Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

Simple, fast, and simultaneous determination of orthophosphate, pyrophosphate, and tripolyphosphate by capillary electrophoresis with capacitively coupled contactless conductivity detection.

This work describes a novel analytical method using capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) for simultaneous, simple, and rapid determination of three inorganic phosphates (orthophosphate, pyrophosphate, and tripolyphosphate) widely used as food additives and in pharmaceutical formulations. A background electrolyte composed of 0.5 mol L-1 acetic acid provided fast separation (around 3.0 min) and good separation efficiency and peak resolution. Linearity in the concentration range of 10-500 mg L-1 was confirmed by the coefficients of determination (R2) higher than 0.99. The limits of detection varied from 0.41 to 0.58 mg L-1. The accuracy of the proposed method was assessed by recovery tests conducted at three concentration levels in tap water samples, food, and personal hygiene products. Recovery values varying from 81% to 118% were achieved, indicating an acceptable accuracy. The proposed CE-C4D successfully determined the three inorganic phosphates in the analyzed samples.

Thienopyrimidine-derived multifunctional fluorescence sensor for the detection of Cu2+, Fe3+, and PPi in different solvents.

Hydrazine substituted thienopyrimidine, a new fluorophore, was used to synthesize a novel Schiff base R1 as a chemosensor via the condensation with p-formyltriphenylamine, and the structure was confirmed using nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) analysis. When treated with Cu2+ in dimethylsulfoxide (DMSO)/H2O buffer, R1 showed a phenomenon of fluorescence quenching, which was reversible with the action of ethylenediaminetetraacetic acid (EDTA). When treated with Fe3+ in dimethylformamide (DMF)/H2O buffer, R1 exhibited the same phenomenon, but fluorescence was recovered with inorganic pyrophosphate (PPi) quantitatively. The complexation ratios for R1-Cu2+ and R1-Fe3+ were both 1:2, which were manifested by MS titrations and corresponding Job's plots. The limits of detection of R1 for Cu2+ and Fe3+ were 3.11 × 10-8 and 1.24 × 10-7 M, respectively. The sensing mechanism of R1 toward Cu2+ and Fe3+ was confirmed using density functional theory calculations and electrostatic potential analysis. Test strips of R1 were fabricated successfully for on-site detection of Cu2+ and Fe3+. In addition, R1 was applied to recognize Cu2+ and Fe3+ in actual water samples with satisfactory recovery.

Synthesis of novel polyethyleneimine-capped silver nanoclusters exhibiting ultraviolet-A fluorescence and their application in multiple sensing.

Cupric ions (Cu2+), pyrophosphate (PPi), and alkaline phosphatase (ALP) are involved in a variety of biochemical processes such as DNA replication, cellular metabolism and play an important role in human growth and development. It is of great significance to establish a method for the sensitive detection of Cu2+, PPi and ALP. In this work, polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) were successfully synthesized by a one-pot method using hydrazine sulfate as reductant, exhibiting a unique strong fluorescence emission in the near-ultraviolet region at ∼339 nm. Since the fluorescence of PEI-AgNCs can be quenched by Cu2+ through inner filtering effect (IFE), then recovered by competitive binding of pyrophosphate and Cu2+, and later weakened again by catalytic hydrolysis of alkaline phosphatase, a sensitive and selective strategy based on the changes of fluorescence "ON" or "OFF" was established to detect Cu2+, PPi and ALP. The LODs of these three analytes were 36 nM, 0.2 µM, and 0.14 U L-1 at a S/N ratio of 3, respectively. A series of logic gate circuits for sensing cupric ions, pyrophosphate, and alkaline phosphatase were successfully constructed. The established methods have the potential for biosensing and environmental analysis and the specific UV-A fluorescence property of PEI-AgNCs may be helpful in photonic and optical areas.

Triazol-Methanaminium-Pillar[5]arene-Functionalized Single Nanochannel for Quantitative Analysis of Pyrophosphate in Water.

