TOWARDS ELIMINATION AND GENETIC MANIPULATION OF ERGOT ALKALOID PRODUCTION IN FUNGAL ENDOPHYTES

Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three putative knockouts were identified. Southern blot analysis of digested genomic DNA indicated that homologous gene replacement at dmaW2 locus took place while dmaW1 was still present. Chromosome separation followed by Southern-blot hybridization showed that the dmaW genes in N. coenophialum are located on different chromosomes. The aim of this study was to obtain a nontoxic endophyte free of marker genes that could be used to inoculate popular tall fescue cultivars. Therefore the Cre/loxP system developed in this study allows reusing the marker gene for sequential transformations. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum, or Epichloë festucae isolates, containing a floxed hygromycin phosphotransferase (hph) gene (loxP::hph::loxP), were transfected with a Crerecombinase expression plasmid and then cultured without selection. The marker was excised in 0.5-2% of the colonies, leaving a single loxP sequence. This strategy will help to reduce the concerns related to field release or commercialization of economically important grasses associated with manipulated fungal strains. It is expected that the technology will likely be adapted and applied in other fungal species. Manipulation of the ergot alkaloid (EA) gene cluster from C. purpurea and C. fusiformis by introducing and expressing its genes in different fungal-grass symbionts was also investigated. Heterologous expression of the ergot alkaloid cluster could result either in the synthesis of compounds similar to the ones produced by the host or in synthesis of novel compounds with new modes of action. Even though the results indicated that several EA genes were expressed in the new symbiota, none of the ergot alkaloids intermediates were detected

Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi

The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala—a triploid hybrid species—were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement

STUDY OF RASPBERRY EXTRACT APPLICATIONS AS TEXTILE COLORANT ON NATURAL FIBERS

The present study deals with the biomordant assisted application of natural extracts obtained from red raspberry (Rubus idaeus L.) fruits onto two different cellulosic supports – flax and bamboo. The study relies on the improvement of multifunctionalities such as colour fastness, washing and rubbing fastness, due to the synergism provided by the co-assistance of both a biomordant, and the complex resulted by inclusion of the pigment molecule, in the cavity of MCT-β-CD; it is well known that natural dye molecule have a good selectivity binding to the hydrophobic monochloro-triazynil-cyclodextrin’s (MCT-β-CD) cavity to form inclusion complexes. An investigation system provided the characterization of the composites: FT-IR spectroscopy stressed the main chemical bonds created between MCT-β-CD as host molecule and guest molecule represented by natural colorant molecule; Brunauer-Emmett-Teller (BET) Surface Area Analysis completes the analysis, proving the positive contribution of MCT-β-cyclodextrin grafting. Dyeing fastness and colour modifications were conclusive for this research. Samples of bamboo knitting are less colorful than those of the flax fabric in terms of high absorption capacity and stability / durability of natural dye applied by inclusion within cyclodextrin’s inner. The results of analysis revealed improvement of washing and rubbing fastness (1-1.5 points). Colour modifications noticed due to the colorant deposition were quantified from up to 3 points

Surgical decompression of arachnoid cysts: A study on 44 pediatric patients

Background: There are yet to clarify some aspects regarding the surgical indications and surgical methods that are at choice for treating ACs. The purpose of the study is to contribute to the ongoing debate on the most appropriate treatment. Materials and methods: We included in the study 44 pediatric patients (aged < 18 years), operated for ACs in the Neurosurgery I Department, of the Emergency Clinical Hospital “Bagdasar-Arseni” in Bucharest, during the period January 2003 to December 2012. The surgical methods were either a craniotomy or cyst shunting. The information regarding these patients was retrospectively assessed based on their clinical and imaging records, and cross checked with the OR protocols. Treatment efficiency was evaluated considering postoperative clinical status, imaging appearance and postoperative complications. Results: 84.1% of the patients reported postoperative disappearance or reduction of symptoms, while 13.6 had the same complaints as prior surgery. Only one patient experienced worsening of symptoms. Follow-up imaging showed cyst disappearance in 29.5% patients and cyst reduction in 61.4% patients. 9.1% had no change in cyst volume. Craniotomy correlated with a better imaging appearance (p=0.02). There was no correlation between improved clinical status and cyst fluid reduction (p=0.6). 9 patients (20.4%), all with temporal cysts, experienced postoperative complications, and 7 of them (15, 9%) needed surgery. There was no severe or permanent dysfunction in any of the patients. 42.8% of the cyst shunting patients suffered postoperative complications, whereas only 10% of the craniotomy patients did. Conclusion: Cyst decompression results in significant clinical improvement and it can be performed with little risk for severe complications. Craniotomy seems to be a better surgical treatment option than cystoperitoneal shunt, with a better neuroimaging appearance and a lower complications rate

The Connections Between Androgens and Adipose Tissue Function in Polycystic Ovary Syndrome Patients

