Species Diversity of and Toxin Production by Gibberella fujikuroi Species Complex Strains Isolated from Native Prairie Grasses in Kansas

Fusarium species from agricultural crops have been well studied with respect to toxin production and genetic diversity, while similar studies of communities from nonagricultural plants are much more limited. We examined 72 Fusarium isolates from a native North American tallgrass prairie and found that Gibberella intermedia (Fusarium proliferatum), Gibberella moniliformis (Fusarium verticillioides), and Gibberella konza (Fusarium konzum) dominated. Gibberella thapsina (Fusarium thapsinum) and Gibberella subglutinans (Fusarium subglutinans) also were recovered, as were seven isolates that could not be assigned to any previously described species on the basis of either morphological or molecular characters. In general, isolates from the prairie grasses produced the same toxins in quantities similar to those produced by isolates of the same species recovered from agricultural hosts. The G. konza isolates produce little or no fumonisins (up to 120 μg/g by one strain), and variable but generally low to moderate amounts of beauvericin (4 to 320 μg/g) and fusaproliferin (50 to 540 μg/g). Toxicity to Artemia salina larvae within most species was correlated with the concentration of either beauvericin or fusaproliferin produced. Organic isolates from some cultures of G. moniliformis were highly toxic towards A. salina even though they produced little, if any, beauvericin or fusaproliferin. Thus, additional potentially toxigenic compounds may be synthesized by G. moniliformis strains isolated from prairie grasses. The Fusarium community from these grasses appears to contain some species not found in surrounding agricultural communities, including some that probably are undescribed, and could be capable of serving as a reservoir for strains of potential agricultural importance.     

Prospective Validation of Immunological Infiltrate for Prediction of Response to Neoadjuvant Chemotherapy in HER2-Negative Breast Cancer – A Substudy of the Neoadjuvant GeparQuinto Trial

Introduction

We have recently described an increased lymphocytic infiltration rate in breast carcinoma tissue is a significant response predictor for anthracycline/taxane-based neoadjuvant chemotherapy (NACT). The aim of this study was to prospectively validate the tumor-associated lymphocyte infiltrate as predictive marker for response to anthracycline/taxane-based NACT.

Patients and Methods

The immunological infiltrate was prospectively evaluated in a total of 313 core biopsies from HER2 negative patients of the multicenter PREDICT study, a substudy of the neoadjuvant GeparQuinto study. Intratumoral lymphocytes (iTuLy), stromal lymphocytes (strLy) as well as lymphocyte-predominant breast cancer (LPBC) were evaluated by histopathological assessment. Pathological complete response (pCR) rates were analyzed and compared between the defined subgroups using the exact test of Fisher.

Results

Patients with lymphocyte-predominant breast cancer (LPBC) had a significantly increased pCR rate of 36.6%, compared to non-LPBC patients (14.3%, p<0.001). LPBC and stromal lymphocytes were significantly independent predictors for pCR in multivariate analysis (LPBC: OR 2.7, p = 0.003, strLy: OR 1.2, p = 0.01). The amount of intratumoral lymphocytes was significantly predictive for pCR in univariate (OR 1.2, p = 0.01) but not in multivariate logistic regression analysis (OR 1.2, p = 0.11).

Conclusion

Confirming previous investigations of our group, we have prospectively validated in an independent cohort that an increased immunological infiltrate in breast tumor tissue is predictive for response to anthracycline/taxane-based NACT. Patients with LPBC and increased stromal lymphocyte infiltration have significantly increased pCR rates. The lymphocytic infiltrate is a promising additional parameter for histopathological evaluation of breast cancer core biopsies.     