Inorganic pyrophosphate (PPi) is an important biological functional anion and plays crucial roles in life science, environmental science, medicine, and chemical process. Quantification of PPi in water has far-reaching significance for life exploration, disease diagnosis, and water pollution control. The label-free quantitative detection of PPi anions with a nanofluidic sensing device based on a conical single nanochannel is demonstrated. The channel surface is functionalized with a synthetic PPi receptor, triazol-methanaminium-functionalized pillar[5]arene (TAMAP5), using carbodiimide coupling chemistry. Due to the specific binding between TAMAP5 and PPi, the functionalized nanochannel can discriminate PPi from other inorganic anions with high selectivity through ionic current recording, even in the presence of various interfering anions. The current response exhibits a linear correlation with PPi concentration in the range from 1 × 10-7 to 1 × 10-4 M with a limit of detection of 6.8 × 10-7 M. A spike-and-recovery analysis of PPi in East Lake water samples indicates that the proposed nanofluidic sensor has the ability to quantitate micromolar concentrations of PPi in environmental water samples.

Portable, quantitative, and sequential monitoring of copper ions and pyrophosphate based on a DNAzyme-Fe3O4 nanosystem and glucometer readout.

In this report, portable, quantitative, and sequential monitoring of copper ions and pyrophosphate (PPi) with a single sensor based on a DNAzyme-Fe3O4 system and glucometer readout was performed. Initially, streptavidin was functionalized on the surface of magnetic Fe3O4 spheres through glutaraldehyde. Then, an invertase-modified DNA Cu substrate was connected to the magnetic Fe3O4 spheres by a specific reaction between streptavidin and biotin. The sensing system was formed by a hybridization reaction between the Cu substrate and Cu enzyme. In the presence of Cu2+, Cu2+ will recognize the Cu DNA substrate and form an "off-on" signal switch, thereby resulting in the separation of invertase from the Fe3O4 nanospheres. PPi recognizes Cu2+ to form a Cu2+-PPi complex, resulting in an "on-off" signal switch. Under optimized conditions, linear detection ranges for Cu2+ and PPi of 0.01-5 and 0.5-10 µM, and detection limits for Cu2+ and PPi of 10 nM and 500 nM, respectively, were obtained. Good selectivity was achieved for the analysis of Cu2+ and PPi. Satisfactory results were achieved for this biosensor during the determination of Cu2+ in real tap samples and PPi in human urine samples. This verified that the sensor is portable and low cost, and can be applied to the sequential monitoring of multiple analytes with a single point-of-care biosensor.

Plasmodium falciparum gametocyte-induced volatiles enhance attraction of Anopheles mosquitoes in the field.

BACKGROUND: Plasmodium parasites manipulate the interaction between their mosquito and human hosts. Patients infected with gametocytes attract anopheline mosquitoes differentially compared to healthy individuals, an effect associated with an increased release of attractive volatile cues. This odour-driven manipulation is partly mediated by the gametocyte-specific metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which induces increased release of select aldehydes and terpenes from red blood cells and results in the enhanced attraction of host-seeking mosquitoes, which are vectors of malaria. This study investigates the effect of the HMBPP-induced volatiles on the attraction of wild Anopheles mosquitoes to humans under field conditions. METHODS: The efficacy of the HMBPP-induced odour blend to attract Anopheles was evaluated in a 4 × 6 Latin rectangular study design indoors using baited Suna traps. Furthermore, to assess the efficacy of the HMBPP-induced odour blend in (1) augmenting the attractiveness of human odour, and (2) attracting Anopheles mosquitoes in the absence of human odour, a two-choice assay using host decoy traps (HDTs) was used and evaluated using binomial generalized regression. RESULTS: Traps baited with the HMBPP-induced odour blend attracted and caught both Anopheles arabiensis and Anopheles pharoensis females in a dose-dependent manner in the presence of background human odour, up to 2.5 times that of an unbaited trap. Given a choice between human odour and human odour laden with the HMBPP-induced odour blend, mosquitoes differentially preferred traps augmented with the HMBPP-induced odour blend, which caught twice as many female An. arabiensis. Traps baited with the HMBPP-induced odour blend but lacking the background of human odour were not effective in attracting and catching mosquitoes. CONCLUSION: The findings of the present study revealed that the HMBPP-induced odour blend, when augmented with human body odour, is attractive to anopheline mosquitoes and could be used as a complementary vector control tool along with existing strategies.

Polyphosphate Chain Length Determination in the Range of Two to Several Hundred P-Subunits with a New Enzyme Assay and 31P NMR.

