Genome sequencing of Porostereum spadiceum to study the degradation of levofloxacin.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications.

BACKGROUND: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization. RESULTS: Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator. CONCLUSIONS: The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

Downregulating hedgehog signaling reduces renal cystogenic potential of mouse models.

Renal cystic diseases are a leading cause of renal failure. Mutations associated with renal cystic diseases reside in genes encoding proteins that localize to primary cilia. These cystoproteins can disrupt ciliary structure or cilia-mediated signaling, although molecular mechanisms connecting cilia function to renal cystogenesis remain unclear. The ciliary gene, Thm1(Ttc21b), negatively regulates Hedgehog signaling and is most commonly mutated in ciliopathies. We report that loss of murine Thm1 causes cystic kidney disease, with persistent proliferation of renal cells, elevated cAMP levels, and enhanced expression of Hedgehog signaling genes. Notably, the cAMP-mediated cystogenic potential of Thm1-null kidney explants was reduced by genetically deleting Gli2, a major transcriptional activator of the Hedgehog pathway, or by culturing with small molecule Hedgehog inhibitors. These Hedgehog inhibitors acted independently of protein kinase A and Wnt inhibitors. Furthermore, simultaneous deletion of Gli2 attenuated the renal cystic disease associated with deletion of Thm1. Finally, transcripts of Hedgehog target genes increased in cystic kidneys of two other orthologous mouse mutants, jck and Pkd1, and Hedgehog inhibitors reduced cystogenesis in jck and Pkd1 cultured kidneys. Thus, enhanced Hedgehog activity may have a general role in renal cystogenesis and thereby present a novel therapeutic target.

Amniocytes can serve a dual function as a source of iPS cells and feeder layers.

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5-7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4(+) or c-Kit(+) amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.

Apc inhibition of Wnt signaling regulates supernumerary tooth formation during embryogenesis and throughout adulthood.

The ablation of Apc function or the constitutive activation of beta-catenin in embryonic mouse oral epithelium results in supernumerary tooth formation, but the underlying mechanisms and whether adult tissues retain this potential are unknown. Here we show that supernumerary teeth can form from multiple regions of the jaw and that they are properly mineralized, vascularized, innervated and can start to form roots. Even adult dental tissues can form new teeth in response to either epithelial Apc loss-of-function or beta-catenin activation, and the effect of Apc deficiency is mediated by beta-catenin. The formation of supernumerary teeth via Apc loss-of-function is non-cell-autonomous. A small number of Apc-deficient cells is sufficient to induce surrounding wild-type epithelial and mesenchymal cells to participate in the formation of new teeth. Strikingly, Msx1, which is necessary for endogenous tooth development, is dispensable for supernumerary tooth formation. In addition, we identify Fgf8, a known tooth initiation marker, as a direct target of Wnt/beta-catenin signaling. These studies identify key mechanistic features responsible for supernumerary tooth formation.

THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia.

Characterization of previously described intraflagellar transport (IFT) mouse mutants has led to the proposition that normal primary cilia are required for mammalian cells to respond to the sonic hedgehog (SHH) signal. Here we describe an N-ethyl-N-nitrosourea-induced mutant mouse, alien (aln), which has abnormal primary cilia and shows overactivation of the SHH pathway. The aln locus encodes a novel protein, THM1 (tetratricopeptide repeat-containing hedgehog modulator-1), which localizes to cilia. aln-mutant cilia have bulb-like structures at their tips in which IFT proteins (such as IFT88) are sequestered, characteristic of Chlamydomonas reinhardtii and Caenorhabditis elegans retrograde IFT mutants. RNA-interference knockdown of Ttc21b (which we call Thm1 and which encodes THM1) in mouse inner medullary collecting duct cells expressing an IFT88-enhanced yellow fluorescent protein fusion recapitulated the aln-mutant cilial phenotype, and live imaging of these cells revealed impaired retrograde IFT. In contrast to previously described IFT mutants, Smoothened and full-length glioblastoma (GLI) proteins localize to aln-mutant cilia. We hypothesize that the aln retrograde IFT defect causes sequestration of IFT proteins in aln-mutant cilia and leads to the overactivated SHH signaling phenotype. Specifically, the aln mutation uncouples the roles of anterograde and retrograde transport in SHH signaling, suggesting that anterograde IFT is required for GLI activation and that retrograde IFT modulates this event.