Conceptual designs for in situ analysis of Mars soil

A goal of this research is to develop conceptual designs for instrumentation to perform in situ measurements of the Martian soil in order to determine the existence and nature of any reactive chemicals. Our approach involves assessment and critical review of the Viking biology results which indicated the presence of a soil oxidant, an investigation of the possible application of standard soil science techniques to the analysis of Martian soil, and a preliminary consideration of non-standard methods that may be necessary for use in the highly oxidizing Martian soil. Based on our preliminary analysis, we have developed strawman concepts for standard soil analysis on Mars, including pH, suitable for use on a Mars rover mission. In addition, we have devised a method for the determination of the possible strong oxidants on Mars

The Role of Integrin-associated Protein “PINCH” in Kidney Development

PINCH (a particularly interesting new cysteine-histidine-rich protein) is a protein that binds with Integrin-linked kinase and Parvin, to form an IPP complex. This complex is formed only when integrin molecules are activated upon interaction with the basement membrane. The IPP complex then acts as a platform for other proteins to come together and regulate cell signaling to and from the extracellular matrix (ECM). This allows for various cellular activities to progress like cell adhesion, migration, proliferation, etc. There are two types of PINCH proteins, PINCH-1 and PINCH-2. Studies from our group have shown that the lack of PINCH-1 in the kidney results in a branching phenotype and smaller kidneys in mice. The cells showed diminished potential to adhere, migrate and proliferate. There is no significant difference in phenotype when PINCH-2 is deleted. In the present study, we use conditional double knockout mice and cells in culture to look at the additive effects of the two PINCH proteins. Stained kidney cross sections will be analyzed and the PINCH null cells will be used in cell adhesion and migration studies. We have preliminary data to show that the deletion of both PINCH-1 and PINCH-2 genes results in a severe developmental phenotype in mouse kidney and corroborates in vitro studies

Getting water from the water of hydration on Mars

Both Viking landers found evidence of water in small concentration in the soils of Mars. Using the gas chromatograph mass spectrometer the soil samples on Mars were heated to 500 C to release the water. This result lead researchers to believe that the water in the soil of Mars was tightly bound in a hydration state. In the laboratory several Mars analog soils and a few bench mark soils were run through a microwave to determine the amount of water released using this method. The results suggest that sufficient water can be obtained using this method to augment the activities of a human base on Mars

Ex Vivo High Salt Activated Tumor-Primed CD4+T Lymphocytes Exert a Potent Anti-Cancer Response

Cell based immunotherapy is rapidly emerging as a promising cancer treatment. A modest increase in salt (sodium chloride) concentration in immune cell cultures is known to induce inflammatory phenotypic differentiation. In our current study, we analyzed the ability of salt treatment to induce ex vivo expansion of tumor-primed CD4 (cluster of differentiation 4)+T cells to an effector phenotype. CD4+T cells were isolated using immunomagnetic beads from draining lymph nodes and spleens from tumor bearing C57Bl/6 mice, 28 days post-injection of Py230 syngeneic breast cancer cells. CD4+T cells from non-tumor bearing mice were isolated from splenocytes of 12-week-old C57Bl/6 mice. These CD4+T cells were expanded ex vivo with five stimulation cycles, and each cycle comprised of treatment with high salt (Δ0.035 M NaCl) or equimolar mannitol controls along with anti-CD3/CD28 monoclonal antibodies for the first 3 days, followed by the addition of interleukin (IL)-2/IL-7 cytokines and heat killed Py230 for 4 days. Ex vivo high salt treatment induced a two-fold higher Th1 (T helper type 1) expansion and four-fold higher Th17 expansion compared to equimolar mannitol treatment. Importantly, the high salt expanded CD4+T cells retained tumor-specificity, as demonstrated by higher in vitro cytotoxicity against Py230 breast cancer cells and reduced in vivo syngeneic tumor growth. Metabolic studies revealed that high salt treatment enhanced the glycolytic reserve and basal mitochondrial oxidation of CD4+T cells, suggesting a role of high salt in enhanced pro-growth anabolic metabolism needed for inflammatory differentiation. Mechanistic studies demonstrated that the high salt induced switch to the effector phenotype was mediated by tonicity-dependent transcription factor, TonEBP/NFAT5. Using a transgenic murine model, we demonstrated that CD4 specific TonEBP/NFAT5 knock out (CD4cre/creNFAT5flox/flox) abrogated the induction of the effector phenotype and anti-tumor efficiency of CD4+T cells following high salt treatment. Taken together, our data suggest that high salt-mediated ex vivo expansion of tumor-primed CD4+T cells could induce effective tumor specific anti-cancer responses, which may have a novel cell-based cancer immunotherapeutic application

