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At least 631 records · Page 35
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

How a Formate Dehydrogenase Responds to Oxygen: Unexpected O 2 Insensitivity of an Enzyme Harboring Tungstopterin, Selenocysteine, and [4Fe–4S] Clusters

The reversible two-electron interconversion of formate and CO 2 is catalyzed by both nonmetallo- and metallo-formate dehydrogenases (FDHs). The latter group comprises molybdenum- or tungsten-containing enzymes with the metal coordinated by two equivalents of a pyranopterin cofactor, a cysteinyl or selenocysteinyl (Sec) ligand supplied by the polypeptide, and a catalytically essential terminal sulfido ligand. In addition, these biocatalysts incorporate one or more [4Fe–4S] clusters for facilitating long-distance electron transfer. However, an interesting dichotomy arises when attempting to understand how the metallo-FDHs react with O 2 . Whereas existing scholarship portrays these enzymes as being unable to perform in air due to extreme O 2 lability of their metal centers, studies dating as far back as the 1930s emphasize that some of these systems exhibit formate oxidase (FOX) activity, coupling formate oxidation to O 2 reduction. Therefore, to reconcile these conflicting views, we explored context-dependent functional linkages between metallo-FDHs and their cognate electron acceptors within the same organism vis-à-vis catalysis under atmospheric O 2 . Here, we report the discovery and characterization of an O 2 -insensitive FDH2 from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) that ligates tungsten, Sec, and four [4Fe–4S] clusters. By advancing a robust expression platform for its recombinant production, we eliminate both the requirement of nitrate or azide during purification and reductive activation with thiols and/or formate prior to catalysis. Because the distinctive spectral signatures of formate-reduced DvH-FDH2 remain invariant under anaerobic and aerobic conditions, we benchmarked the enzyme activity in air, identifying CO 2 as the catalytic product. Full reaction progress curve analysis discloses a high catalytic efficiency when probed with a high-potential artificial electron acceptor. Furthermore, we show that DvH-FDH2 enables near-stoichiometric hydrogen peroxide production without superoxide release to achieve O 2 insensitivity. Notably, simultaneous electron transfer to cytochrome c and O 2 reveals that metal-based electron bifurcation is operational in this system. Taken together, our work proves the co-occurrence of redox bifurcated FDH and FOX activities within a metalloenzyme scaffold. These findings set the stage for uncovering previously unknown O 2 -insensitive flavin-based electron bifurcation mechanisms, as well as for developing authentic formate/air biofuel cells, engineering O 2 -stable FDHs and biohybrid metallocatalysts, and discerning formate bioenergetics of gut microbiota.

13C NMR↗
DOE OSTI2014

Comparison of Subantarctic Mode Water and Antarctic Intermediate Water formation rates in the South Pacific between NCAR-CCSM4 and observations

Average formation rates for Subantarctic Mode (SAMW) and Antarctic Intermediate Waters (AAIW) in the South Pacific are calculated from the National Center for Atmospheric Research Community Climate System Model version 4 (NCAR-CCSM4), using chlorofluorocarbon inventories. CFC-12 inventories and formation rates are compared to ocean observations. CCSM4 accurately simulates the southeast Pacific as the main formation region for SAMW and AAIW. CCSM4 formation rates for SAMW are 3.4 Sv, about half of the observational rate. Shallow mixed layers and a thinner SAMW in CCSM4 are responsible for lower formation rates. A formation rate of 8.1 Sv for AAIW in CCSM4 is higher than observations. Higher inventories in CCSM4 in the southwest and central Pacific, and higher surface concentrations are the main reasons for higher formation rates of AAIW. This comparison of model and observations is useful for understanding the uptake and transport of other gases, e.g., CO2 by the model.

antarctic↗
DOE OSTI2017

Structure and decomposition of the silver formate Ag(HCO{sub 2})

