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At least 1,657 records · Page 92

Downregulation of hsa{sub c}irc{sub 0}007534 suppresses breast cancer cell proliferation and invasion by targeting miR-593/MUC19 signal pathway

Highlights: • We reported circular RNA, has{sub c}irc{sub 0}007534 in breast cancer (BC). • We reported mircro-RNA, miR-593 in breast cancer. • Has{sub c}irc{sub 0}007534 sponges miR-593 in BC. • MiR-593 directly targets MUC19 in BC. • Has{sub c}irc{sub 0}007534/miR-593/MUC19 axis plays critical role in BC pathogenesis. Circular RNAs (circRNAs) are important non-coding RNAs that are reportedly involved in the progression of diverse human cancers through their action as a microRNA (miRNA) sponge. However, the exact roles of circRNAs in breast cancer (BC) remain largely unknown. The present data demonstrate the significantly upregulated expression of hsa{sub c}irc{sub 0}007534 circRNA in BC tissues and cell lines. In contrast, miR-593 expression was significantly downregulated. Knockdown of hsa{sub c}irc{sub 0}007534 inhibited BC cell proliferation, colony formation, and invasion, and promoted apoptosis in BC cells. Moreover, hsa{sub c}irc{sub 0}007534 was demonstrated to be a sponge of miR-593, and expression of miR-593 in BC cells was negatively correlated with hsa{sub c}irc{sub 0}007534. MUC19 expression was markedly increased in BC tissues and cell lines, and the 3-UTR of MUC19 was targeted by miR-593. The expression of MUC19 was negatively regulated by miR-593 in BC cells. Our findings suggest an oncogenic role for hsa{sub c}irc{sub 0}007534 in BC by acting as a miR-593 sponge to promote MUC19 expression.

60 APPLIED LIFE SCIENCES↗

Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c

Highlights: • Minocycline blocked cytochrome c-mediated peroxidative permeabilization of liposomes. • Minocycline prevented cytochrome c binding to cardiolipin-containing liposomes. • Minocycline suppressed cardiolipin-stimulated peroxidase activity of cytochrome c. • This could be relevant to minocycline inhibition of cyt c release from mitochondria. Peroxidase activity of cytochrome c stimulated by interaction of the protein with cardiolipin is considered to be involved in the induction of mitochondrial apoptosis associated with cytochrome c release from mitochondria. In model systems, this activity has been previously shown to cause permeabilization of cardiolipin-containing membranes. Here, we found that the antibiotic minocycline, known to have neuroprotective properties, inhibited both the binding of cyt c to cardiolipin-containing membranes and the cyt c/H{sub 2}O{sub 2}-induced liposome permeabilization. The results could be relevant to inhibition of cyt c release from mitochondria exerted by minocycline.

60 APPLIED LIFE SCIENCES↗

The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

The proton-driven ATP synthase (F O F 1 ) is comprised of two rotary, stepping motors (F O and F 1 ) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded F O c-ring. Proton transport by F O is firmly coupled to the rotation of the c-ring relative to other F O subunits (ab 2 ). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure. Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.

36 MATERIALS SCIENCE↗

Impact of water vapour and carbon dioxide on surface composition of C{sub 3}A polymorphs studied by X-ray photoelectron spectroscopy

The surface specific analytical method, X-ray photoelectron spectroscopy (XPS), has been used to study the effects of water vapour and CO{sub 2} on the cubic and orthorhombic polymorphs of C{sub 3}A. Significant differences between the two polymorphs were observed in the XPS spectra. Upon exposure to water vapour, both polymorphs produced C{sub 4}AH{sub 13} on their surfaces. Additionally, the sodium-doped o-C{sub 3}A developed NaOH and traces of C{sub 3}AH{sub 6} on its surface. Subsequent carbonation yielded mono carboaluminate on both polymorphs. Large amounts of Na{sub 2}CO{sub 3} also formed on the surface of o-C{sub 3}A as a result of carbonation of NaOH. Furthermore, the extent of carbonation was much more pronounced for o-C{sub 3}A{sub o} than for c-C{sub 3}A.

