Development of an optimized activatable MMP-14 targeted SPECT imaging probe

Matrix metalloproteinase-14 (MT1-MMP or MMP-14) is a membrane-associated protease implicated in a variety of tissue remodeling processes and a molecular hallmark of select metastatic cancers. The ability to detect MMP-14 in vivo would be useful in studying its role in pathologic processes and may potentially serve as a guide for the development of targeted molecular therapies. Four MMP-14 specific probes containing a positively charged cell penetrating peptide (CPP) d-arginine octamer (r₈) linked with a MMP-14 peptide substrate and attenuating sequences with glutamate (8e, 4e) or glutamate-glycine (4eg and 4egg) repeating units were modeled using an AMBER force field method. The probe with 4egg attenuating sequence exhibited the highest CPP/attenuator interaction, predicting minimized cellular uptake until cleaved. The in vitro MMP-14-mediated cleavage studies using the human recombinant MMP-14 catalytic domain revealed an enhanced cleavage rate that directly correlated with the linearity of the embedded peptide substrate sequence. Successful cleavage and uptake of a technetium-99m labeled version of the optimal probe was demonstrated in MMP-14 transfected human breast cancer cells. Two-fold reduction of cellular uptake was found in the presence of a broad spectrum MMP inhibitor. The combination of computational chemistry, parallel synthesis and biochemical screening, therefore, shows promise as a set of tools for developing new radiolabeled probes that are sensitive to protease activity.     

Is a cooperative approach to seaweed farming effectual? An analysis of the seaweed cluster project (SCP), Malaysia

Seaweed (Kappaphycus spp.) farming has been practised in Malaysia since the late 1970s following government policy incentives (training and farming inputs). However, numerous governance, economic, environmental, technological and sociocultural challenges have limited the industry from achieving its full potential. The Seaweed Cluster Project (SCP) was introduced in 2012 to address some of these challenges. We sought to evaluate the effectiveness of the SCP in delivering its central objectives of increasing seaweed production, optimising the farming area, improving seaweed quality and farming efficiency, raising farmers’ income, and reducing the environmental impact of seaweed farming. Community and industry perceptions of the SCP were obtained from seven communities using a mixed-methods approach based on face-to-face semi-structured interviews, focus group discussions, household surveys, observation and secondary data. Views on the SCP outcomes were generally negative, including low take-up rates by indigenous people, poor stakeholder participation in decision-making, limited acceptance of new technologies, economic vulnerability, a complex marketing system, and low social cohesion of seaweed farming communities. Positive perceptions included recognition that the SCP confers high social status upon a community, reduces operating costs, and facilitates the production of certified seaweed. The SCP’s problems are linked to poor multi-level governance, weak market mechanisms and unintegrated community development. The study concludes with five recommendations to improve the SCP: promote the participation of indigenous people; legalise existing migrant farmers; strengthen local seaweed cooperative organisations; provide entrepreneurship skills to farmers; and fully integrate stakeholders into decision-making.     

Comparative proteomic studies in Rhodospirillum rubrum grown under different nitrogen conditions

Forty-four differentially expressed proteins have been identified in the photosynthetic diazotroph Rhodospirillum rubrum grown anaerobic and photoheterotrophically, with different nitrogen sources, using 2D-PAGE and MALDI-TOF, from gels containing an average of 679 +/- 52 (in N(+)) and 619 +/- 37 (in N(-)) protein spots for each gel. A higher level of expression was found under nitrogen-rich growth, for proteins involved in carbon metabolism (reductive tricarboxylic acid cycle, CO(2) fixation, and poly-beta-hydroxybutyrate metabolism) and amino acid metabolism. The key enzymes RuBisCO and alpha-ketoglutarate synthase were found to be present in higher amounts in nitrogen-rich conditions. Ntr and Nif regulated proteins, such as glutamine synthetase and nitrogenase, were, as expected, induced under nitrogen-fixing conditions and glutamate dehydrogenase was down regulated. A novel 2Fe-2S ferredoxin with unknown function was identified from nitrogen-fixing cultures. In addition to differential expression, two of the identified proteins revealed variable p I values in response to the nitrogen source used.     

