Oxygen isotope ratios of PO4: An inorganic indicator of enzymatic activity and P metabolism and a new biomarker in the search for life

The distinctive relations between biological activity and isotopic effect recorded in biomarkers (e.g., carbon and sulfur isotope ratios) have allowed scientists to suggest that life originated on this planet nearly 3.8 billion years ago. The existence of life on other planets may be similarly identified by geochemical biomarkers, including the oxygen isotope ratio of phosphate (δ18Op) presented here. At low near-surface temperatures, the exchange of oxygen isotopes between phosphate and water requires enzymatic catalysis. Because enzymes are indicative of cellular activity, the demonstration of enzyme-catalyzed PO4–H2O exchange is indicative of the presence of life. Results of laboratory experiments are presented that clearly show that δ18OP values of inorganic phosphate can be used to detect enzymatic activity and microbial metabolism of phosphate. Applications of δ18Op as a biomarker are presented for two Earth environments relevant to the search for extraterrestrial life: a shallow groundwater reservoir and a marine hydrothermal vent system. With the development of in situ analytical techniques and future planned sample return strategies, δ18Op may provide an important biosignature of the presence of life in extraterrestrial systems such as that on Mars.

The current biodiversity extinction event: Scenarios for mitigation and recovery

The current massive degradation of habitat and extinction of species is taking place on a catastrophically short timescale, and their effects will fundamentally reset the future evolution of the planet's biota. The fossil record suggests that recovery of global ecosystems has required millions or even tens of millions of years. Thus, intervention by humans, the very agents of the current environmental crisis, is required for any possibility of short-term recovery or maintenance of the biota. Many current recovery efforts have deficiencies, including insufficient information on the diversity and distribution of species, ecological processes, and magnitude and interaction of threats to biodiversity (pollution, overharvesting, climate change, disruption of biogeochemical cycles, introduced or invasive species, habitat loss and fragmentation through land use, disruption of community structure in habitats, and others). A much greater and more urgently applied investment to address these deficiencies is obviously warranted. Conservation and restoration in human-dominated ecosystems must strengthen connections between human activities, such as agricultural or harvesting practices, and relevant research generated in the biological, earth, and atmospheric sciences. Certain threats to biodiversity require intensive international cooperation and input from the scientific community to mitigate their harmful effects, including climate change and alteration of global biogeochemical cycles. In a world already transformed by human activity, the connection between humans and the ecosystems they depend on must frame any strategy for the recovery of the biota.

Frontiers of finance: Evolution and efficient markets

In this review article, we explore several recent advances in the quantitative modeling of financial markets. We begin with the Efficient Markets Hypothesis and describe how this controversial idea has stimulated a number of new directions of research, some focusing on more elaborate mathematical models that are capable of rationalizing the empirical facts, others taking a completely different tack in rejecting rationality altogether. One of the most promising directions is to view financial markets from a biological perspective and, specifically, within an evolutionary framework in which markets, instruments, institutions, and investors interact and evolve dynamically according to the “law” of economic selection. Under this view, financial agents compete and adapt, but they do not necessarily do so in an optimal fashion. Evolutionary and ecological models of financial markets is truly a new frontier whose exploration has just begun.

Toward a plant genomics initiative: Thoughts on the value of cross-species and cross-genera comparisons in the grasses

Comparative genomics offers unparalleled opportunities to integrate historically distinct disciplines, to link disparate biological kingdoms, and to bridge basic and applied science. Cross-species, cross-genera, and cross-kingdom comparisons are proving key to understanding how genes are structured, how gene structure relates to gene function, and how changes in DNA have given rise to the biological diversity on the planet. The application of genomics to the study of crop species offers special opportunities for innovative approaches for combining sequence information with the vast reservoirs of historical information associated with crops and their evolution. The grasses provide a particularly well developed system for the development of tools to facilitate comparative genetic interpretation among members of a diverse and evolutionarily successful family. Rice provides advantages for genomic sequencing because of its small genome and its diploid nature, whereas each of the other grasses provides complementary genetic information that will help extract meaning from the sequence data. Because of the importance of the cereals to the human food chain, developments in this area can lead directly to opportunities for improving the health and productivity of our food systems and for promoting the sustainable use of natural resources.

