Statin-Induced Necrotizing Autoimmune Myopathy: Case Report of a Patient under Chronic Treatment.

Introduction: 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors are widely used worldwide to treat dyslipidaemia and prevent cardiovascular events. Statins can cause a wide variety of muscle injuries ranging from myalgia to severe rhabdomyolysis. In most cases, these symptoms are mild and self-limiting and do not require specific treatment besides drug withdrawal. Statin-induced autoimmune necrotizing myopathy (SINAM) is a rare but potentially fatal complication, characterized by the subacute onset of progressive proximal muscle weakness and considerably high creatine phosphokinase (CK) levels in patients exposed to statins. The diagnosis is supported by the presence of antibodies HMGCR, which allows the differentiation from other forms of necrotizing autoimmune myopathies. Symptoms usually progress even after statin discontinuation and can determine severe muscle damage. Summary. We describe the case of a 77-year-old man who developed SINAM after 5 years of statin use. He suffered from muscle functional impairment mainly involving proximal lower limb muscles which progressed to the point that he almost became bedridden. Initial treatment with prednisone alone was not effective, and he required a combination therapy with steroids, methotrexate, and intravenous immunoglobulins. After 5 months of therapy and rehabilitation, he showed complete laboratory response and muscle strength recovery. Conclusion: Recognizing SINAM is paramount in order to promptly start treatment and avoid permanent muscle damage. Using a combination therapy from the beginning could contribute to a better outcome. Prompt statin cessation, categorization of the muscle disease by autoantibody testing, imaging, and histology, exclusion of malignancy, and anti-inflammatory therapy with corticosteroids, antimetabolites, immunoglobulins, and in some cases rituximab are currently accepted approaches to this entity.

Environmental DNA detects biodiversity and ecological features of phytoplankton communities in Mediterranean transitional waters.

Climate changes and anthropogenic pressures are causing a biodiversity decline in terms of species number and genetic diversity, reducing the adaptability and evolvability of natural communities. Transitional water ecosystems are more sensitive to habitat reduction and degradation and, thus, are more exposed to biodiversity declines requiring biodiversity monitoring programs for their conservation. Environmental DNA (eDNA) metabarcoding represents a high-throughput tool for biodiversity assessment that is facilitating data collection for biodiversity monitoring. In this study, we applied, for the first time, eDNA metabarcoding in a Mediterranean coastal lagoon to assess the ecological features of eukaryotic phytoplankton communities. We sampled water in seven different lagoon sites and amplified the extracted DNA with primers targeting the variable region 4 (V4) of the 18S rRNA gene marker. The results demonstrated the validity of eDNA studies to provide insights into lagoon phytoplankton composition, establish the structure and spatial variation of phytoplankton communities, and evaluate its correlation to abiotic factors. Finally, the genetic distances analysis suggests that the different spatial distribution of OTUs, at least for the Tetraselmis genus, reflects the genetic background.

A Novel Sphingomonas sp. Isolated from Argan Soil for the Polyhydroxybutyrate Production from Argan Seeds Waste.

Polyhydroxybutyrate (PHB) is a biodegradable bio-based polymer synthesized by microorganisms under unfavorable conditions from agro-industrial residues as a source of carbon. These aspects make the bio-based polymer attractive for the mass production of biodegradable plastics, and a definitive replacement for petroleum-based plastics. The aim of this work was to characterize the putative PHB-producing bacterium 1B isolated from the argan soil, to identify the polymer produced, and quantify the PHB production using argan seeds waste. DNA extraction, PCR, and Sanger sequencing were conducted for the molecular identification of strain 1B; the residual biomass and the PHB quantification were measured and compared in the presence of simple sugars and pretreated argan seeds waste. The 1B growth and PHB synthesis were optimized by selecting physical and nutritional parameters: temperature, incubation time, pH, NaCl concentration, and nitrogen sources concentrations. A preliminary characterization of the bio-based polymer extracted was conducted by UV-Visible spectrophotometry and FTIR analysis. The strain 1B was identified as belonging to the genus Sphingomonas. The PHB final yield was higher in a growth culture enriched with argan waste (3.06%) than with simple sugars. The selected conditions for the bacterial optimal growth incremented the PHB final yield to 6.13%, while the increase in the argan residue concentration from 1 to 3% in a larger culture volume led to the PHB final yield of 8.16%. UV-Visible spectrophotometry of the extracted sample reported a remarkable peak at 248 nm, as well as FTIR spectra analysis, showed peaks at 1728 and 1282 wavenumber/cm. Both preliminary characterizations demonstrated that the extracted sample is the bio-based polymer polyhydroxybutyrate. The results reported in this work reveal how the costless available argan seeds can be used for polyhydroxybutyrate production using a novel Sphingomonas species.

