Lonely Points in Simplices.

Given a lattice L ⊆ Z m and a subset A ⊆ R m , we say that a point in A is lonely if it is not equivalent modulo L to another point of A. We are interested in identifying lonely points for specific choices of L when A is a dilated standard simplex, and in conditions on L which ensure that the number of lonely points is unbounded as the simplex dilation goes to infinity.

The probabilistic termination tool amber.

We describe the Amber tool for proving and refuting the termination of a class of probabilistic while-programs with polynomial arithmetic, in a fully automated manner. Amber combines martingale theory with properties of asymptotic bounding functions and implements relaxed versions of existing probabilistic termination proof rules to prove/disprove (positive) almost sure termination of probabilistic loops. Amber supports programs parametrized by symbolic constants and drawing from common probability distributions. Our experimental comparisons give practical evidence of Amber outperforming existing state-of-the-art tools.

Membrane Sphingolipids Regulate the Fitness and Antifungal Protein Susceptibility of Neurospora crassa.

The membrane sphingolipid glucosylceramide (GlcCer) plays an important role in fungal fitness and adaptation to most diverse environments. Moreover, reported differences in the structure of GlcCer between fungi, plants and animals render this pathway a promising target for new generation therapeutics. Our knowledge about the GlcCer biosynthesis in fungi is mainly based on investigations of yeasts, whereas this pathway is less well characterized in molds. We therefore performed a detailed lipidomic profiling of GlcCer species present in Neurospora crassa and comprehensively show that the deletion of genes encoding enzymes involved in GlcCer biosynthesis affects growth, conidiation and stress response in this model fungus. Importantly, our study evidences that differences in the pathway intermediates and their functional role exist between N. crassa and other fungal species. We further investigated the role of GlcCer in the susceptibility of N. crassa toward two small cysteine-rich and cationic antimicrobial proteins (AMPs), PAF and PAFB, which originate from the filamentous ascomycete Penicillium chrysogenum. The interaction of these AMPs with the fungal plasma membrane is crucial for their antifungal toxicity. We found that GlcCer determines the susceptibility of N. crassa toward PAF, but not PAFB. A higher electrostatic affinity of PAFB than PAF to anionic membrane surfaces might explain the difference in their antifungal mode of action.

The Value of a Novel Panel of Cervical Cancer Biomarkers for Triage of HPV Positive Patients and for Detecting Disease Progression.

In the era of primary vaccination against HPV and at the beginning of the low prevalence of cervical lesions, introduction of screening methods that can distinguish between low- and high-grade lesions is necessary in order to maintain the positive predictive value of screening. This case-control study included 562 women who attended cervical screening or were referred for colposcopy and 140 disease free controls, confirmed by histology and/or cytology. The cases were stratified by age. Using routine exfoliated liquid based cytological samples RT-PCR measurements of biomarker genes, high-risk HPV testing and liquid based cytology were performed and used to evaluate different testing protocols including sets of genes/tests with different test cut-offs for the diagnostic panels. Three new panels of cellular biomarkers for improved triage of hrHPV positive women (diagnostic panel) and for prognostic assessment of CIN lesions were proposed. The diagnostic panel (PIK3AP1, TP63 and DSG3) has the potential to distinguish cytologically normal hrHPV+ women from hrHPV+ women with CIN2+. The prognostic gene panels (KRT78, MUC5AC, BPIFB1 and CXCL13, TP63, DSG3) have the ability to differentiate hrHPV+ CIN1 and carcinoma cases. The diagnostic triage panel showed good likelihood ratios for all age groups. The panel showed age-unrelated performance and even better diagnostic value under age 30, a unique feature among the established cervical triage tests. The prognostic gene-panels demonstrated good discriminatory power and oncogenic, anti-oncogenic grouping of genes. The study highlights the potential for the gene expression panels to be used for diagnostic triage and lesion prognostics in cervical cancer screening.

Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila.

UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. Here, we show that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. Our data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans.

Checkpoints couple transcription network oscillator dynamics to cell-cycle progression.

BACKGROUND: The coupling of cyclin dependent kinases (CDKs) to an intrinsically oscillating network of transcription factors has been proposed to control progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. The transcription network regulates the temporal expression of many genes, including cyclins, and drives cell-cycle progression, in part, by generating successive waves of distinct CDK activities that trigger the ordered program of cell-cycle events. Network oscillations continue autonomously in mutant cells arrested by depletion of CDK activities, suggesting the oscillator can be uncoupled from cell-cycle progression. It is not clear what mechanisms, if any, ensure that the network oscillator is restrained when progression in normal cells is delayed or arrested. A recent proposal suggests CDK acts as a master regulator of cell-cycle processes that have the potential for autonomous oscillatory behavior. RESULTS: Here we find that mitotic CDK is not sufficient for fully inhibiting transcript oscillations in arrested cells. We do find that activation of the DNA replication and spindle assembly checkpoints can fully arrest the network oscillator via overlapping but distinct mechanisms. Further, we demonstrate that the DNA replication checkpoint effector protein, Rad53, acts to arrest a portion of transcript oscillations in addition to its role in halting cell-cycle progression. CONCLUSIONS: Our findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.