Crystal structure of the silver formate Ag(HCO{sub 2}) has been determined (orthorhombic, sp.gr. Pccn, a=7.1199(5), b=10.3737(4), c=6.4701(3)Å, V=477.88(4) Å{sup 3}, Z=8). The structure contains isolated formate ions and the pairs Ag{sub 2}{sup 2+} which form the layers in (001) planes (the shortest Ag–Ag distances is 2.919 in the pair and 3.421 and 3.716 Å between the nearest Ag atoms of adjacent pairs). Silver formate is unstable compound which decompose spontaneously vs time. Decomposition was studied using Rietveld analysis of the powder diffraction patterns. It was concluded that the diffusion of Ag atoms leads to the formation of plate-like metal particles as nuclei in the (100) planes which settle parallel to (001) planes of the silver formate matrix. - Highlights: • Silver formate Ag(HCO{sub 2}) was synthesized and characterized. • Layered packing of Ag-Ag pairs in the structure was found. • Decomposition of Ag(HCO{sub 2}) and formation of metal phase were studied. • Rietveld-refined micro-structural characteristics during decomposition reveal the space relationship between the matrix structure and forming Ag phase REPLACE with: Space relationship between the matrix structure and forming Ag phase.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering

Conversion of CO 2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO 2 , has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin-Benson-Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. Further, the metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ..delta..phcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

First-principles characterization of formate and carboxyl adsorption on the stoichiometric CeO2(111) and CeO2(110) surfaces

Molecular adsorption of formate and carboxyl on the stoichiometric CeO2(111) and CeO2(110) surfaces was studied using periodic density functional theory (DFT+U) calculations. Two distinguishable adsorption modes (strong and weak) of formate are identified. The bidentate configuration is more stable than the monodentate adsorption configuration. Both formate and carboxyl bind at the more open CeO2(110) surface are stronger. The calculated vibrational frequencies of two adsorbed species are consistent with experimental measurements. Finally, the effects of U parameters on the adsorption of formate and carboxyl over both CeO2 surfaces were investigated. We found that the geometrical configurations of two adsorbed species are not affected by using different U parameters (U=0, 5, and 7). However, the calculated adsorption energy of carboxyl pronouncedly increases with the U value while the adsorption energy of formate only slightly changes (<0.2 eV). The Bader charge analysis shows the opposite charge transfer occurs for formate and carboxyl adsorption where the adsorbed formate is negatively charge whiled the adsorbed carboxyl is positively charged. Interestingly, with the increasing U parameter, the amount of charge is also increased. This work was supported by the Laboratory Directed Research and Development (LDRD) project of the Pacific Northwest National Laboratory (PNNL) and by a Cooperative Research and Development Agreement (CRADA) with General Motors. The computations were performed using the Molecular Science Computing Facility in the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL), which is a U.S. Department of Energy national scientific user facility located at PNNL in Richland, Washington. Part of the computing time was also granted by the National Energy Research Scientific Computing Center (NERSC)

adsorption↗
DOE OSTI2026

Base-Free Catalytic Transfer Hydrogenation of Alkyl Formates and Organic Esters at Mild Temperature

The pincer-ligated ruthenium complex ( iPr PN H P)Ru(CO)H 2 ( iPr PN H P = ( i Pr 2 PC 2 H 4 ) 2 NH) is an active catalyst for the transfer hydrogenation of alkyl formates (HCO 2 R) and organic esters (RCO 2 R′) to the corresponding alcohols under base-free reaction conditions at mild temperatures. Specifically, a range of alkyl formate esters were reduced to MeOH and the corresponding alcohols in high yields using ( iPr PN H P)Ru(CO)H 2 as the catalyst and isopropanol ( i PrOH) as the hydrogen donor at 30 °C. The first step in the process is the metal-catalyzed transesterification of the alkyl formate with i PrOH to generate isopropyl formate, which is then reduced. The use of i PrOH as the hydrogen donor is crucial. ( iPr PN H P)Ru(CO)H 2 can also catalyze the transfer hydrogenation of a broad range of organic esters, including cyclic, acyclic, heteroatom-substituted, and long-chain bio-derived esters, to the corresponding alcohols in good yields using ethanol (EtOH) as the hydrogen donor at 55 °C. Computational studies were used to elucidate the proposed pathway for alkyl formate reduction and the underlying reasons why i PrOH is the most effective hydrogen donor for alkyl formate reduction, while EtOH is optimal for organic ester reduction. Overall, this work describes a highly active catalyst for alkyl formate and organic ester transfer hydrogenation and provides mechanistic insight into the factors responsible for the strong catalytic performance. Finally, these findings will be valuable for designing catalysts for both transfer hydrogenation and related reactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Central star formation in double-peak, gas-rich radio galaxies