Genome-Wide Massively Parallel Sequencing of Formaldehyde Fixed-Paraffin Embedded (FFPE) Tumor Tissues for Copy-Number- and Mutation-Analysis

Background

Cancer re-sequencing programs rely on DNA isolated from fresh snap frozen tissues, the preparation of which is combined with additional preservation efforts. Tissue samples at pathology departments are routinely stored as formalin-fixed and paraffin-embedded (FFPE) samples and their use would open up access to a variety of clinical trials. However, FFPE preparation is incompatible with many down-stream molecular biology techniques such as PCR based amplification methods and gene expression studies.

Methodology/Principal Findings

Here we investigated the sample quality requirements of FFPE tissues for massively parallel short-read sequencing approaches. We evaluated key variables of pre-fixation, fixation related and post-fixation processes that occur in routine medical service (e.g. degree of autolysis, duration of fixation and of storage). We also investigated the influence of tissue storage time on sequencing quality by using material that was up to 18 years old. Finally, we analyzed normal and tumor breast tissues using the Sequencing by Synthesis technique (Illumina Genome Analyzer, Solexa) to simultaneously localize genome-wide copy number alterations and to detect genomic variations such as substitutions and point-deletions and/or insertions in FFPE tissue samples.

Conclusions/Significance

The application of second generation sequencing techniques on small amounts of FFPE material opens up the possibility to analyze tissue samples which have been collected during routine clinical work as well as in the context of clinical trials. This is in particular important since FFPE samples are amply available from surgical tumor resections and histopathological diagnosis, and comprise tissue from precursor lesions, primary tumors, lymphogenic and/or hematogenic metastases. Large-scale studies using this tissue material will result in a better prediction of the prognosis of cancer patients and the early identification of patients which will respond to therapy.     

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation.

In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.     

The habitat functional response links seasonal third‐order selection to second‐order landscape characteristics

Determining how animals respond to differences in resource availabilities across spatiotemporal extents is critical to our understanding of organism distributions. Variations in resource distribution leading to changes in spatial arrangements across landscapes are indicative of a habitat functional response. Our goal was to assess how resource availabilities influenced both second‐order (i.e., home ranging behavior) and third‐order (i.e., habitat or resource selection) selection by feral pigs (Sus scrofa) in an agricultural landscape. We defined agriculturally based seasons to estimate home range characteristics using autocorrelated kernel density estimation within each season. We then modeled home range size as a function of resource availability (i.e., resource selection analyses) to determine whether individual behaviors were predicted by shifts in home ranging behavior. Both home range analyses and resource selection analyses indicated seasonal differences in selection for agricultural resources as availabilities changed, suggesting second‐ and third‐order selection is mechanistically linked through a habitat functional response.     

High resolution genetic and physical mapping of the mouse asebia locus: A key gene locus for sebaceous gland differentiation

The asebia (ab) mutation in the mouse is an autosomal recessive trait with hypoplastic sebaceous glands. As a first step toward cloning the ab gene, we report here the genetic mapping of the ab locus with respect to Chromosome 19 microsatellite markers. 644 backcross progeny were generated by mating (CAST/EiJ × DBA/1LacJ-ab^2J/ab^2J) F₁ heterozygous females and parental ab^2J/ab^2J mutant males. Our results located the ab gene to an interval of 1.6 cM on mouse Chromosome 19 defined by flanking markers D19Mit11 and D19Mit53/D19Mit27, and identified a tightly linked polymorphic marker, D19Mit67, that co-segregates with the mutation in the backcross progeny examined. This places ab locus 4 cM distal to its present position as indicated in Mouse Genome Database at The Jackson Laboratory. We have also mapped a yeast artificial chromosome (YAC) contig in this locus interval which suggests the ab interval to be less than one megabase of DNA.     

Protein hydration studied with homonuclear 3D1H NMR experiments

Summary Homonuclear 3D¹H NOESY-TOCSY and 3D¹H ROESY-TOCSY experiments were used to resolve and assign nuclear Overhauser effect (NOE) cross peaks between the water signal and individual polypeptide proton resonances in H₂O solutions of the basic pancreatic trypsin inhibitor. Combined with a novel, robust water-suppression technique, positive and negative intermolecular NOEs were detected at 4°C. The observation of positive NOEs between water protons and protein protons enables more precise estimates of the very short residence times of the water molecules in the hydration sites on the protein surface.     

