The Anopheles gambiae adult midgut peritrophic matrix proteome

Malaria is a devastating disease. For transmission to occur, Plasmodium, the causative agent of malaria, must complete a complex developmental cycle in its mosquito vector. Thus, the mosquito is a potential target for disease control. Plasmodium ookinetes, which develop within the mosquito midgut, must first cross the midgut's peritrophic matrix (PM), a thick extracellular sheath that completely surrounds the blood meal. The PM poses a partial, natural barrier against parasite invasion of the midgut and it is speculated that modifications to the PM may lead to a complete barrier to infection. However, such strategies require thorough characterization of the structure of the PM. Here, we describe for the first time, the complete PM proteome of the main malaria vector, Anopheles gambiae. Altogether, 209 proteins were identified by mass spectrometry. Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. Lastly, we provide a model for the putative interactions among the proteins identified in this study.     

Fast deuterium access to the buried magnesium/manganese site in cytochrome c oxidase.

The recently determined crystal structures of bacterial and bovine cytochrome c oxidases show an area of organized water within the protein immediately above the active site where oxygen chemistry occurs. A pathway for exit of protons or water produced during turnover is suggested by possible connections of this aqueous region to the exterior surface. A non-redox-active Mg(2+) site is located in the interior of this region, and our previous studies [Florens, L., Hoganson, C., McCracken, J., Fetter, J., Mills, D., Babcock, G. T., and Ferguson-Miller, S. (1998) in Phototropic Prokaryotes (Peschek, G. A., Loeffelhard, W., and Schmetterer, G., Eds.) Kluwer Academic/Plenum, New York] have shown that the protons of water molecules that coordinate the metal can be exchanged within minutes of mixing with (2)H(2)O. Here we examine the extent and rate of deuterium exchange, using a combination of rapid freeze-quench and electron spin echo envelope modulation (ESEEM) analysis of Mn(2+)-substituted cytochrome c oxidase, which retains full activity. In the oxidized enzyme at room temperature, deuterium exchange at the Mn(2+) site occurs in less than 11 ms, which corresponds to an apparent rate constant higher than 3000 s(-1). The extent of deuterium substitution is dependent on the concentration of (2)H(2)O in the sample, indicative of rapid equilibrium, with three inner sphere (2)H(2)O exchanged per Mn(2+). This indicates that the water ligands of the Mn(2+)/Mg(2+) site, or the protons of these waters, can exchange with bulk solvent at a rate consistent with a role for this region in product release during turnover.     

Formation of P1, P2-dinucleoside 5′-pyrophosphates under potentially prebiological conditions

Summary Organic pyrophosphates such as UppA and NAD are formed when a solution containing a nucleotide, a nucleoside 5-polyphosphate, Mg²⁺ and imidazole are allowed to dry out. We suggest that this synthesis may have occured concurrently with oligonucleotide formation.Abbreviations Im Imidazole - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - U uridine - p_nA adenosine 5-poly-phosphate containing n phosphate residues - pU uridine 5-phosphate - AppA P₁,P₂-diadenosine 5-pyrophosphate - UppA P₁-(uridine 5)-P₂-(adenosine 5)-pyrophosphate - ImpA adenosine 5-phosphorimidazolide - NMN nicotinamide mononucleotide - NAD nicotinamide-adenine dinucleotide     

A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.     

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia

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Nephele streaming: stream processing under QoS constraints at scale

The ability to process large numbers of continuous data streams in a near-real-time fashion has become a crucial prerequisite for many scientific and industrial use cases in recent years. While the individual data streams are usually trivial to process, their aggregated data volumes easily exceed the scalability of traditional stream processing systems. At the same time, massively-parallel data processing systems like MapReduce or Dryad currently enjoy a tremendous popularity for data-intensive applications and have proven to scale to large numbers of nodes. Many of these systems also provide streaming capabilities. However, unlike traditional stream processors, these systems have disregarded QoS requirements of prospective stream processing applications so far. In this paper we address this gap. First, we analyze common design principles of today’s parallel data processing frameworks and identify those principles that provide degrees of freedom in trading off the QoS goals latency and throughput. Second, we propose a highly distributed scheme which allows these frameworks to detect violations of user-defined QoS constraints and optimize the job execution without manual interaction. As a proof of concept, we implemented our approach for our massively-parallel data processing framework Nephele and evaluated its effectiveness through a comparison with Hadoop Online. For an example streaming application from the multimedia domain running on a cluster of 200 nodes, our approach improves the processing latency by a factor of at least 13 while preserving high data throughput when needed.     

Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore

The kinetochore is a complex multiprotein structure located at centromeres that is essential for proper chromosome segregation. Budding-yeast Cse4 is an essential evolutionarily conserved histone H3 variant recruited to the centromere by an unknown mechanism. We have identified Scm3, an inner kinetochore protein that immunopurifies with Cse4. Scm3 is essential for viability and localizes to all centromeres. Construction of a conditional SCM3 allele reveals that depletion results in metaphase arrest, with duplicated spindle poles, short spindles, and unequal DNA distribution. The metaphase arrest is mediated by the mitotic spindle checkpoint being dependent on Mad1 and the Aurora kinase B homolog Ipl1. Scm3 interacts with both Ndc10 and Cse4 and is essential to establish centromeric chromatin after DNA replication. In addition, Scm3 is required to maintain kinetochore function throughout the cell cycle. We propose a model in which Ndc10/Scm3 binds to centromeric DNA, which is in turn essential for targeting Cse4 to centromeres.