The respective contributions of gas accretion, galaxy interactions, and mergers to the mass assembly of galaxies, as well as the evolution of their molecular gas and star-formation activity are still not fully understood. In a recent work, a large sample of double-peak (DP) emission-line galaxies have been identified from the SDSS. While the two peaks could represent two kinematic components, they may be linked to the large bulges that their host galaxies tend to have. Star-forming DP galaxies display a central star-formation enhancement and have been discussed as compatible with a sequence of recent minor mergers. In order to probe merger-induced star-formation mechanisms, we conducted observations of the molecular-gas content of 35 star-forming DP galaxies in the upper part of the main sequence (MS) of star formation (SF) with the IRAM 30 m telescope. Including similar galaxies 0.3 dex above the MS and with existing molecular-gas observations from the literature, we finally obtained a sample of 52 such galaxies. We succeeded in fitting the same kinematic parameters to the optical ionised- and molecular-gas emission lines for ten (19%) galaxies. We find a central star-formation enhancement resulting most likely from a galaxy merger or galaxy interaction, which is indicated by an excess of gas extinction found in the centre. This SF is traced by radio continuum emissions at 150 MHz, 1.4 GHz, and 3 GHz, all three of which are linearly correlated in log with the CO luminosity with the same slope. The 52 DP galaxies are found to have a significantly larger amount of molecular gas and longer depletion times, and hence a lower star-formation efficiency, than the expected values at their distance of the MS. The large bulges in these galaxies might be stabilising the gas, hence reducing the SF efficiency. This is consistent with a scenario of minor mergers increasing the mass of bulges and driving gas to the centre. We also excluded the inwards-directed gas migration and central star-formation enhancement as the origin of a bar morphology. Hence, these 52 DP galaxies could be the result of recent minor mergers that funnelled molecular gas towards their centre, triggering SF, but with moderate efficiency.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2015

Core formation and core composition from coupled geochemical and geophysical constraints

The formation of Earth’s core left behind geophysical and geochemical signatures in both the core and mantle that remain to this day. Seismology requires that the core be lighter than pure iron and therefore must contain light elements, and the geochemistry of mantle-derived rocks reveals extensive siderophile element depletion and fractionation. Both features are inherited from metal–silicate differentiation in primitive Earth and depend upon the nature of physiochemical conditions that prevailed during core formation. To date, core formation models have only attempted to address the evolution of core and mantle compositional signatures separately, rather than seeking a joint solution. Here we combine experimental petrology, geochemistry, mineral physics and seismology to constrain a range of core formation conditions that satisfy both constraints. We find that core formation occurred in a hot (liquidus) yet moderately deep magma ocean not exceeding 1,800 km depth, under redox conditions more oxidized than present-day Earth. This new scenario, at odds with the current belief that core formation occurred under reducing conditions, proposes that Earth’s magma ocean started oxidized and has become reduced through time, by oxygen incorporation into the core. As a result, this core formation model produces a core that contains 2.7–5% oxygen along with 2–3.6% silicon, with densities and velocities in accord with radial seismic models, and leaves behind a silicate mantle that matches the observed mantle abundances of nickel, cobalt, chromium, and vanadium.