Currently, 31P NMR is the only analytical method that quantitatively determines the average chain length of long inorganic polyphosphate (>80 P-subunits). In this study, an enzyme assay is presented that determines the average chain length of polyphosphate in the range of two to several hundred P-subunits. In the enzyme assay, the average polyP chain length is calculated by dividing the total polyphosphate concentration by the concentration of the polyphosphate chains. The total polyphosphate is determined by enzymatic polyphosphate hydrolysis with Saccharomyces cerevisiae exopolyphosphatase 1 and S. cerevisiae inorganic pyrophosphatase 1, followed by colorimetric orthophosphate detection. Because the exopolyphosphatase leaves one pyrophosphate per polyphosphate chain, the polyphosphate chain concentration is assayed by coupling the enzymes exopolyphosphatase (polyP into pyrophosphate), ATP sulfurylase (pyrophosphate into ATP), hexokinase (ATP into glucose 6-phosphate), and glucose 6-phosphate dehydrogenase (glucose 6-phosphate into NADPH), followed by fluorometric NADPH detection. The ability of 31P NMR and the enzyme assay to size polyP was demonstrated with polyP lengths in the range from 2 to ca. 280 P-subunits (no polyP with a longer chain length was available). The small deviation between methods (-4 ± 4%) indicated that the new enzyme assay performed accurately. The limitations of 31P NMR (i.e., low throughput, high sample concentration, expensive instrument) are overcome by the enzyme assay that is presented here, which allows for high sample throughput and requires only a commonly available plate reader and micromole per liter concentrations of polyphosphate.

Smart probe for simultaneous detection of copper ion, pyrophosphate, and alkaline phosphatase in vitro and in clinical samples.

Wilson's disease (WD), which might lead to acute liver failure, is an inherited disorder characterized by accumulation of copper (Cu2+) in the brain, the liver, and other vital organs. In the clinic, decreased serum alkaline phosphatase (ALP) concentration is used for WD diagnosis. But to the best of our knowledge, using a fluorescent probe to simultaneously detect multiple factors in WD (e.g., Cu2+, pyrophosphate (PPi), and ALP) has not been reported. Herein, we rationally designed a fluorescent switch (E)-8-((4-methylbenzylidene)amino)napthalen-1-amine (L) and successfully applied it for sequential and selective detections of Cu2+, PPi, and ALP in vitro, in living cells and synovial fluid samples with "Off," "On," and "Off" fluorescence signals, respectively. Considering the obvious correlations among Cu2+, PPi, and ALP in WD, we envision that our fluorescent probe L could be applied to in vitro diagnosing WD in the near future.

Signal-on fluorescence assay for pyrophosphate ions based on DNA-stabilized silver nanoclusters.

Pyrophosphate anion (P2 O7 4- , PPi) is considered as a potential biomarker for arthritic diseases because high levels of PPi may result in calcium pyrophosphate dehydrate crystal deposition diseases. In this study, a simple fluorescence method for PPi was demonstrated by organic integration of the efficient fluorescence quenching ability of copper ions to DNA-scaffolded silver nanoclusters and the strong affinity of PPi towards copper ions. This simple fluorescence sensor showed a low detection limit (0.28 µM based on signal/noise = 3) towards the detection of PPi. Practical application of this method was also validated by detection of PPi in the synovial fluid.

2D-Metal-Organic-Framework-Nanozyme Sensor Arrays for Probing Phosphates and Their Enzymatic Hydrolysis.

The detection of phosphates and their enzymatic hydrolysis is of great importance because of their essential roles in various biological processes and numerous diseases. Compared with individual sensors for detecting one given phosphate at a time, sensor arrays are able to discriminate multiple phosphates simultaneously. Although nanomaterial-based sensor arrays have shown great promise for the discrimination of phosphates, very few of them have been explored for probing phosphates involved enzymatic hydrolysis. To fill this gap, herein we fabricated two-dimensional-metal-organic-framework (2D-MOF)-nanozyme-based sensor arrays by modulating their peroxidase-mimicking activity with various phosphates, including AMP, ADP, ATP, pyrophosphate (PPi), and phosphate (Pi). The sensor arrays were used to successfully discriminate the five phosphates not only in aqueous solutions but also in biological samples. The practical application of the sensor arrays was then validated with blind samples, where 30 unknown samples containing phosphates were accurately identified. Moreover, the sensor arrays were successfully applied to probing hydrolytic processes involving ATP and PPi that are catalyzed by apyrase and PPase, respectively. This work demonstrates a nanozyme-based sensor array as a convenient and reliable analytical platform for probing phosphates and their related enzymatic processes, which could be applied to other analytes and enzymatic reactions.

