ITS-1 versus ITS-2 pyrosequencing: a comparison of fungal populations in truffle-grounds

International audienceIn a recent study pyrosequencing of the ribosomal internal transcribed spacer-1 (ITS-1) has validated the effectiveness of such technology in the survey of soil fungal diversity. Here we compare the two ITS regions, ITS-1 and ITS-2, of the fungal populations occurring in Tuber melanosporum/Quercus pubescens truffle grounds and sampled in two areas, one devoid of vegetation ("burned", bade in French) where T melanosporum fruiting bodies are usually collected, and outside the brule. TS1F/IT52 and ITS3/ITS4 were used respectively for the amplification of the ITS-1 and ITS-2 regions. Two amplicon libraries were built, one for inside and the other for outside. A set of 15.788 reads was obtained. After the removal of low quality sequences, 3568 and 3156 sequences were obtained from inside the brule with the ITS-1 and ITS-2 primers respectively. The sequences obtained from outside the brule were 4490 with the ITS-1 primers and 2432 with the ITS-2 primers. Most of the sequences obtained for both ITS fragments could be attributed to fungal organisms. The pair of primers, ITS1-F/ITS2, was more selective, producing fewer non-fungal sequences (1% inside, 3% outside), in addition to a higher number of sequences, than the pair ITS3/ITS4 (6% inside, 11% outside). Although differences are present in the taxa percentages between ITS-1 and ITS-2, both reveal that Ascomycota were the dominant fungal phylum and that their number decreased moving from inside the brule to outside, while the number of Basidiomycota increased. Taken together, both the short ITS-1 and ITS-2 reads obtained by the high throughput 454 sequencing provide adequate information for taxon assignment and are suitable to correlate the dynamics of the fungal populations to specific environments

Milk Thistle (Silybum Marianum L.) as a Novel Multipurpose Crop for Agriculture in Marginal Environments: A Review

Milk thistle (Silybum marianum (L.) Gaertn.) is a versatile crop that has adapted to the broadly different soil and environmental conditions throughout all continents. To date, the fruits (\u201cseeds\u201d) of the plant are the only reliable source of silymarin, which, given its recognized therapeutic effects and its many present and potential uses, has led to a significant re-discovery and enhancement of the crop in recent years. Overall, although many studies have been carried out globally on the bioactivity, phytochemistry, and genetics of milk thistle, few and discontinuous research activity has been conducted on its basic agronomy as well as on the farm opportunities offered by the cultivation of this species. However, the multiple potential uses of the plant and its reduced need for external inputs suggest that milk thistle can perfectly fit among the most interesting alternative crops, even for marginal environments. The growing interest in natural medicine, the increasing popularity of herbal dietary supplements, and the multiple possibilities for livestock feeding are all arguments supporting the idea that in many rural areas, this crop could represent a significant tool for enhancing and stabilizing farm income. However, several issues still have to be addressed. The species retains some morphological and physiological traits belonging to non-domesticated plants, which make the application of some common agronomic practices challenging. Furthermore, the lack of reliable field data devoted to the definition of suitable cropping protocols represents a major constraint on the spread of this crop among farmers. This review has therefore focused on updating information on the main morphological and phytochemical traits of the crop and its agronomic characteristics and novel uses. Several gaps in technical knowledge have been addressed, and further goals for experimental activity have been outlined in order to guide farmers eager to cope with the cultivation of such a challenging and resource-rich crop

Biochar enhances root development and aloin content of mature leaves in containerized Aloe arborescens Mill

The leaves of the medicinal plant Aloe arborescens Mill. Asphodelaceae) contain significant amounts of bioactive metabolites, including aloin (a mixture of the two diastereoisomers, aloin A and aloin B), aloesin, isoaloeresin D, and aloenin A. The presence of these metabolites varies considerably depending on the plant’s growth conditions, including the used growing substrate. In recent years, there has been growing interest in using biochar for potted plants cultivation. However, there is currently no available information regarding the suitability of biochar for the containerized cultivation of A. arborescens. A pot experiment was conducted with the hypothesis that biochar could influence the growth and phytochemistry of A. arborescens. The growing medium was supplied with increasing proportions of biochar (1: 100% commercial substrate; 2: mixed 50%(v/v) substrate; 3: 100% conifers wood biochar). Over the course of three years, the plants were closely monitored, and several key growth parameters were measured, including plant height, stem diameter, number and weight of leaves, and the number of suckers. After the first year, the content of selected active metabolites wasassessed. This evaluation also involved a comparison of the respective levels in the leaves taken from the apical, median, and basal sections of the stem. The leaves collected from the median section of plants were found to be larger and exhibited the highest percentage of spikes, epidermis, and gel on fresh weight. As a general trend, it was observed that in plants cultivated within the highest amount of biochar, the leaves collected from the intermediate stem portion contained the highest quantity of secondary metabolites

