Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Pentosinane, a Post-Translational Modification of Human Proteins with Underappreciated Stability.

Pentosinane is a structurally complex nonenzymatic post-translational modification of proteins believed to be present in all living things. It falls into the category of advanced glycation end products (AGEs) and is structurally related to the other AGEs pentosidine and glucosepane. Although pentosidine and glucosepane have been widely studied for their role in wide-ranging conditions (e.g., diabetes mellitus, Alzheimer's disease, and human aging), relatively little is known about pentosinane. Interestingly, previous reports have suggested that pentosidine may derive from pentosinane. The (patho)physiological significance of pentosinane in humans is largely unexplored. As a first step to address this knowledge gap, we report herein the first total synthesis of pentosinane. Our synthesis is high yielding (1.7% over seven steps), concise, and enantioselective, and it leverages a strategy for synthesizing 2,5-diaminoimidazoles previously developed by our lab. Access to synthetic pentosinane has allowed us to perform additional studies showing that its oxidation to pentosidine is both pH and oxygen dependent and is substantially slower under physiological conditions than previously believed. Additionally, pentosinane rapidly decomposes under harshly acidic conditions typically employed for pentosidine isolation. Taken together, these results suggest that pentosinane is likely to be more abundant in vivo than previously appreciated. We believe these results represent a critical step toward illuminating the role(s) of pentosinane in human biology.

Paired riparian water table monitoring to quantify hydraulic loading to a saturated buffer.

The use of saturated buffers for reducing NO3-N loads from tile-drained croplands is increasing in the US Midwest and there is a need to develop options for estimating reductions at riparian sites. In this study, we present a paired water table monitoring approach to estimate hydraulic and NO3-N loading into a saturated buffer in eastern Iowa. One well was located within the saturated buffer (treatment) and a second well was installed in the same section of the riparian buffer but without the saturated buffer (control). Over a season of monitoring, water table depths were remarkably consistent between the two wells but the water table beneath the saturated buffer was consistently 0.22 m higher than the non-saturated buffer control. The increase in water table height increased the amount of water discharged from a 162 m long buffer by 468.2 m3/year and, assuming concentration reduction of 15 mg/l, resulted in a N reduction of approximately 7 kg. Although more work is needed to document this paired monitoring approach elsewhere, the method may hold promise for inexpensively quantifying the performance of conservation practices at landowner-led sites.

Use of high-resolution ground conductivity measurements for denitrifying conservation practice placement.

Accurate field-scale maps of soil properties including features such as texture, soil organic matter (SOM) content, and hydraulic conductivity are essential for proper placement of conservation practices that utilize anoxic soil environments for denitrification. However, in many cases, soil maps inaccurately represent subsoil properties and can mislead managers about where to install new practices. Non-invasive methods of subsoil property analysis including electromagnetic induction techniques are a potentially efficient method for improving existing field-scale soil maps. In this study, we quantified the accuracy of existing soil maps in an agricultural field in north-central Iowa. Of 60 soil cores collected and reclassified, 19 were identified as taxadjunct at the soil series level primarily due to hydrologic indicators and soil particle size. We assessed the correlation among physical and chemical soil properties measured in-lab and geophysical responses measured in-field. We identified significant correlation of SOM and sand to electrical conductivity for individual core and mean soil series data. From this analysis, we developed a conservation practice suitability map and evaluated the potential for field-scale geophysical investigations to serve as a new tool for agricultural conservation planning and placement of site-specific denitrifying conservation practices. Study results suggest that incorporating a geophysical conductivity investigation into conservation planning may improve understanding of critical soil properties beyond those ascertained with limited soil borings.

Age-related changes in the physical properties, cross-linking, and glycation of collagen from mouse tail tendon.

Collagen is a structural protein whose internal cross-linking critically determines the properties and functions of connective tissue. Knowing how the cross-linking of collagen changes with age is key to understanding why the mechanical properties of tissues change over a lifetime. The current scientific consensus is that collagen cross-linking increases with age and that this increase leads to tendon stiffening. Here, we show that this view should be reconsidered. Using MS-based analyses, we demonstrated that during aging of healthy C57BL/6 mice, the overall levels of collagen cross-linking in tail tendon decreased with age. However, the levels of lysine glycation in collagen, which is not considered a cross-link, increased dramatically with age. We found that in 16-week-old diabetic db/db mice, glycation reaches levels similar to those observed in 98-week-old C57BL/6 mice, while the other cross-links typical of tendon collagen either decreased or remained the same as those observed in 20-week-old WT mice. These results, combined with findings from mechanical testing of tendons from these mice, indicate that overall collagen cross-linking in mouse tendon decreases with age. Our findings also reveal that lysine glycation appears to be an important factor that contributes to tendon stiffening with age and in diabetes.

Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks.

Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.

Quantifying the effectiveness of a saturated buffer to reduce tile NO3-N concentrations in eastern Iowa.

Agricultural drainage tiles are primary contributors to NO3-N export from Iowa croplands. Saturated buffers are a relatively new conservation practice that diverts tile water into a distribution tile installed in a riparian buffer parallel to a stream with the intent of enhancing NO3-N processing within the buffer. In this study, tile NO3-N concentration reductions were characterized through two different saturated buffers at a working farm site in eastern Iowa. Study objectives were to (1) evaluate the hydrogeology and water quality patterns in the saturated buffer and (2) quantify the reduction in tile NO3-N concentration from the saturated buffer installation. Results showed that the two saturated buffers are reducing NO3-N concentrations in tile drainage water from input concentrations of approximately 15 mg/l to levels < 1.5 mg/l at the streamside well locations. The reduction occurs rapidly in the fine-textured and organic-rich alluvial soils with most of the reduction occurring within 1.5 m of the distribution line. Denitrification is hypothesized as being primarily responsible for the concentration reductions based on soil and water chemistry conditions, completion of a geophysical survey (quantifying low potential for N loss to deeper aquifers), and comparisons to other similar Iowa sites. The study provides more assurance to new adopters that this practice can be installed in many areas throughout the Midwestern Cornbelt region.

One-Step Synthesis of 2,5-Diaminoimidazoles and Total Synthesis of Methylglyoxal-Derived Imidazolium Crosslink (MODIC).

Here we describe a general method for the synthesis of 2,5-diaminoimidazoles, which involves a thermal reaction between α-aminoketones and substituted guanylhydrazines without the need for additives. As one of the few known ways to access the 2,5-diaminoimidazole motif, our method greatly expands the number of reported diaminoimidazoles and further supports our previous observations that these compounds spontaneously adopt the non-aromatic 4(H) tautomer. The reaction works successfully on both cyclic and acyclic amino ketone starting materials, as well as a range of substituted guanylhydrazines. Following optimization, the method was applied to the efficient synthesis of the advanced glycation end product (AGE) methylglyoxal-derived imidazolium crosslink (MODIC). We expect that this method will enable rapid access to a variety of biologically important 2,5-diaminoimidazole-containing products.

Generation and characterization of antibodies against arginine-derived advanced glycation endproducts.

Although antibodies reagents have been widely employed for studying advanced glycation end-products (AGEs), these materials have been produced using complex mixtures of immunogens. Consequently, their epitope specificity remains unknown. Here we have generated the first antibodies capable of recognizing each of the three isomers of the methylglyoxal hydroimidazolones (MG-Hs) by using chemical synthesis to create homogenous immunogens. Furthermore, we have thoroughly characterized the epitope specificity of both our antibodies and that of two existing monoclonals by implementing a direct ELISA protocol employing synthetic MG-H antigens. Finally, we employed the reported anti-MG-H antibodies to the detection of MG-Hs in cellular systems using immunofluorescence microscopy. These studies have demonstrated that anti-MG-H1 and anti-MG-H3 staining is concentrated within the nucleus, while anti-MG-H2 affords only minimal signal. These observations are consistent with reported formation preferences for MG-Hs, and may suggest novel nuclear targets for non-enzymatic posttranslational modification. The antibody reagents reported herein, as well as the strategy employed for their creation, are likely to prove useful for the immunochemical study of AGEs in biological systems.

A three-step process to facilitate the annulation of polycyclic aromatic hydrocarbons.

A new efficient three-step process to annulate polycyclic aromatic hydrocarbons (PAHs) has been developed, providing access to PAHs with saturated rings that under current chemical methods would be difficult to produce in an efficient manner. This method relies on a palladium-catalyzed cross-coupling reaction of various brominated PAHs with cyclohexanone to yield α-arylated ketones, which are converted to regiospecific vinyl triflates and cyclized by a palladium-catalyzed intramolecular arene-vinyl triflate coupling to produce PAHs with incorporated saturated rings or "tetrahydroindeno-annulated" PAHs.