Improvement of intracellular β-galactosidase production in fed-batch culture of Kluyveromyces fragilis

Four fed-batch control strategies were evaluated to improve the specific lactase activity of Kluyveromyces fragilis. Control strategies tested included DO-stat control, exponential feeding, exponential feeding with manual feedback control and corrected feed-forward control. Each was implemented with standard sensors (i.e., temperature, dissolved oxygen and pH sensors) commonly installed in fermenters. The highest specific activity was obtained using the corrected feed-forward control strategy, a strategy incorporating a novel method for on-line estimation of specific growth rate. The control strategy was able to operate effectively to a final cell density of 69 g dry wt l^–1 with a specific lactase activity of 2 U mg^–1 cell dry wt.     

Marine dinoflagellates show induced life-history shifts to escape parasite infection in response to water-borne signals

Many dinoflagellate species form dormant resting cysts as a part of their life cycle, and in some freshwater species, hatching of these cysts can be delayed by the presence of water-borne signals from grazing zooplankton. Some marine dinoflagellates can form temporary cysts, which may function to resist unfavourable short-term environmental conditions. We investigated whether the marine dinoflagellate Alexandrium ostenfeldii is able to induce an increased resistance to the parasitic flagellate Parvilucifera infectans by forming temporary cysts. We performed several laboratory experiments where dinoflagellates were exposed either to direct contact with parasites or to filtered water from cultures of parasite-infected conspecifics (parasite-derived signals). Infection by P. infectans is lethal to motile A. ostenfeldii cells, but temporary cysts were more resistant to parasite infection. Furthermore, A. ostenfeldii induced a shift in life-history stage (from motile cells to temporary cysts) when exposed to parasite-derived water-borne signals. The response was relaxed within a couple of hours, indicating that A. ostenfeldii may use this behaviour as a short-term escape mechanism to avoid parasite infection. The results suggest that intraspecies chemical communication evoked by biotic interactions can be an important mechanism controlling life-history shifts in marine dinoflagellates, which may have implications for the development of toxic algal blooms.     

Baculovirus apoptotic suppressor P49 is a substrate inhibitor of initiator caspases resistant to P35 in vivo

Caspases play a critical role in the execution of metazoan apoptosis and are thus attractive therapeutic targets for apoptosis-associated diseases. Here we report that baculovirus P49, a homolog of pancaspase inhibitor P35, prevents apoptosis in invertebrates by inhibiting an initiator caspase that is P35 insensitive. Consequently P49 blocked proteolytic activation of effector caspases at a unique step upstream from that affected by P35 but downstream from inhibitor of apoptosis Op-IAP. Like P35, P49 was cleaved by and stably associated with its caspase target. Ectopically expressed P49 blocked apoptosis in cultured cells from a phylogenetically distinct organism, Drosophila melanogaster. Furthermore, P49 inhibited human caspase-9, demonstrating its capacity to affect a vertebrate initiator caspase. Thus, P49 is a substrate inhibitor with a novel in vivo specificity for a P35-insensitive initiator caspase that functions at an evolutionarily conserved step in the caspase cascade. These data indicate that activated initiator caspases provide another effective target for apoptotic intervention by substrate inhibitors.     

Genetic footprints reveal geographic patterns of expansion in Fennoscandian red foxes

Population expansions of boreal species are among the most substantial ecological consequences of climate change, potentially transforming both structure and processes of northern ecosystems. Despite their importance, little is known about expansion dynamics of boreal species. Red foxes (Vulpes vulpes) are forecasted to become a keystone species in northern Europe, a process stemming from population expansions that began in the 19th century. To identify the relative roles of geographic and demographic factors and the sources of northern European red fox population expansion, we genotyped 21 microsatellite loci in modern and historical (1835–1941) Fennoscandian red foxes. Using Bayesian clustering and Bayesian inference of migration rates, we identified high connectivity and asymmetric migration rates across the region, consistent with source‐sink dynamics, whereby more recently colonized sampling regions received immigrants from multiple sources. There were no clear clines in allele frequency or genetic diversity as would be expected from a unidirectional range expansion from south to north. Instead, migration inferences, demographic models and comparison to historical red fox genotypes suggested that the population expansion of the red fox is a consequence of dispersal from multiple sources, as well as in situ demographic growth. Together, these findings provide a rare glimpse into the anatomy of a boreal range expansion and enable informed predictions about future changes in boreal communities.     

Expression of the Na+/H+ antiporter gene (g1-nhaC) of alkaliphilic Bacillus sp. G1 in Escherichia coli

A Na(+)/H(+) antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na(+)/H(+) antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine-Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCl, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na(+) extrusion at lower pH values and in pH homeostasis at pH 10 under Na(+)-limiting conditions.