Identity and systematic position of Paradinium poucheti and other Paradinium-like parasites of marine copepods based on morphology and nuclear-encoded SSU rDNA

Paradinium and Paradinium-like parasites were detected in various copepod hosts collected in the NW Mediterranean Sea, the North Atlantic Ocean, and the Godth?bsfjord (Greenland). The identity and systematic position of the parasitic, plasmodial protist Paradinium was investigated on the basis of SSU rDNA and morphology. SSU rDNA sequences were obtained from 3 specimens of Paradinium poucheti isolated from their cyclopoid copepod host, Oithona similis. In addition, a comparable sequence was obtained from a hitherto undescribed species of Paradinium from the harpactacoid copepod Euterpina acutifrons. Finally, SSU rDNA sequences were acquired from 2 specimens of a red plasmodial parasite (RP parasite) isolated from Clausocalanus sp. Both morphological and SSU rDNA sequence data supported that P. poucheti and Paradinium sp. are closely related organisms. In phylogenetic analyses based on SSU rDNA sequences, Paradinium spp. clustered with sequences from an uncultured eukaryote clone from the Pacific Ocean and two sequences from haplosporidian-like parasites of shrimps, Pandalus spp. This Paradinium clade branched as a sister group to a clade comprising the Haplosporidia and the Foraminifera. The RP parasite had a superficial morphological resemblance to Paradinium and has previously been interpreted as a member of this genus. However, several morphological characters contradict this and SSU rDNA sequence data disagree with the RP parasite and Paradinium being related. The phylogenetic analyses suggested that the RP parasite is a fast-evolved alveolate and a member of the so-called marine alveolate Group I (MAGI) and emerging data now suggest that this enigmatic group may, like the syndinian dinoflagellates, consist of heterotrophic parasites.     

RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins

Accumulation of repair proteins on damaged chromosomes is required to restore genomic integrity. However, the mechanisms of protein retention at the most destructive chromosomal lesions, the DNA double-strand breaks (DSBs), are poorly understood. We show that RNF8, a RING-finger ubiquitin ligase, rapidly assembles at DSBs via interaction of its FHA domain with the phosphorylated adaptor protein MDC1. This is accompanied by an increase in DSB-associated ubiquitylations and followed by accumulation of 53BP1 and BRCA1 repair proteins. Knockdown of RNF8 or disruption of its FHA or RING domains impaired DSB-associated ubiquitylation and inhibited retention of 53BP1 and BRCA1 at the DSB sites. In addition, we show that RNF8 can ubiquitylate histone H2A and H2AX, and that its depletion sensitizes cells to ionizing radiation. These data suggest that MDC1-mediated and RNF8-executed histone ubiquitylation protects genome integrity by licensing the DSB-flanking chromatin to concentrate repair factors near the DNA lesions.     

KL4 Peptide Induces Reversible Collapse Structures on Multiple Length Scales in Model Lung Surfactant

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).     

Development of antibiotic resistance and up-regulation of the antimutator gene pfpI in mutator Pseudomonas aeruginosa due to inactivation of two DNA oxidative repair genes (mutY, mutM)

Prevention and correction of oxidative DNA lesions in Pseudomonas aeruginosa is ensured by the DNA oxidative repair system (GO). Single inactivation of mutT, mutY and mutM involved in GO led to elevated mutation rates (MRs) that correlated to increased development of resistance to antibiotics. In this study, we constructed a double mutant in mutY and mutM (PAOMY-Mgm) and characterized the phenotype and the gene expression profile using microarray and RT-PCR. PAOMY-Mgm presented 28-fold increases in MR compared with wild-type reference strain PAO1. In comparison, the PAOMYgm (mutY) single mutant showed only a fivefold increase, whereas the single mutant PAOMMgm (mutM) showed a nonsignificant increase in MR compared with PAO1 and the single mutants. Mutations in the regulator nfxB leading to hyperexpression of MexCD-OprJ efflux pump were found as the mechanism of resistance to ciprofloxacin in the double mutant. A better fitness of the mutator compared with PAO1 was found in growth competition experiments in the presence of ciprofloxacin at concentrations just below minimal inhibitory concentration. Up-regulation of the antimutator gene pfpI, that has been shown to provide protection to oxidative stress, was found in PAOMY-Mgm compared with PAO1. In conclusion, we showed that MutY and MutM are cooperating in the GO of P. aeruginosa, and that oxidative DNA lesions might represent an oxidative stress for the bacteria.     

