Genetic Variability of Microcystin Biosynthesis Genes in Planktothrix as Elucidated from Samples Preserved by Heat Desiccation during Three Decades

Historic samples of phytoplankton can provide information on the abundance of the toxigenic genotypes of cyanobacteria in dependence on increased or decreased eutrophication. The analysis of a time-series from preserved phytoplankton samples by quantitative PCR (qPCR) extends observation periods considerably. The analysis of DNA from heat-desiccated samples by qPCR can be aggravated by point substitutions or the fragmentation of DNA introduced by the high temperature. In this study, we analyzed whether the heat desiccation of the cellular material of the cyanobacterium Planktothrix sp. introduced potential errors to the template DNA that is used for qPCR within (i) 16S rDNA and phycocyanin genes and (ii) the mcyA gene indicative of the incorporation of either dehydrobutyrine (Dhb) or N-methyl-dehydroalanine (Mdha) in position 7, and (ii) the mcyB gene, which is indicative of homotyrosine (Hty) in position 2 of the microcystin (MC) molecule. Due to high temperature desiccation, the deterioration of the DNA template quality was rather due to fragmentation than due to nucleotide substitutions. By using the heat-desiccated samples of Lake Zürich, Switzerland the abundance of the Dhb, Mdha and Hty genotypes was determined during three decades (1977-2008). Despite major changes in the trophic state of the lake resulting in a major increase of the total Planktothrix population density, the proportion of these genotypes encoding the synthesis of different MC congeners showed high stability. Nevertheless, a decline of the most abundant mcyA genotype indicative of the synthesis of Dhb in position 7 of the MC molecule was observed. This decline could be related to the gradual incline in the proportion of a mutant genotype carrying a 1.8kbp deletion of this gene region. The increase of this mcyA (Dhb) gene deletion mutant has been minor so far, however, and likely did not affect the overall toxicity of the population.     

Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent

An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cell-cell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the transmission mode and differed depending on the window of action during the entry process, underscoring that powerful combinations of bnAbs are needed for in vivo application.     

Life‐history responses of a freshwater rotifer to copper pollution

In organisms with dormant stages, life‐history responses to past pollution can be studied retrospectively. Here, we study such responses in a rotifer (Brachionus calyciflorus) from the once heavily copper‐polluted Lake Orta (Italy). We extracted resting eggs from sediments, established clonal lineages from hatchlings, and exposed newborns of these lineages to one of three copper concentrations that each mimicked a specific period in the lake''s pollution history. For each rotifer, we daily collected life‐table data. We then estimated treatment‐specific vital rates and used a stage‐structured population model to project population growth rate λ. We also estimated elasticities of λ to vital rates and contributions of vital rates to observed Δλ between copper treatments. As expected, λ decreased with increasing copper concentration. This decrease resulted mostly from a decline in juvenile survival rate (S_J ) and partly from a decline in the survival rate of asexually reproducing females (S_A ). Maturation rate, and with one exception fecundity, also declined but did not contribute consistently to Δλ. λ was most elastic to S_J and S_A , indicating that survival rates were under stronger selection than maturation rate and fecundity. Together, our results indicate that variation in juvenile survival is a key component in the rotifers’ copper response. The consistent decrease in S_J with increasing copper stress and the sensitivity of λ to that decrease also suggest that juvenile survival is a useful indicator of population performance under environmental pollution.     

Treatment of poor-risk myelodysplastic syndromes and acute myeloid leukemia with a combination of 5-azacytidine and valproic acid

5-azacytidine (AZA) has become standard treatment for patients with higher-risk myelodysplastic syndrome (MDS). Response rate is about 50% and response duration is limited. Histone deactylase (HDAC) inhibitors are attractive partners for epigenetic combination therapy. We treated 24 patients with AZA (100?mg/m(2), 5?days) plus valproate (VPA; continuous dosing, trough serum level 80-110?μg/ml). According to WHO classification, 5 patients had MDS, 2 had MDS/MPD, and 17 had acute myeloid leukemia (AML). Seven patients (29%) had previously received intensive chemotherapy, and five had previous HDAC inhibitor treatment. The overall response rate was 37% in the entire cohort but significantly higher (57%) in previously untreated patients, especially those with MDS (64%). Seven (29%) patients achieved CR (29%) and two PR (8%), respectively. Hematological CR was accompanied by complete cytogenetic remission according to conventional cytogenetics in all evaluable cases. Some patients also showed complete remission according to FISH on bone marrow mononuclear cells and CD34(+) peripheral blood cells, as well as by follow-up of somatic mitochondrial DNA mutations. Four additional patients achieved at least marrow remissions. Factors influencing response were AML (vs. MDS), marrow blast count, pretreatment, transfusion dependency, concomitant medication with hydroxyurea, and valproic acid (VPA) serum level. This trial is the first to assess the combination of AZA plus VPA without additional ATRA. A comparatively good CR rate, relatively short time to response, and the influence of VPA serum levels on response suggest that VPA provided substantial additional benefit. However, the importance of HDAC inhibitors in epigenetic combination therapy can only be proven by randomized trials.     