Distinct Biochemical and Topological Properties of the 31- and 27-Kilodalton Plasma Membrane Intrinsic Protein Subgroups from Red Beet1

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387–392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

Intervention of carbohydrate recognition by proteins and nucleic acids.

Carbohydrates in biological systems are often associated with specific recognition and signaling processes leading to important biological functions and diseases. Considerable efforts have been directed toward understanding and mimicking the recognition processes and developing effective agents to control the processes. The pace of discovery research in glycobiology and development of carbohydrate-based therapeutics, however, has been relatively slow due to the lack of appropriate strategies and methods available for carbohydrate-related research. This review summarizes some of the most recent developments in the field, with particular emphasis on work from our laboratories regarding the use of chemoenzymatic strategies to tackle the carbohydrate recognition problem. Highlights include the study of selectin-carbohydrate and aminoglycoside-RNA interactions and development of agents for the intervention of these recognition processes.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

Selection for genes encoding secreted proteins and receptors.

Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

Mutations in the rotated abdomen locus affect muscle development and reveal an intrinsic asymmetry in Drosophila.

In bilateral animals, the left and right sides of the body usually present asymmetric structures, the genetic bases of whose generation are still largely unknown [CIBA Foundation (1991) Biological Asymmetry and Handedness, CIBA Foundation Symposium 162 (Wiley, New York), pp. 1-327]. In Drosophila melanogaster, mutations in the rotated abdomen (rt) locus cause a clockwise helical rotation of the body. Even null alleles are viable but exhibit defects in embryonic muscle development, rotation of the whole larval body, and helical staggering of cuticular patterns in abdominal segments of the adult. rotated abdomen is expressed in the embryonic mesoderm and midgut but not in the ectoderm; it encodes a putative integral membrane glycoprotein (homologous to key yeast mannosyltransferases). Mesodermal cells defective in O-glycosylation lead to an impaired larval muscular system. We propose that the staggering of the adult abdominal segments would be a consequence of the relaxation of intrinsic rotational torque of muscle architecture, preventing the colateral alignment of the segmental histoblast cells during their proliferation at metamorphosis.

Polynitrosylated proteins: characterization, bioactivity, and functional consequences.

Chemical modification of proteins is a common theme in their regulation. Nitrosylation of protein sulfhydryl groups has been shown to confer nitric oxide (NO)-like biological activities and to regulate protein functions. Several other nucleophilic side chains -- including those with hydroxyls, amines, and aromatic carbons -- are also potentially susceptible to nitrosative attack. Therefore, we examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules. Chemical analysis and spectroscopic studies show that nitrosation reactions are sustained at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being the most reactive functionality. The exemplary protein, BSA, in the presence of a 1-, 20-, 100-, or 200-fold excess of nitrosating equivalents removes 0.6 +/- 0.2, 3.2 +/- 0.4, 18 +/- 4, and 38 +/- 10, respectively, moles of NO equivalents per mole of BSA from the reaction medium; spectroscopic evidence shows the proportionate formation of a polynitrosylated protein. Analogous reaction of tissue-type plasminogen activator yields comparable NO protein stoichiometries. Disruption of protein tertiary structure by reduction results in the preferential nitrosylation of up to 20 thus-exposed thiol groups. The polynitrosylated proteins exhibit antiplatelet and vasodilator activity that increases with the degree of nitrosation, but S-nitroso derivatives show the greatest NO-related bioactivity. Studies on enzymatic activity of tissue-type plasminogen activator show that polynitrosylation may lead to attenuated function. Moreover, the reactivity of tyrosine residues in proteins raises the possibility that NO could disrupt processes regulated by phosphorylation. Polynitrosylated proteins were found in reaction mixtures containing interferon-gamma/lipopolysaccharide-stimulated macrophages and in tracheal secretions of subjects treated with NO gas, thus suggesting their physiological relevance. In conclusion, multiple sites on proteins are susceptible to attack by nitrogen oxides. Thiol groups are preferentially modified, supporting the notion that S-nitrosylation can serve to regulate protein function. Nitrosation reactions sustained at additional nucleophilic centers may have (patho)physiological significance and suggest a facile route by which abundant NO bioactivity can be delivered to a biological system, with specificity dictated by protein substrate.

Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities.