Production of a defensin-like antifungal protein NFAP from Neosartorya fischeri in Pichia pastoris and its antifungal activity against filamentous fungal isolates from human infections.

Neosartorya fischeri NRRL 181 isolate secretes a defensin-like antifungal protein (NFAP) which has a remarkable antifungal effect against ascomycetous filamentous fungi. This protein is a promising antifungal agent of biotechnological value; however in spite of the available knowledge of the nature of its 5'-upstream transcriptional regulation elements, the bulk production of NFAP has not been resolved yet. In this study we carried out its heterologous expression in the yeast Pichia pastoris and investigated the growth inhibition effect exerted by the heterologous NFAP (hNFAP) on filamentous fungal isolates from human infections compared with what was caused by the native NFAP. P. pastoris KM71H transformant strain harboring the pPICZαA plasmid with the mature NFAP encoding gene produced the protein. The final yield of the hNFAP was sixfold compared to the NFAP produced by N. fischeri NRRL 181. Based on the signal dispersion of the amide region, it was proven that the hNFAP exists in folded state. The purified hNFAP effectively inhibited the growth of fungal isolates belonging to the Aspergillus and to the Fusarium genus, but all investigated zygomycetous strain proved to be insusceptible. There was no significant difference between the growth inhibition effect exerted by the native and the heterologous NFAP. These data indicated that P. pastoris KM71H can produce the NFAP in an antifungally active folded state. Our results provide a base for further research, e.g., investigation the connection between the protein structure and the antifungal activity using site directed mutagenesis.

Investigation of the antimicrobial effect of Neosartorya fischeri antifungal protein (NFAP) after heterologous expression in Aspergillus nidulans.

Neosartorya fischeri antifungal protein (NFAP) is a ß-defensin-like peptide produced by the N. fischeri NRRL 181 isolate. In this study, we investigated the manifestation of the antimicrobial effect of NFAP via heterologous expression of the nfap gene in an NFAP-sensitive fungus, Aspergillus nidulans. Heterologous expression of the nfap gene was carried out in A. nidulans CS2902 using a pAMA1-based autonomous replicative vector construct. The effect of the produced NFAP on the germination of A. nidulans conidia was investigated by scanning electron microscopy (SEM), and by DAPI and Calcofluor white (CFW) staining. 2',7'-Dichlorodihydrofluorescein diacetate staining and an Annexin V-FITC Apoptosis Detection kit were used to reveal the accumulation of reactive oxygen species (ROS) and the possible apoptotic, necrotic effect. The impact of mono- and divalent cations on the antimicrobial activity of NFAP was also examined. Transformants expressing the nfap gene showed reduced hyphal growth compared with the untransformed strain. This effect was absent in the presence of mono- and divalent cations (50 and 100 mM KCl, Mg(2)SO(4), Na(2)SO(4)). Delayed and abnormal germination was observed in the case of transformants. Conidia developed short branching germination tubes with swollen tips. The great majority of germinating conidia were destroyed after 8 h of cultivation, although a few survived and developed into abnormal hyphae. Damage in the organization of the cell wall, the destruction of chitin filaments and the accumulation of nuclei at the broken hyphal tips were detected by SEM, DAPI and CFW staining. The accumulation of ROS and more frequent apoptotic, necrotic events were also observed in the case of the NFAP-producing A. nidulans strain.

Antifungal peptides homologous to the Penicillium chrysogenum antifungal protein (PAF) are widespread among Fusaria.

Putative antifungal peptide encoding genes containing Penicillium chrysogenum antifungal protein (PAF) characteristic amino acid motifs were identified in 15 Fusarium isolates, representing 10 species. Based on the predicted sequences of mature peptides, discrepancy in one, two or three amino acids was observed between them. Phylogenetic investigations revealed that they show high amino acid sequence similarity to PAF and they belong to the group of fungal derived antifungal peptides with PAF-cluster. Ten from the 15 partially purified <10 kDa peptide fraction of Fusarium ferment broths showed antifungal activity. The presence of approximately 6.3 kDa molecular weight peptides was detected in all of the antifungally active ferment broths, and this peptide was isolated and purified from Fusarium polyphilaidicum. The minimal inhibitiory concentrations of F. polyphilaidicum antifungal protein (FPAP) were determined against different filamentous fungi, yeasts and bacteria. Filamentous fungal species were the most susceptible to FPAF, but some yeasts were also slightly sensitive.

