Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25

The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4⁺ T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma.     

Phytoplankton spring blooms in the southern Baltic Sea--spatio-temporal development and long-term trends

The seasonal and long-term development of the phytoplanktonspring bloom in different regions of the southern Baltic Seawas investigated on the basis of monitoring data. The developmentof a spring bloom starts when the upper mixed layer becomesshallower than the euphotic zone, as proved also by a mesocosmexperiment. This already happens in March in Mecklenburg Bightand the western part of the Arkona Sea, leading to a diatombloom, but only in April in the Bornholm Sea, increasingly givingrise to a dinoflagellate bloom. The new production of the springphytoplankton may be calculated from the decrease in nutrientsduring spring. In comparison with the Redfield ratio, phosphorusis taken up in excess (N:P = 9.2–10.2). The consumptionof silicate in spring has been reduced in the southern Balticproper since 1989, pointing to a decline in diatoms. The increasein chlorophyll a in the Bornholm and the southern Gotland Seasis related to eutrophication, whereas the decrease in diatomsin favour of the dinoflagellates is related to mild winters.The lack of deep-reaching circulation after mild winters maybe one reason for the suppression of the non-motile diatoms.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Carotid baroreflex control of leg vascular conductance at rest and during exercise.

We sought to test the hypothesis that the carotid baroreflex (CBR) alters mean leg blood flow (LBF) and leg vascular conductance (LVC) at rest and during exercise. In seven men and one woman, 25 +/- 2 (SE) yr of age, CBR control of LBF and LVC was determined at rest and during steady-state one-legged knee extension exercise at approximately 65% peak O(2) uptake. The application of 5-s pulses of +40 Torr neck pressure and -60 Torr neck suction significantly altered mean arterial pressure (MAP) and LVC both at rest and during exercise. CBR-mediated changes in MAP were similar between rest and exercise (P > 0.05). However, CBR-mediated decreases in LVC (%change) to neck pressure were attenuated in the exercising leg (16.4 +/- 1.6%) compared with rest (33 +/- 2.1%) and the nonexercising leg (23.7 +/- 1.9%) (P < 0.01). These data suggest CBR control of blood pressure is partially mediated by changes in leg vascular tone both at rest and during exercise. Furthermore, despite alterations in CBR-induced changes in LVC during exercise, CBR control of blood pressure was well maintained.     

Novel Alkane Hydroxylase Gene (alkB) Diversity in Sediments Associated with Hydrocarbon Seeps in the Timor Sea,Australia

