The sources of dissolved inorganic carbon (DIC) used to produce scleractinian coral skeletons are not understood. Yet this knowledge is essential for understanding coral biomineralization and assessing the potential impacts of ocean acidification on coral reefs. Here we use skeletal boron geochemistry to reconstruct the DIC chemistry of the fluid used for coral calcification. We show that corals concentrate DIC at the calcification site substantially above seawater values and that bicarbonate contributes a significant amount of the DIC pool used to build the skeleton. Corals actively increase the pH of the calcification fluid, decreasing the proportion of DIC present as CO 2 and creating a diffusion gradient favouring the transport of molecular CO 2 from the overlying coral tissue into the calcification site. Coupling the increases in calcification fluid pH and [DIC] yields high calcification fluid [CO 3 2 À ] and induces high aragonite saturation states, favourable to the precipitation of the skeleton.

Episodes of prolonged drought coupled with heat waves (i.e. drought and heat combination) can have a devastating impact on agricultural production and crop yield. It is therefore not surprising that improving tolerance to drought and heat combination has been a major goal for breeders and biotech companies. Although much is known about the physiological and molecular responses of vegetative tissues to a combination of drought and heat stress, less is known about the impact of this stress combination on yield and different yield components. Here, we used a meta-analysis approach to synthesize results from over 120 published case studies of crop responses to combined drought and heat stress. Our findings reveal that drought and heat stress combination significantly impacts yield by decreasing harvest index, shortening the life cycle of crops, and altering seed number, size and composition. Furthermore, these impacts are more severe when the stress combination is applied during the reproductive stage of plants. We further identify differences in how legumes and cereals respond to the stress combination and reveal that utilizing C3 or C4 metabolism may not provide an advantage to plants during stress combinations. Taken together our study highlights a need to focus future studies, as well as breeding efforts, on crop responses to drought and heat combination at the reproductive stage of different crop species.

No abstract

A combination of drought and heat stress, occurring at the vegetative or reproductive growth phase of many different crops can have a devastating impact on yield. In soybean (Glycine max), a considerable effort has been made to develop genotypes with enhanced yield production under conditions of drought or heat stress. However, how these genotypes perform in terms of growth, physiological responses, and most importantly seed production, under conditions of drought and heat combination is mostly unknown. Here, we studied the impact of water deficit and heat stress combination on the physiology, seed production, and yield per plant of two soybean genotypes, Magellan and Plant Introduction (PI) 548313, that differ in their reproductive responses to heat stress. Our findings reveal that although PI 548313 produced more seeds than Magellan under conditions of heat stress, under conditions of water deficit, and heat stress combination its seed production decreased. Because the number of flowers and pollen germination of PI 548313 remained high under heat or water deficit and heat combination, the reduced seed production exhibited by PI 548313 under the stress combination could be a result of processes that occur at the stigma, ovaries and/or other parts of the flower following pollen germination.

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant ( ) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.

Engineered protein therapeutics offer advantages, including strong target affinity, selectivity, and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease upregulated with tumor progression, associated with poor prognosis, and implicated in tumor growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biologic inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity, and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350,000-fold greater specificity, and 83-fold improved proteolytic stability vs wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V/mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds.

Protein kinase B/Akt (PKB) is an anti-apoptotic protein kinase that has strongly elevated activity in human malignancies. We therefore initiated a program to develop PKB inhibitors, "Aktstatins". We screened about 500 compounds for PKB inhibitors, using a radioactive assay and an ELISA assay that we established for this purpose. These compounds were produced as combinatorial libraries, designed using the structure of the selective PKA inhibitor H-89 as a starting point. We have identified a successful lead compound, which inhibits PKB activity in vitro and in cells overexpressing active PKB. The new compound shows reversed selectivity to H-89: In contrast to H-89, which inhibits PKA 70 times better than PKB, the new compound, NL-71-101, inhibits PKB 2.4-fold better than PKA. The new compound, but not H-89, induces apoptosis in tumor cells in which PKB is amplified. We have identified structural features in NL-71-101 that are significant for the specificity and that can be used for future development and optimization of PKB inhibitors.

Light enhanced calcification (LEC) is a well-documented phenomenon in reef-building corals. The main mechanism proposed for LEC is that photosynthetic CO 2 uptake by the algal symbionts elevates the pH and thus enhances calcification. We evaluated the role of light and of photosynthesis on calcification by assessing the response of the corals Porites lutea and Acropora variabilis to different components of the light spectrum. Calcification and photosynthesis of both species decreased under "lagoon" blue, green and red light (peaks at 500, 550, and 600 nm, respectively). However, blue light (peak at 455 nm) enhanced calcification rates of P. lutea and A. variabilis (up to 4.1-and 10.5-fold of dark values, respectively) reaching levels comparable to those measured under full spectrum illumination. However, contrary to our expectations, photosynthetic oxygen production was considerably reduced under blue light, to the extent that it remained below the compensation point even under illumination as high as 400 µmol photons m −2 s −1. It is the first time that a direct effect of light not mediated by the photosynthetic process has been demonstrated to trigger LEC in corals. We propose that blue light signaling, and animal receptors thereof may be involved in the enhancement of calcification by hermatypic corals.