ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down‐regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.     

Actinorhizal Alder Phytostabilization Alters Microbial Community Dynamics in Gold Mine Waste Rock from Northern Quebec: A Greenhouse Study

Phytotechnologies are rapidly replacing conventional ex-situ remediation techniques as they have the added benefit of restoring aesthetic value, important in the reclamation of mine sites. Alders are pioneer species that can tolerate and proliferate in nutrient-poor, contaminated environments, largely due to symbiotic root associations with the N₂-fixing bacteria, Frankia and ectomycorrhizal (ECM) fungi. In this study, we investigated the growth of two Frankia-inoculated (actinorhizal) alder species, A. crispa and A. glutinosa, in gold mine waste rock from northern Quebec. Alder species had similar survival rates and positively impacted soil quality and physico-chemical properties in similar ways, restoring soil pH to neutrality and reducing extractable metals up to two-fold, while not hyperaccumulating them into above-ground plant biomass. A. glutinosa outperformed A. crispa in terms of growth, as estimated by the seedling volume index (SVI), and root length. Pyrosequencing of the bacterial 16S rRNA gene for bacteria and the ribosomal internal transcribed spacer (ITS) region for fungi provided a comprehensive, direct characterization of microbial communities in gold mine waste rock and fine tailings. Plant- and treatment-specific shifts in soil microbial community compositions were observed in planted mine residues. Shannon diversity and the abundance of microbes involved in key ecosystem processes such as contaminant degradation (Sphingomonas, Sphingobium and Pseudomonas), metal sequestration (Brevundimonas and Caulobacter) and N₂-fixation (Azotobacter, Mesorhizobium, Rhizobium and Pseudomonas) increased over time, i.e., as plants established in mine waste rock. Acetate mineralization and most probable number (MPN) assays showed that revegetation positively stimulated both bulk and rhizosphere communities, increasing microbial density (biomass increase of 2 orders of magnitude) and mineralization (five-fold). Genomic techniques proved useful in investigating tripartite (plant-bacteria-fungi) interactions during phytostabilization, contributing to our knowledge in this field of study.     

The relationship of bionts and taphonomic processes in molluscan taphofacies formation on the continental shelf and slope: eight-year trends: Gulf of Mexico and Bahamas

The Shelf and Slope Experimental Taphonomy Initiative (SSETI) deployed a suite of molluscan species in environments covering a range of depths and sediment types in the Bahamas and on the Gulf of Mexico continental shelf and upper slope for 8 years. Taphonomic state rarely correlated with the distribution of biont guilds among environments. The preservable and nonpreservable biont guilds were also routinely orthogonal. Several coincidences of taphonomic trait and biont guild occurred, including green discoloration that consistently co-occurred with boring algae and bacterial films associated with the development of chalkiness and a soft shell surface. Environments of preservation (EOPs) of disparate taphonomic signature and biont guild complement occur in similar sediment types and environments with similar rates of burial. A paucity of biont coverage is no more a reliable indicator of rapid burial than is a limited degree of shell degradation. The suggestion that preservable bionts might protect shells from taphonomic processes is not well supported. Certain EOP groups are delineated from others most readily by a combination of biont guild and taphonomic trait. Thus, biont guilds augment taphonomic analysis in differentiating EOPs. Shell preservational state, including taphonomic signature and biont coverage, is influenced in a complex way by environment. The analysis confirms an expectation that the diversity of EOPs should be greater in shallow water. Clustering of EOPs reveals that visually distinctive environments may be taphonomically and biotically similar. Visually similar environments may be quite disparate in taphonomic state and biont complement. EOPs grouped by similarity in taphonomic signature and biont coverage very likely define geographically widespread biological and taphonomic regimes, which, however, are everywhere restricted locally in areal dimension.     

Autochthonous death assemblages from chemoautotrophic communities at petroleum seeps: Palaeoproduction,energy flow,and implications for the fossil record

