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Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections. Biosynthesis of glycosylphosphatidylinositol (GPI) is essential for the integrity of the fungal cell wall. Here, the authors show that the natural product jawsamycin inhibits GPI biosynthesis by targeting a subunit of the fungal UDP-glycosyltransferase, and displays pronounced activity against pathogenic fungi of the order Mucorales.

• Previous studies have shown that arbuscular mycorrhizal fungi (AMF) can influence plant diversity and ecosystem productivity. However, little is known about the effects of AMF and different AMF taxa on other important community properties such as nutrient acquisition, plant survival and soil structure. • We established experimental grassland microcosms and tested the impact of AMF and of different AMF taxa on a number of grassland characteristics. We also tested whether plant species benefited from the same or different AMF taxa in subsequent growing seasons. • AMF enhanced phosphorus acquisition, soil aggregation and survival of several plant species, but AMF did not increase total plant productivity. Moreover, AMF increased nitrogen acquisition by some plant species, but AMF had no effect on total N uptake by the plant community. Plant growth responses to AMF were temporally variable and some plant species obtained the highest biomass with different AMF in different years. Hence the results indicate that it may be beneficial for a plant to be colonized by different AMF taxa in different seasons. • This study shows that AMF play a key role in grassland by improving plant nutrition and soil structure, and by regulating the make-up of the plant community.

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.

The Haplobank contains over 100,000 individually reversibly mutagenized, barcoded, mouse embryonic cell lines; proof-of-principle experiments were used to search for genes that are required for rhinovirus infection and angiogenesis using forward and reverse genetic screens, respectively. A biobank of genomic proportions Heterogeneity among clones of mouse embryonic stem cells, as well as the presence of two copies of the genomes in such cells, can impede genomic screens. Josef Penninger and colleagues have prepared a biobank of more than 100,000 clones that target nearly 17,000 genes in a conditional and reversible fashion. They showcase the power and versatility of the 'Haplobank' for studying the role of essential genes in mouse embryonic stem cells, to uncover genes that mediate the rhinovirus-induced inflammatory response and to identify novel genes that control angiogenesis and the specification of blood vessel lineage. The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance 1 , 2 that can confound analyses and undermine reproducibility 3 . Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance1,2 that can confound analyses and undermine reproducibility3. Here, we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid murine embryonic stem cell (mESC) lines targeting 16,950 genes with genetically bar-coded, conditional and reversible mutations. This Haplobank is the largest resource of hemi-/homozygous mutant mESCs to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We utilize Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mESCs, and to identify novel genes that control sprouting angiogenesis and blood vessel lineage specification. Further, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor required for cytotoxicity by rhinoviruses, which cause the common cold. Thus, Haplobank clones and revertible technologies enable high-throughput, reproducible functional annotation of the genome.

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (S accharomyces cerevisiae —Erg26p, Homo sapiens —NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound. FR171456 is a bioactive chemical produced by some microorganisms. Here, the authors identify the enzyme NSDHL of the sterol synthesis pathway as the molecular target of FR171456, rendering it the first compound to specifically target this class of enzyme in yeast and mammalian cells.

PurposeTo determine the risk factors, morbidity, and quality of life associated with same-day surgery in gynecologic oncology.MethodsIn this prospective study, patients with a good performance status and an indication for a simple procedure [diagnostic laparoscopy (peritoneal carcinomatosis index, biopsy, and Port-A-Cath), adnexectomy or fimbriectomy] and a complex procedure [total hysterectomy, omentectomy with peritoneal staging, pelvic lymphadenectomy (or sentinel lymph node (SLN)), and/or paraaortic lymphadenectomy by minimally invasive surgery] were included. Univariate logistic regression analysis, multivariate analysis, and the Wilcoxon signed-rank test were used in the statistical analyses.ResultsThere were 171 consecutive surgeries (55 complex and 116 simple procedures). The rate of readmissions on the same day and at 30 days postoperatively was, respectively, 8% versus 3% with simple procedures and 16% versus 11% with complex procedures. We modified our procedure to decrease complications by prescribing prophylactic low molecular weight heparin (LMWH) the next day at 8 am at home instead of the evening before discharge (fewer vaginal hematomas after hysterectomy: 2 hematomas/5 hysterectomies with LMWH on the same day versus 0/22 with LMWH on the next day, P = 0.03). The following factors were significantly associated with readmission: complex procedure (odds ratio [OR] 4.25, 95% confidence interval [CI] 1.66–10.85, P = 0.003), absence of an exsufflation drain (OR 2.96, 95% CI 1.19–7.31, P = 0.019), and end of surgery after 2 pm (OR 5.82, 95% CI 2.13–15.94, P = 0.001).ConclusionWe modified our protocol to decrease complications (vaginal hematomas after hysterectomy) by prescribing prophylactic LMWH the next day at 8 am at home instead of the evening before discharge.

The aim of this study was to enhance the transdermal absorption of the highly active progestin gestodene from matrix type transdermal delivery systems (TDDS) by formation of prodrugs with improved matrix solubility. Gestodene esters were synthesized via acylation of the drug with the respective carboxylic anhydrides. Subsequently TDDS were produced using the solvent cast method. Selected formulations were examined with in vitro diffusion experiments using skin of nude mice. One prodrug, gestodene caproate proved to be an oil at ambient temperature and showed a very high solubilty of over 10.5% in the TDDS matrix. Within in vitro penetration studies using those systems the prodrug exhibited a significantly higher transdermal penetration rate than gestodene from reference systems. Furthermore, the prodrug was hydrolyzed to the parent drug to a high extent during the passage of the skin. Designing prodrugs to the requirements of matrix TDDS is an efficient way of enhancing the transdermal drug flux rate.