IFNL3 polymorphisms and HCV infection in patients with beta thalassemia

Background and relationale for the study. Genome-wide association studies have identified host genetic variation to be critical for spontaneous clearance and treatment response in patients infected with hepatitis C virus. Recently, the role of the IFNL3 polymorphisms in influencing the spontaneous clearance of HCV, the response to interferon and the progression of liver fibrosis, was also demonstrated in patients with thalassemia major infected by genotype 1b. In the present study we retrospectively analyzed 368 anti-HCV positive patients with beta-thalassemia at two Italian major centers in Cagliari and Torino. Results. C/C variant of polymorphism rs12979860 was related to response to interferon treatment and, above all, to spontaneous clearance of the virus. However, the positive predictive power was stronger for viral persistence than spontaneous clearance and in such respect the TT allele was more predictive than CC. The methylation associated polymorphism rs4803221 had independent effects with respect to rs12979860 and the haplotype tagged by SNP rs12979860 and rs4803221 significantly could improve the viral clearance prediction in infected patients. Neither necroinflammation or bilirubin values in the chronic phase of the hepatitis C were related to IFNL3 polymorphisms. No relation among IFNL3 polymorphisms and fibrosis stage directly shown by the liver biopsy was found. Conclusions. Also in thalassemia the SNPs on chromosome 19q13 closely associates with spontaneous and treatment-induced HCV clearance. The viral clearance prediction is significantly improved by the haplotype tagged by SNP rs12979860 and rs4803221. Neither necroinflammation, bilirubin values or fibrosis stage seem to be related to IFNL3 polymorphisms

X-linked genodermatoses from diagnosis to tailored therapy

Background: Genodermatoses are rare heterogeneous genetic skin diseases with multiorgan involvement. They severely impair an individual's well-being and can also lead to early death. Methods: During the progress of this review, we have implemented a targeted research approach, diligently choosing the most relevant and exemplary articles within the subject matter. Our method entailed a systematic exploration of the scientific literature to ensure a compre-hensive and accurate compilation of the available sources. Results: Among genodermatoses, X-linked ones are of particular importance and should always be considered when pediatric males are affected. Regardless of other syndromic forms without prevalence of skin symptoms, X-linked genodermatoses can be classified in three main groups: keratinization defects, pigmentation defects, and inflammatory skin diseases. Typical examples are dyskeratosis congenita, keratosis follicularis spinulosa decalvans, hypohidrotic ectodermal dysplasia, chondrodysplasia punctata, hypohidrotic ectodermal dysplasia, incontinentia pigmenti, chronic granulomatous disease, CHILD syndrome and ichthyosis. In this field, genetic diagnosis of the specific disease is important, also considering that numerous clinical trials of orphan drugs and genetic therapies are being proposed for these rare genetic diseases. Conclusions: Thus, this chapter starts from clinical to molecular testing and ends with a review of all clinical trials on orphan drugs and gene therapy for genodermatoses

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases

Omics sciences and precision medicine in melanoma

Background: This article provides an overview of the application of omics sciences in melanoma research. The name omics sciences refers to the large-scale analysis of biological molecules like DNA, RNA, proteins, and metabolites. Methods: In the course of this review, we have adopted a focu-sed research strategy, meticulously selecting the most pertinent and emblematic articles related to the topic. Our methodology included a systematic examination of the scientific literature to guarantee a thorough and precise synthesis of the existing sources. Results: With the advent of high-throughput technologies, omics have become an essential tool for understanding the complexity of melanoma. In this article, we discuss the different omics approaches used in melanoma research, including genomics, transcriptomics, proteomics, and metabolomics. We also highlight the major findings and insights gained from these studies, including the identification of new therapeutic targets and the development of biomarkers for diagnosis and prognosis. Finally, we discuss the challenges and future directions in omics-based melanoma research, including the integration of multiple omics data and the development of personalized medicine approaches. Conclusions: Overall, this article emphasizes the importance of omics science in advancing our understanding of melanoma and its potential for improving patient outcomes

Genetic characteristics of 234 Italian patients with macular and cone/cone-rod dystrophy

