Rationally designed turn promoting mutation in the amyloid-β peptide sequence stabilizes oligomers in solution

Enhanced production of a 42-residue beta amyloid peptide (Aβ(42)) in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD). The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs) in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the Aβ(42) peptide (Aβ(42)Nle35p37) that appear to organize Aβ(42) into stable oligomers. 2D NMR on the Aβ(42)Nle35p37 peptide revealed the occurrence of two β-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the Aβ(21-43)Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.     

Enzymatic depilation of animal hide: identification of elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a depilating protease

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme.     

Molecular implication of PP2A and Pin1 in the Alzheimer's disease specific hyperphosphorylation of Tau

Background

Tau phosphorylation and dephosphorylation regulate in a poorly understood manner its physiological role of microtubule stabilization, and equally its integration in Alzheimer disease (AD) related fibrils. A specific phospho-pattern will result from the balance between kinases and phosphatases. The heterotrimeric Protein Phosphatase type 2A encompassing regulatory subunit PR55/Bα (PP2A_T55α) is a major Tau phosphatase in vivo, which contributes to its final phosphorylation state. We use NMR spectroscopy to determine the dephosphorylation rates of phospho-Tau by this major brain phosphatase, and present site-specific and kinetic data for the individual sites including the pS202/pT205 AT8 and pT231 AT180 phospho-epitopes.

Methodology/Principal Findings

We demonstrate the importance of the PR55/Bα regulatory subunit of PP2A within this enzymatic process, and show that, unexpectedly, phosphorylation at the pT231 AT180 site negatively interferes with the dephosphorylation of the pS202/pT205 AT8 site. This inhibitory effect can be released by the phosphorylation dependent prolyl cis/trans isomerase Pin1. Because the stimulatory effect is lost with the dimeric PP2A core enzyme (PP2A_D) or with a phospho-Tau T231A mutant, we propose that Pin1 regulates the interaction between the PR55/Bα subunit and the AT180 phospho-epitope on Tau.

Conclusions/Significance

Our results show that phosphorylation of T231 (AT180) can negatively influence the dephosphorylation of the pS202/pT205 AT8 epitope, even without an altered PP2A pool. Thus, a priming dephosphorylation of pT231 AT180 is required for efficient PP2A_T55α-mediated dephosphorylation of pS202/pT205 AT8. The sophisticated interplay between priming mechanisms reported for certain Tau kinases and the one described here for Tau phosphatase PP2A_T55α may contribute to the hyperphosphorylation of Tau observed in AD neurons.     

Explicit coordination to prevent congestion in data center networks

Large cluster-based cloud computing platforms increasingly use commodity Ethernet technologies, such as Gigabit Ethernet, 10GigE, and Fibre Channel over Ethernet (FCoE), for intra-cluster communication. Traffic congestion can become a performance concern in the Ethernet due to consolidation of data, storage, and control traffic over a common layer-2 fabric, as well as consolidation of multiple virtual machines (VMs) over less physical hardware. Even as networking vendors race to develop switch-level hardware support for congestion management, we make the case that virtualization has opened up a complementary set of opportunities to reduce or even eliminate network congestion in cloud computing clusters. We present the design, implementation, and evaluation of a system called XCo, that performs explicit coordination of network transmissions over a shared Ethernet fabric to proactively prevent network congestion. XCo is a software-only distributed solution executing only in the end-nodes. A central controller uses explicit permissions to temporally separate (at millisecond granularity) the transmissions from competing senders through congested links. XCo is fully transparent to applications, presently deployable, and independent of any switch-level hardware support. We present a detailed evaluation of our XCo prototype across a number of network congestion scenarios, and demonstrate that XCo significantly improves network performance during periods of congestion. We also evaluate the behavior of XCo for large topologies using NS3 simulations.     

Membrane progesterone receptor alpha as a potential prognostic biomarker for breast cancer survival: a retrospective study

Classically, the actions of progesterone (P4) are attributed to the binding of nuclear progesterone receptor (PR) and subsequent activation of its downstream target genes. These mechanisms, however, are not applicable to PR- or basal phenotype breast cancer (BPBC) due to lack of PR in these cancers. Recently, the function of membrane progesterone receptor alpha (mPRα) in human BPBC cell lines was studied in our lab. We proposed that the signaling cascades of P4→mPRα pathway may play an essential role in controlling cell proliferation and epithelial mesenchymal transition (EMT) of breast cancer. Using human breast cancer tissue microarrays, we found in this study that the average intensity of mPRα expression, but not percentage of breast cancer with high level of mPRα expression (mPRα-HiEx), was significantly lower in the TNM stage 4 patients compared to those with TNM 1-3 patients; and both average intensities of mPRα expression and mPRα-HiEx rates were significantly higher in cancers negative for ER, as compared with those cancers with ER+. However, after adjusting for age at diagnosis and/or TNM stage, only average intensities of mPRα expression were associated with ER status. In addition, we found that the rates of mPRα-HiEx were significantly higher in cancers with epithelial growth factor receptor-1 (EGFR+) and high level of Ki67 expression, indicating positive correlation between mPRα over expression and EGFR or Ki67. Further analysis indicated that both mPRα-HiEx rate and average intensity of mPRα expression were significantly higher in HER2+ subtype cancers (i.e. HER2+ER-PR-) as compared to ER+ subtype cancers. These data support our hypothesis that P4 modulates the activities of the PI3K and cell proliferation pathways through the caveolar membrane bound growth factor receptors such as mPRα and growth factor receptors. Future large longitudinal studies with larger sample size and survival outcomes are necessary to confirm our findings.     

Design and synthesis of 4-alkynyl pyrazoles as inhibitors of PDE4: a practical access via Pd/C-Cu catalysis

The design and synthesis of 4-alkynyl pyrazole derivatives has led to the identification of new class of PDE4 inhibitors. All these compounds were accessed for the first time via a facile Pd/C-CuI-PPh(3) mediated C-C bond forming reaction between an appropriate pyrazole iodide and various terminal alkynes. In vitro PDE4B inhibitory properties and molecular modeling studies of some of the compounds synthesized indicated that 4-alkynyl pyrazole could be a promising template for the discovery of novel PDE4 inhibitors.     

[18F](R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-fluoroethoxy)-2,5-dimethyl phenylamino)pyrazin-2(1H)-one: Introduction of N3-phenylpyrazinones as potential CRF-R1 PET imaging agents

Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10 was evaluated for potential development as PET radioligand. Compound [¹⁸F]10 was prepared with high radiochemical purity and showed promising binding properties in rat brain imaging experiments.     

Lead optimization of purine based orally bioavailable Mps1 (TTK) inhibitors

Efforts to optimize biological activity, novelty, selectivity and oral bioavailability of Mps1 inhibitors, from a purine based lead MPI-0479605, are described in this Letter. Mps1 biochemical activity and cytotoxicity in HCT-116 cell line were improved. On-target activity confirmation via mechanism based G2/M escape assay was demonstrated. Physico-chemical and ADME properties were optimized to improve oral bioavailability in mouse.