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Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000–58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism. [ABSTRACT FROM AUTHOR]

The naturally occurring sulphur-containing histidine derivative, ergothioneine (EGT), exhibits potent antioxidant properties and has been proposed to confer human health benefits. Although it is only produced by select fungi and prokaryotes, likely to protect against environmental stress, the GRAS organism Saccharomyces cerevisiae does not produce EGT naturally. Herein, it is demonstrated that the recombinant expression of a single gene, Aspergillus fumigatus egtA, in S. cerevisiae results in EgtA protein presence which unexpectedly confers complete EGT biosynthetic capacity. Both High Performance Liquid Chromatography (HPLC) and LC–mass spectrometry (MS) analysis were deployed to detect and confirm EGT production in S. cerevisiae. The localisation and quantification of the resultant EGT revealed a significantly (p < 0.0001) larger quantity of EGT was extracellularly present in culture supernatants than intracellularly accumulated in 96 h yeast cultures. Methionine addition to cultures improved EGT production. The additional expression of two candidate cysteine desulfurases from A. fumigatus was thought to be required to complete EGT biosynthesis, namely AFUA_2G13295 and AFUA_3G14240, termed egt2a and egt2b in this study. However, the co-expression of egtA and egt2a in S. cerevisiae resulted in a significant decrease in the observed EGT levels (p < 0.05). The AlphaFold prediction of A. fumigatus EgtA 3-Dimensional structure illuminates the bidomain structure of the enzyme and the opposing locations of both active sites. Overall, we clearly show that recombinant S. cerevisiae can biosynthesise and secrete EGT in an EgtA-dependent manner which presents a facile means of producing EGT for biotechnological and biomedical use. [ABSTRACT FROM AUTHOR]

Armillaria mellea is a major plant pathogen. Yet, the strategies the organism uses to infect susceptible species, degrade lignocellulose and other plant material and protect itself against plant defences and its own glycodegradative arsenal are largely unknown. Here, we use a combination of gel and MS-based proteomics to profile A. mellea under conditions of oxidative stress and changes in growth matrix. 2-DE and LC-MS/MS were used to investigate the response of A. mellea to H2O2 and menadione/FeCl3 exposure, respectively. Several proteins were detected with altered abundance in response to H2O2, but not menadione/FeCl3 (i.e., valosin-containing protein), indicating distinct responses to these different forms of oxidative stress. One protein, cobalamin-independent methionine synthase, demonstrated a common response in both conditions, which may be a marker for a more general stress response mechanism. Further changes to the A. mellea proteome were investigated using MS-based proteomics, which identified changes to putative secondary metabolism (SM) enzymes upon growth in agar compared to liquid cultures. Metabolomic analyses revealed distinct profiles, highlighting the effect of growth matrix on SM production. This establishes robust methods by which to utilize comparative proteomics to characterize this important phytopathogen. [ABSTRACT FROM AUTHOR]

The Phytophthora genus includes some of the most devastating plant pathogens. Here we report draft genome sequences for three ubiquitous Phytophthora species—Phytophthora chlamydospora, Phytophthora gonapodyides, and Phytophthora pseudosyringae. Phytophthora pseudosyringae is an important forest pathogen that is abundant in Europe and North America. Phytophthora chlamydospora and Ph. gonapodyides are globally widespread species often associated with aquatic habitats. They are both regarded as opportunistic plant pathogens. The three sequenced genomes range in size from 45 Mb to 61 Mb. Similar to other oomycete species, tandem gene duplication appears to have played an important role in the expansion of effector arsenals. Comparative analysis of carbohydrate-active enzymes (CAZymes) across 44 oomycete genomes indicates that oomycete lifestyles may be linked to CAZyme repertoires. The mitochondrial genome sequence of each species was also determined, and their gene content and genome structure were compared. Using mass spectrometry, we characterised the extracellular proteome of each species and identified large numbers of proteins putatively involved in pathogenicity and osmotrophy. The mycelial proteome of each species was also characterised using mass spectrometry. In total, the expression of approximately 3000 genes per species was validated at the protein level. These genome resources will be valuable for future studies to understand the behaviour of these three widespread Phytophthora species. [ABSTRACT FROM AUTHOR]

Although the pan-genome concept originated in prokaryote genomics, an increasing number of eukaryote species pan-genomes have also been analysed. However, there is a relative lack of software intended for eukaryote pan-genome analysis compared to that available for prokaryotes. In a previous study, we analysed the pan-genomes of four model fungi with a computational pipeline that constructed pan-genomes using the synteny-dependent Pan-genome Ortholog Clustering Tool (PanOCT) approach. Here, we present a modified and improved version of that pipeline which we have called Pangloss. Pangloss can perform gene prediction for a set of genomes from a given species that the user provides, constructs and optionally refines a species pan-genome from that set using PanOCT, and can perform various functional characterisation and visualisation analyses of species pan-genome data. To demonstrate Pangloss's capabilities, we constructed and analysed a species pan-genome for the oleaginous yeast Yarrowia lipolytica and also reconstructed a previously-published species pan-genome for the opportunistic respiratory pathogen Aspergillus fumigatus. Pangloss is implemented in Python, Perl and R and is freely available under an open source GPLv3 licence via GitHub. [ABSTRACT FROM AUTHOR]

The force-vector theory contends that horizontal exercises are more specific to horizontal sports skills. In this context, the focus is on horizontal force production relative to the global coordinate frame. However, according to the principle of dynamic correspondence, the direction of force relative to the athlete is more important, and thus the basis for the force-vector theory is flawed. The purpose of this study was therefore to test the force-vector theory. According to the force-vector theory, hip thrust is a horizontally loaded exercise, and so hip thrust training would be expected to create greater improvements in horizontal jump performance than vertical jump performance. Eleven collegiate female athletes aged 18–24 years completed a 14-week hip thrust training programme. Pre and post testing was used to measure the following: vertical squat jump, vertical countermovement jump, horizontal squat jump, horizontal countermovement jump and hip thrust 3 repetition maximum (3RM). Subjects improved their 3 repetition maximum hip thrust performance by 33.0% (d = 1.399, p < 0.001, η2 = 0.784) and their vertical and horizontal jump performance (improvements ranged from 5.4–7.7%; d = 0.371–0.477, p = 0.004, η2 = 0.585). However, there were no differences in the magnitude of the improvement between horizontal and vertical jumping (p = 0.561, η2 = 0.035). The results of this study are contrary to the predictions of the force-vector theory. Furthermore, this paper concludes with an analysis of the force-vector theory, presenting the mechanical inconsistencies in the theory. Coaches should use the well established principle of dynamic correspondence in order to assess the mechanical similarity of exercises to sports skills. [ABSTRACT FROM AUTHOR]