Comparison of diatom communities in remote high-mountain lakes using index B and cluster analysis

Six remote high-mountain lakes in SE Switzerland were characterized on the basis of their water chemistry and the composition of their epilithic diatom communities. The index B (Renberg & Hellberg, 1982), calculated by summarizing the relative frequencies of occurrence in all five pH classes defined by Hustedt (1938/39), was found to be well correlated with pH values measured from August to October (5.8 to 7.0). Four lakes with pH values from 6.3 to 6.6 could not be distinguished from one another by means of the index B despite some differences in their physical, chemical and biological characteristics. However, it was possible to separate them by cluster analysis using the relative frequencies of the diatom species. We conclude that much ecological information is lost when applying the pH classification of Hustedt (1938/39).     

Der Geruchssinn des Kartoffelkäfers (Leptinotarsa decemlineata Say)

Zusammenfassung Am Kartoffelkäfer wurden Freiland- und Laboratoriumsbeobachtungen und Versuche zur Frage seines Geruchsvermögens angestellt. Normale augenlackierte Tiere fanden kleine Kartoffelblattstückchen im Durchschnitt nach 4 min. Käfer, denen beiderseits bis drei Antennenglieder fehlten, brauchten bis 10 min Laufzeit. Nach Amputation von vier und mehr Gliedern fanden sie den Köder nicht mehr.Einseitig antennenamputierte Käfer machen im diffusen Duftfeld Manegebewegungen.In unbewegter Luft hält der laufende Käfer oft inne und wittert durch rasches Antennenbewegen und seitliches Abspreizen der Antennen, sowie durch Fächeln mit den Vorderbeinen.Die beiden mit gleicher duftfreier Luft durchströmten Röhren des Olfaktometers erhalten gleichstarken Besuch. Beide Geschlechter und beide Generationen verhalten sich gleich. Im Luftstrom des Olfaktometers wird nicht gefächelt. Die relative Feuchtigkeit der Luft beeinflußt die Wahl nicht entscheidend.Tiere mit erhaltenen Tastern können nach Verlust der beiden letzten Antennenglieder die nach Kartoffelkraut duftende Röhre nicht mehr von der duftfreien unterscheiden. Dagegen wählen Tiere mit amputierten Tastern noch nach Verlust von beiderseits drei Antennengliedern richtig. Vielleicht senkt Wegfall der Tasterfunktion die Schwelle der Geruchsorgane. Sinngemäß umgekehrt reagieren die Käfer auf abweisende Stoffe (Nelkenöl).Die Antennen tragen drei Arten von Sensillen, nämlich zweierlei Tasthaare und sehr viel kleinere Härchen, die nur auf den fünf Endgliedern der Antenne vorkommen. Ihre prozentuale Verteilung auf dieser und die Ergebnisse der Olfaktometerversuche führen zu dem sicheren Schluß, daß die letztgenannten kleinen feinen Härchen Riechhaare sind. Ihre Geruchswahrnehmungen lenken den Kartoffelkäfer zum Futter.Die flaschenförmigen Drüsen sind wahrscheinlich Schmierdrüsen; sie sind überall im Körperchitin verteilt.Kiefer- und Lippentaster besitzen neben Tasthaaren weitere Formen von Chemorezeptoren, versenkte Haare und sensillae basiconicae; letztere sind wahrscheinlich Geschmacksorgane.Auf den Tarsen wurden keine Chemorezeptoren gefunden.     

Quantitative Measurement of Melanoma Spread in Sentinel Lymph Nodes and Survival

Background

Sentinel lymph node spread is a crucial factor in melanoma outcome. We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.

Methods and Findings

We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology. For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100. None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells. We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations. At a median follow-up of 49 mo (range 3–123 mo), 138 patients (13.4%) had died from melanoma. Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61–2.01, for a 10-fold increase in DCCD + 1). Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02–2.58). Upon multivariable testing DCCD was a stronger predictor of death than histopathology. The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.

Conclusions

Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death. A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up. If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically. Please see later in the article for the Editors'' Summary