36 MATERIALS SCIENCE↗

Effects of pH on the nano/micro structure of calcium silicate hydrate (C-S-H) under sulfate attack

Calcium silicate hydrate (C-S-H), the most important Portland cement hydration product, determines the mechanical properties and durability of cementitious materials. In marine environment, C-S-H often suffers from sulfate attack – one of the most common and severe degradations for concrete. pH is considered as a critical factor in determining the deterioration behavior of C-S-H during sulfate attack, of which the significance may be overlooked. This study focused on the role of pH on the deterioration of C-S-H in terms of the composition and nano/micro structure under sulfate attack. The results show that lowering pH aggravates the decalcification of C-S-H, whereas a strong alkaline condition is beneficial to increase the resistance to deterioration. An increase in the mean chain length together with the proportion of large pores (>10 nm) is observed when C-S-H is subjected to low-pH sulfate attack (pH = 10–12), resulting in a relatively loose structure. The deep analysis on defective tobermorite model reveals that calcium at the interlayer of C-S-H nanostructure is readily removed under sulfate attack, thereby improving the potential of Si-O-Si groups formation and increases the mean chain length of C-S-H.

36 MATERIALS SCIENCE↗

Planning Evaluation of C-Arm Cone Beam CT Angiography for Target Delineation in Stereotactic Radiation Surgery of Brain Arteriovenous Malformations

Purpose: Stereotactic radiation surgery (SRS) is one of the therapeutic modalities currently available to treat cerebral arteriovenous malformations (AVM). Conventionally, magnetic resonance imaging (MRI) and MR angiography (MRA) and digital subtraction angiography (DSA) are used in combination to identify the target volume for SRS treatment. The purpose of this study was to evaluate the use of C-arm cone beam computed tomography (CBCT) in the treatment planning of SRS for cerebral AVMs. Methods and Materials: Sixteen consecutive patients treated for brain AVMs at our institution were included in this retrospective study. Prior to treatment, all patients underwent MRA, DSA, and C-arm CBCT. All images were coregistered using the GammaPlan planning system. AVM regions were delineated independently by 2 physicians using either C-arm CBCT or MRA, resulting in 2 volumes: a CBCT volume (VCBCT) and an MRA volume (V{sub MRA}). SRS plans were generated based on the delineated regions. Results: The average volume of treatment targets delineated using C-arm CBCT and MRA were similar, 6.40 cm{sup 3} and 6.98 cm{sup 3}, respectively (P=.82). However, significant regions of nonoverlap existed. On average, the overlap of the MRA with the C-arm CBCT was only 52.8% of the total volume. In most cases, radiation plans based on V{sub MRA} did not provide adequate dose to the region identified on C-arm CBCT; the mean minimum dose to V{sub CBCT} was 29.5%, whereas the intended goal was 45% (P<.001). The mean volume of normal brain receiving 12 Gy or more in C-arm CBCT-based plans was not greater than in the MRA-based plans. Conclusions: Use of C-arm CBCT images significantly alters the delineated regions of AVMs for SRS planning, compared to that of MRA/MRI images. CT-based planning can be accomplished without increasing the dose to normal brain and may represent a more accurate definition of the nidus, increasing the chances for successful obliteration.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Microstructure and mechanical properties of titanium aluminum carbides neutron irradiated at 400–700 °C

Here, this work reports the first mechanical properties of Ti 3 AlC 2 -Ti 5 Al 2 C 3 materials neutron irradiated at ~400, 630 and 700 °C at a fluence of 2 × 10 25 n m -2 (E > 0.1 MeV) or a displacement dose of ~2 dpa. After irradiation at ~400 °C, anisotropic swelling and loss of 90% flexural strength was observed. After irradiation at ~630–700 °C, properties were unchanged. Microcracking and kinking-delamination had occurred during irradiation at ~630–700 °C. Further examination showed no cavities in Ti 3 AlC 2 after irradiation at ~630 °C, and MX and A lamellae were preserved. However, disturbance of (0004) reflections corresponding to M-A layers was observed, and the number density of line/planar defects was ~10 23 m -3 of size 5–10 nm. HAADF identified these defects as antisite Ti Al atoms. Finally, Ti 3 AlC 2 -Ti 5 Al 2 C 3 shows abrupt dynamic recovery of A-layers from ~630 °C, but a higher temperature appears necessary for full recovery.