58 GEOSCIENCES↗
DOE OSTI2017

Age of Jupiter inferred from the distinct genetics and formation times of meteorites

The age of Jupiter, the largest planet in our Solar System, is still unknown. Gas-giant planet formation likely involved the growth of large solid cores, followed by the accumulation of gas onto these cores. Thus, the gas-giant cores must have formed before dissipation of the solar nebula, which likely occurred within less than 10 My after Solar System formation. Although such rapid accretion of the gas-giant cores has successfully been modeled, until now it has not been possible to date their formation. Here, using molybdenum and tungsten isotope measurements on iron meteorites, we demonstrate that meteorites derive from two genetically distinct nebular reservoirs that coexisted and remained spatially separated between ~1 My and ~3–4 My after Solar System formation. The most plausible mechanism for this efficient separation is the formation of Jupiter, opening a gap in the disk and preventing the exchange of material between the two reservoirs. As such, our results indicate that Jupiter’s core grew to ~20 Earth masses within <1 My, followed by a more protracted growth to ~50 Earth masses until at least ~3–4 My after Solar System formation. Furthermore, Jupiter is the oldest planet of the Solar System, and its solid core formed well before the solar nebula gas dissipated, consistent with the core accretion model for giant planet formation.

58 GEOSCIENCES↗
DOE OSTI2020

Induced Metal-free Star Formation around a Massive Black Hole Seed

The direct formation of a massive black hole is a potential seeding mechanism of the earliest observed supermassive black holes. We investigate how the existence of a massive black hole seed impacts the ionization and thermal state of its pre-galactic host halo and subsequent star formation. We show that its X-ray radiation ionizes and heats the medium, enhancing H 2 formation in shielded regions, within the nuclear region in the span of a million years. The enhanced molecular cooling triggers the formation of a ~10 4 M⊙ metal-free stellar cluster at a star formation efficiency of ~0.1% in a single event. Star formation occurs near the edges of the H ii region that is partially ionized by X-rays; thus, the initial size depends on the black hole properties and surrounding environment. Furthermore, the simulated metal-free galaxy has an initial half-light radius of ~10 pc but expands to ~50 pc after 10 million years because of the outward velocities of their birth clouds. Supernova feedback then quenches any further star formation for tens of millions of years, allowing the massive black hole to dominate the spectrum once the massive metal-free stars die.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2024

GraphTango: A Hybrid Representation Format for Efficient Streaming Graph Updates and Analysis

Abstract Streaming graph processing performs batched updates and analytics on a time-evolving graph. The underlying representation format of the graph largely determines the throughputs of these updates and analytics phases. Existing representation formats usually employ variations of hash tables or adjacency lists. However, a recent study showed that the adjacency-list-based approaches perform poorly on heavy-tailed graphs, and the hash table-based approaches suffer on short-tailed graphs. We propose GraphTango, a hybrid representation format that provides excellent update and analytics throughput regardless of the graph’s degree distribution. GraphTango dynamically switches among three different formats based on a vertex’s degree: (i) Low-degree vertices store the edges directly with the neighborhood metadata, confining accesses to a single cache line, (2) Medium-degree vertices use adjacency lists, and (3) High-degree vertices use hash tables as well as adjacency lists. In this case, the adjacency list provides fast traversal during the analytics phase, while the hash table provides constant-time lookups during the update phase. We further optimized the performance by designing an open-addressing-based hash table that fully utilizes every fetched cache line. In addition, we developed a thread-local lock-free memory pool that allows fast growing/shrinking of the adjacency lists and hash tables in a multi-threaded environment. We evaluated GraphTango with the help of the SAGA-Bench framework and compared it with four other representation formats: Stinger, Degree-aware Robin Hood Hashing, and two adjacency list-based formats with different workload balancing scheme. On average, GraphTango provides 4.5x higher insertion throughput, 3.2x higher deletion throughput, and 1.1x higher analytics throughput over the next best format. Furthermore, we integrated GraphTango with the state-of-the-art graph processing frameworks DZiG and RisGraph. Compared to the vanilla DZiG and vanilla RisGraph , [ GraphTango + DZiG ] and [ GraphTango + RisGraph ] reduces the average batch processing time by 2.3x and 1.5x, respectively.

