Discrimination of three genetically close Aspergillus species by using high resolution melting analysis applied to indoor air as case study.

peer reviewedBACKGROUND: Indoor air pollution caused by fungal contamination is suspected to have a public health impact. Monitoring of the composition of the indoor airborne fungal contaminants is therefore important. To avoid problems linked to culture-dependent protocols, molecular methods are increasingly being proposed as an alternative. Among these molecular methods, the polymerase chain reaction (PCR) and the real-time PCR are the most frequently used tools for indoor fungal detection. However, even if these tools have demonstrated their appropriate performance, some of them are not able to discriminate between species which are genetically close. A solution to this could be the use of a post-qPCR high resolution melting (HRM) analysis, which would allow the discrimination of these species based on the highly accurate determination of the difference in melting temperature of the obtained amplicon. In this study, we provide a proof-of-concept for this approach, using a dye adapted version of our previously developed qPCR SYBR(R)Green method to detect Aspergillus versicolor in indoor air, an important airborne fungus in terms of occurrence and cause of health problems. Despite the good performance observed for that qPCR method, no discrimination could previously be made between A. versicolor, Aspergillus creber and Aspergillus sydowii. METHODS: In this study, we developed and evaluated an HRM assay for the discrimination between A. versicolor, Aspergillus creber and Aspergillus sydowii. RESULTS: Using HRM analysis, the discrimination of the 3 Aspergillus species could be made. No false positive, nor false negatives were observed during the performance assessment including 20 strains of Aspergillus. The limit of detection was determined for each species i.e., 0.5 pg of gDNA for A. creber and A. sydowii, and 0.1 pg of gDNA for A. versicolor. The HRM analysis was also successfully tested on environmental samples. CONCLUSION: We reported the development of HRM tools for the discrimination of A. versicolor, A. creber and A. sydowii. However, this study could be considered as a study case demonstrating that HRM based on existing qPCR assays, allows a more accurate identification of indoor air contaminants. This contributes to an improved insight in the diversity of indoor airborne fungi and hence, eventually in the causal link with health problems

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

International audienceObjective. Zinc ions are essential for the formation of hexameric insulin and hormone crystallisation. Correspondingly, a non-synonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. Here, we describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. Research Design and Methods. Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection, or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles, were undertaken using standard protocols. Results. ZnT8(-/-) mice displayed age, sex and diet-dependent abnormalities in glucose tolerance, insulin secretion and body weight. Islets isolated from null mice had reduced granule zinc content, and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion and insulin crystal dissolution, as assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8(-/-) islets. Insulin processing was normal. Molecular modelling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn(2+) transport activity than W325 ZnT8 by fluorescence-based assay. Discussion and conclusions. ZnT8 is required for normal insulin crystallisation and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risk

Structure-Preserving Signatures on Equivalence Classes From Standard Assumptions

Structure-preserving signatures on equivalence classes (SPS-EQ) introduced at ASIACRYPT 2014 are a variant of SPS where a message is considered as a projective equivalence class, and a new representative of the same class can be obtained by multiplying a vector by a scalar. Given a message and corresponding signature, anyone can produce an updated and randomized signature on an arbitrary representative from the same equivalence class. SPS-EQ have proven to be a very versatile building block for many cryptographic applications. In this paper, we present the first EUF-CMA secure SPS-EQ scheme under standard assumptions. So far only constructions in the generic group model are known. One recent candidate under standard assumptions are the weakly secure equivalence class signatures by Fuchsbauer and Gay (PKC\u2718), a variant of SPS-EQ satisfying only a weaker unforgeability and adaption notion. Fuchsbauer and Gay show that this weaker unforgeability notion is sufficient for many known applications of SPS-EQ. Unfortunately, the weaker adaption notion is only proper for a semi-honest (passive) model and as we show in this paper, makes their scheme unusable in the current models for almost all of their advertised applications of SPS-EQ from the literature. We then present a new EUF-CMA secure SPS-EQ scheme with a tight security reduction under the SXDH assumption providing the notion of perfect adaption (under malicious keys). To achieve the strongest notion of perfect adaption under malicious keys, we require a common reference string (CRS), which seems inherent for constructions under standard assumptions. However, for most known applications of SPS-EQ we do not require a trusted CRS (as the CRS can be generated by the signer during key generation). Technically, our construction is inspired by a recent work of Gay et al. (EUROCRYPT\u2718), who construct a tightly secure message authentication code and translate it to an SPS scheme adapting techniques due to Bellare and Goldwasser (CRYPTO\u2789)

Controlling complexity: the clinical relevance of mouse complex genetics.

Experimental animal models are essential to obtain basic knowledge of the underlying biological mechanisms in human diseases. Here, we review major contributions to biomedical research and discoveries that were obtained in the mouse model by using forward genetics approaches and that provided key insights into the biology of human diseases and paved the way for the development of novel therapeutic approaches

Exploiting the Advantages of Molecular Tools for the Monitoring of Fungal Indoor Air Contamination: First Detection of Exophiala jeanselmei in Indoor Air of Air-Conditioned Offices