DNA Barcode Gap Analysis for Multiple Marker Genes for Phytoplankton Species Biodiversity in Mediterranean Aquatic Ecosystems.

The implementation of DNA metabarcoding and environmental DNA (eDNA) to the biodiversity assessment and biomonitoring of aquatic ecosystems has great potential worldwide. However, DNA metabarcoding and eDNA are highly reliant on the coverage of the DNA barcode reference libraries that are currently hindered by the substantial lack of reference sequences. The main objective of this study was to analyze the current coverage of DNA barcode reference libraries for phytoplankton species of the aquatic Mediterranean ecoregion in the southeast of Italy (Apulia Region) in order to assess the applicability of DNA metabarcoding and eDNA in this area. To do so, we investigated three main DNA barcode reference libraries, BOLD Systems, GenBank and SILVA, for the availability of DNA barcodes of the examined phytoplankton species. The gap analysis was conducted for three molecular gene markers, 18S, 16S and COI. The results showed a considerable lack of barcodes for all three markers. However, among the three markers, 18S had a greater coverage in the reference libraries. For the 18S gene marker, the barcode coverage gap across the three types of ecosystems examined was 32.21-39.68%, 60.12-65.19% for the 16S marker gene, and 72.44-80.61 for the COI marker gene. Afterwards, the interspecific genetic distance examined on the most represented molecular marker, 18S, was able to distinguish 80% of the species mined for lakes and 70% for both marine and transitional waters. Conclusively, this work highlights the importance of filling the gaps in the reference libraries, and constitutes the basis towards the advancement of DNA metabarcoding and eDNA application for biodiversity assessment and biomonitoring.

Characterization of wheat lacking B-type starch granules.

The physicochemical and agronomic properties of a new form of bread wheat lacking B-type starch granules (BlessT) were assessed. Three BlessT mutant lines made by combining homoeologous deletions of BGC1, a gene responsible for the control of B-granule content, were compared with two sibling lines with normal starch phenotype and the parent line, cv. Paragon. Quantification of starch granule size and number in developing grain confirmed the lack of small, B-type starch granules throughout development in BlessT. Most starch, flour, grain and loaf characteristics did not vary between BlessT and the wild type sibling controls. However, BlessT starches had higher water absorption, reduced grain hardness and higher protein content, and dough made from BlessT flour required more water and had increased elasticity. Despite the lack of B-granules, BlessT lines do not display a significant decrease in total starch content suggesting that it should be possible to produce commercial wheat varieties that lack B-type starch granules without compromising yield. These findings support the potential utility of this novel type of wheat as a specialist crop in applications ranging from bread making and alcohol production to improved industrial starch products.

Testing the Influence of Incomplete DNA Barcode Libraries on Ecological Status Assessment of Mediterranean Transitional Waters.

The ecological assessment of European aquatic ecosystems is regulated under the framework directives on strategy for water and marine environments. Benthic macroinvertebrates are the most used biological quality element for ecological assessment of rivers, coastal-marines, and transitional waters. The morphological identification of benthic macroinvertebrates is the current tool for their assessment. Recently, DNA-based tools have been proposed as effective alternatives. The main current limits of DNA-based applications include the incompleteness of species recorded in the DNA barcode reference libraries and the primers bias. Here, we analysed the influence of the incompleteness of DNA barcode databases on species diversity indices, ecological indicators, and ecological assessment in transitional waters of the southeast Mediterranean, taking into account the availability of commonly sequenced and deposited genomic regions for listed species. The ecological quality status assigned through the potential application of both approaches to the analysed transitional water ecosystems was different in 27% of sites. We also analysed the inter-specific genetic distances to evaluate the potential application of the DNA metabarcoding method. Overall, this work highlights the importance to expand the barcode databases and to analyse, at the regional level, the gaps in the DNA barcodes.

A carbohydrate-binding protein, B-GRANULE CONTENT 1, influences starch granule size distribution in a dose-dependent manner in polyploid wheat.

