Functional proteomics and biochemsitry: working together to unravel mitochondrial phenomena of African trypansomes

Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of the mitochondrial model organism T. brucei and characterized its proteome. Our results show that the trypanosomal MOM proteome consists of 82 proteins. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. In mammalian cells, a putative tethering complex was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved.

Human NR5A1/SF-1 mutations show decreased activity on BDNF (brain-derived neurotrophic factor), an important regulator of energy balance: testing impact of novel SF-1 mutations beyond steroidogenesis

CONTEXT Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. OBJECTIVE To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. PATIENTS 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. METHODS SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). RESULTS Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. CONCLUSIONS Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations.

Networks of Information Exchange Under Uncertainty: The Case of Unconventional Gas Development in the United Kingdom

Modern policy-making is increasingly influenced by different types of uncertainty. Political actors are supposed to behave differently under the context of uncertainty then in “usual” decision-making processes. Actors exchange information in order to convince other actors and decision-makers, to coordinate their lobbying activities and form coalitions, and to get information and learn on the substantive issue. The literature suggests that preference similarity, social trust, perceived power and functional interdependence are particularly important drivers of information exchange. We assume that social trust as well as being connected to scientific actors is more important under uncertainty than in a setting with less uncertainty. To investigate information exchange under uncertainty analyze the case of unconventional shale gas development in the UK from 2008 till 2014. Our study will rely on statistical analyses of survey data on a diverse set of actors dealing with shale gas development and regulation in the UK.

Low Energy Neutral Atoms from the Heliosheath

In the heliosheath beyond the termination shock, low energy (<0.5 keV) neutral atoms are created by charge exchange with interstellar neutrals. Detecting these neutrals from Earth's orbit is difficult because their flux is reduced substantially by ionization losses as they propagate from about 100 to 1 AU and because there are a variety of other signals and backgrounds that compete with this weak signal. Observations from IBEX-Lo and -Hi from two opposing vantage points in Earth's orbit established a lower energy limit of about 0.1 keV on measurements of energetic neutral atoms (ENAs) from the heliosphere and the form of the energy spectrum from about 0.1 to 6 keV in two directions in the sky. Below 0.1 keV, the detailed ENA spectrum is not known, and IBEX provides only upper limits on the fluxes. However, using some assumptions and taking constraints on the spectrum into account, we find indications that the spectrum turns over at an energy between 0.1 and 0.2 keV.

Simulating Energy Transfer and Upconversion in β-NaYF 4 : Yb 3+ , Tm 3+

The design of upconversion phosphors with higher quantum yield requires a deeper understanding of the detailed energy transfer and upconversion processes between active ions inside the material. Rate equations can model those processes by describing the populations of the energy levels of the ions as a function of time. However, this model presents some drawbacks: energy migration is assumed to be infinitely fast, it does not determine the detailed interaction mechanism (multipolar or exchange), and it only provides the macroscopic averaged parameters of interaction. Hence, a rate equation model with the same parameters cannot correctly predict the time evolution of upconverted emission and power dependence under a wide range of concentrations of active ions. We present a model that combines information about the host material lattice, the concentration of active ions, and a microscopic rate equation system. The extent of energy migration is correctly taken into account because the energy transfer processes are described on the level of the individual ions. This model predicts the decay curves, concentration, and excitation power dependences of the emission. This detailed information can be used to predict the optimal concentration that results in the maximum upconverted emission.

Functional proteomics and biochemistry: working together to unravel mitochondrial phenomena of African trypanosomes

Eukaryotic cells are compartmentalized into membrane-bound organelles in order to provide sheltered reaction rooms for various specific processes. Organelles are not randomly distributed in a cell or operate isolated from each other. At the contrary — some organelles are closely linked and their functions are tightly orchestrated. The most well-known example of two such organelles acting in concert are the ER and the mitochondrion that work together in order to coordinate cellular lipid biosynthesis, maintain Ca2+-homeostasis, regulate mitochondrial division and control mitochondrial/ER shape as well as to synchronize the movement of these organelles within a cell. To study the mitochondrion and its interface to the ER requires a simplified mitochondrial system. African trypanosomes represent such a system. The unicellular parasite that causes devastating diseases in humans and animals has only one large mitochondrion that does not undergo fission/fusion events except for the context of cell division. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the protozoan. Central to the understanding of how mitochondria control their morphology, communicate with their surroundings and manage exchange of metabolites and transport of biopolymers (proteins, RNAs) is the mitochondrial outer membrane (MOM), as the MOM defines the boundary of the organelle. Recently, we have purified the MOM of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal MOM proteome consists of 82 proteins, two thirds of which have never been associated with mitochondria before. Among these, we identified novel factors required to regulate mitochondrial morphology and the long-elusive protein import machinery of T. brucei. A comparison with the MOM proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. One of these is the Miro-GTPase Gem1. In yeast, this Ca2+-EF-Hand containing polypeptide is thought to be involved in a protein complex that physically tethers the mitochondrion to the ER. Interestingly, a putative tethering complex in mammalian cells was linked to the mitochondrial fusion/fission machinery. Thus, the concept of a protein complex-mediated connection seems to be a general and conserved feature. We are currently investigating, if such a protein complex exists in T. brucei and if the trypanosomal Gem1 protein is involved. This ER-subdomain associated with mitochondria has been termed mitochondria-associated ER-membranes or MAM. The MAM has recently been implicated to play a key role in Alzheimer’s disease. It is therefore of broad and general interest to establish other eukaryotic model systems in order to investigate the MAM-MOM connection in more detail.