Few studies reported that androgens levels could be among the regulators of adipose tissue hormones. Polycystic ovary syndrome (PCOS) is characterized by both increased adiposity and hyperandrogenism, but the relationship between androgens levels and adipokines in PCOS has not been well characterized. Our aim was to study the relationship between leptin, adiponectin, total testosterone, sex hormone binding globulin (SHBG) and free androgen index (FAI) in PCOS patients. We conducted a cross-sectional study on 131 PCOS patients (mean age 24 [6] yrs, mean body mass index (BMI) 25.8 [10.44] kg/m2) diagnosed based on Rotterdam Consensus criteria. All the patients were evaluated by clinical, paraclinical and hormonal exam. HOMA-IR was calculated for all the patients. Leptin was positively associated with age (p<0.05), BMI (p<0,0001), waist-hip ratio (WHR) (p<0.0001), waist circumference (WC) (p<0,0001), HOMA-IR (p<0,0001), insulinemia (p<0.0001) and FAI (p<0,0001) and negatively with SHBG (p<0.0001). Adiponectin was negatively associated with age (p<0.05), BMI (p<0,0001), WC (p<0,0001), WHR (p<0,0001), HOMA-IR (p<0,0001), insulinemia (p<0.0001) and FAI (p<0,005) and positively associated with SHBG (p<0,0001). Both adipokines were not correlated with total testosterone. The association between serum leptin and FAI/SHBG was lost after adjustment for age and body mass index. In turn the relationship between leptin and FAI, but not SHBG was independent of HOMA-IR. Circulating adiponectin was associated with SHBG independently of adiposity and HOMA-IR, but the association with FAI was lost after adjustment for HOMA-IR. In conlusion, circulating adipokines are correlated with FAI and SHBG serum levels. This association seems to be mediated by adiposity for leptin. Although the link between adiponectin and FAI is probably due to insulin resistance, SHBG seems to directly modulate adiponectin production

Chromosome-End Knockoff Strategy to Reshape Alkaloid Profiles of a Fungal Endophyte

Molecular genetic techniques to precisely eliminate genes in asexual filamentous fungi require the introduction of a marker gene into the target genome. We developed a novel strategy to eliminate genes or gene clusters located in subterminal regions of chromosomes, and then eliminate the marker gene and vector backbone used in the transformation procedure. Because many toxin gene clusters are subterminal, this method is particularly suited to generating nontoxic fungal strains. We tested this technique on Epichloë coenophiala, a seed-transmissible symbiotic fungus (endophyte) of the important forage grass, tall fescue (Lolium arundinaceum). The endophyte is necessary for maximal productivity and sustainability of this grass but can produce ergot alkaloids such as ergovaline, which are toxic to livestock. The genome sequence of E. coenophiala strain e19 revealed two paralogous ergot alkaloid biosynthesis gene clusters, designated EAS1 and EAS2. EAS1 was apparently subterminal, and the lpsB copy in EAS2 had a frame-shift mutation. We designed a vector with a fungal-active hygromycin phosphotransferase gene (hph), an lpsA1 gene fragment for homologous recombination at the telomere-distal end of EAS1, and a telomere repeat array positioned to drive spontaneous loss of hph and other vector sequences, and to stabilize the new chromosome end. We transformed E. coenophiala with this vector, then selected “knockoff” endophyte strains, confirmed by genome sequencing to lack 162 kb of a chromosome end including most of EAS1, and also to lack vector sequences. These ∆EAS1 knockoff strains produced no detectable ergovaline, whereas complementation with functional lpsB restored ergovaline production

Genetics, Genomics and Evolution of Ergot Alkaloid Diversity

The ergot alkaloid biosynthesis system has become an excellent model to study evolutionary diversification of specialized (secondary) metabolites. This is a very diverse class of alkaloids with various neurotropic activities, produced by fungi in several orders of the phylum Ascomycota, including plant pathogens and protective plant symbionts in the family Clavicipitaceae. Results of comparative genomics and phylogenomic analyses reveal multiple examples of three evolutionary processes that have generated ergot-alkaloid diversity: gene gains, gene losses, and gene sequence changes that have led to altered substrates or product specificities of the enzymes that they encode (neofunctionalization). The chromosome ends appear to be particularly effective engines for gene gains, losses and rearrangements, but not necessarily for neofunctionalization. Changes in gene expression could lead to accumulation of various pathway intermediates and affect levels of different ergot alkaloids. Genetic alterations associated with interspecific hybrids of Epichloë species suggest that such variation is also selectively favored. The huge structural diversity of ergot alkaloids probably represents adaptations to a wide variety of ecological situations by affecting the biological spectra and mechanisms of defense against herbivores, as evidenced by the diverse pharmacological effects of ergot alkaloids used in medicine

Laparoscopic Sleeve Gastrectomy Improves Reproductive Hormone Levels in Morbidly Obese Males -A Series of 28 Cases

Rezumat Gastrectomia longitudinalã laparoscopicã îmbunãtãåeaete nivelul hormonilor reproductivi la bãrbaåii cu obezitate morbidã -o serie de 28 cazuri Introducere: Bãrbaåii obezi prezintã frecvent reduceri ale hormonilor androgeni ce pot fi modificate dupã scãderea ponderalã obåinutã prin chirurgie bariatricã. Gastrectomia longitudinalã laparoscopicã (GLL) a fost folositã frecvent în ultimul deceniu pentru tratarea obezitãåii severe. Scopul studiului a fost evaluarea modificãrilor hormonilor reproductivi dupã GLL

Congenital Abnormalities of the Fetal Heart

Congenital heart defects (CHDs) are the most frequent congenital malformations, the costliest hospital admissions for structural defects and the leading cause of infant general and malformations related mortality. Fetal echocardiography represents a skilled ultrasound examination, because of the complexity, physiological and structural particularities of the fetal heart. The efficiency of the cardiac scan is reported with great variation, depending on the scanning protocol, examiner experience and equipment quality but CHDs remains among the most frequently missed congenital abnormalities

Plant-Symbiotic Fungi as Chemical Engineers: Multi-Genome Analysis of the Clavicipitaceae Reveals Dynamics of Alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses