Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production—A New Approach for Shortening Protein Production Development Timelines

Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell-based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli-based open cell-free synthesis (OCFS) system that is optimized for predictable high-yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high-level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte-macrophage colony-stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide-bonded biotherapeutic proteins. Biotechnol. Bioeng. 2011; 108:1570–1578. © 2011 Wiley Periodicals, Inc

Molecular mechanism for kinesin-1 direct membrane recognition

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1\u2013dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1\u2013mediated organelle transport

Caribbean Corals in Crisis: Record Thermal Stress, Bleaching, and Mortality in 2005

BACKGROUND The rising temperature of the world's oceans has become a major threat to coral reefs globally as the severity and frequency of mass coral bleaching and mortality events increase. In 2005, high ocean temperatures in the tropical Atlantic and Caribbean resulted in the most severe bleaching event ever recorded in the basin. METHODOLOGY/PRINCIPAL FINDINGS Satellite-based tools provided warnings for coral reef managers and scientists, guiding both the timing and location of researchers' field observations as anomalously warm conditions developed and spread across the greater Caribbean region from June to October 2005. Field surveys of bleaching and mortality exceeded prior efforts in detail and extent, and provided a new standard for documenting the effects of bleaching and for testing nowcast and forecast products. Collaborators from 22 countries undertook the most comprehensive documentation of basin-scale bleaching to date and found that over 80% of corals bleached and over 40% died at many sites. The most severe bleaching coincided with waters nearest a western Atlantic warm pool that was centered off the northern end of the Lesser Antilles. CONCLUSIONS/SIGNIFICANCE Thermal stress during the 2005 event exceeded any observed from the Caribbean in the prior 20 years, and regionally-averaged temperatures were the warmest in over 150 years. Comparison of satellite data against field surveys demonstrated a significant predictive relationship between accumulated heat stress (measured using NOAA Coral Reef Watch's Degree Heating Weeks) and bleaching intensity. This severe, widespread bleaching and mortality will undoubtedly have long-term consequences for reef ecosystems and suggests a troubled future for tropical marine ecosystems under a warming climate.This work was partially supported by salaries from the NOAA Coral Reef Conservation Program to the NOAA Coral Reef Conservation Program authors. NOAA provided funding to Caribbean ReefCheck investigators to undertake surveys of bleaching and mortality. Otherwise, no funding from outside authors' institutions was necessary for the undertaking of this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Lake salinization drives consistent losses of zooplankton abundance and diversity across coordinated mesocosm experiments

Human-induced salinization increasingly threatens inland waters; yet we know little about the multifaceted response of lake communities to salt contamination. By conducting a coordinated mesocosm experiment of lake salinization across 16 sites in North America and Europe, we quantified the response of zooplankton abundance and (taxonomic and functional) community structure to a broad gradient of environmentally relevant chloride concentrations, ranging from 4 to ca. 1400 mg Cl- L-1. We found that crustaceans were distinctly more sensitive to elevated chloride than rotifers; yet, rotifers did not show compensatory abundance increases in response to crustacean declines. For crustaceans, our among-site comparisons indicate: (1) highly consistent decreases in abundance and taxon richness with salinity; (2) widespread chloride sensitivity across major taxonomic groups (Cladocera, Cyclopoida, and Calanoida); and (3) weaker loss of functional than taxonomic diversity. Overall, our study demonstrates that aggregate properties of zooplankton communities can be adversely affected at chloride concentrations relevant to anthropogenic salinization in lakes.Peer reviewe

Depletion of B2 but Not B1a B Cells in BAFF Receptor-Deficient ApoE−/− Mice Attenuates Atherosclerosis by Potently Ameliorating Arterial Inflammation

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE−/− mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE−/− mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R−/− ApoE−/− (BaffR.ApoE DKO) and BAFF-R+/+ApoE−/− (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE−/− mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation

Widespread variation in salt tolerance within freshwater zooplankton species reduces the predictability of community-level salt tolerance

The salinization of freshwaters is a global threat to aquatic biodiversity. We quantified variation in chloride (Cl-) tolerance of 19 freshwater zooplankton species in four countries to answer three questions: (1) How much variation in Cl- tolerance is present among populations? (2) What factors predict intraspecific variation in Cl- tolerance? (3) Must we account for intraspecific variation to accurately predict community Cl- tolerance? We conducted field mesocosm experiments at 16 sites and compiled acute LC(50)s from published laboratory studies. We found high variation in LC(50)s for Cl- tolerance in multiple species, which, in the experiment, was only explained by zooplankton community composition. Variation in species-LC50 was high enough that at 45% of lakes, community response was not predictable based on species tolerances measured at other sites. This suggests that water quality guidelines should be based on multiple populations and communities to account for large intraspecific variation in Cl- tolerance.Peer reviewe

Current water quality guidelines across North America and Europe do not protect lakes from salinization

Human-induced salinization caused by the use of road deicing salts, agricultural practices, mining operations, and climate change is a major threat to the biodiversity and functioning of freshwater ecosystems. Yet, it is unclear if freshwater ecosystems are protected from salinization by current water quality guidelines. Leveraging an experimental network of land-based and in-lake mesocosms across North America and Europe, we tested how salinization-indicated as elevated chloride (C-) concentration-will affect lake food webs and if two of the lowest Cl- thresholds found globally are sufficient to protect these food webs. Our results indicated that salinization will cause substantial zooplankton mortality at the lowest Cl- thresholds established in Canada (120 mg Cl-/L) and the United States (230 mg Cl-/L) and throughout Europe where Cl- thresholds are generally higher. For instance, at 73% of our study sites, Cl- concentrations that caused a >= 50% reduction in cladoceran abundance were at or below Cl thresholds in Canada, in the United States, and throughout Europe. Similar trends occurred for copepod and rotifer zooplankton. The loss of zooplankton triggered a cascading effect causing an increase in phytoplankton biomass at 47% of study sites. Such changes in lake food webs could alter nutrient cycling and water clarity and trigger declines in fish production. Current Cl- thresholds across North America and Europe clearly do not adequately protect lake food webs. Water quality guidelines should be developed where they do not exist, and there is an urgent need to reassess existing guidelines to protect lake ecosystems from human-induced salinization.Peer reviewe

The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7–dependent siRNAs require Dicer. We identify that the eri-6/7–dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3–4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7–dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA–associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation

Association Rate Constants of Ras-Effector Interactions Are Evolutionarily Conserved

Evolutionary conservation of protein interaction properties has been shown to be a valuable indication for functional importance. Here we use homology interface modeling of 10 Ras-effector complexes by selecting ortholog proteins from 12 organisms representing the major eukaryotic branches, except plants. We find that with increasing divergence time the sequence similarity decreases with respect to the human protein, but the affinities and association rate constants are conserved as predicted by the protein design algorithm, FoldX. In parallel we have done computer simulations on a minimal network based on Ras-effector interactions, and our results indicate that in the absence of negative feedback, changes in kinetics that result in similar binding constants have strong consequences on network behavior. This, together with the previous results, suggests an important biological role, not only for equilibrium binding constants but also for kinetics in signaling processes involving Ras-effector interactions. Our findings are important to take into consideration in system biology approaches and simulations of biological networks