Potential for managing pool levels in a flood-control reservoir to increase nitrate-nitrogen load reductions.

Few strategies are available to reduce nitrate-nitrogen (NO3 -N) loads at larger landscape scales, but flood control reservoirs are known to reduce riverine loads. In this study, we evaluated the potential to increase nitrogen (N) loss at Lake Red Rock, a large reservoir located in central Iowa, by evaluating the inundation of sediments deposited at the reservoir inflow. Sediment samples were collected at 51 locations in the lower delta region and analyzed for particle size and nutrient content. Nitrogen loss rates in delta sediments were determined from laboratory assays, and satellite imagery was used to develop a rating curve to quantify land area inundated within the delta. The daily mass of NO3 -N reduced with delta inundation was estimated by applying the mean N 24-h loss rate (0.66 g N m2 day-1 ) by the area of inundation (m2 ). Results indicated that raising pool elevations to inundate more of the delta would result in greater N losses, ranging from 2 to 377 Mg per year. Potential N loss of 102 Mg achieved by increasing pool stage by 0.5 m would be equivalent to installing nearly 650 edge-of-field practices in the watershed. Although more work is needed to integrate with an existing environmental pool management plan, study results indicate that reservoir management could achieve N reductions at a novel landscape scale.

Response of Cancer Stem Cells and Human Skin Fibroblasts to Picosecond-Scale Electron Irradiation at 1010 to 1011 Gy/s.

PURPOSE: This study aimed to demonstrate for the first time the possibility of irradiating biological cells with gray (Gy)-scale doses delivered over single bursts of picosecond-scale electron beams, resulting in unprecedented dose rates of 1010 to 1011 Gy/s. METHODS AND MATERIALS: Cancer stem cells and human skin fibroblasts were irradiated with MeV-scale electron beams from a laser-driven source. Doses up to 3 Gy per pulse with a high spatial uniformity (coefficient of variance, 3%-6%) and within a timescale range of 10 to 20 picoseconds were delivered. Doses were characterized during irradiation and were found to be in agreement with Monte Carlo simulations. Cell survival and DNA double-strand break repair dynamics were studied for both cell lines using clonogenic assay and 53BP1 foci formation. The results were compared with reference x-rays at a dose rate of 0.49 Gy/min. RESULTS: Results from clonogenic assays of both cell lines up to 3 Gy were well fitted by a linear quadratic model with α = (0.68 ± 0.08) Gy-1 and ß = (0.01 ± 0.01) Gy-2 for human skin fibroblasts and α = (0.51 ± 0.14) Gy-1 and ß = (0.01 ± 0.01) Gy-2 for cancer stem cells. Compared with irradiation at 0.49 Gy/min, our experimental results indicate no statistically significant difference in cell survival rate for doses up to 3 Gy despite a significant increase in the α parameter, which may reflect more complex damage. Foci measurements showed no significant difference between irradiation at 1011 Gy/s and at 0.49 Gy/min. CONCLUSIONS: This study demonstrates the possibility of performing radiobiological studies with picosecond-scale laser-generated electron beams at ultrahigh dose rates of 1010 to1011 Gy/s. Preliminary results indicate, within statistical uncertainties, a significant increase of the α parameter, a possible indication of more complex damage induced by a higher density of ionizing tracks.

Dissolved inorganic and organic carbon export from tile-drained midwestern agricultural systems.

While carbon is a critically important natural element cycling through the soil profile of agricultural systems, few studies have examined the flux of dissolved organic carbon (OC) and inorganic carbon (IC) through artificially-drained cropped fields. In this study, we monitored eight tile outlets, nine groundwater wells and the receiving stream during a March to November period in 2018 to quantify subsurface IC and OC flux from tiles and groundwater to a perennial stream from a single cropped field in north-central Iowa. Results showed that carbon export from the field was dominated by IC losses through subsurface drainage tiles that were 20× higher than dissolved OC concentration in tiles, groundwater and in Hardin Creek. IC loads from tiles comprised approximately 96 % of the total carbon export. Detailed soil sampling within the field quantified TC stocks to a 1.2 m depth (246,514 kg/ha), and based on the maximum annual rate of IC loss from the field (553 kg/ha per year), we estimated that approximately 0.23 % of the TC content (0.32 % of the TOC content and 0.70 % of the TIC content) of the shallow soils was lost in a single year. Loss of dissolved carbon from the field is likely offset by reduced tillage and additions of lime. Study results suggest that attention should be given to improved monitoring of aqueous total carbon export from fields for accurate accounting of carbon sequestration performance.