Proteomic Profiling of Triple-negative Breast Carcinomas in Combination With a Three-tier Orthogonal Technology Approach Identifies Mage-A4 as Potential Therapeutic Target in Estrogen Receptor Negative Breast Cancer

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)⁻ and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.Breast cancer, although a very heterogeneous disease, can be divided into three therapeutically relevant fundamental disease entities, simply based on estrogen receptor (ER) and human epidermal growth factor receptor 2 (Her2)¹ status (i.e. ER⁺ and/or Her2⁺, and ER⁻Her2⁻), as the major currently available breast cancer therapeutic options are based on the ability to target these proteins. Hormone receptor positive and hormone receptor negative breast cancers are disease entities with distinct morphological, genetic and biological behavior (1). Hormone receptor negative tumors, which constitute ∼30% of primary breast cancers, tend to be high-grade, more frequently BRCA1 and TP53 mutated, and, more importantly, are not amenable to endocrine therapy. Her2 is amplified in ∼18–20% of breast cancers, and is more frequently observed in hormone receptor negative tumors. Her2 amplification is associated with worse prognosis (higher rate of recurrence and mortality) in patients with newly diagnosed breast cancer who do not receive any adjuvant systemic therapy. Her2 status is also predictive for several systemic therapies, particularly for agents that target Her2. The development of a humanized monoclonal antibody against Her2 (trastuzumab) has resulted in reduction of the risk of recurrence and mortality in patients with Her2 amplification (2, 3). Although trastuzumab is considered one of the most effective targeted therapies currently available in oncology, a significant number of patients with Her2-overexpressing breast cancer do not benefit from it (4, 5).Breast tumors that do not express ER, PgR, or Her2 (ER⁻ PgR⁻ Her2⁻), as determined by immunohistochemistry (IHC), are generally referred to as triple negative breast cancers (TNBCs), and they are not candidates for targeted therapies (endocrine therapy or trastuzumab). Although TNBCs account for a relatively small proportion of breast cancer cases (10–15%), they are responsible for a disproportionate number of breast cancer deaths. TNBC tumors form a recognizable prognostic group of breast cancer with aggressive behavior that currently lacks the benefit of available systemic therapy (6–8). Given the need to develop molecular criteria to reproducibly categorize molecular breast tumor subtypes at the protein level and the lack of targeted therapies available to treat patients bearing TNBCs, we have implemented a systematic proteomics approach to identify, characterize, and evaluate proteins present in triple-negative tumors that could constitute an appropriate therapeutic target for the clinical management of this group of patients. To this end, based on the analysis of 78 individual TNBC samples, we have established a large, cumulative, 2D-PAGE database of proteins expressed by TNBCs, including some that could be of potential therapeutic value. Comparison of this TNBC protein database with protein databases of other breast cancer subtypes previously established by our laboratory allowed us to single out a number of proteins preferentially expressed in TNBCs for which targeted therapeutics exist. In this report we further focused on the characterization of one such target, the cancer/testis antigen, melanoma-associated antigen 4 - Mage-A4.Cancer/testis antigens (CTAs) are expressed in a large variety of tumor types, whereas their expression in normal tissues is restricted to male germ cells, which are immune-privileged because of their lack of or low expression of human leukocyte antigen (HLA) molecules (9). Several studies have shown the existence of natural cellular and humoral responses against some CTAs, indicating that they are appropriate targets for vaccine-based cancer immunotherapy (10–12). So far, the use of CTAs in immunotherapeutic approaches to cancer treatment has been tested in more than 60 early phase clinical trials, with varying success, and a few candidate products have reached late-stage clinical trials. One such candidate vaccine, Astuprotimut-R (GSK-249553), a Mage-A3 antigen-specific cancer immunotherapeutic agent, is currently under clinical evaluation by GlaxoSmithKline in the largest-ever treatment trial in lung cancer, called MAGRIT (Mage-A3 as Adjuvant nonsmall cell lunG canceR ImmunoTherapy) (13).At present, CTAs comprise about 150 members, more than half of which are encoded by large, recently expanded families on chromosome X (14; see also CTDatabase at www.cta.lncc.br; last accessed 01.09.2012). These genes are organized into clusters and have undergone rapid evolution, possibly because of positive selection. The biological functions of CTAs are not fully understood, but emerging evidence suggest that they direct the proliferation, differentiation, and survival of human germ line cells and may have similar effect in cancer cells. Mage-A4 protein belongs to the Mage-A family of CT antigens. The Mage-A family is composed by 12 proteins (14, 15) and many members of the Mage-A family of CTAs have been associated with cancer, including breast cancer (14, 16, 17). However, past studies reported mostly on MAGE genes rather than protein expression, or on the expression of Mage protein families and not on any given specific protein.In this paper we describe the identification of Mage-A4 in breast tumor biopsies using 2D PAGE coupled with MS proteomics, and follow the protein localization from the tumor cells, to the tumor microenvironment, and to the serum of a patient. Using a three-tier orthogonal technology approach that combined 2D PAGE silver staining coupled with MS, with 2D Western blotting, and IHC, we showed that high level Mage-A4 expression in breast tumors occurs almost exclusively in the receptor negative disease (TNBC and Her2⁺ER⁻PgR⁻). The existence of immunotherapeutic approaches targeting MAGE protein family members (Mage-A4 specific or with broader specificity) and the fact that we were able to detect its presence in serum suggest novel management options for patients bearing Mage-A4 positive TNBCs and Her2⁺ER⁻PgR⁻ tumors.     