Interferon tau (IFN tau), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFN alpha species (IFN alpha s). Ovine IFN tau has antiviral activity similar to that of human IFN alpha A on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFN tau was found to differ from IFN alpha A in that is was > 30-fold less toxic to MDBK cells at high concentrations. Excess IFN tau did not block the cytotoxicity of IFN alpha A on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 125I-IFN tau had a Kd of 3.90 x 10(-10) M for receptor on MDBK cells, whereas that of 125I-IFN alpha A was 4.45 x 10(-11) M. Consistent with the higher binding affinity, IFN alpha A was severalfold more effective than IFN tau in competitive binding against 125I-IFN tau to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFN alpha A, with the higher binding affinity, was more toxic than IFN tau. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Stat1 alpha and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N terminal of IFN tau and IFN alpha with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare'' with respect to certain biological properties, and we speculate that IFNs may induce a concentration-dependent selective association of receptor subunits.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Multi-agent system for control of robots inspired on the distributed activity and hormonal regulation of humans

Robotics is an emerging field with great activity. Robotics is a field that presents several problems because it depends on a large number of disciplines, technologies, devices and tasks. Its expansion from perfectly controlled industrial environments toward open and dynamic environment presents a many new challenges. New uses are, for example, household robots or professional robots. To facilitate the low cost, rapid development of robotic systems, reusability of code, its medium and long term maintainability and robustness are required novel approaches to provide generic models and software systems who develop paradigms capable of solving these problems. For this purpose, in this paper we propose a model based on multi-agent systems inspired by the human nervous system able to transfer the control characteristics of the biological system and able to take advantage of the best properties of distributed software systems. Specifically, we model the decentralized activity and hormonal variation.

Subsidence damage assessment of a Gothic church using differential interferometry and field data

The Santas Justa and Rufina Gothic church (fourteenth century) has suffered several physical, mechanical, chemical, and biochemical types of pathologies along its history: rock alveolization, efflorescence, biological activity, and capillary ascent of groundwater. However, during the last two decades, a new phenomenon has seriously affected the church: ground subsidence caused by aquifer overexploitation. Subsidence is a process that affects the whole Vega Baja of the Segura River basin and consists of gradual sinking in the ground surface caused by soil consolidation due to a pore pressure decrease. This phenomenon has been studied by differential synthetic aperture radar interferometry techniques, which illustrate settlements up to 100 mm for the 1993–2009 period for the whole Orihuela city. Although no differential synthetic aperture radar interferometry information is available for the church due to the loss of interferometric coherence, the spatial analysis of nearby deformation combined with fieldwork has advanced the current understanding on the mechanisms that affect the Santas Justa and Rufina church. These results show the potential interest and the limitations of using this remote sensing technique as a complementary tool for the forensic analysis of building structures.

(Table1) Sedimentation rates and age-control points form ODP Hole 152-919A

Planktic d18O and d13C records and point count records of biogenic, volcanic, and nonvolcanic terrigenous [ice-rafted debris (IRD)] sediment components from Hole 919A in the Irminger basin, northern North Atlantic provide a comprehensive dataset from which a paleoceanographic reconstruction for the last 630 kyr has been developed. The paleoceanographic evolution of the Irminger basin during this time contains both long-term patterns and significant developmental steps. One long-term pattern observed is the persistent deposition of hematite-stained ice-rafted debris. This record suggests that the modern and late Pleistocene discharges of icebergs from northern redbed regions to the Irminger Sea lie in the low end of the range observed over the last 630 kyr. In addition, Arctic front fluctuations appear to have been the main controlling factor on the long-term accumulation patterns of IRD and planktic biogenic groups. The Hole 919A sediment record also contains a long-term association between felsic volcanic ash abundances and light d18O excursions in both interglacial and glacial stages, which suggests a causal link between deglaciations and explosive Icelandic eruptions. A significant developmental step in the paleoceanographic reconstruction based on benthic evidence was for diminished supply of Denmark Strait Overflow Water (DSOW) beginning at ~380 ka, possibly initiated by the influx of meltwater from broad-scale iceberg discharges along the east Greenland coast. There is also planktic evidence of a two-step cooling of sea surface conditions in the Irminger basin, first at ~338-309 ka and later at ~211-190 ka, after which both glacials and interglacials were colder as the Arctic front migrated southeast of Site 919. In addition to offering these findings, this reconstruction provides a longer-term geologic context for the interpretation of more recent paleoceanographic events and patterns of deposition from this region.