Flavonglykoside in Blüten vonPaeonia arborea undPaeonia suffruticosa

Summary Three glycosides have been isolated fromPaeonia arborea: kaempferol-3--glucoside-7--glucoside (Paeonoside), apigenin-7--glucoside, and apigenin-7-rhamnoglucoside (Rhoifolin).Paeonia suffruticosa also contains these three compounds but its main glycoside is kaempferol-3--glucoside (astragalin), which is present inPaeonia arborea only in traces.     

SALMFamide2 and serotonin immunoreactivity in the nervous system of some acoels (Xenacoelomorpha)

Acoel worms are simple, often microscopic animals with direct development, a multiciliated epidermis, a statocyst, and a digestive parenchyma instead of a gut epithelium. Morphological characters of acoels have been notoriously difficult to interpret due to their relative scarcity. The nervous system is one of the most accessible and widely used comparative features in acoels, which have a so‐called commissural brain without capsule and several major longitudinal neurite bundles. Here, we use the selective binding properties of a neuropeptide antibody raised in echinoderms (SALMFamide2, or S2), and a commercial antibody against serotonin (5‐HT) to provide additional characters of the acoel nervous system. We have prepared whole‐mount immunofluorescent stainings of three acoel species: Symsagittifera psammophila (Convolutidae), Aphanostoma pisae, and the model acoel Isodiametra pulchra (both Isodiametridae). The commissural brain of all three acoels is delimited anteriorly by the ventral anterior commissure, and posteriorly by the dorsal posterior commissure. The dorsal anterior commissure is situated between the ventral anterior commissure and the dorsal posterior commissure, while the statocyst lies between dorsal anterior and dorsal posterior commissure. S2 and serotonin do not co‐localise, and they follow similar patterns to each other within an animal. In particular, S2, but not 5‐HT, stains a prominent commissure posterior to the main (dorsal) posterior commissure. We have for the first time observed a closed posterior loop of the main neurite bundles in S. psammophila for both the amidergic and the serotonergic nervous system. In I. pulchra, the lateral neurite bundles also form a posterior loop in our serotonergic nervous system stainings.     

A genetically selected cyclic peptide inhibitor of BCL6 homodimerization

We report an inhibitor of the homodimeric protein-protein interaction of the BCL6 oncoprotein, identified from a genetically encoded SICLOPPS library of 3.2 million cyclic hexapeptides in combination with a bacterial reverse two-hybrid system. This cyclic peptide is shown to bind the BTB domain of BCL6, disrupts its homodimerization, and subsequent binding of the SMRT2 corepressor peptide.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Ultrastructure of spermatogenesis and mature spermatozoa in the flatworm Prosthiostomum siphunculus (Polycladida,Cotylea)

This is the first study investigating spermatogenesis and spermatozoan ultrastructure in the polyclad flatworm Prosthiostomum siphunculus. The testes are numerous and scattered as follicles ventrally between the digestive ramifications. Each follicle contains the different stages of sperm differentiation. Spermatocytes and spermatids derive from a spermatogonium and the spermatids remain connected by intercellular bridges. Chromatoid bodies are present in the cytoplasm of spermatogonia up to spermatids. During early spermiogenesis, a differentiation zone appears in the distal part of spermatids. A ring of microtubules extends along the entire sperm shaft just beneath the cell membrane. An intercentriolar body is present and gives rise to two axonemes, each with a 9 + “1” micro‐tubular pattern. Development of the spermatid leads to cell elongation and formation of a filiform, mature spermatozoon with two free flagella and with cortical microtubules along the sperm shaft. The flagella exit the sperm shaft at different levels, a finding common for acotyleans, but so far unique for cotylean polyclads. The Golgi complex produces numerous electron‐dense bodies of two types and of different sizes. These bodies are located around a perinuclear row of mitochondria. The elongated nucleus extends almost along the entire sperm body. The nucleus is wide in the proximal part and becomes narrow going towards the distal end. Thread‐like chromatin mixed with electron‐dense intranuclear spindle‐shaped bodies are present throughout nucleus. The general sperm ultrastructure, the presence of intranuclear bodies and a second type of cytoplasmic electron‐dense bodies may provide characters useful for phylogenetic analysis.     

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.     

Association of reactive oxygen species-mediated signal transduction with in vitro apoptosis sensitivity in chronic lymphocytic leukemia B cells

Background

Chronic lymphocytic leukemia (CLL) is a B cell malignancy with a variable clinical course and unpredictable response to therapeutic agents. Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in signaling biology in the context of molecular changes occurring in malignancies. In this study SCNP was used to identify proteomic profiles associated with in vitro apoptotic responsiveness of CLL B cells to fludarabine, as a basis for ultimately linking these with clinical outcome.

Methodology/Principal Finding

SCNP was used to quantify modulated-signaling of B cell receptor (BCR) network proteins and in vitro F-ara-A mediated apoptosis in 23 CLL samples. Of the modulators studied the reactive oxygen species, hydrogen peroxide (H₂O₂), a known intracellular second messenger and a general tyrosine phosphatase inhibitor stratified CLL samples into two sub-groups based on the percentage of B cells in a CLL sample with increased phosphorylation of BCR network proteins. Separately, in the same patient samples, in vitro exposure to F-ara-A also identified two sub-groups with B cells showing competence or refractoriness to apoptotic induction. Statistical analysis showed that in vitro F-ara-A apoptotic proficiency was highly associated with the proficiency of CLL B cells to undergo H₂O₂-augmented signaling.

Conclusions/Significance

This linkage in CLL B cells among the mechanisms governing chemotherapy-induced apoptosis increased signaling of BCR network proteins and a likely role of phosphatase activity suggests a means of stratifying patients for their response to F-ara-A based regimens. Future studies will examine the clinical applicability of these findings and also the utility of this approach in relating mechanism to function of therapeutic agents.