cerebral arterial walls. MeLioN blocked TNF-α activation and NF-κB signaling pathway. In the Sylvian cistern, co-localization was found between the CD68-positive macrophage infiltrations and the MeLioN distributions detected on Prussian Blue and T2* gradient-echo MRI, suggesting the role of macrophage harboring MeLioN.Conclusions: The macrophage infiltration into the arterial wall plays a critical role in the MMP-9 secretion. MeLioN, designed for ION-mediated melittin delivery, effectively prevents IADE formation by suppressing macrophage-mediated inflammations and MMP activity. MeLioN can be a promising strategy preventing IADE development in high-risk populations.     

Metformin treatment of diverse Caenorhabditis species reveals the importance of genetic background in longevity and healthspan extension outcomes

Metformin, the most commonly prescribed anti‐diabetes medication, has multiple reported health benefits, including lowering the risks of cardiovascular disease and cancer, improving cognitive function with age, extending survival in diabetic patients, and, in several animal models, promoting youthful physiology and lifespan. Due to its longevity and health effects, metformin is now the focus of the first proposed clinical trial of an anti‐aging drug—the Targeting Aging with Metformin (TAME) program. Genetic variation will likely influence outcomes when studying metformin health effects in human populations. To test for metformin impact in diverse genetic backgrounds, we measured lifespan and healthspan effects of metformin treatment in three Caenorhabditis species representing genetic variability greater than that between mice and humans. We show that metformin increases median survival in three C. elegans strains, but not in C. briggsae and C. tropicalis strains. In C. briggsae, metformin either has no impact on survival or decreases lifespan. In C. tropicalis, metformin decreases median survival in a dose‐dependent manner. We show that metformin prolongs the period of youthful vigor in all C. elegans strains and in two C. briggsae strains, but that metformin has a negative impact on the locomotion of C. tropicalis strains. Our data demonstrate that metformin can be a robust promoter of healthy aging across different genetic backgrounds, but that genetic variation can determine whether metformin has positive, neutral, or negative lifespan/healthspan impact. These results underscore the importance of tailoring treatment to individuals when testing for metformin health benefits in diverse human populations.