36 MATERIALS SCIENCE↗

Effects of neutron irradiation of Ti 3 SiC 2 and Ti 3 AlC 2 in the 121–1085 °C temperature range

Herein we report on the formation of defects in response to neutron irradiation of polycrystalline Ti 3 SiC 2 and Ti 3 AlC 2 samples exposed to doses of 0.14±0.01, 1.6±0.1, and 3.4±0.1 displacements per atom (dpa) at irradiation temperatures of 121±12, 735±6 and 1085±68 °C. After irradiation to 0.14 dpa at 121 °C and 735 °C, black spots are observed in both Ti 3 SiC 2 and Ti 3 AlC 2 . After irradiation to 1.6 and 3.4 dpa at 735 °C, basal dislocation loops, with a Burgers vector of b = ½ [0001] are observed in Ti 3 SiC 2 , with loop diameters of 21±6 and 30±8 nm for 1.6 dpa and 3.4 dpa, respectively. In Ti3AlC2, larger dislocation loops, 75±34 nm in diameter are observed after 3.4 dpa at 735 °C, in addition to stacking faults. Impurity particles of TiC, as well as stacking fault TiC platelets in the MAX phases, are seen to form extensive dislocation loops under all conditions. Voids are observed at grain boundaries and within stacking faults after 3.4 dpa irradiation, with extensive void formation in the TiC regions at 1085 °C. Remarkably, denuded zones on the order of 1 µm are observed in Ti 3 SiC 2 after irradiation to 3.4 dpa at 735 °C. Small grains, 3-5 µm in diameter, are damage free after irradiation at 1085 °C at this dose. The presence of the A-layer in the MAX phases is seen to provide enhanced irradiation tolerance. Based on these results, and up to 3.41 dpa, Ti 3 SiC 2 remains a promising candidate for high temperature nuclear applications.

36 MATERIALS SCIENCE↗

TiO{sub 2} nanobelts with a uniform coating of g-C{sub 3}N{sub 4} as a highly effective heterostructure for enhanced photocatalytic activities

A novel g-C{sub 3}N{sub 4}/TiO{sub 2} nanobelt (NB) heterostructure was successfully designed and prepared. The as-prepared g-C{sub 3}N{sub 4}/TiO{sub 2} NB heterostructure exhibited high photocatalytic activity not only in the photodegradation of Rhodamine B (RhB) but also in photocatalytic H{sub 2} production. The g-C{sub 3}N{sub 4}/TiO{sub 2} NB heterostructure with a mass ratio of 1:1 demonstrated the best performance in the photodegradation of RhB, whereas a mass ratio of 3:1 demonstrated the highest H{sub 2} production rate of 46.6 μmol h{sup −1} in photocatalytic H{sub 2} production. We conclude that the synergistic effect between g-C{sub 3}N{sub 4} and TiO{sub 2} NBs promotes the photogenerated carrier separation in space. This valuable insight into the rational architectural design of nanostructure-based photocatalysts is expected to shed light on other photocatalytic reaction systems in the future. - Graphical abstract: A novel strategy to fabricate the g-C{sub 3}N{sub 4}/TiO{sub 2} nanobelt (NB) heterostructures was reported. The g-C{sub 3}N{sub 4}/TiO{sub 2} NB heterostructures exhibited highly effective photocatalytic activities for photodegradation of Rhodamine B and H{sub 2} production. - Highlights: • A novel strategy to fabricate the g-C{sub 3}N{sub 4}/TiO{sub 2} NB heterostructures was reported. • The heterostructure exhibited high catalytic activity in photodegradation of RhB. • The heterostructure showed good H{sub 2} productivity in photocatalytic water splitting. • The synergistic effect between g-C{sub 3}N{sub 4} and TiO{sub 2} NBs are important. • This study shows that the heterostructure can be an effective photocatalyst.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A microporous metal–organic framework for selective C{sub 2}H{sub 2} and CO{sub 2} separation

A quartzlike metal–organic framework with interesting one dimensional channel has been synthesized. It exhibits considerable acetylene and carbon dioxide uptake of 41.5 and 24.6 cm{sup 3} g{sup −1}, respectively, and relatively high selectivity for separation of C{sub 2}H{sub 2}/C{sub 2}H{sub 4}, C{sub 2}H{sub 2}/CH{sub 4}, CO{sub 2}/CH{sub 4} and CO{sub 2}/N{sub 2} at ambient condition. - Graphical abstract: A quartzlike topology metal−organic framework exhibits relatively high selectivity for separation of C{sub 2}H{sub 2}/C{sub 2}H{sub 4}, C{sub 2}H{sub 2}/CH{sub 4}, CO{sub 2}/CH{sub 4} and CO{sub 2}/N{sub 2} at ambient condition. Display Omitted - Highlights: • We firstly explored the selective adsorption of C{sub 2}H{sub 2} and CO{sub 2} over other gases in QMOF-1. • QMOF-1 shows considerable adsorption capacity for C{sub 2}H{sub 2} and CO{sub 2}. • Selectivity for C{sub 2}H{sub 2}/C{sub 2}H{sub 4}, C{sub 2}H{sub 2}/CH{sub 4}, CO{sub 2}/CH{sub 4} and CO{sub 2}/N{sub 2} mixtures were evaluated.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Semiorganic nonlinear optical material: Crystal growth, structure and optical properties of NaB(L-C{sub 4}H{sub 4}O{sub 5}){sub 2}