Enzymatic quantification and length determination of polyphosphate down to a chain length of two.

Polyacrylamide gel electrophoresis, being the current method of choice for length determination of inorganic polyphosphate (polyP), requires a sequencing apparatus, relies on commercially not available polyP length standards and yields only a chain length distribution. State of the art polyP quantification involves enzymatic hydrolysis of polyP to orthophosphate with the Saccharomyces cerevisiae exopolyphosphatase 1 (scPpx1p) and subsequent colorimetric orthophosphate detection. Because scPpx1p leaves one pyrophosphate per polyP, short chain polyPs are only partially detected. To overcome this analytical limitation, a method involving both the scPpx1p and the S. cerevisiae inorganic pyrophosphatase (scIpp1p) is proposed. Differential enzymatic hydrolysis of polyP with scPpx1p, and a combination of scIpp1p and scPpx1p allows not only for comprehensive quantification of polyP (excluding cyclic polyP) down to a chain length of two, but also absolute average chain length determination in the range of two to approximately 80. An optimized one-reagent method for rapid (2 min) orthophosphate quantification is part of the assay. Biological phosphorous containing molecules at equimolar phosphorous concentrations regarding polyP do not interfere. The method requires 1.5 µg polyP and calls only for a plate reader. This is the first enzymatic method for simultaneous average polyP chain length determination as well as comprehensive quantification.

A simple and ultrasensitive fluorescence assay for single-nucleotide polymorphism.

In this report, a simple, label-free and highly efficient nucleic acid amplification technique is developed for ultrasensitive detection of single-nucleotide polymorphism (SNP). Briefly, a designed padlock probe is first circularized by a DNA ligase when it perfectly complements to a mutant gene. Then, the mutant gene functions as a primer to initiate branched rolling circle amplification reaction (BRCA), generating a large number of branched DNA strands and a lot of pyrophosphate molecules which is equivalent to the number of nucleotides consumed. With the addition of a terpyridine-Zn(II) complex, pyrophosphate molecules can be sensitively detected owing to the formation of a fluorescent terpyridine-Zn(II)-pyrophosphate complex. The fluorescence intensity is directly associated with the content of the mutant gene in a sample solution. On the other hand, the circulation of the padlock probe is prohibited when it hybridizes with the wild-type gene. In this assay, the accumulative nature of the BRCA process produces a detection limit of 0.1 pM and an excellent selectivity factor of 1000 toward SNP. As little as 0.1% mutant in the wild-type gene can be successfully detected. The simple procedure, high sensitivity, and high selectivity of this assay offer a potentially viable alternative for routine SNP analysis. Graphical abstract A simple and label-free fluorescence assay for SNP detection by coupling BRCA with selective fluorescence detection of pyrophosphate using the terpyridine-Zn(II) complex.

Live-Cell Pyrophosphate Imaging by in Situ Hot-Spot Generation.

Controlling the electromagnetic hot-spot generation is essential for surface-enhanced Raman scattering (SERS) assays. Current hot-spot-based SERS assays have been extensively studied in solutions or on substrates. However, probing biospecies by controlling the hot-spot assembly in living systems has not been demonstrated thus far. Herein, we report a background-free SERS probe for imaging pyrophosphate (PPi), a biochemically significant anion, in living cells. Intracellular PPi is able to induce the nanoparticle dimerization, thus creating an intense electromagnetic hot spot and dramatically enhancing the signal of the Raman reporters residing in the hot spot. More impressively, the reporter we used in this study provides a strong and sharp single peak in the cellular Raman-silent region (1800-2800 cm-1), thus eliminating the possible background interference. This strategy could be readily extended to detect other biomarkers by only replacing the recognition ligands.