Ahmed, Alif↗
DOE OSTI2017

Adduct Formation in ESI/MS by Mobile Phase Additives

Adduct formation is a common ionization method in electrospray ionization mass spectrometry (ESI/MS). However, this process is poorly understood and complicated to control. We demonstrate possibilities to control adduct formation via mobile phase additives in ESI positive mode for 17 oxygen and nitrogen bases. Mobile phase additives were found to be a very effective measure for manipulating the formation efficiencies of adducts. An appropriate choice of additive may increase sensitivity by up to three orders of magnitude. In general, sodium adduct [M + Na]{sup +} and protonated molecule [M + H]{sup +} formation efficiencies were found to be in good correlation; however, the former were significantly more influenced by mobile phase properties. Although the highest formation efficiencies for both species were observed in water/acetonitrile mixtures not containing additives, the repeatability of the formation efficiencies was found to be improved by additives. It is concluded that mobile phase additives are powerful, yet not limiting factors, for altering adduct formation. .

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2025

Glass formation during combinatorial sputtering in binary alloys

Glass formation is a complex phenomenon influenced by thermodynamic and kinetic aspects, which are often controlled by extrinsic contributions. While bulk metallic glasses are typically multicomponent alloys, binary alloys offer a simplified approach to studying glass formation. In this study, we fabricated 57 binary alloy systems through combinatorial sputtering, where each alloy system is represented in 66 different alloys. We developed an automated analysis to determine structure and composition using X-ray diffraction and energy-dispersive X-ray spectroscopy for over 3700 alloys. We found that ∼17 % of the alloys form glasses under the estimated cooling rate during sputtering of ∼10 8 K/s. Data analysis revealed that commonly used factors like atomic size ratio and heat of mixing are ineffective in predicting glass formation. However, the crystal structure mismatch of the alloys’ elements emerged as the strongest indicator of glass formation under sputtering conditions of binary alloys. Here, the differences in glass formation under slow cooling rates used for bulk glass formation and the here observed glass formation under rapid cooling rates are discussed.

Binary alloys↗
DOE OSTI2017

Rac1 regulates sepsis-induced formation of platelet-derived microparticles and thrombin generation

Dysfunctional coagulation aggravates clinical outcome in patients with sepsis. The aim of this study was to define the role of Rac-1 in the formation of platelet-derived microparticles (PMPs) and thrombin generation (TG) in abdominal sepsis. Male C57BL/6 mice underwent cecal ligation and puncture (CLP). Scanning electron microscopy and flow cytometry were used to quantify PMPs. TG was determined by use of a fluorimetric assay. It was found that CLP increased Rac1 activity in platelets, which was abolished by administration of the Rac1 inhibitor NSC23766. Sepsis-induced TG in vivo was reflected by reduced capacity of plasma from septic animals to generate thrombin ex vivo. Administration of NSC23766 increased peak and total TG in plasma from CLP mice indicating that Rac-1 regulates sepsis-induced formation of thrombin. The number of circulating PMPs was markedly elevated in animals with abdominal sepsis. Treatment with NSC23766 significantly decreased formation of PMPs in septic mice. Platelet activation in vitro caused release of numerous MPs. Notably, NSC23766 abolished PMP formation in activated platelets in vitro. These findings suggest that Rac-1 regulates PMP formation and TG in sepsis and that inhibition of Rac1 activity could be a useful target to inhibit dysfunctional coagulation in abdominal sepsis. - Highlights: • Rac-1 regulates platelet-derived microparticles formation in abdominal sepsis. • Rac-1 mediates formation of thrombin generation in abdominal sepsis. • Targeting Rac-1 activity might protect against coagulation dysfunction in sepsis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