Graphical abstract: - Highlights: • Semiorganic NLO material NaB(L-C{sub 4}H{sub 4}O{sub 5}){sub 2} has been grown with sizes up to 8 × 5 × 2 mm{sup 3}. • It crystallizes in the noncentrosymmetric Monoclinic space group P2. • Thermal analysis demonstrated NaB(L-C{sub 4}H{sub 4}O{sub 5}){sub 2} are thermally stable up to 280. • NaB(L-C{sub 4}H{sub 4}O{sub 5}){sub 2} has an absorption edge about 220 nm in the UV region. • It exhibits a NLO efficiency of about two times that of KDP (KH{sub 2}PO{sub 4}) standard. - Abstract: Single crystal of the semiorganic NLO material NaB(L-C{sub 4}H{sub 4}O{sub 5}){sub 2} has been grown with sizes up to 8 × 5 × 2 mm{sup 3} by the slow evaporation method. The crystal structure of this compound was determined by single crystal X-ray diffraction. It crystallizes in the Monoclinic space group P2 with lattice parameters a = 5.4045(11) Å, b = 11.692(2) Å, c = 9.4773(19) Å and Z = 2. Thermal analysis demonstrated NaB(L-C{sub 4}H{sub 4}O{sub 5}){sub 2} are thermally stable up to 280 °C. NaB(L-C{sub 4}H{sub 4}O{sub 5}){sub 2} exhibits an absorption edge about 220 nm in the UV region. The powder second-harmonic generation (SHG) measurement performed on the ground crystal indicates that it has a NLO efficiency of about 2 times that of KDP (KH{sub 2}PO{sub 4}) standard. In addition, the origin of stronger SHG effect was discussed.

36 MATERIALS SCIENCE↗

Synthesis and characterization of g-C{sub 3}N{sub 4}/Cu{sub 2}O composite catalyst with enhanced photocatalytic activity under visible light irradiation

The prepared g-C{sub 3}N{sub 4}/Cu{sub 2}O composite exhibited the enhanced photocatalytic activity under visible-light irradiation due to the stronger ability in separation of electron–hole pairs, which was proven by the transient photocurrent measurement. - Highlights: • The coupled Cu{sub 2}O with g-C{sub 3}N{sub 4} of narrow-band-gap semiconductor has been designed. • g-C{sub 3}N{sub 4}/Cu{sub 2}O is prepared via an alcohol-aqueous based on chemical precipitation method. • g-C{sub 3}N{sub 4}/Cu{sub 2}O exhibits the enhanced photocatalytic activity under visible-light. • The enhanced photocatalytic activity is proven by the transient photocurrent test. • A mechanism for the visible-light-driven photocatalysis of g-C{sub 3}N{sub 4}/Cu{sub 2}O is revealed. - Abstract: To overcome the drawback of low photocatalytic efficiency brought by electron–hole pairs recombination and narrow photo-response range, a novel g-C{sub 3}N{sub 4}/Cu{sub 2}O composite photocatalyst was designed and prepared successfully. Compared with bare Cu{sub 2}O and g-C{sub 3}N{sub 4}, the g-C{sub 3}N{sub 4}/Cu{sub 2}O composite exhibited significantly enhanced photocatalytic activity for acid orange-II (AO-II) degradation under visible light irradiation. Based on energy band positions, the mechanism of enhanced visible-light photocatalytic activity was proposed.