Chemistry and decomposition of litter from Populus tremuloides Michaux grown at elevated atmospheric CO2 and varying N availability

It has been hypothesized that greater production of total nonstructural carbohydrates (TNC) in foliage grown under elevated atmospheric carbon dioxide (CO₂) will result in higher concentrations of defensive compounds in tree leaf litter, possibly leading to reduced rates of decomposition and nutrient cycling in forest ecosystems of the future. To evaluate the effects of elevated atmospheric CO₂ on litter chemistry and decomposition, we performed a 111 day laboratory incubation with leaf litter of trembling aspen (Populus tremuloides Michaux) produced at 36 Pa and 56 Pa CO₂ and two levels of soil nitrogen (N) availability. Decomposition was quantified as microbially respired CO₂ and dissolved organic carbon (DOC) in soil solution, and concentrations of nonstructural carbohydrates, N, carbon (C), and condensed tannins were monitored throughout the incubation. Growth under elevated atmospheric CO₂ did not significantly affect initial litter concentrations of TNC, N, or condensed tannins. Rates of decomposition, measured as both microbially respired CO₂ and DOC did not differ between litter produced under ambient and elevated CO₂. Total C lost from the samples was 38 mg g^?1 litter as respired CO₂ and 138 mg g^?1 litter as DOC, suggesting short‐term pulses of dissolved C in soil solution are important components of the terrestrial C cycle. We conclude that litter chemistry and decomposition in trembling aspen are minimally affected by growth under higher concentrations of CO₂.     

Alkaline phosphatase. A dominant enzyme of microvillus structure of cephalopod photoreceptors

The rhabdomeres of cephalopod photoreceptors, which are built up mainly of rhodopsin and phospholipid molecules, show a very high alkaline phosphatase activity. The enzyme has been partially characterized in purified rhodopsin vesicle fractions of the rhabdomeres by the following kinetic data: pH optimum 8.7; activation energy 9100 cal·m^?1; $\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 2.5}$ μmol·min^?1·mg^?1; $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 1.5·10}{}^{\mathrm{?4}}\text{M}$; its activity depends on Mg²⁺. There is good evidence that the alkaline phosphatase is a membrane-bound enzyme with receptor sites presumably located on the inside of the membrane. This enzyme has not been purified but its high activity compared to that of other known alkalin phosphatases (see Table I) indicates that each mirovillus, the structural unit of the rhabdomere, contains 1–20 enzyme molecules. This finding supports the hypothesis that the alkaline phosphatase is involved in the biochemical amplification process of excitation, or adaptation.     

Abundance and distribution of anaerobic protozoa and their contribution to methane production in Lake Cadagno (Switzerland)

Abstract In the uupermost layers of the anoxic sediment in Lake Cadagno, 9 different species of anaerobic protozoa were identified. The total number of these organisms was about 580 cells·ml⁻¹ sediment. Most pf these protozoa contained endosymbiotic methanogenic bacteria which in total amounted to 10⁶ methanogens·ml⁻¹ sediment. In addition to the methanogenic endosymbionts, cells of Metopus setosus and Caenomorpha lata also contained a non-fluorescent bacterial rod inside the cytoplasm. In some individual cells of C. lata this second type of endosymbiotic bacterium was sometimes the only endosymbiont observed. Contrary to earlier suggestions, anaerobic protozoa do not seem to play a major role in methane production at least in Lake Cadagno. No significant methane production due to the anaerobic protozoa and their methanogenic endosymbionts was found in situ. Isolated ciliates and amoebae produced methane at 12°C, but not at 6°C, probably as a result of temperature limitation. In the sediment of Lake Cadagno sulfate reduction seemed to be the dominant terminal degradation process.