Death assemblages produced by chemoautotrophic communities at cold seeps represent a type of autochthonous accumulation that is difficult to differentiate from other heterotrophic autochthonous communities using taphonomic characteristics. We test the hypothesis that cold‐seep assemblages can be discriminated by unique biological or community attributes rather than taphonomic attributes. To test this hypothesis, we compared several cold seeps on the Louisiana upper continental slope to heterotrophic sites on the Louisiana slope and to a putative seep site in the middle‐late Campanian Pierre Shale near Pueblo, Colorado. Seep assemblages are characterized by a unique tier and guild structure, size‐frequency composition, and animal density that together identify the palaeoenergetics structure of these communities and distinguish them from the other assemblages of the shelf and slope. All seep assemblages were dominated by primary consumers, whereas the heterotrophic assemblage was dominated by carnivores. Carnivore dominance seems to be typical of shelf (or euhaline) death assemblages. Seep assemblages, in contrast, retain the theoretically‐expected rarity of predaceous forms in fossil assemblages. Epifauna and semi‐infauna dominate the tier structure of the heterotrophic assemblage as is typical for continental shelf and slope assemblages. The infaunal tier was unusually well represented in most petroleum seep assemblages. Local enrichment of food resources and the dominance of shelled primary consumers explain the guild and tier structure of seep assemblages. Hindcasting of energy demand (palaeoingestion) and an estimate of sedimentation rate confirms that energy demand by the community exceeds the supply from planktonic rain in seep communities. Thus, seep assemblages can be recognized using biological attributes where taphonomic analysis is ambiguous.     

Structures of apomyoglobin's various acid-destabilized forms

The structures and the cold and hot melting thermodynamics of the acid- and salt-destabilized states of horse heart apomyoglobin (apoMb), including the E (extended) and various I forms, are studied using probes of tertiary structure (tryptophan fluorescence and FTIR spectroscopy) and secondary structure (far-UV CD and FTIR spectroscopy). These forms likely resemble early structures in the folding of the largely helical protein. Both the I and E forms retain the AGH core whereby the two ends of the protein are tied together with sufficient numbers of tertiary contacts, involving a number of hydrophobic residues, to show cooperative melting. The melting thermodynamics of E and I are distinctly different. E contains no other tertiary structure and probably little other secondary structure apart from the core. The more destabilized E form appears to contain "random" buried runs of polypeptide backbone which convert to alpha-helix in the I form(s). Most interestingly, E consists not of a single structure but is composed of a heterogeneous mixture of conformations, all showing corelike cooperative melting characteristics, and consisting presumably of varying contacts between the A portion of apomyoglobin and the G-H hairpin. These results bear on the energy landscape and structural features of the early part of apomyoglobin's folding pathway.     

Contributions of amino acid side chains to the kinetics and thermodynamics of the bivalent binding of protein L to Ig kappa light chain

Protein L is a bacterial surface protein with 4-5 immunoglobulin (Ig)-binding domains (B1-B5), each of which appears to have two binding sites for Ig, corresponding to the two edges of its beta-sheet. To verify these sites biochemically and to probe their relative contributions to the protein L-Ig kappa light chain (kappa) interaction, we compared the binding of PLW (the Y47W mutant of the B1 domain) to that of mutants designed to disrupt binding to sites 1 and 2, using gel filtration, BIAcore surface plasmon resonance, fluorescence titration, and solid-phase radioimmunoassays. Gel filtration experiments show that PLW binds kappa both in 1:1 complexes and multivalently, consistent with two binding sites. Covalent dimers of the A20C and V51C mutants of PLW were prepared to eliminate site 1 and site 2 binding, respectively; both the A20C and V51C dimers bind kappa in 1:1 complexes and multivalently, indicating that neither site 1 nor site 2 is solely responsible for kappa binding. The A20R mutant was designed computationally to eliminate site 1 binding while preserving site 2 binding; consistent with this design, the A20R mutant binds kappa in 1:1 complexes but not multivalently. To probe the contributions of amino acid side chains to binding, we prepared 75 point mutants spanning nearly every residue of PLW; BIAcore studies of these mutants revealed two binding-energy "hot spots" consistent with sites 1 and 2. These data indicate that PLW binds kappa at both sites with similar affinities (high nanomolar), with the strongest contributions to the binding energy from Tyr34 (site 2) and Tyr36 (site 1). Compared to other protein-protein complexes, the binding is insensitive to amino acid substitutions at these sites, consistent with the large number of main chain interactions relative to side chain interactions. The strong binding of protein L to Ig kappa light chains of various species may result from the ambidextrous binding of the B1-B5 domains and the unimportance of specific side chain interactions.     

Treatment of Menorrhagia with Tranexamic Acid. A Double-blind Trial

In a double-blind trial tranexamic acid (Cyclokapron) 1 g. four times a day for the first four days of menstruation, significantly decreased menstrual blood loss in women with menorrhagia for which no organic cause had been found. No difference in side-effects was noted between the active and placebo treatment.     

New developments in developmental biology

A report on the 15th International Society of Developmental Biologists Congress, Sydney, Australia, 3-7 September 2005.