Determination of the overall volumetric mass transfer coefficient kLa,by a temperature dependent change of gas solubility

Summary The solubility of oxygen in the liquid phase of a bioreactor was changed by a ramp change of temperature, and k_La was determined from the resulting return to equilibrium of dissolved oxygen activity. The maximum k_La that can be measured by this method in a standard laboratory scale bioreactor is 145 h^–1 corresponding to a temperature change rate of 320°C h^–1.Nomenclature p Difference between p_G and p_L (% saturation) - T Ramp change of temperature (°C) - E Temperature-compensated output from the oxygen electrode (A) - E_u Uncompensated output from the oxygen electrode (A) - k_La Overall volumetric mass transfer coefficient (h^–1) - k_La_Tm Overall volumetric mass transfer coefficient at temperature T_m (h^–1) - P_G Dissolved oxygen activity in equilibrium with the gas phase (% saturation) - p_L Dissolved oxygen activity (% saturation) - p_Lm Dissolved oxygen activity at time t_m (% saturation) - t Time (h) - t_m Time of maximum p (h) - T Temperature (°C) - T_cal Calibration temperature of the oxygen electrode (°C) - T_m Final temperature after a temperature shift (°C) - T_n Temperature at time t_n     

Phosphorus depletion in the rhizosphere as influenced by soil moisture

To study the influence of soil moisture on phosphorus (P) depletion in the rhizosphere, maize (Zea mays cv. Trak) was pre-grown in vermiculite filled-PVC tubes for 9 days and then the plants with the tubes were transplanted into soil columns maintained at two soil moisture levels () of 0.14 and 0.20 cm³ cm^–3 for 10 days. The soil columns were separated at 1 cm depth by a nylon screen of 53 m inner mesh size, into 1 cm soil layer above and 3 cm soil column below screen. A root mat developed over the screen, but root hairs only could penetrate it. Regardless of the soil moisture level in the columns, and adequate and equal water and nutrients supply was maintained via wicks from an external nutrient solution to the plant roots in vermiculite. After 10 days, the soil columns were separated from the root mats, quickly frozen in liquid nitrogen and sliced into thin layers (0.2mm) using a refrigerated microtome to give soil samples at defined distances from the root mats for analyses. Lower soil moisture (=0.14) resulted in narrower and steeper depletion profile of 0.5 M NaHCO₃ extractable P (NaHCO₃-P_i) as compared to higher soil moisture (=0.20). Depletion of P in soil solution in the immediate vicinity of root mats did not differ much but the extension of the depletion zones was 0.10 cm at =0.14 and 0.20 cm at =0.20. The depletion up to 0.05cm with =0.14 and up to 0.07 cm with =0.20 was uniform, and may be attributed to the depletion in the root hair zone. Beyond the root hair zones, the theory of diffusion and mass flow was able to explain the observed differences in shape and extent of the P depletion profiles at the two soil moisture levels.     

The Larval Eye of Nannochoristid Scorpionflies (Insecta,Mecoptera)

Abstract The stemmata of last–instar Nannochoristalarvae are compound eyes composed of 10 or more ommatidia. Each ommatidium has four Semper cells, four distal and four proximal retinula cells which form a cruciform and layered rhabdom. The ommatidia are separated by epidermal cells (possibly rudimentary pigment cells). Corneal lenses are lacking. At the posterior edge, aberrant stemma units may be present which lack a dioptric apparatus and have a star–shaped rhabdom composed of at least six retinula cells. The stemmata of Nannochoristaappear to be derived from stemmata of the Panorpa-type (Mecoptera-Panorpidae). Differences between the stemmata of Nannochoristaand Panorpacan be explained as adaptations to aquatic life (flat cornea) or as regression. A compound larval eye is ascribed to the ground plan of the Mecoptera sensu latoand is considered a genuine plesiomorphy. The identical basic number (seven) of stemmata in the Neuropteroid/Coleoptera assemblage, Amphiesmenoptera and some Mecoptera (Bittacidae, Boreidae) is attributed to parallel evolution.     

Colorless Sulfur Bacteria, Beggiatoa spp. and Thiovulum spp., in O2 and H2S Microgradients

The interactions between colorless sulfur bacteria and the chemical microgradients at the oxygen-sulfide interface were studied in Beggiatoa mats from marine sediments and in Thiovulum veils developing above the sediments. The gradients of O₂, H₂S, and pH were measured by microelectrodes at depth increments of 50 μm. An unstirred boundary layer in the water surrounding the mats and veils prevented microturbulent or convective mixing of O₂ and H₂S. The two substrates reached the bacteria only by molecular diffusion through the boundary layer. The bacteria lived as microaerophiles or anaerobes even under stirred, oxic water. Oxygen and sulfide zones overlapped by 50 μm in the bacterial layers. Both compounds had concentrations in the range of 0 to 10 μmol liter⁻¹ and residence times of 0.1 to 0.6 s in the overlapping zone. The sulfide oxidation was purely biological. Diffusion calculations showed that formation of mats on solid substrates or of veils in the water represented optimal strategies for the bacteria to achieve a stable microenvironment, a high substrate supply, and an efficient competition with chemical sulfide oxidation. The continuous gliding movement of Beggiatoa cells in mats or the flickering motion of Thiovulum cells in veils were important for the availability of both O₂ and H₂S for the individual bacteria.