Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus

Keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning, is the leading indication for corneal transplantation in the developed world. Genome-wide association studies have identified common SNPs 100 kb upstream of ZNF469 strongly associated with corneal thickness. Homozygous mutations in ZNF469 and PR domain-containing protein 5 (PRDM5) genes result in brittle cornea syndrome (BCS) Types 1 and 2, respectively. BCS is an autosomal recessive generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Some individuals with heterozygous PRDM5 mutations demonstrate a carrier ocular phenotype, which includes a mildly reduced corneal thickness, keratoconus and blue sclera. We hypothesized that heterozygous variants in PRDM5 and ZNF469 predispose to the development of isolated keratoconus. We found a significant enrichment of potentially pathologic heterozygous alleles in ZNF469 associated with the development of keratoconus (P = 0.00102) resulting in a relative risk of 12.0. This enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients represents a significant mutational load and highlights ZNF469 as the most significant genetic factor responsible for keratoconus identified to date.

Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants

Purpose:The aim of this study was to determine whether mutations in mitochondrial DNA play a role in high-pressure primary open-angle glaucoma (OMIM 137760) by analyzing new data from massively parallel sequencing of mitochondrial DNA.
Methods:Glaucoma patients with high-tension primary open-angle glaucoma and ethnically matched and age-matched control subjects without glaucoma were recruited. The entire human mitochondrial genome was amplified in two overlapping fragments by long-range polymerase chain reaction and used as a template for massively parallel sequencing on an Ion Torrent Personal Genome Machine. All variants were confirmed by conventional Sanger sequencing.
Results:Whole-mitochondrial genome sequencing was performed in 32 patients with primary open-angle glaucoma from India (n = 16) and Ireland (n = 16). In 16 of the 32 patients with primary open-angle glaucoma (50% of cases), there were 22 mitochondrial DNA mutations consisting of 7 novel mutations and 8 previously reported disease-associated sequence variants. Eight of 22 (36.4%) of the mitochondrial DNA mutations were in complex I mitochondrial genes.
Conclusion:Massively parallel sequencing using the Ion Torrent Personal Genome Machine with confirmation by Sanger sequencing detected a pathogenic mitochondrial DNA mutation in 50% of the primary open-angle glaucoma cohort. Our findings support the emerging concept that mitochondrial dysfunction results in the development of glaucoma and, more specifically, that complex I defects play a significant role in primary open-angle glaucoma pathogenesis.

Small RNAs from plants, bacteria and fungi within the order Hypocreales are ubiquitous in human plasma

Background

The human microbiome plays a significant role in maintaining normal physiology. Changes in its composition have been associated with bowel disease, metabolic disorders and atherosclerosis. Sequences of microbial origin have been observed within small RNA sequencing data obtained from blood samples. The aim of this study was to characterise the microbiome from which these sequences are derived.

Abundant non-human small RNA sequences were identified in plasma and plasma exosomal samples. Assembly of these short sequences into longer contigs was the pivotal novel step in ascertaining their origin by BLAST searches. Most reads mapped to rRNA sequences. The taxonomic profiles of the microbes detected were very consistent between individuals but distinct from microbiomes reported at other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Many contigs were from plants, presumably of dietary origin.  In addition, extremely abundant small RNAs derived from human Y RNAs were detected.

A characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status. The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

Just a matter of time: Fungi and roots significantly and rapidly aggregate soil over four decades along the Tagliamento River, NE Italy