Determination of trace metal concentration in compost, DAP, and TSP fertilizers by neutron activation analysis (NAA) and insights from density functional theory calculations.

Leaching of toxic metals from fertilizers is a growing concern in an agricultural country like Bangladesh due to the serious consequences in health and food chain. Fertilizers used in farming fields and nurseries (plant sales outlet) in the mid-southern part of Bangladesh were collected for the determination of toxic metals. This study employed the neutron activation method and a relative standardization approach. Three standard/certified reference materials, namely NIST coal fly ash 1633b, IAEA-Soil-7, and IAEA-SL-1 (lake sediment), were considered for elemental quantification. Concentration of As (2.63-16.73 mg/kg), Cr (40.93-261.77 mg/kg), Sb (0.47-63.58 mg/kg), Th (1.44-19.16 mg/kg), and U (1.90-209.41 mg/kg) were determined in fertilizers. High concentrations of Cr, Sb, and U were detected in some compost and phosphate fertilizers (TSP and diammonium phosphate (DAP)) in comparison with the IAEA/European market standard and other studies. Quantum mechanical calculations were performed to understand the molecular level interaction of CrO3, Sb2O3, and AsO3, with DAP by employing density functional theory with the B3LYP/SDD level of theory. Our results indicated that CrO3 and Sb2O3 have strong binding affinity with DAP compared to AsO3, which supports the experimental results. These compounds attached to the phosphate group through covalent-like bonding with oxygen. The frontier molecular orbital calculation indicated that HOMO-LUMO gap of the AsO3-DAP (5.46 eV) and Sb2O3-DAP (6.48 eV) complexes are relatively lower than the CrO3-DAP, which indicates that As and Sb oxides are chemically more prone to attach with the phosphate group of DAP fertilizer.

Influence of Humic Acids Extracted from Peat by Different Methods on Functional Activity of Macrophages in Vitro.

We studied activation of macrophages with humic acids extracted from peat of large deposits in the Tomsk region by two extraction methods: by hydroxide or sodium pyrophosphate. Humic acid of lowland peat types containing large amounts of aromatic carbon, phenolic and alcohol groups, carbohydrate residues and ethers, irrespectively of the extraction methods contained LPS admixture that probably determines their activating properties. Humic acid of upland peat types characterized by high content of carbonyl, carboxyl, and ester groups enhance NO production and reduce arginase expression, but these effects were minimized when sodium hydroxide was used as an extraction solvent. Pyrophosphate samples of the upland peat types were characterized by aromaticity and diversity of functional groups and have a significant advantage because of they induce specific endotoxin-independent stimulating action on antigen presenting cells.

Copper-Mediated DNA-Scaffolded Silver Nanocluster On-Off Switch for Detection of Pyrophosphate and Alkaline Phosphatase.

We present a new copper-mediated on-off switch for detecting either pyrophosphate (PPi) or alkaline phosphatase (ALP) based on DNA-scaffolded silver nanoclusters (DNA/AgNCs) templated by a single-stranded sequence containing a 15-nt polythymine spacer between two different emitters. The switch is based on three favorable properties: the quenching ability of Cu(2+) for DNA/AgNCs with excitation at 550 nm; the strong binding capacity of Cu(2+) and PPi; and the ability of ALP to transform PPi into orthophosphate (Pi). The change in fluorescence of DNA/AgNCs depends on the concentrations of Cu(2+), PPi, and ALP. Copper(II) acts as a mediator to interact specifically with the Probe, while PPi and ALP convert the signal of the Probe by removing and recovering Cu(2+), operating as an on-off switch. In the presence of Cu(2+) only, DNA/AgNCs exhibit low fluorescence because the combination of Cu(2+) and DNA template disturbs the precise formation of DNA/AgNCs. When PPi is added to the system containing Cu(2+), free DNA template is obtained due to the stronger interaction of PPi and Cu(2+), leading to a significant fluorescence increase (ON state) which depends on the concentration of PPi. Further addition of ALP results in the release of free Cu(2+) via ALP-catalysis of hydrolysis of PPi into Pi, thereby returning the system to the low fluorescence OFF state. The switch allows the analysis of either PPi or ALP by observation of the fluorescence status, with the detection limit of 112.69 nM and 0.005 U/mL for PPi and ALP, respectively. The AgNCs on-off switch provides the advantages of simple design, convenient operation, and low experimental cost without need of chemical modification, organic dyes, or separation procedures.