36 MATERIALS SCIENCE↗

Metal-free g-C{sub 3}N{sub 4} photocatalyst by sulfuric acid activation for selective aerobic oxidation of benzyl alcohol under visible light

Highlights: • A novel visible-light-driven acid-modified g-C{sub 3}N{sub 4} was prepared. • The texture, electronic and surface property were tuned by acid modification. • Acid-modified g-C{sub 3}N{sub 4} shows much higher activity for photocatalytic activity. • Acid sites on the surface of g-C{sub 3}N{sub 4} favor efficient charge separation. - Abstract: In this work, modification of graphitic carbon nitride photocatalyst with acid was accomplished with a facile method through reflux in different acidic substances. The g-C{sub 3}N{sub 4}-based material was found to be a metal-free photocatalyst useful for the selective oxidation of benzyl alcohol with dioxygen as the oxidant under visible light irradiation. Acid modification had a significant influence on the photocatalytic performance of g-C{sub 3}N{sub 4}. Among all acid tested, sulfuric acid-modified g-C{sub 3}N{sub 4} showed the highest catalytic activity and gave benzaldehyde in 23% yield for 4 h under visible light irradiation, which was about 2.5 times higher than that of g-C{sub 3}N{sub 4}. The acid modification effectively improved surface area, reduced structural size, enlarged band gap, enhanced surface chemical state, and facilitated photoinduced charge separation, contributing to the enhanced photocatalytic activity. It is hoped that our work can open promising prospects for the utilization of metal free g-C{sub 3}N{sub 4}-based semiconductor as visible-light photocatalyst for selective organic transformation.

36 MATERIALS SCIENCE↗

Matrix structure evolution and thermo-mechanical properties of carbon fiber-reinforced Al{sub 2}O{sub 3}-SiC-C castable composites

Highlights: • Carbon fibers are formed in Al{sub 2}O{sub 3}-SiC-C castable composites under the action of nano Ni. • Starting growth temperature is 900 °C and growth mechanism agrees with V–S model. • The high temperature strength of composites can be increased by above 40%. • The thermal shock resistance can be enhanced by above 20%. - Abstract: The spalling and corrosion during the thermal cycles are the main causes of the damages observed in Al{sub 2}O{sub 3}-SiC-C castable composites that are used in molten-iron system. Using the catalyst of nano Ni and ball pitch in the matrix, Al{sub 2}O{sub 3}-SiC-C castable composites were prepared with the anti-oxidant addition of silicon. The results indicate that the high temperature of the Al{sub 2}O{sub 3}-SiC-C castable composites can be increased by above 42%, and the thermal shock resistance can be enhanced by above 20% because the ball pitch is carbonized and releases C{sub x}H{sub y} vapor, which can be pyrolized to carbon atoms and subsequently deposited into carbon fibers under the catalyst action. The starting temperature of carbon fiber growth is approximately 900 °C, and their diameter and aspect ratio can increase with the rising temperature. The in-situ generation of carbon fibers in Al{sub 2}O{sub 3}-SiC-C castable composites can significantly improve the fibers’ thermo-mechanical properties.

36 MATERIALS SCIENCE↗

Solid-state reaction mediated microstructural evolution in a spark plasma sintered in situ Ti–B 4 C composite

For this study, a novel porosity-free in situ Ti–B 4 C composite was fabricated via spark plasma sintering (SPS). Detailed analysis of the phase evolution, coupling results from XRM, XRD and SEM-EBSD-EDS, indicate that primarily TiB 2 precipitates formed due to the in-situ reactions between the boron-carbide and titanium powders. The precipitation of TiB 2 resulted in the formation of a graphitic C-rich thin layer circumscribing the partially reacted B 4 C particles. Further progression of the in-situ reaction leads to the out-diffusion of the excess carbon from the B 4 C particles, across the graphite and TiB 2 phases, forming TiC upon reacting with the titanium matrix. Therefore, the final microstructure primarily consisted of TiB 2 , TiC, and partially reacted B 4 C phases, with small amounts of TiB, α-Ti, and graphite. Furthermore, the microstructure in these SPS processed in situ composites appeared to be the product of a solid-state Ti–B 4 C diffusion couple, substantially different from their fusion-based additively manufactured counterparts. Nano-indentation tests revealed a remarkably high average hardness of ~25 GPa for this SPS-processed Ti–B 4 C composite and comparable (with literature) phase-specific hardness and modulus values for the constituent TiB 2 , TiC, and B 4 C phases.

36 MATERIALS SCIENCE↗

Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13 C metabolic flux analysis

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no 13 C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in 13 C metabolic flux analysis ( 13 C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated 13C-MFA using the optimal tracers [1,2- 13 C]glucose, [1,6- 13 C]glucose, [1,2- 13 C]xylose and [5- 13 C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U- 13 C]glucose and [U-13C]xylose tracers. We demonstrate that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO 2 originates from biomass turnover. Using knockout strains, we also demonstrate that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH2, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.