Fluvial islands are emergent landforms which form at the interface between the permanently inundated areas of the river channel and the more stable areas of the floodplain as a result of interactions between physical river processes, wood and riparian vegetation. These highly dynamical systems are ideal to study soil structure development in the short to medium term, a process in which soil biota and plants play a substantial role. We investigated soil structure development on islands along a 40 year chronosequence within a 3 km island-braided reach of the Tagliamento River, Northeastern Italy. We used several parameters to capture different aspects of the soil structure, and measured biotic (e.g., fungal and plant root parameters) and abiotic (e.g. organic carbon) factors expected to determine the structure. We estimated models relating soil structure to its determinants, and, in order to confer statistical robustness to our results, we explicitly took into account spatial autocorrelation, which is present due to the space for time substitution inherent in the study of chronosequences and may have confounded results of previous studies. We found that, despite the eroding forces from the hydrological and geomorphological dynamics to which the system is subject, all soil structure variables significantly, and in some case greatly increased with site age. We interpret this as a macroscopic proxy for the major direct and indirect binding effects exerted by root variables and extraradical hyphae of arbuscular mycorrhizal fungi (AMF). Key soil structure parameters such as percentage of water stable aggregates (WSA) can double from the time the island landform is initiated (mean WSA = 30%) to the full 40 years (mean WSA = 64%) covered by our chronosequence. The study demonstrates the fundamental role of soil biota and plant roots in aggregating soils even in a system in which intense short to medium term physical disturbances are common.

Water and Temperature Relations of Growth of the Entomogenous Fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus

The effects of temperature (5-50°C), water availability (0.998-0.88 water activity, aw), and aw × temperature interactions (15-45°C) on growth of three entomogenous fungi, Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus, were evaluated on a Sabouraud dextrose-based medium modified with the ionic solute KCl, the non-ionic solute glycerol, and an inert solute, polyethylene glycol (PEG) 600. The temperature ranges for growth of B. bassiana, M. anisopliae, and P. farinosus were 5-30, 5-40, and 5-30°C, and optimum growth temperatures were 25, 30, and 20°C, respectively. All three species grew over a similar aw range (0.90-0.998) at optimum temperatures for growth. However, there were significant interspecies variations in growth rates on media modified with each of the three aw-modifying solutes. Growth aw optima ranged between 0.99 and 0.97 on KCl-, glycerol-, and PEG 600-modified media for M. anisopliae and P. farinosus. B. bassiana grew optimally at 0.998 aw, regardless of aw. Comprehensive two-dimensional profiles of aw × temperature relations for growth of these three species were constructed for the first time. The results are discussed in relation to the environmental limits that determine efficacy of entomogenous fungi as biocontrol agents in nature. © 1999 Academic Press.

Effects of KCl concentration on accumulation of acyclic sugar alcohols and trehalose in conidia of three entomopathogenic fungi

Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were grown on Sabouraud Dextrose Agar (SDA) modified with KCl to give a range of water activity (a(w)) from 0.938 to 0.998. Growth of all three species was optimal at 0.983 a(w) and growth occurred over the a(w) range tested. Acyclic sugar alcohol (polyol) and trehalose content of conidia was determined by HPLC and found to vary with species and a(w). Conidia of B. bassiana and P. farinosus were found to contain totals of 1.5% and 2.3% polyols respectively at 0.998 a(w), and double these amounts at <0.950 a(w). Conidia of M. anisopliae contained from 5.7% to 6.8% polyols at each a(w) tested. In conidia of all three species the predominant polyol was mannitol. The lower molecular weight polyols, arabitol and erythritol, were found to accumulate at reduced a(w). Small amounts of glycerol were present in conidia of each species; <15% total polyols. Conidia of B. bassiana and M. anisopliae contained about 0.5% trehalose from 0.970 to 0.998 a(w), but only trace amounts below 0.950 a(w). Conidia of P. farinosus contained 2.1% trehalose at 0.998 a(w) and this decreased to <0.1% below 0.950 a(w). Potential to manipulate the endogenous reserves of conidia of these biological control agents to enhance viability and desiccation tolerance is discussed.

Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi

The entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were cultured on solid agar media containing different carbohydrate components (glycerol, glucose, trehalose or starch) at concentrations of ≤ 142.7 g added carbon 1-1 for 30 d at 25°C. The water activity (a(w)) of the media ranged from 0.925 to 0.998. Growth of M. anisopliae and P. farinosus was stimulated between 0.975 and 0.995 a(w) on glucose media and that of P. farinosus at 0. 975 a(w) on glycerol media. At < 0.970 a(w), growth of each fungal species was significantly reduced (P < 0.05). Polyhydroxy alcohols (polyols) and trehalose were extracted from conidia produced on different media and quantified using HPLC. Total polyol content of conidia produced on glucose media varied between 5.2 and 52.2 mg g-1 for B. bassiana, 77.3 and 90.3 mg g-1 for M. anisopliae, and 26.7 and 76.1 mg g-1 for P. farinosus. The amounts of specific polyols in conidia varied significantly from media of different glucose concentrations. Mannitol was the predominant polyol in conidia of all three species, with conidia of M. anisopliae, for example, containing as much as 75.2 mg mannitol g-1 when cultured on glucose media. The amount of the lower molecular mass polyols glycerol and erythritol was greater in conidia produced on glucose media with > 50.0 g added carbon 1-1 than that in conidia produced at lower glucose concentrations. Conidia contained between 10.8 and 20.8 mg glycerol plus erythritol g-1 on glucose media with 142.7 g added carbon 1-1, depending on species. Conversely, conidia of B. bassiana and P. farinosus contained maximum amounts of trehalose ( ≤ 23.5 mg g-1) when produced on glucose media with < 50.0 g added carbon l-1, and trehalose content was considerably less at higher glucose concentrations. There were accumulations of glycerol and erythritol in conidia of all three species when grown on glycerol media with > 25.0 g added carbon 1-1; conidia of B. bassiana contained up to 154.0 mg glycerol plus erythritol g-1. hen B. bassiana and P. farinosus were grown on trehalose media, conidia contained up to 222.1 mg trehalose g-1. By contrast, conidia of M. anisopliae contained < 17.0 mg trehalose g-1 under all conditions tested. The water availability of solutions of different polyols is discussed in relation to their potential to act in osmotic adjustment during germination. The ability to manipulate polyol and trehalose content of fungal propagules may be critical in enhancing the storage life and efficacy of biological control agents.

The application of TiO2 photocatalysis for disinfection of water contaminated with pathogenic micro-organisms: a review

The use of semiconductor photocatalysis for treatment of water and air has been the topic of intense research activity over the past 20 years. This powerful process has also been extended to the disinfection of environments contaminated with pathogenic micro-organisms. This review summarizes recent developments concerned with the photocatalytic treatment of water contaminated with pathogenic micro-organisms presenting a potential hazard to animals and human beings.

A comparison of the effectiveness of TiO2 photocatalysis and UVA photolysis for the destruction of three pathogenic micro-organisms

TiO₂ photocatalysis has demonstrated efficacy as a treatment process for water contaminated with chemical pollutants. When exposed to UVA light TiO₂ also demonstrates an effective bactericidal activity. The mechanism of this process has been reported to involve attack by valence band generated hydroxyl radicals. In this study when three common bacterial pathogens, Escherichia coli, Salmonella enterica serovar Enteritidis and Pseudomonas aeruginosa, were exposed to TiO₂ and UVA light a substantial decrease in bacterial numbers was observed. Control experiments in which all three pathogens were exposed to UVA light only resulted in a similar reduction in bacterial numbers. Moreover, exposure to UVA light alone resulted in the production of a smaller than average colony phenotype among the surviving bacteria, for all three pathogens examined, a finding which was not observed following treatment with UVA and TiO₂. Small slow growing colonies have been described for several pathogenic bacteria and are referred to as small colony variants. Several studies have demonstrated an association between small colony variants and persistent, recurrent and antibiotic resistant infections. We propose that the production of small colony variants of pathogenic bacteria following UVA treatment of drinking water may represent a health hazard. As these small colony variants were not observed with the UVA/TiO₂ system this potential hazard is not a risk when using this technology. It would also appear that the bactericidal mechanism is different with the UVA/TiO₂ process compared to when UVA light is used alone.