A Disassembly Strategy for Imaging Endogenous Pyrophosphate in Mitochondria by Using an Fe(III) -salen Complex.

Inorganic pyrophosphate (PPi) is produced from nucleoside triphosphates in important biosynthetic reactions and is considered a diagnostic marker for various diseases, such as cancer, crystal deposition disease, and arthritis. Traditional methods for biological PPi detection rely on off-line analytics after sample destruction. Molecular probes for imaging this biologically important analyte with temporal and spatial control in living cells are currently in demand. Herein, we report an Fe(III) -salen complex as the first small reaction-based probe for endogenous mitochondrial PPi following a disassembly approach. Significantly, we successfully applied this complex for the detection of increased cellular PPi levels, and its performance was not affected by the presence of mitochondrial ATP in living cells.

Naked-eye sensitive detection of alkaline phosphatase (ALP) and pyrophosphate (PPi) based on a horseradish peroxidase catalytic colorimetric system with Cu(ii).

In this paper, a novel colorimetric method for the detection of alkaline phosphatase (ALP) and pyrophosphate (PPi) was designed based on a Cu(2+)-horseradish peroxidase (HRP)-3,3',5,5'-tetra-methylbenzidine (TMB)-H2O2 system. In the presence of ALP, l-ascorbic acid-2-phosphate (AAP) could be hydrolyzed to ascorbic acid which could reduce Cu(2+) to Cu(+) to inhibit the enzymatic activity of HRP in the colorimetric system. The change in absorbance was found to be proportional to the ALP concentration with a linear detection range and a limit of detection of 5.4 mU mL(-1). In the presence of PPi, because Cu(2+) was chelated by PPi, the conversion of Cu(ii) by AA was effectively inhibited. The color of the HRP-TMB-H2O2 system with Cu(2+) showed blue. The HRP-TMB-H2O2 system with the Cu(2+) colorimetric system could also detect PPi with a satisfying result. In summary, this method possesses sensitivity, reproducibility, and cost-effectiveness without labelling and separation and the use of a colorimetric method is more in line with the requirements of on-site detection and green chemistry.

Iron and Carbon Dynamics during Aging and Reductive Transformation of Biogenic Ferrihydrite.

Natural organic matter is often associated with Fe(III) oxyhydroxides, and may be stabilized as a result of coprecipitation or sorption to their surfaces. However, the significance of this association in relation to Fe and C dynamics and biogeochemical cycling, and the mechanisms responsible for organic matter stabilization as a result of interaction with minerals under various environmental conditions (e.g., pH, Eh, etc.) are not entirely understood. The preservation of mineral-bound OM may be affected by OM structure and mineral identity, and bond types between OM and minerals may be central to influencing the stability, transformation and composition of both organic and mineral components under changing environmental conditions. Here we use bulk and submicron-scale spectroscopic synchrotron methods to examine the in situ transformation of OM-bearing, biogenic ferrihydrite stalks (Gallionella ferruginea-like), which formed following injection of oxygenated groundwater into a saturated alluvial aquifer at the Rifle, CO field site. A progression from oxidizing to reducing conditions during an eight-month period triggered the aging and reductive transformation of Gallionella-like ferrihydrite stalks to Fe (hydroxy)carbonates and Fe sulfides, as well as alteration of the composition and amount of OM. Spectromicroscopic measurements showed a gradual decrease in reduced carbon forms (aromatic/alkene, aliphatic C), a relative increase in amide/carboxyl functional groups and a significant increase in carbonate in the stalk structures, and the appearance of organic globules not associated with stalk structures. Biogenic stalks lost ∼30% of their initial organic carbon content. Conversely, a significant increase in bulk organic matter accompanied these transformations. The character of bulk OM changed in parallel with mineralogical transformations, showing an increase in aliphatic, aromatic and amide functional groups. These changes likely occurred as a result of an increase in microbial activity, or biomass production under anoxic conditions. By the end of this experiment, a substantial fraction of organic matter remained in identifiable Fe containing stalks, but carbon was also present in additional pools, for example, organic matter globules and iron carbonate minerals.