Cyclin-dependent kinases are regulators and effectors of oscillations driven by a transcription factor network.

During embryonic cell cycles, B-cyclin-CDKs function as the core component of an autonomous oscillator. Current models for the cell-cycle oscillator in nonembryonic cells are slightly more complex, incorporating multiple G1, S phase, and mitotic cyclin-CDK complexes. However, periodic events persist in yeast cells lacking all S phase and mitotic B-cyclin genes, challenging the assertion that cyclin-CDK complexes are essential for oscillations. These and other results led to the proposal that a network of sequentially activated transcription factors functions as an underlying cell-cycle oscillator. Here we examine the individual contributions of a transcription factor network and cyclin-CDKs to the maintenance of cell-cycle oscillations. Our findings suggest that while cyclin-CDKs are not required for oscillations, they do contribute to oscillation robustness. A model emerges in which cyclin expression (thereby, CDK activity) is entrained to an autonomous transcriptional oscillator. CDKs then modulate oscillator function and serve as effectors of the oscillator.

Isolation and characterization of Neosartorya fischeri antifungal protein (NFAP).

A novel 6.6 kDa antifungal peptide (NFAP) from the culture supernatant of the mold, Neosartorya fischeri (anamorf: Aspergillus fischerianus), and its encoding gene were isolated in this study. NFAP is a small, basic and cysteine-rich protein consisting of 57 amino acid residues. It shows 37.9-50% homology to similar proteins described in literature from Aspergillus clavatus, Aspergillus giganteus, Aspergillus niger, and Penicillium chrysogenum. The in silico presumed tertiary structure of NFAP, e.g. the presence of five antiparallel ß-sheet connected with filaments, and stabilized by three disulfide bridges, is very similar to those of the defensin-like molecules. NFAP exhibited growth inhibitory action against filamentous fungi in a dose-dependent manner, and maintained high antifungal activity within broad pH and temperature ranges. Furthermore, it exhibited relevant resistance to proteolysis. All these characteristics make NFAP a promising candidate for further in vitro and in vivo investigations aiming at the development of new antifungal compounds.

The karyopherin Kap95 and the C-termini of Rfa1, Rfa2, and Rfa3 are necessary for efficient nuclear import of functional RPA complex proteins in Saccharomyces cerevisiae.

Nuclear protein import in eukaryotic cells is mediated by karyopherin proteins, which bind to specific nuclear localization signals on substrate proteins and transport them across the nuclear envelope and into the nucleus. Replication protein A (RPA) is a nuclear protein comprised of three subunits (termed Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae) that binds single-stranded DNA and is essential for DNA replication, recombination, and repair. RPA associates with two different karyopherins in yeast, Kap95, and Msn5/Kap142. However, it is unclear which of these karyopherins is responsible for RPA nuclear import. We have generated GFP fusion proteins with each of the RPA subunits and demonstrate that these Rfa-GFP chimeras are functional in yeast cells. The intracellular localization of the RPA proteins in live cells is similar in wild-type and msn5Δ deletion strains but becomes primarily cytoplasmic in cells lacking functional Kap95. Truncating the C-terminus of any of the RPA subunits results in mislocalization of the proteins to the cytoplasm and a loss of protein-protein interactions between the subunits. Our data indicate that Kap95 is likely the primary karyopherin responsible for RPA nuclear import in yeast and that the C-terminal regions of Rfa1, Rfa2, and Rfa3 are essential for efficient nucleocytoplasmic transport of each RPA subunit.

Inhibitory effects of cysteine and cysteine derivatives on germination of sporangiospores and hyphal growth of different Zygomycetes.

The in vitro antifungal activity of cysteine (D- and L-cysteine) and its four derivatives (L-cysteine-methyl-ester, N-acetyl-cysteine, N-isobutyryl-D-cysteine, and N-isobutyryl-L-cysteine) were investigated on 20 fungal isolates representing 16 genera (Absidia, Actinomucor, Backusella, Gilbertella, Micromucor, Mortierella, Mucor, Mycotypha, Phycomyces, Rhizomucor, Rhizopus, Saksenaea, Syncephalastrum, Thamnostylum, Umbellopsis, and Zygorynchus). The inhibitory potential of different concentrations of these compounds, ranging from 0.625 to 10 mM, were investigated on the germination of sporangiospores as well as on hyphal extension, using broth microdilution method and agar plate test. Treatment with cysteine and its derivatives resulted in a strong inhibition in most studied strains. At 10 mM of compounds, complete blockage of growth was observed for some isolates. Sensitive species exhibited severe changes in colony morphology in the presence of 10 mM L-cysteine, N-acetyl-cysteine, and N-isobutyryl-L-cysteine. Microscopic observations revealed that 10 mM N-acetyl-cysteine induced dramatic modifications in the structural organization of the hyphae. Results suggest that cysteine and its derivatives have a therapeutic potential against fungal infections caused by Zygomycetes species.