Charge exchange in cometary coma: Discovery of H- ions in the solar wind close to comet 67P/Churyumov-Gerasimenko

As Rosetta was orbiting comet 67P/Churyumov-Gerasimenko, the Ion and Electron Sensor detected negative particles with angular distributions like those of the concurrently measured solar wind protons but with fluxes of only about 10% of the proton fluxes and energies of about 90% of the proton energies. Using well-known cross sections and energy-loss data, it is determined that the fluxes and energies of the negative particles are consistent with the production of H- ions in the solar wind by double charge exchange with molecules in the coma.

Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

Functional analysis of BLAP75, a BLM-associated protein

Bloom syndrome (BS) is an autosomal recessive disorder characterized by dwarfism, immunodeficiency, impaired fertility, and most importantly, early development of a broad range of cancers. The hallmark of BS cells is hyper-recombination, characterized by a drastically elevated frequency of sister chromatid exchange (SCE). BLM, the gene mutated in BS, encodes a DNA helicase of the RecQ protein family. BLM is thought to participate in several DNA transactions and to interact with many proteins involved in DNA replication, recombination, and repair. However, the precise function of BLM and the BLM-dependent anti-tumor mechanism remain obscure. ^ A novel protein, BLAP75 (BLM-associated polypeptide, 75KD), was identified to form an evolutionarily conserved complex with BLM and DNA topoisomerase IIIα (Topo IIIα). Our work demonstrates that loss of BLAP75 destabilized BLM and Topo IIIα proteins. BLAP75 colocalized with BLM in subnuclear foci in response to DNA damage and the recruitment of BLM to these foci was BLAP75-dependent. Moreover, depletion of BLAP75 by siRNA resulted in an elevated SCE rate similar to cells depleted of BLM by siRNA. In addition, RNAi-mediated silencing of BLAP75 greatly diminished cell viability. This cellular deficiency was rescued by expression of wild type BLAP75 but not BLAP75 with mutated conserved domain III, which abrogated the interaction between BLAP75, BLM and Topo IIIα, suggesting that the integrity of BLM-Topo IIIα-BLAP75 complex might be critical for cell survival. Finally, I found that BLAP75 was phosphorylated during mitosis and upon various DNA-damaging agents, implying that BLAP75 might also function in mitosis and DNA damage response. ^ Taken together, this study has defined BLAP75 as an integral component of the BLM complex to maintain genome stability. Our findings provide insights into the molecular mechanisms of the BLM helicase pathway and tumorigenesis process associated with these mechanisms. ^

Spectroscopic and functional investigations of the AMPA subtype of ionotropic glutamate receptors

Ion channels play a crucial role in the functioning of different systems of the body because of their ability to bridge the cell membrane and allow ions to pass in and out of the cell. Ionotropic glutamate receptors are one class of these important proteins and have been shown to be critical in propagating synaptic transmission in the central nervous system and in other diverse functions throughout the body. Because of their wide-ranging effects, this family of receptors is an important target for structure-function investigations to understand their mechanism of action. ^ α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one subtype of glutamate receptors and have been shown to be the primary receptors involved in rapid excitatory signaling in the central nervous system. Agonist binding to the extracellular ligand binding domain of these receptors causes various conformational changes that culminate in formation of the ion channel. Previous structural investigations have provided important information about their mechanism of action, including uncovering a relationship between the degree of cleft closure in the binding domain and activation of the receptor. However, what question remains unanswered is how specific interactions between the agonist and the protein interplay with cleft closure to mediate receptor activation. ^ To investigate this question, I applied a multiscale approach to investigate the effects of agonist binding on various levels. Vibrational spectroscopy was utilized to investigate molecular-level interactions in the binding pocket, and fluorescence resonance energy transfer (FRET) was employed to measure cleft closure in the isolated ligand binding domain. The results of these studies in the isolated binding domain were then correlated to activation of the full receptor. These investigations showed a relationship between the strength of the interaction at the α-amine group of the agonist and extent of receptor activation, where a stronger interaction correlated to a larger activation, which was upheld even when the extent of cleft closure did not correlate to activation. These results show that this interaction at the α-amine group is critical in mediating the allosteric mechanism of activation and provide a bit more insight into how agonist binding is coupled to channel gating in AMPA receptors. ^

Health information exchange: Using metrics to address quality of care and return on investment