Two-hundred and thirty-four Italian patients with a clinical diagnosis of macular, cone and cone-rod dystrophies (MD, CD, and CRD) were examined using next-generation sequencing (NGS) and gene sequencing panels targeting a specific set of genes, Sanger sequencing and—when necessary—multiplex ligation-dependent probe amplification (MLPA) to diagnose the molecular cause of the aforementioned diseases. When possible, segregation analysis was performed in order to confirm unsolved cases. Each patient’s retinal phenotypic characteristics were determined using focal and full-field ERGs, perimetry, spectral domain optical coherence tomography and fundus autofluorescence. We identified 236 potentially causative variants in 136 patients representing the 58.1% of the total cohort, 43 of which were unpublished. After stratifying the patients according to their clinical suspicion, the diagnostic yield was 62.5% and 53.8% for patients with MD and for those with CD/CRD, respectively. The mode of inheritance of all cases confirmed by genetic analysis was 70% autosomal recessive, 26% dominant, and 4% X-linked. The main cause (59%) of both MD and CD/CRD cases was the presence of variants in the ABCA4 gene, followed by variants in PRPH2 (9%) and BEST1 (6%). A careful morpho-functional evaluation of the phenotype, together with genetic counselling, resulted in an acceptable diagnostic yield in a large cohort of Italian patients. Our study emphasizes the role of targeted NGS to diagnose MDs, CDs, and CRDs, as well as the clinical usefulness of segregation analysis for patients with unsolved diagnosis

Acute Delta Hepatitis in Italy spanning three decades (1991–2019): Evidence for the effectiveness of the hepatitis B vaccination campaign

Updated incidence data of acute Delta virus hepatitis (HDV) are lacking worldwide. Our aim was to evaluate incidence of and risk factors for acute HDV in Italy after the introduction of the compulsory vaccination against hepatitis B virus (HBV) in 1991. Data were obtained from the National Surveillance System of acute viral hepatitis (SEIEVA). Independent predictors of HDV were assessed by logistic-regression analysis. The incidence of acute HDV per 1-million population declined from 3.2 cases in 1987 to 0.04 in 2019, parallel to that of acute HBV per 100,000 from 10.0 to 0.39 cases during the same period. The median age of cases increased from 27 years in the decade 1991-1999 to 44 years in the decade 2010-2019 (p < .001). Over the same period, the male/female ratio decreased from 3.8 to 2.1, the proportion of coinfections increased from 55% to 75% (p = .003) and that of HBsAg positive acute hepatitis tested for by IgM anti-HDV linearly decreased from 50.1% to 34.1% (p < .001). People born abroad accounted for 24.6% of cases in 2004-2010 and 32.1% in 2011-2019. In the period 2010-2019, risky sexual behaviour (O.R. 4.2; 95%CI: 1.4-12.8) was the sole independent predictor of acute HDV; conversely intravenous drug use was no longer associated (O.R. 1.25; 95%CI: 0.15-10.22) with this. In conclusion, HBV vaccination was an effective measure to control acute HDV. Intravenous drug use is no longer an efficient mode of HDV spread. Testing for IgM-anti HDV is a grey area requiring alert. Acute HDV in foreigners should be monitored in the years to come

Genetic Screening in a Large Cohort of Italian Patients Affected by Primary Lymphedema Using a Next Generation Sequencing (NGS) Approach

Primary lymphedema is a rare inherited condition characterized by swelling of body tissues caused by accumulation of fluid, especially in the lower limbs. In many patients, primary lymphedema has been associated with variations in a number of genes involved in the development and maintenance of the lymphatic system. In this study, we performed a genetic screening in patients affected by primary lymphedema using a next generation sequencing (NGS) approach. With this technology, based on a custom-made oligonucleotide probe library, we were able to analyze simultaneously in each patient all the coding exons of 10 genes (FLT4, FOXC2, CCBE1, GJC2, MET, HGF, GATA2, SOX18, VEGFC, KIF11) associated with primary lymphedema. In the study population, composed of 45 familial and 71 sporadic cases, we identified the presence of rare variants with a potential pathogenic effect in 33% of subjects. Overall, we found a total of 36 different rare nucleotidic alterations, 30 of which had not been previously described. Among these, we identified 23 mutations that we considered most likely to be disease causing. Patients with an FLT4 or FOXC2 alteration accounted for the largest percentage of the sample, followed by MET, HGF, KIK11, GJC2 and GATA2. No alterations were identified in SOX18, VEGFC, and CCBE1 genes. In conclusion, we showed that NGS technology can be successfully applied to perform molecular screening of lymphedema-associated genes in large cohort of patients with a reasonable effort in terms of cost, work, and time