Mars Atmospheric Oxidant Sensor (MAOS): An In-Situ Heterogeneous Chemistry Analysis

We describe a chemometric array sensor, the Mars Atmospheric Oxidant Sensor (MAOS, pronounced "mouse '') that is designed measure the oxidation rate of thin films on the martian surface. We select films that are sensitive to particular types of oxidants, that represent key elements in the martian soil, or that emulate prebiotic materials. Concern that naturally arising martian oxidants may have destroyed evidence of ancient life on Mars was raised by the Viking mission in the 1970's. The possibility that oxidants may limit the viability of biologica1 habitats is particularly timely in light of recent suggestions of contemporary flowing water on Mars. By controlling the temperature of the films, as well as their exposure to dust and ultraviolet light, MAOS will discriminate among leading hypotheses for oxidant production. MAOS weighs 55 grams, fits in a 6 x 7 x 2 cm envelope, and uses 250 mW power. Much of the enabling technology was developed for the MOx experiment, lost on the Russian Mars '96 mission

Oligodeoxynucleotides ODN 2006 and M362 Exert Potent Adjuvant Effect through TLR-9/-6 Synergy to Exaggerate Mammaglobin-A Peptide Specific Cytotoxic CD8+T Lymphocyte Responses against Breast Cancer Cells

Mammaglobin-A (MamA) is overexpressed in 40–80% of all human breast cancers. Recent phase I clinical trials of the MamA DNA vaccine showed encouraging safety outcomes. However, this vaccine elicited only a modest increase in MamA specific CD8+T lymphocyte (CTL) activation. As vaccine adjuvants play a critical role in enhancing the immunotherapeutic efficiency of vaccines, we tested the potential role of three synthetic CpG oligodeoxynucleotides (ODN2216—class A ODN, ODN2006—class B ODN, and ODN M362—class C ODN) to further enhance MamA specific CTL responses. Towards this, naïve CD8+T cells were obtained from healthy HLA-A2+ human donors. The HLA-A2 specific immunodominant epitope of MamA, MamA2.1 (LIYDSSLCDL), was utilized to activate naïve CD8+T cells. The THP-1 (HLA-A2+) cells were used as antigen presenting cells to stimulate naïve CD8+T cells along with (or without) co-treatment of various ODNs mentioned above. Activation of naïve CD8+T cells with the MamA2.1 peptide along with ODNs demonstrated enhanced MamA specific CTL mediated cytotoxicity on AU565 (HLA-A+/MamA+) breast cancer cells following co-treatment with ODN2006 and M362 compared to ODN2216 or MamA2.1 peptide alone. However, no significant cytotoxicity was noted upon treatment of MamA2.1 activated CTLs on MCF7 (HLA-A+/MamA−) cells, suggesting that the activation of CTLs is specific to the MamA antigen. Functional characterization studies demonstrated specific IL-12 mediated cross-talk between TLR-6 and -9 in THP-1 cells following stimulation with ODN2006 and M362, which was critical for the final cytotoxic activation of CD8+T lymphocytes. Based on these data, we conclude that ODN2006 and ODN M362 exerted a strong adjuvant effect through induction of the initial innate immune response through TLR9 upregulation followed by enhanced MamA specific CTL dependent adaptive immune responses. Our current data provide evidence for the application of Class-B/-C-CpG-ODNs as potential vaccine adjuvants towards enhancing the success of MamA based breast cancer vaccination

Implications of the differing roles of the beta 1 and beta 3 transmembrane and cytosplasmic domains for integrin function