Hydrocarbon seeps provide inputs of petroleum hydrocarbons to widespread areas of the Timor Sea. Alkanes constitute the largest proportion of chemical components found in crude oils, and therefore genes involved in the biodegradation of these compounds may act as bioindicators for this ecosystem''s response to seepage. To assess alkane biodegradation potential, the diversity and distribution of alkane hydroxylase (alkB) genes in sediments of the Timor Sea were studied. Deduced AlkB protein sequences derived from clone libraries identified sequences only distantly related to previously identified AlkB sequences, suggesting that the Timor Sea maybe a rich reservoir for novel alkane hydroxylase enzymes. Most sequences clustered with AlkB sequences previously identified from marine Gammaproteobacteria though protein sequence identities averaged only 73% (with a range of 60% to 94% sequence identities). AlkB sequence diversity was lower in deep water (>400 m) samples off the continental slope than in shallow water (<100 m) samples on the continental shelf but not significantly different in response to levels of alkanes. Real-time PCR assays targeting Timor Sea alkB genes were designed and used to quantify alkB gene targets. No correlation was found between gene copy numbers and levels of hydrocarbons measured in sediments using sensitive gas chromatography-mass spectrometry techniques, probably due to the very low levels of hydrocarbons found in most sediment samples. Interestingly, however, copy numbers of alkB genes increased substantially in sediments exposed directly to active seepage even though only low or undetectable concentrations of hydrocarbons were measured in these sediments in complementary geochemical analyses due to efficient biodegradation.Alkanes are saturated hydrocarbons that are widespread in marine environments due to a variety of anthropogenic and natural sources. They constitute the major fraction of hydrocarbon components found in crude oils and refined petroleum and are also produced by various marine organisms (e.g., zooplankton) as cellular components (2, 44). Alkanes are considered as pollutants, with short-chained alkanes acting as solvents toward cellular membranes and other lipid components (34) while longer-chained alkanes may contribute to the formation of oil films and slicks that may limit nutrient and oxygen exchange (21). Importantly, alkanes also serve as important carbon and energy sources for some microorganisms. In marine environments, alkanes succumb to various removal and dispersal processes such as dissolution, photochemical oxidation, evaporation, adsorption, and sedimentation. However, the greatest removal pathway for alkanes in marine sediments is via biodegradation by bacteria (13). This mechanism also mediates the transfer of oil-derived carbon to higher trophic levels (28, 37), and therefore these bacteria have an important role in carbon cycling in environments subject to long-term inputs of hydrocarbons such as marine seep-associated ecosystems. Alkane biodegradation is mediated by a diverse range of marine bacteria using various electron acceptors although degradation generally proceeds at greater rates under aerobic conditions than under anaerobic conditions, where the process is relatively slow (8, 26).In the presence of oxygen, well-characterized alkane oxidation pathways are initiated by an activation step whereby oxygen is introduced to the alkane substrate before further catabolic steps can proceed. A number of oxygen-dependent alkane hydroxylase enzyme systems have been discovered that catalyze this initial step including the soluble di-iron methane monooxygenases and the membrane-bound copper-containing methane monooxygenases, both of which act upon short-chain alkanes (i.e., C₁ up to C₈). Integral membrane non-heme iron alkane hydroxylases (the alk system) that are related to the well-characterized AlkB of Pseudomonas putida GPo1 (also known as Pseudomonas oleovorans TF4-1 I) act upon longer-chain alkanes (i.e., C₅ to C₁₆) (40). Other systems exist that include alkane-hydroxylating cytochrome P450 enzymes in addition to other enzyme systems that are known to exist based purely on chemical analyses of metabolites formed during alkane degradation experiments (22, 25, 29); however, knowledge pertaining to the enzymes and genes involved as well as their importance in the environment is limited. Only recently have genes involved in the degradation of long-chain alkanes (e.g., C₃₂ and C₃₆) been identified in Acinetobacter sp. strain DSM 17874 (39) though there is no information about the presence or importance of such enzymes in the environment.Although various chemical and microbiological aspects of petroleum oil and alkane biodegradation in marine systems have been relatively well studied, there is a general lack of knowledge concerning the diversity or abundance of the functional genes involved. The biochemical and molecular aspects of alkB genes and the enzymes they encode have been relatively well studied, and this has enabled the development of molecular tools for the study of alkB genes in the environment (19). Elevated levels of hydrocarbons or the introduction of hydrocarbons to environments has been shown to increase gene copy numbers, indicating the potential use of alkB genes as bioindicators of oil pollution and/or biodegradation (16, 33, 36, 43). However, to date only one study has used culture-independent molecular methods to examine the diversity of alkB genes in a marine environment (20), and no studies have examined hydrocarbon-degrading genes where natural hydrocarbon seepage occurs.In this study, the diversity and relative abundance of alkB genes were examined in sediments of the Timor Sea, a region where natural seeps are sources of widespread petroleum hydrocarbons. It was hypothesized that (i) novel alkB genes may exist in this unique tropical marine environment, (ii) that variations in gene diversity would be found in sediments with different hydrocarbon levels, and (iii) that the abundance of certain alkB gene types may reflect the levels of measured hydrocarbons in sediments, and therefore this assay could be used as a complementary tool for monitoring petroleum inputs into sediments of the Timor Sea.     

<Emphasis Type="Italic">mab-31</Emphasis> and the TGF-β pathway act in the ray lineage to pattern <Emphasis Type="Italic">C. elegans</Emphasis>male sensory rays

Background  

C. elegans TGF-β-like Sma/Mab signaling pathway regulates both body size and sensory ray patterning. Most of the components in this pathway were initially identified by genetic screens based on the small body phenotype, and many of these mutants display sensory ray patterning defect. At the cellular level, little is known about how and where these components work although ray structural cell has been implicated as one of the targets. Based on the specific ray patterning abnormality, we aim to identify by RNAi approach additional components that function specifically in the ray lineage to elucidate the regulatory role of TGF-β signaling in ray differentiation.     

Genome sequencing of a single cell of the widely distributed marine subsurface Dehalococcoidia,phylum Chloroflexi

Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated ‘DEH-J10'' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO₂ or oxidation of organics completely to CO₂. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.     

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

Background

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data.

Results

Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution.

Conclusions

While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.     

Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.