Liquid–liquid phase separation of Tau by self and complex coacervation

The liquid–liquid phase separation (LLPS) of Tau has been postulated to play a role in modulating the aggregation property of Tau, a process known to be critically associated with the pathology of a broad range of neurodegenerative diseases including Alzheimer''s Disease. Tau can undergo LLPS by homotypic interaction through self‐coacervation (SC) or by heterotypic association through complex‐coacervation (CC) between Tau and binding partners such as RNA. What is unclear is in what way the formation mechanisms for self and complex coacervation of Tau are similar or different, and the addition of a binding partner to Tau alters the properties of LLPS and Tau. A combination of in vitro experimental and computational study reveals that the primary driving force for both Tau CC and SC is electrostatic interactions between Tau‐RNA or Tau‐Tau macromolecules. The liquid condensates formed by the complex coacervation of Tau and RNA have distinctly higher micro‐viscosity and greater thermal stability than that formed by the SC of Tau. Our study shows that subtle changes in solution conditions, including molecular crowding and the presence of binding partners, can lead to the formation of different types of Tau condensates with distinct micro‐viscosity that can coexist as persistent and immiscible entities in solution. We speculate that the formation, rheological properties and stability of Tau droplets can be readily tuned by cellular factors, and that liquid condensation of Tau can alter the conformational equilibrium of Tau.     

Identification of Genes Transcriptionally Responsive to the Loss of MLL Fusions in MLL-Rearranged Acute Lymphoblastic Leukemia

Introduction

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year) is characterized by high relapse rates and a dismal prognosis. To facilitate the discovery of novel therapeutic targets, we here searched for genes directly influenced by the repression of various MLL fusions.

Methods

For this, we performed gene expression profiling after siRNA-mediated repression of MLL-AF4, MLL-ENL, and AF4-MLL in MLL-rearranged ALL cell line models. The obtained results were compared with various already established gene signatures including those consisting of known MLL-AF4 target genes, or those associated with primary MLL-rearranged infant ALL samples.

Results

Genes that were down-regulated in response to the repression of MLL-AF4 and MLL-ENL appeared characteristically expressed in primary MLL-rearranged infant ALL samples, and often represented known MLL-AF4 targets genes. Genes that were up-regulated in response to the repression of MLL-AF4 and MLL-ENL often represented genes typically silenced by promoter hypermethylation in MLL-rearranged infant ALL. Genes that were affected in response to the repression of AF4-MLL showed significant enrichment in gene expression profiles associated with AF4-MLL expressing t(4;11)+ infant ALL patient samples.

Conclusion

We conclude that the here identified genes readily responsive to the loss of MLL fusion expression potentially represent attractive therapeutic targets and may provide additional insights in MLL-rearranged acute leukemias.     

Proteomic and microarray analyses of the Dictyostelium Zak1-GSK-3 signaling pathway reveal a role in early development

GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathway's functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.     

A Drosophila gene encoding a DEAD box RNA helicase can suppress loss of wee1/mik1 function in Schizosaccharomyces pombe

We describe a screen to isolate cDNAs encoding Drosophila mitosis inhibitors capable of suppressing the mitotic catastrophe phenotype resulting in Schizosaccharomyces pombe from the combination of the weel-50 mutation with either a deletion allele of mil1, or with overexpression of cdc25 ⁺. One plasmid was isolated which could suppress the temperature sensitive lethality of both these strains. The cDNA in this plasmid encodes a protein highly homologous to the DEAD-box family of ATP-dependent RNA helicases, rather than to protein kinases as might be expected. It is possible that the RNA helicase described here may regulate entry into mitosis by down regulating the expression of other genes whose activity may be rate-limiting for entry into mitosis.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

Deficiency in Clonogenic Endometrial Mesenchymal Stem Cells in Obese Women with Reproductive Failure – a Pilot Study

Objectives

The mechanisms of obesity associated reproductive complications remain poorly understood. Endometrial mesenchymal stem-cells are critical for cyclic renewal and uterine function. Recently, W5C5⁺ cells, with high clonogenicity, capable of producing endometrial stroma in vivo, have been described. We sought to investigate the abundance and cloning efficiency of W5C5⁺ and W5C5⁻ endometrial cells in relation to Body Mass Index, age and reproductive outcome.

Design

W5C5⁺ and W5C5⁻ cells were purified from mid-luteal endometrial biopsies (n = 54) by magnetic bead separation and subjected to in vitro colony-forming assays.

Results

First trimester pregnancy losses were significantly higher in obese subjects (n = 12) compared to overweight (n = 20) and subjects with normal Body Mass Index (n = 22) (P<0.05, P<0.01, respectively). W5C5⁺ cells (%) were significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). W5C5⁺ cloning efficiency was significantly lower in obese subjects compared to overweight and subjects with normal Body Mass Index (P<0.05, respectively). W5C5⁻ cloning efficiency was significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). Body Mass Index was significantly negatively correlated with W5C5⁺ cloning efficiency and W5C5⁻ cloning efficiency (P<0.01, respectively), and positively correlated with first trimester loss (P<0.01). We found no significant results with age (P>0.05).

Conclusions

Our observations suggest that the regenerative capacity and plasticity of the endometrium of obese women is suboptimal, which in turn may account for the increased risk of reproductive complications associated with obesity.     

Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR

EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)-polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18-21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.     

Can Amphipod Behavior Help to Predict Chronic Toxicity of Sediments?

Amphipods are widely used in both acute and chronic (sub-lethal) sediment tests. Acute sediment tests provide relatively rapid results, but may fail to detect moderately toxic contaminants that are bound to the sediment, whereas chronic life-cycle tests are rarely performed as they are time consuming and expensive. Observations during chronic testing of oil-contaminated sediment suggested that there may be a link between the behavior of the marine amphipod Corophium volutator and reduction in growth rate. Behavior tests were performed with six individual amphipods per treatment using sediment spiked with weathered Forties oil with burrowing time, re-emergence from sediment, and activity prior to burrowing as endpoints. Further behavior tests were used to predict the chronic toxicity of sediments spiked with three crude oils each with a dominant unresolved complex mixture of hydrocarbons (UCM). The effect of sediment type on behavior was also investigated. The results suggested that although the behavior test could not be used alone as a viable alternative to sediment toxicity tests, it could prove useful as an adjunct to acute tests, and help select sediments that deserve further investigation.     

A novel mechanism of ligand binding and release in the odorant binding protein 20 from the malaria mosquito Anopheles gambiae

Anopheles gambiae mosquitoes that transmit malaria are attracted to humans by the odor molecules that emanate from skin and sweat. Odorant binding proteins (OBPs) are the first component of the olfactory apparatus to interact with odorant molecules, and so present potential targets for preventing transmission of malaria by disrupting the normal olfactory responses of the insect. AgamOBP20 is one of a limited subset of OBPs that it is preferentially expressed in female mosquitoes and its expression is regulated by blood feeding and by the day/night light cycles that correlate with blood‐feeding behavior. Analysis of AgamOBP20 in solution reveals that the apo‐protein exhibits significant conformational heterogeneity but the binding of odorant molecules results in a significant conformational change, which is accompanied by a reduction in the conformational flexibility present in the protein. Crystal structures of the free and bound states reveal a novel pathway for entrance and exit of odorant molecules into the central‐binding pocket, and that the conformational changes associated with ligand binding are a result of rigid body domain motions in α‐helices 1, 4, and 5, which act as lids to the binding pocket. These structures provide new insights into the specific residues involved in the conformational adaptation to different odorants and have important implications in the selection and development of reagents targeted at disrupting normal OBP function.