Identification and replication of three novel myopia common susceptibility gene loci on chromosome 3q26 using linkage and linkage disequilibrium mapping

Refractive error is a highly heritable quantitative trait responsible for considerable morbidity. Following an initial genome-wide linkage study using microsatellite markers, we confirmed evidence for linkage to chromosome 3q26 and then conducted fine-scale association mapping using high-resolution linkage disequilibrium unit (LDU) maps. We used a preliminary discovery marker set across the 30-Mb region with an average SNP density of 1 SNP/15 kb (Map 1). Map 1 was divided into 51 LDU windows and additional SNPs were genotyped for six regions (Map 2) that showed preliminary evidence of multi-marker association using composite likelihood. A total of 575 cases and controls selected from the tails of the trait distribution were genotyped for the discovery sample. Malecot model estimates indicate three loci with putative common functional variants centred on MFN1 (180,566 kb; 95% confidence interval 180,505–180, 655 kb), approximately 156 kb upstream from alternate-splicing SOX2OT (182,595 kb; 95% CI 182,533–182,688 kb) and PSARL (184,386 kb; 95% CI 184,356–184,411 kb), with the loci showing modest to strong evidence of association for the Map 2 discovery samples (p<10⁻⁷, p<10⁻¹⁰, and p=0.01, respectively). Using an unselected independent sample of 1,430 individuals, results replicated for the MFN1 (p=0.006), SOX2OT (p=0.0002), and PSARL (p=0.0005) gene regions. MFN1 and PSARL both interact with OPA1 to regulate mitochondrial fusion and the inhibition of mitochondrial-led apoptosis, respectively. That two mitochondrial regulatory processes in the retina are implicated in the aetiology of myopia is surprising and is likely to provide novel insight into the molecular genetic basis of common myopia.     

SAMHD1 protects cancer cells from various nucleoside-based antimetabolites

Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies.     

Development of a Markerless Knockout Method for Actinobacillus succinogenes

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes''s auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.     

Arabinogalactan protein-rich cell walls,paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria

Background and Aims

Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae.

Methods

Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs).

Key Results

Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem.

Conclusions

The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.