Inactivation of <Emphasis Type="Italic">Dicer1</Emphasis> in Steroidogenic factor 1-positive cells reveals tissue-specific requirement for <Emphasis Type="Italic">Dicer1</Emphasis>in adrenal,testis, and ovary

Background  

The synthesis of microRNA (miRNA) is a multi-step process that requires the action of the ribonuclease Dicer1. Dicer1 is responsible for the final processing of miRNA and has been implicated in cellular processes such as proliferation, apoptosis, and differentiation. Mouse embryos lacking Dicer1 die in early embryogenesis. In this study, we investigated whether Dicer1 is required for development of adrenal, testis, and ovary in mouse embryos.     

Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases

Molecular evolution has always been a subject of discussions, and researchers are interested in understanding how proteins with similar scaffolds can catalyze different reactions. In the superfamily of serine penicillin-recognizing enzymes, d-alanyl-d-alanine peptidases and β-lactamases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while β-lactamases hydrolyze penicillins very fast, leading to their inactivation, these molecules inhibit d-alanyl-d-alanine peptidases by forming stable covalent penicilloyl enzymes. In cyanobacteria, we have discovered a new family of penicillin-binding proteins (PBPs) presenting all the sequence features of class A β-lactamases but having a six-amino-acid deletion in the conserved Ω-loop and lacking the essential Glu166 known to be involved in the penicillin hydrolysis mechanism. With the aim of evolving a member of this family into a β-lactamase, PBP-A from Thermosynechococcus elongatus has been chosen because of its thermostability. Based on sequence alignments, introduction of a glutamate in position 158 of the shorter Ω-loop afforded an enzyme with a 50-fold increase in the rate of penicillin hydrolysis. The crystal structures of PBP-A in the free and penicilloylated forms at 1.9 Å resolution and of L158E mutant at 1.5 Å resolution were also solved, giving insights in the catalytic mechanism of the proteins. Since all the active-site elements of PBP-A-L158E, including an essential water molecule, are almost perfectly superimposed with those of a class A β-lactamase such as TEM-1, the question why our mutant is still 5 orders of magnitude less active as a penicillinase remains and our results emphasize how far we are from understanding the secrets of enzymes. Based on the few minor differences between the active sites of PBP-A and TEM-1, mutations were introduced in the L158E enzyme, but while activities on d-Ala-d-Ala mimicking substrates were severely impaired, further improvement in penicillinase activity was unsuccessful.     

Von Economo neurons in the anterior insula of the macaque monkey

The anterior insular cortex (AIC) and its unique spindle-shaped von Economo neuron (VEN) emerged within the last decade as having a potentially major role in self-awareness and social cognition in humans. Invasive examination of the VEN has been precluded so far by the assumption that this neuron occurs among primates exclusively in humans and great apes. Here, we demonstrate the presence of the VEN in the agranular anterior insula of the macaque monkey. The morphology, size, laminar distribution, and proportional distribution of the monkey VEN suggest that it is at least a primal anatomical homolog of the human VEN. This finding sheds new light on the phylogeny of the VEN and AIC. Most importantly, it offers new and much-needed opportunities to investigate the primal connections and physiology of a neuron that could be crucial for human self-awareness, social cognition, and related neuropsychiatric disorders.     

The GCP3-interacting proteins GIP1 and GIP2 are required for γ-tubulin complex protein localization, spindle integrity, and chromosomal stability

Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of γ-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with gamma-tubulin complex protein3 (GCP3), GCP3-interacting protein1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation.     

Development and Validation of an Immunoassay for Quantification of Topoisomerase I in Solid Tumor Tissues

Background

Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors.

Methodology/Principal Findings

We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998.