In Triticeae endosperm (e.g. wheat and barley), starch granules have a bimodal size distribution (with A- and B-type granules) whereas in other grasses the endosperm contains starch granules with a unimodal size distribution. Here, we identify the gene, BGC1 (B-GRANULE CONTENT 1), responsible for B-type starch granule content in Aegilops and wheat. Orthologues of this gene are known to influence starch synthesis in diploids such as rice, Arabidopsis, and barley. However, using polyploid Triticeae species, we uncovered a more complex biological role for BGC1 in starch granule initiation: BGC1 represses the initiation of A-granules in early grain development but promotes the initiation of B-granules in mid grain development. We provide evidence that the influence of BGC1 on starch synthesis is dose dependent and show that three very different starch phenotypes are conditioned by the gene dose of BGC1 in polyploid wheat: normal bimodal starch granule morphology; A-granules with few or no B-granules; or polymorphous starch with few normal A- or B-granules. We conclude from this work that BGC1 participates in controlling B-type starch granule initiation in Triticeae endosperm and that its precise effect on granule size and number varies with gene dose and stage of development.

Predictors of Effectiveness of Anakinra in Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: To seek predictors of therapeutic response to the interleukin (IL)-1 inhibitor anakinra in children with systemic-onset juvenile idiopathic arthritis (sJIA). METHODS: The clinical charts of all patients with sJIA who were newly treated with anakinra at our center between 2004 and 2017 were reviewed retrospectively. Predictors included baseline demographic, clinical, and laboratory variables as well as previous or concomitant therapies. The effectiveness of anakinra was assessed at 1 year after treatment start. Complete clinical response (CCR) was defined as absence of fever, physician's global assessment ≤ 1, count of active joints ≤ 1, negative C-reactive protein, and ≥ 75% reduction of corticosteroid dose. According to the intention-to-treat principle, patients who had anakinra discontinued before 1 year for any reasons other than disease remission were classified as nonresponders. Statistics included univariate and multivariable analyses. RESULTS: Of the 62 patients included in the study, 24 (39%) met the criteria for CCR at 1 year, whereas 38 (61%) did not. On multivariable analysis, independent correlations with achievement of CCR were identified for shorter disease duration, lower active joint count, higher ferritin level, and greater activity of systemic manifestations. The area under the curve of the model was 0.83. CONCLUSION: Our findings help to delineate the clinical profile of patients with sJIA who are more likely to benefit from IL-1 blockade. They also underscore the need for studies aimed at examining the therapeutic role of early IL-1 inhibition and to identify biomarkers predicting response to either IL-1 or IL-6 antagonists.

Down-regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato.

Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during the colonization of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre-penetration phase and during conidiation at the end of the infection cycle, but are repressed during the biotrophic phase of tomato colonization. It has been suggested that the tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, whilst retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over-produce cladofulvin during the biotrophic growth phase. Non-producers infected tomato in a similar manner to the wild-type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over-producers caused strong necrosis and desiccation of tomato leaves, which, in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low-temperature stress. Overall, this study demonstrates that the repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, whereas its production is important for survival outside its host.

Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum.

Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen Elsinoë fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production. Here, we report the characterization of the CfPKS1 gene cluster to resolve this ambiguity. Activation of the regulator CfTSF1 specifically induced the expression of CfPKS1 and CfRDT1, but not of CfPRF1. These co-regulated genes that define the CfPKS1 gene cluster are orthologous to genes involved in 1,3-dihydroxynaphthalene (DHN) melanin biosynthesis in other fungi. Heterologous expression of CfPKS1 in Aspergillus oryzae yielded 1,3,6,8-tetrahydroxynaphthalene, a typical precursor of DHN melanin. Δcfpks1 deletion mutants showed similar altered pigmentation to wild type treated with DHN melanin inhibitors. These mutants remained virulent on tomato, showing this gene cluster is not involved in pathogenicity. Altogether, our results showed that the CfPKS1 gene cluster is involved in the production of DHN melanin and suggests that elsinochrome production in E. fawcettii likely involves another gene cluster.

Transfer of a starch phenotype from wild wheat to bread wheat by deletion of a locus controlling B-type starch granule content.

Our previous genetic analysis of a tetraploid wild wheat species, Aegilops peregrina, predicted that a single gene per haploid genome, Bgc-1, controls B-type starch granule content in the grain. To test whether bread wheat (Triticum aestivum L.) has orthologous Bgc-1 loci, we screened a population of γ-irradiated bread wheat cv. Paragon for deletions of the group 4 chromosomes spanning Bgc-1. Suitable deletions, each encompassing ~600-700 genes, were discovered for chromosomes 4A and 4D. These two deletions are predicted to have 240 homoeologous genes in common. In contrast to single deletion mutant plants, double deletion mutants were found to lack B-type starch granules. The B-less grains had normal A-type starch granule morphology, normal overall starch content, and normal grain weight. In addition to variation in starch granule size distribution, the B-less wheat grains differed from controls in grain hardness, starch swelling power, and amylose content. We believe that these B-less wheat plants are the only Triticeae cereals available that combine substantial alterations in starch granule size distribution with minimal impact on starch content.