59 BASIC BIOLOGICAL SCIENCES↗

13 C-metabolic flux analysis of Clostridium ljungdahlii illuminates its core metabolism under mixotrophic culture conditions

Carbon dioxide-fixing acetogenic bacteria (acetogens) utilizing the Wood-Ljungdahl Pathway (WLP) play an important role in CO 2 fixation in the biosphere and in the development of biological processes – alone or in cocultures, under both autotrophic and mixotrophic conditions – for production of chemicals and fuels. To date, limited work has been reported in experimentally validating and quantifying reaction fluxes of their core metabolic pathways. Here, the core metabolic model of the acetogen Clostridium ljungdahlii was interrogated using 13 C-metabolic flux analysis ( 13 C-MFA), which required the development of a new defined culture medium. Autotrophic, heterotrophic, and mixotrophic growth in defined medium was possible by adding 1 mM methionine to replace yeast extract. Our 13 C-MFA found an incomplete TCA cycle and inactive core pathways/reactions, notably those of the oxidative pentose phosphate pathway, Entner-Doudoroff pathway, and malate dehydrogenase. 13 C-MFA during mixotrophic growth using the parallel tracers [1– 13 C]fructose, [1,2– 13 C]fructose, [1,2,3– 13 C]fructose, and [U– 13 C]asparagine found that externally supplied CO 2 contributed the majority of carbon consumed. All internally-produced CO 2 from the catabolism of asparagine and fructose was consumed by the WLP. While glycolysis of fructose was active, it was not a major contributor to overall production of ATP, NADH, and acetyl-CoA. Gluconeogenic reactions were active despite the availability of organic carbon. Asparagine was catabolized equally via conversion to threonine and subsequent cleavage to produce acetaldehyde and glycine, and via deamination to fumarate and then the anaplerotic conversion of malate to pyruvate. Both pathways for asparagine catabolism produced acetyl-CoA, either directly via pyruvate or indirectly via the WLP. Cofactor stoichiometry based on our data predicted an essentially zero flux through the ferredoxin-dependent transhydrogenase (Nfn) reaction. Instead, nearly all of NADPH generated from the hydrogenase reaction was consumed by the WLP. Reduced ferredoxin produced by the hydrogenase reaction and glycolysis was mostly used for ATP generation via the RNF/ATPase system, with the remainder consumed by the WLP. NADH produced by RNF/ATPase was entirely consumed via the WLP.

13C metabolic flux analysis↗

Optical 14 C Tracing for Biological and Pharmaceutical Applications Using Two-Color Cavity Ringdown Spectroscopy

Laser-based 14 C quantitation has been proposed as a more affordable, higher-throughput, table-top alternative to accelerator mass spectrometry (AMS). Here, we demonstrate the feasibility of a mid-IR 14 C detector based on two-color cavity ringdown spectroscopy (2C-CRDS) for low-level 14 C isotope tracing in biological studies. The 2C-CRDS technique quantifies the sample 14 C content by measuring the 14 CO 2 absorption signals from the combusted samples with mid-IR lasers. With 2C-CRDS, we previously demonstrated the most sensitive and accurate optical measurements of 14 CO 2 . The current detection sensitivity and quantitation accuracy of the instrument, at a few parts per quadrillion (where a quadrillion = 10 15 ) 14 C/C mole fraction, is competitive against AMS. Here, by applying the 2C-CRDS 14 C sensor to two applications relevant to 14 C-labeled biochemical analysis and pharmaceutical studies, we demonstrate sub-fCi level (where 1 fCi = 10 –15 Ci) quantitation of sample 14 C activity, with a minimum sample-size requirement of 3 mg of carbon. The current measurement throughput, ~25 min/sample, is largely limited by the sampling efficiency of the online combustion and CO 2 processing interface to the 2C-CRDS instrument. The possibility of a significantly improved measurement throughput of a few minutes per sample is suggested by the results of a flow-through 14 CO 2 sampling scheme. In conclusion, this improved measurement efficiency, combined with the relatively low cost and compact size of a 2C-CRDS sensor, could potentially revolutionize high-sensitivity 14 C tracing in biological, pharmaceutical, and clinical studies.

60 APPLIED LIFE SCIENCES↗