An analysis of fluff formation in metallic fuel via data analyzes from EBR-II experiments and BISON fuel code modeling

During the operation of EBR-II, it was found that a highly porous structure (over 40% area fraction) formed at the top of several fuel columns. Previous work has shown that this structure, designated fluff in this paper, contains a significant fraction of fuel elements (e.g., U and Pu) which could potentially impact neutronics. This work aims in analyzing the formation mechanism of this microstructure so its impact can be incorporated into future metallic fuel modeling codes and algorithms. This paper details a preliminary examination into the formation mechanisms of fluff by performing qualitative and statistical analysis of EBR-II experimental data. Additionally, the operating conditions that have the greatest impact on fluff formation were determined based on this data set. Also, BISON fuel code simulations were used to help postulate potential fluff formation mechanisms. From this analysis it was found that the largest contributors to fluff formation were fuel burnup and composition, with fluff formation exhibiting a roughly linear positive correlation with increasing burnup and a negative correlation with increasing Pu content. It was also found that higher pin operating temperatures decreased fluff formation but only for U-10Zr fuels.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
DOE OSTI2013

Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis and mineralisation produced by GSK-3 inhibition. • In rats, 3 GSK-3 inhibitors produced a unique serum bone turnover biomarker profile. • Enhanced bone formation was seen within 7 to 14 days of compound treatment in rats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Study on the role of Hsa-miR-31-5p in hypertrophic scar formation and the mechanism

Highlights: • The level of miR31-5p in HS was higher than in NS. • MiR31-5p plays a central role for hypoxia induced ECM synthesis and contraction. • Blocking miR31-5p inhibits HIF-1a activation in hypoxic via upregulating FIH. Hypertrophic scar (HS) formation is associated with the fibrosis of fibrocytes caused by excessive extracellular matrix (ECM) synthesis and deposition, the initial event of HS formation. Our high throughput screen of miRNA expression profiles identified hsa-miR31-5p, whose transcription level was most differentially in normal skin fibroblasts (NS) and HS among other miRNAs. The level of hsa-miR31-5p in HS was significantly higher than in NS. In-vitro functional experiments showed hsa-miR31-5p knockdown remarkably suppressed the proliferation of hypertrophic scar fibroblasts (HSFBs) under hypoxia, promoted cell invasion, and inhibited the expression of Collagen I and III and Fibronectin (FN), suggesting that hsa-miR31-5p knockdown effectively reduces HS formation caused by excessive ECM synthesis and deposition in HSFBs under hypoxia. Mechanism study showed that the regulation of HS formation by hsa-miR31-5p was mediated by its target gene, factor-inhibiting HIF-1 (FIH): under hypoxia, hsa-miR31-5p down-regulated FIH and thus increased the level of hypoxia inducible factor-1α (HIF-1α), which subsequently activated the HIF-1α fibrosis regulation pathway in HSFBs, and stimulated the proliferation and ECM synthesis in HSFBs, eventually resulting in fibrosis and scar formation. The data also show that knockdown of hsa-miR31-5p in HSFBs impaired the trend of increased proliferation, reduced invasion and excessive ECM synthesis and deposition caused by HIF-1a activation under hypoxia through upregulating FIH, indicating that knockdown of hsa-miR31-5p effectively inhibits the formation of HS. In conclusion, hsa-miR31 -5p plays an important role in HS formation by inhibiting FIH and regulating the HIF-1α pathway. Therefore, hsa-miR31 -5p may be a novel therapeutic target for HS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

RalGPS2 is involved in tunneling nanotubes formation in 5637 bladder cancer cells

Highlights: • RalGPS2 is expressed in 5637 cancer cells and is essential for their growth. • RalGPS2 is required for TNT formation. • The overexpression of RalGPS2 PH domain markedly increased the number and length of TNT. • Interaction between RalA and Sec5 is required for TNT formation in 5637 cells. • RalGPS2 interacts with LST1 and RalA leading to TNT formation. RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

60 APPLIED LIFE SCIENCES↗