In vitro activity of phenothiazines and their combinations with amphotericin B against Zygomycetes causing rhinocerebral zygomycosis.

The in vitro antifungal activities of two phenothiazine (PTZ) compounds, trifluoperazine (TFP) and chlorpromazine (CPZ) separately and in combination with amphotericin B (AMB) were tested against eight fungal isolates known to be possible agents of rhinocerebral zygomycosis. While both PTZs individually had antifungal effects against these filamentous fungi, only the antifungal activity of TFP increased in presence of AMB. TFP and AMB acted synergistically and caused full inhibition of all strains tested except for Absidia glauca. In contrast, CPZ was found to act antagonistically with AMB with all of studied isolates.

Transcription networks and cyclin/CDKs: the yin and yang of cell cycle oscillators.

It is widely accepted that the central cell cycle oscillator is based on cyclin/CDK complexes. In a recent study, we challenged this model, and showed that a transcription factor network can drive correctly-timed periodic events independent of cyclin/CDK functions. The transcription factor network oscillator was revealed in budding yeast cells deleted for all six B-cyclin genes. These cells undergo successive rounds of budding, and continue to activate the bulk of the cell cycle-regulated genes on schedule, even though they are unable to replicate DNA or enter mitosis. These findings led us to propose that the cell cycle is regulated by the coupled activities of a transcription network oscillator and the CDK oscillator. Here, we discuss the transcription network oscillator in the context of the historical view that CDKs form the central cell cycle oscillator. We also discuss the role of transcription networks in controlling temporal programs in a variety of biological systems and their ability to function as oscillators. Finally, we consider how the transcription network oscillator collaborates with CDKs and checkpoint mechanisms to control the ordered events of the cell cycle.

Intrinsic and cyclin-dependent kinase-dependent control of spindle pole body duplication in budding yeast.

Centrosome duplication must be tightly controlled so that duplication occurs only once each cell cycle. Accumulation of multiple centrosomes can result in the assembly of a multipolar spindle and lead to chromosome mis-segregation and genomic instability. In metazoans, a centrosome-intrinsic mechanism prevents reduplication until centriole disengagement. Mitotic cyclin/cyclin-dependent kinases (CDKs) prevent reduplication of the budding yeast centrosome, called a spindle pole body (SPB), in late S-phase and G2/M, but the mechanism remains unclear. How SPB reduplication is prevented early in the cell cycle is also not understood. Here we show that, similar to metazoans, an SPB-intrinsic mechanism prevents reduplication early in the cell cycle. We also show that mitotic cyclins can inhibit SPB duplication when expressed before satellite assembly in early G1, but not later in G1, after the satellite had assembled. Moreover, electron microscopy revealed that SPBs do not assemble a satellite in cells expressing Clb2 in early G1. Finally, we demonstrate that Clb2 must localize to the cytoplasm in order to inhibit SPB duplication, suggesting the possibility for direct CDK inhibition of satellite components. These two mechanisms, intrinsic and extrinsic control by CDK, evoke two-step system that prevents SPB reduplication throughout the cell cycle.

Molecular beacon-based real-time PCR method for detection of 15 high-risk and 5 low-risk HPV types.

Detection of HPV infections requires a robust time-effective single-step method for efficient screening. A molecular beacon-based one-step multiplex real-time PCR system was developed to detect 15 high-risk (HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and 5 low-risk HPV types (HPV types 6, 11, 42, 43, 44). Molecular beacons detecting high-risk types are 5'-FAM-3'-DABCYL-labelled, molecular beacons for low-risk detection are 5'-TET-3'-DABCYL-labelled, while the internal control added before sample DNA extraction is detected by a 5'-FAM-TexasRed-3'-DABCYL wavelength-shifting molecular beacon. Accordingly, fluorescent data for HPV detection are collected at 530 nm for high-risk types, 560 nm in case of low-risk types and the reaction internal control is detected at 610 nm on a Roche LightCycler 2.0 instrument. The sensitivity for detected types varies between 22 and 700 copies/reaction. The clinical performance was tested on 161 clinical sample DNAs. The MB-RT PCR results were compared to the typing results obtained by the L1F/L1R PCR and hybridization-based system described previously, and the concordance rate between the two systems was 89.44%. The favorable characteristics shown by this multiplex single-step real-time HPV detection system make this promising approach worthy for further development and application for clinical screening.