Integrins are transmembrane receptors composed of alpha and beta subunits. Although most integrins contain beta 1, canonical activation mechanisms are based on studies of the platelet integrin, allb beta 3. Its inactive conformation is characterized by the association of the alpha llb transmembrane and cytosolic domain (TM/CT) with a tilted beta 3 TM/CT that leads to activation when disrupted. We show significant structural differences between beta 1 and beta 3 TM/CT in bicelles. Moreover, the 'snorkeling' lysine at the TM/CT interface of beta subunits, previously proposed to regulate alpha llb beta 3 activation by ion pairing with nearby lipids, plays opposite roles in beta 1 and beta 3 integrin function and in neither case is responsible for TM tilt. A range of affinities from almost no interaction to the relatively high avidity that characterizes alpha llb beta 3 is seen between various alpha subunits and beta 1 TM/CTs. The alpha llb beta 3-based canonical model for the roles of the TM/CT in integrin activation and function clearly does not extend to all mammalian integrins

Membrane-Type 4 Matrix Metalloproteinase (MT4-MMP) Modulates Water Homeostasis in Mice

MT4-MMP is a membrane-type metalloproteinase (MMP) anchored to the membrane by a glycosyl-phosphatidylinositol (GPI) motif. GPI-type MT-MMPs (MT4- and MT6-MMP) are related to other MT-MMPs, but their physiological substrates and functions in vivo have yet to be identified. In this manuscript we show that MT4-MMP is expressed early in kidney development, as well as in the adult kidney, where the highest levels of expression are found in the papilla. MT4-MMP null mice had minimal renal developmental abnormalities, with a minor branching morphogenesis defect in early embryonic kidney development and slightly dysmorphic collecting ducts in adult mice. Interestingly, MT4-MMP null mice had higher baseline urine osmolarities relative to wild type controls, but these animals were able to concentrate and dilute their urines normally. However, MT4-MMP-null mice had decreased daily water intake and daily urine output, consistent with primary hypodipsia. MT4-MMP was shown to be expressed in areas of the hypothalamus considered important for regulating thirst. Thus, our results show that although MT4-MMP is expressed in the kidney, this metalloproteinase does not play a major role in renal development or function; however it does appear to modify the neural stimuli that modulate thirst

The Thermal Electrical Conductivity Probe (TECP) for Phoenix

The Thermal and Electrical Conductivity Probe (TECP) is a component of the Microscopy, Electrochemistry, and Conductivity Analyzer (MECA) payload on the Phoenix Lander. TECP will measure the temperature, thermal conductivity and volumetric heat capacity of the regolith. It will also detect and quantify the population of mobile H2O molecules in the regolith, if any, throughout the polar summer, by measuring the electrical conductivity of the regolith, as well as the dielectric permittivity. In the vapor phase, TECP is capable of measuring the atmospheric H2O vapor abundance, as well as augment the wind velocity measurements from the meteorology instrumentation. TECP is mounted near the end of the 2.3 m Robotic Arm, and can be placed either in the regolith material or held aloft in the atmosphere. This paper describes the development and calibration of the TECP. In addition, substantial characterization of the instrument has been conducted to identify behavioral characteristics that might affect landed surface operations. The greatest potential issue identified in characterization tests is the extraordinary sensitivity of the TECP to placement. Small gaps alter the contact between the TECP and regolith, complicating data interpretation. Testing with the Phoenix Robotic Arm identified mitigation techniques that will be implemented during flight. A flight model of the instrument was also field tested in the Antarctic Dry Valleys during the 2007-2008 International Polar year.

Chemistry and Mineralogy of Antarctica Dry Valley Soils: Implications for Mars

The Antarctic Dry Valleys (ADV) comprise the largest ice-free region of Antarctica. Precipitation almost always occurs as snow, relative humidity is frequently low, and mean annual temperatures are about -20 C. The ADV soils have previously been categorized into three soil moisture regimes: subxerous, xerous and ultraxerous, based on elevation and climate influences. The subxerous regime is predominately a coastal zone soil, and has the highest average temperature and precipitation, while the ultraxerous regime occurs at high elevation (>1000 m) and have very low temperature and precipitation. The amounts and types of salts present in the soils vary between regions. The nature, origin and significance of salts in the ADV have been previously investigated. Substantial work has focused on soil formation in the ADVs, however, little work has focused on the mineralogy of secondary alteration phases. The dominant weathering process in the ADV region is physical weathering, however, chemical weathering has been well documented. The objective of this study was to characterize the chemistry and mineralogy, including the alteration mineralogy, of soils from two sites, a subxerous soil in Taylor Valley, and an ultraxerous soil in University Valley. The style of aqueous alteration in the ADVs may have implications for pedogenic processes on Mars