Conclusions/Significance

Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase in eukaryotes, and essential for DNA replication. By applying serial extractions to mammalian cells synchronized by release from quiescence, we reveal dynamic changes to the sub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase, identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix. The data distinguish 3 states that correspond to loose association with chromatin prior to DNA replication, transient highly stable binding to the nuclear-matrix coincident with initiation, and a post-initiation phase when MCM2 remains tightly associated with chromatin but not the nuclear-matrix. The data suggests that functional MCM complex loading takes place at the nuclear-matrix.     

7-Sulfonamido-3-benzazepines as potent and selective 5-HT2C receptor agonists: Hit-to-lead optimization

New 7-sulfonamido-3-benzazepines 3 are disclosed as 5-HT_2C receptor agonists. Appropriate substitution of the amino group (R¹R²N–) gave compounds that were potent 5-HT_2C agonists with minimal activation of the 5-HT_2A and 5-HT_2B receptors. Furthermore, representative examples had excellent in vitro ADME properties and good selectivity over ion channel activity.     

Design and synthesis of pyridazinone-based 5-HT2C agonists

The SAR of a series of pyridazinone derived 5-HT_2C agonists has been explored and resulted in identification of a compound with excellent levels of 5-HT_2C functional agonism and selectivity over 5-HT_2A and 5-HT_2B. This compound displayed good in vivo efficacy in pre-clinical models of stress urinary incontinence, despite having physiochemical properties commensurate with impaired CNS penetration.     

Plant TPX2 and related proteins

At the onset of mitosis, microtubules form a bipolar spindle around the prophase nucleus. TPX2 is phosphorylated during mitosis and acts as a spindle assembly factor that nucleates microtubules in the close vicinity of chromosomes, independent of the centrosomes. Furthermore, it activates the kinase Aurora A and targets the Xenopus kinesin-like protein 2 to spindle poles. We have characterized the plant orthologue of TPX2 that possesses all identified functional domains of its animal counterpart. Moreover, we have demonstrated that it is exported before nuclear envelope breakdown and that its activity around the nuclear envelope is essential for prospindle assembly. Here, we compare the sequences of several characterized TPX2 domains, allowing us to define TPX2. We propose that true TPX2 orthologues share simultaneously all these conserved domains and that other proteins possessing only some of these functional blocks may be considered as TPX2-related proteins.Key words: mitosis, microtubules, spindle assembly, TPX2 signature, targeting domains, Prosite motifs, evolution     

Evaluation of Performance of Introduced Yam Bean (<Emphasis Type="Italic">Pachyrhizus spp.</Emphasis>) in Three Agro-Ecological Zones of Rwanda

The yam bean (Pachyrizhus spp) was recently introduced as a root crop with high-yield potential, considerable protein and micro-nutrient concentration to investigate its potential for food production in Rwanda. Except for Chuin types (Pachyrizhus tuberosus) which have high storage root dry matter (RDM) (26 to 36%), most accessions are consumed raw and are reported to have low RDM. The present study aimed to evaluate and identify adapted high yielding yam bean accessions in major agro-ecological zones of Rwanda. Field experiments with 22 accessions were conducted in 2012 at three research sites representing the major agro-ecologies of Rwanda. Strict reproductive pruning was followed to enhance fresh storage root yields. Across locations, ANOVA indicated highly significant differences (p < 0.01) for genotypes (G), locations (L), seasons (S) and G x L effects for storage root yield, vine yield and harvest index and accounted for 21.88%, 43.41%, 1.43% and 13.25% of the treatment sum of squares, respectively. The GGE bi-plot revealed that EC209018 is high yielding but unstable. However, genotypes, AC209034, AC209035 and EC209046, were outstanding in terms of adaptation and relative stability across the 3 locations, suggesting consistent root yields irrespective of location and environmental conditions. The GGE scatter plot showed that all genotypes formed one mega-environment for storage root yield (Karama, Musanze and Rubona) and two mega-environments for biomass yield (Karama and Rubona as one mega-environment and Musanze the second one). This study revealed that Karama is the most suitable environment for evaluation and selection of yam bean for yield components in Rwanda.