Systematic Investigation of FLOWERING LOCUS T-Like Poaceae Gene Families Identifies the Short-Day Expressed Flowering Pathway Gene, TaFT3 in Wheat (Triticum aestivum L.).

To date, a small number of major flowering time loci have been identified in the related Triticeae crops, bread wheat (Triticum aestivum), durum wheat (T. durum), and barley (Hordeum vulgare). Natural genetic variants at these loci result in major phenotypic changes which have adapted crops to the novel environments encountered during the spread of agriculture. The polyploid nature of bread and durum wheat means that major flowering time loci in which recessive alleles confer adaptive advantage in related diploid species have not been readily identified. One such example is the PPD-H2 flowering time locus encoded by FLOWERING LOCUS T 3 (HvFT3) in the diploid crop barley, for which recessive mutant alleles confer delayed flowering under short day (SD) photoperiods. In autumn-sown barley, such alleles aid the repression of flowering over the winter, which help prevent the development of cold-sensitive floral organs until the onset of inductive long day (LD) photoperiods the following spring. While the identification of orthologous loci in wheat could provide breeders with alternative mechanisms to fine tune flowering time, systematic identification of wheat orthologs of HvFT3 has not been reported. Here, we characterize the FT gene families in six Poaceae species, identifying novel members in all taxa investigated, as well as FT3 homoeologs from the A, B and D genomes of hexaploid (TaFT3) and tetraploid wheat. Sequence analysis shows TaFT3 homoeologs display high similarity to the HvFT3 coding region (95-96%) and predicted protein (96-97%), with conservation of intron/exon structure across the five cereal species investigated. Genetic mapping and comparative analyses in hexaploid and tetraploid wheat find TaFT3 homoeologs map to the long arms of the group 1 chromosomes, collinear to HvFT3 in barley and FT3 orthologs in rice, foxtail millet and brachypodium. Genome-specific expression analyses show FT3 homoeologs in tetraploid and hexaploid wheat are upregulated under SD photoperiods, but not under LDs, analogous to the expression of HvFT3. Collectively, these results indicate that functional wheat orthologs of HvFT3 have been identified. The molecular resources generated here provide the foundation for engineering a novel major flowering time locus in wheat using forward or reverse genetics approaches.

Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization.

Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the ß-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families.

Regulation of secondary metabolite production in the fungal tomato pathogen Cladosporium fulvum.

Cladosporium fulvum is a non-obligate biotrophic fungal tomato pathogen for which fifteen secondary metabolite (SM) gene clusters were previously identified in its genome. However, most of these SM biosynthetic pathways remain cryptic during growth in planta and in different in vitro conditions. The sole SM produced in vitro is the pigment cladofulvin. In this study, we attempted to activate cryptic pathways in order to identify new compounds produced by C. fulvum. For this purpose, we manipulated orthologues of the global regulators VeA, LaeA and HdaA known to regulate SM biosynthesis in other fungal species. In C. fulvum, deleting or over-expressing these regulators yielded no new detectable SMs. Yet, quantification of cladofulvin revealed that CfHdaA is an activator whilst CfVeA and CfLaeA seemed to act as repressors of cladofulvin production. In the wild type strain, cladofulvin biosynthesis was affected by the carbon source, with highest production under carbon limitation and traces only in presence of saccharose. Repression of cladofulvin production by saccharose was dependent on both CfVeA and CfLaeA. Deletion of CfVeA or CfLaeA caused production of sterile mycelia, whilst Δcfhdaa deletion mutants sporulated, suggesting that cladofulvin production is not linked to asexual reproduction. Profiling the transcription of these regulators showed that CfHdaA-mediated regulation of cladofulvin production is independent of both CfVeA and CfLaeA. Our data suggest CfLaeA directly affects cladofulvin production whilst the effect of CfVeA is indirect, suggesting a role for CfLaeA outside of the Velvet complex. In conclusion, our results showed that regulation of SM production in C. fulvum is different from other fungi and indicate that manipulation of global regulators is not a universal tool to discover new fungal natural products.