Health Information Exchange (HIE) will play a key part in our nation’s effort to improve healthcare. The evidence of HIEs transformational role in healthcare delivery systems is quite limited. The lack of such evidence led us to explore what exists in the healthcare industry that may provide evidence of effectiveness and efficiency of HIEs. The objective of the study was to find out how many fully functional HIEs are using any measurements or metrics to gauge impact of HIE on quality improvement (QI) and on return on investment (ROI).^ A web-based survey was used to determine the number of operational HIEs using metrics for QI and ROI. Our study highlights the fact that only 50 percent of the HIEs who responded use or plan to use metrics. However, 95 percent of the respondents believed HIEs improve quality of care while only 56 percent believed HIE showed positive ROI. Although operational HIEs present numerous opportunities to demonstrate the business model for improving health care quality, evidence to document the impact of HIEs is lacking. ^

(Table 1) Biomass (as energy) of different elements of the planktonic community in the Equatorial Pacific in the 0-150 m layer

An analysis was made of composition and content of nutrients, salts, particulate and dissolved organic matter, and various plankton groups in a series of samples collected by a 140-liter sampling bottle to depth up to 150 m at 4 equatorial stations between 97° and 154°W. Large and small phytoplankton, bacteria (aggregated and dispersed), heterotrophic flagellates, infusorians, radiolarians, foraminifers, fine filter-feeders, small and large, mostly herbivorous copepods, cyclopoids, predatory calanoids, and other predators were investigated separately. Trophic relations between these elements are established from personal and published data, and rate of their metabolism and some other physiological parameters are determined. Such functional characteristics as extent of satisfaction of food requirements of organisms belonging to various trophic groups, intensity of trophic relations, balance between production and consumption by individual elements of the community, ecological efficiency, and net and specific production of the groups distinguished, of individual trophic levels, of total zooplankton, and of the community as a whole are calculated. Variations of these characteristics along the equator with decreasing upwelling intensity are examined and their possible causes and mechanisms are discussed.

Introducing Nuclear Energy to High-School Students: The Spanish Young Generation in Nuclear (Jóvenes Nucleares) Lectures

One of the main goals of Spanish Young Generation (JJNN) is to spread knowledge about nuclear energy, not only pointing out its advantages and its role in our society, but also trying to correct some of the ideas that are due to the biased information and to the lack of knowledge. With this goal in mind, lectures were given in several high schools, aimed at students ranging from 14 to 18 years old. This paper explains the experience accumulated during those talks and the conclusions that can be drawn, so as to better focus the communication about nuclear energy, especially the one aimed at a young public. In order to evaluate the degree of knowledge and information on a specific topic of a given group of individuals, statistical methods must be used. At the beginning of each lecture (and sometimes at the end, in order to evaluate the impact of the talk) the students were submitted to a short survey conducted by Spanish Young Generation. It consisted in eight questions, dealing with the relation between the main environmental issues (global warming, acid rain, radioactive waste…) and nuclear energy. The answers can be surprising, especially for professionals of the nuclear field who, since they are so familiar with this topic, often forget that this is just the case of a minority of people. A better knowledge of the degree of information of a given group enables to focus and personalize the communication. Another communication tool is the direct contact with students: it starts with their questions, which can then lead to a small debate. If the surveys inform about the topics they are unaware of, the direct exchange with them enables to find the most effective way to provide them the information. Of course, it depends a lot on the public attending the talk (age, background…) and on the debate following the talk: a good communication, adapted to the public, is necessary. Therefore, the outcome of the performed exercise is that Spanish teenagers have still a lack of knowledge about nuclear energy. We can learn that items that are evident for nuclear young professionals are unknown for high school teenagers

Electronic property analysis of O-doped Cu3SbS3

The ternary Cu-Sb-S semiconductors are considered to be sustainable and potential alternative absorber materials in thin film photovoltaic applications. In these compounds, several phases may coexist, albeit in different proportions depending on experimental growth conditions. Additionally, the photovoltaic efficiency could be increased with isoelectronic doping. In this work we analyze the electronic properties of O-doped Cu3SbS3 in two structures: the wittichenite and the skinnerite. We use first-principles within the density functional formalism with two different exchange-correlation potentials. In addition, we estimate the potential of these compounds for photovoltaic applications.

Functional roles of HypC and HupK in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum.

Some rhizobia induce a hydrogen (H2)-uptake system with a [NiFe] hydrogenase along with nitrogenase to recover part of the energy lost as H2. Biosynthesis of NiFe hydrogenases is a process that ocurrs in the cytoplasm, where a number of auxiliary proteins (products of hup and hyp genes) are required to synthesize and insert the metal cofactors into the enzyme structural units. Although HypC is expressed in all hydrogenase systems, HupF and HupK are found only in bacteria that express the hydrogenase in the presence of oxygen (O2). Co-purification experiments have demonstrated HypC-HupK and HypC-HupL interactions. Results have shown that some conserved residues from HypC and HupK play a protective role of hydrogenase against the presence of O2.