Today, indoor air pollution is considered a public health issue. Among the impacting pollutants, indoor airborne fungi are increasingly highlighted. Most of the monitoring protocols are culture-based, but these are unable to detect the uncultivable and/or dead fraction or species suppressed by fast-growing fungi, even though this fraction could impact health. Among the contaminants suspected to be part of this fraction, Exophiala jeanselmei is an interesting case study. Known to be pathogenic, this black yeast grows in humid environments such as air-conditioning systems, where it has been previously detected using classical culture-based methods. However, until now, this fungus was never detected in indoor air in contact with these air-conditioning systems. This study shows the first detection of E. jeanselmei in indoor air collected from offices in contact with contaminated air-conditioning reservoirs. While its presence in indoor air could not be demonstrated with culture-based methods, it was found by real-time PCR and massive parallel sequencing. The latter also allowed obtaining a broader view on the fungal diversity in the tested samples. Similar approaches were applied on water samples collected from the conditioning reservoirs to trace the source of contamination. The comparison of results obtained with both methods confirmed that the molecular tools could improve indoor air monitoring, especially of dead and/or uncultivable contaminants or when competition between species could occur

Développement d’outils moléculaires de détection des moisissures présentes dans l’air intérieur afin de déterminer leur impact sur la santé publique

Currently, contamination of the indoor environment by fungi is suggested to be a public health problem, although scientific evidence on the causal link is still limited. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical monitoring methods, based on cultivation and microscopic identification, have some limitations. For example, uncultivable or dead fungi (“unknown” fraction) cannot be identified, although they could have an impact on human health. In this context, molecular tools seem to be a valuable alternative. In this PhD work, different molecular tools were developed, from simplex to multiplex, to detect and identify indoor airborne fungi. The goal was to improve the detection of fungal contaminants, including the “unknown” fraction, as compared to the currently used classical monitoring methods. The necessary air sampling and DNA extraction protocols, adapted to the downstream molecular monitoring methods have also been developed. Through the application of the developed tools to specific case studies, we aimed to improve the current knowledge on fungal contamination. At first, we developed a specific ITS-based SYBR®green real-time PCR (qPCR) assay for Aspergillus versicolor, a species frequently observed in indoor air and known to be allergenic. Additionally, an ITS-based qPCR assay was developed for the specific detection of Exophiala jeanselmei, a pathogenic yeast suspected to be a part of the “unknown fraction”. The performance of these qPCR methods was assessed. This comparison demonstrated that SYBR®green qPCR assays can be used as a molecular alternative for monitoring of contaminated samples while eliminating the need for culturing and thereby considerably decreasing the required analysis time. However, qPCR has some limitations especially concerning the discrimination of genetically close species and multiplexing. The first issue was addressed through the use of post-qPCR high resolution melting (HRM) analysis, providing a proof-of-concept for this approach, using 3 closely related Aspergillus, i.e., A. versicolor, Aspergillus creber and Aspergillus sydowii. This HRM tool will allow a more accurate monitoring of these closely related indoor air contaminants, thereby contributing to an improved insight in the causal link between the specific presence of these species and health issues. The multiplexing issue was overcome through a Luminex xMAP® assay, developed for the simultaneous detection of the 10 most frequently in indoor air found fungi. All the species identified with the classical method were also detected with the xMAP® assay, however in a shorter time frame, and using less sample material. This assay will improve the communication with the involved medical team and the patient. To provide scientific evidence for the causal link between indoor airborne fungi and health problems, the full diversity needs however to be identified. This cannot be achieved by using a targeted assay. Therefore, next generation sequencing (NGS) could offer a valuable alternative as an open approach multiplex monitoring method. An NGS-based metagenomics approach was used to investigate the “unknown” agents in air samples of offices in contact with air-conditioning reservoirs and showed the first detection of E. jeanselmei in indoor air. Finally, a metagenomics analysis was performed to investigate the indoor airborne fungal diversity in contaminated residences in Brussels where people with health problems were living. This demonstrated that NGS could contribute to improved data concerning the indoor airborne fungal diversity, as compared to the currently used classical methods. The methods developed in this PhD work and the insights obtained are a first step for a better understanding of the causal link between indoor airborne fungi and public health.mycoMOLAI

Thirty years of Mus spretus: a promising future

Extensive genetic polymorphisms in Mus spretus have ensured its widespread use in many areas of genetics. With the recent increase in the number of single nucleotide polymorphisms available for laboratory mouse strains, M. spretus is becoming less appealing, in particular for genetic mapping. Although M. spretus mice are aggressive and poor breeders, they have a bright future because they provide phenotypes unobserved in laboratory strains, and tools are available for modifying their genome and dissecting the genetic architecture of complex traits. Furthermore, they provide information on fundamental genetic questions, such as the details of evolution of genomes and speciation. Here, we examine the use of M. spretus from these perspectives. The impending completion of the M. spretus genome sequence will synergize these advantages

Development and performance assessment of a luminex xMAP(R) direct hybridization assay for the detection and identification of indoor air fungal contamination.

Considered as a public health problem, indoor fungal contamination is generally monitored using classical protocols based on culturing. However, this culture dependency could influence the representativeness of the fungal population detected in an analyzed sample as this includes the dead and uncultivable fraction. Moreover, culture-based protocols are often time-consuming. In this context, molecular tools are a powerful alternative, especially those allowing multiplexing. In this study a Luminex xMAP(R) assay was developed for the simultaneous detection of 10 fungal species which are most frequently in indoor air and that may cause health problems. This xMAP(R) assay was found to be sensitive, i.e. its limit of detection is ranging between 0.05 and 0.01 ng of gDNA. The assay was subsequently tested with environmental air samples which were also analyzed with a classical protocol. All the species identified with the classical method were also detected with the xMAP(R) assay, however in a shorter time frame. These results demonstrate that the Luminex xMAP(R) fungal assay developed in this study could contribute to the improvement of public health and specifically to the indoor fungal contamination treatment