Reconnaissance of Oxygenic Denitrifiers in Agriculturally Impacted Soils.

Row crop production in the agricultural Midwest pollutes waterways with nitrate, and exacerbates climate change through increased emissions of nitrous oxide and methane. Oxygenic denitrification processes in agricultural soils mitigate nitrate and nitrous oxide pollution by short-circuiting the canonical pathway to avoid nitrous oxide formation. Furthermore, many oxygenic denitrifiers employ a nitric oxide dismutase (nod) to create molecular oxygen that is used by methane monooxygenase to oxidize methane in otherwise anoxic soils. The direct investigation of nod genes that could facilitate oxygenic denitrification processes in agricultural sites is limited, with no prior studies investigating nod genes at tile drainage sites. Thus, we performed a reconnaissance of nod genes at variably saturated surface sites, and within a variably to fully saturated soil core in Iowa to expand the known distribution of oxygenic denitrifiers. We identified new nod gene sequences from agricultural soil and freshwater sediments in addition to identifying nitric oxide reductase (qNor) related sequences. Surface and variably saturated core samples displayed a nod to 16S rRNA gene relative abundance of 0.004% to 0.1% and fully saturated core samples had relative nod gene abundance of 1.2%. The relative abundance of the phylum Methylomirabilota increased from 0.6% and 1% in the variably saturated core samples to 3.8% and 5.3% in the fully saturated core samples. The more than 10-fold increase in relative nod abundance and almost 9-fold increase in relative Methylomirabilota abundance in fully saturated soils suggests that potential oxygenic denitrifiers play a greater nitrogen cycling role under these conditions. IMPORTANCE The direct investigation of nod genes in agricultural sites is limited, with no prior studies investigating nod genes at tile drains. An improved understanding of nod gene diversity and distribution is significant to the field of bioremediation and ecosystem services. The expansion of the nod gene database will advance oxygenic denitrification as a potential strategy for sustainable nitrate and nitrous oxide mitigation, specifically for agricultural sites.

Targeting the Expanded TCF4/Fuchs' Endothelial Corneal Dystrophy CUG Repeat with Morpholino Peptide Conjugates.

Fuchs' corneal endothelial dystrophy (FECD) is a major cause of vision loss. Corneal transplantation is the only effective curative treatment, but this surgery has limitations. A pharmacological intervention would complement surgery and be beneficial for many patients. FECD is caused by an expanded CUG repeat within intron 2 of the TCF4 RNA. Agents that recognize the expanded repeat can reverse the splicing defects associated with the disease. Successful drug development will require diverse strategies for optimizing the efficacy of anti-CUG oligomers. In this study, we evaluate anti-CUG morpholinos conjugated to cyclic cell penetrating peptides. The morpholino domain of the conjugate is complementary to the repeat, while the peptide has been optimized for import across cell membranes. We show that morpholino conjugates can enter corneal endothelial cells and block the CUG RNA foci associated with the disease. These experiments support morpholino peptide conjugates as an approach for developing anti-CUG therapies for FECD.

Generation and Characterization of Anti-Glucosepane Antibodies Enabling Direct Detection of Glucosepane in Retinal Tissue.

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

Glycation and Serum Albumin Infiltration Contribute to the Structural Degeneration of Bioprosthetic Heart Valves.

Valvular heart diseases are associated with significant cardiovascular morbidity and mortality, and often require surgical and/or percutaneous repair or replacement. Valve replacement is limited to mechanical and biological prostheses, the latter of which circumvent the need for lifelong anticoagulation but are subject to structural valve degeneration (SVD) and failure. Although calcification is heavily studied, noncalcific SVD, which represent roughly 30% of BHV failures, is relatively underinvestigated. This original work establishes 2 novel and interacting mechanisms-glycation and serum albumin incorporation-that occur in clinical valves and are sufficient to induce hallmarks of structural degeneration as well as functional deterioration.

A low glycemic diet protects disease-prone Nrf2-deficient mice against age-related macular degeneration.

Age-related macular degeneration (AMD) is a major blinding disease, affecting over 14% of the elderly. Risk for AMD is related to age, diet, environment, and genetics. Dietary modulation of AMD risk is a promising treatment modality, but requires appropriate animal models to demonstrate advantages of diet. Mice lacking the antioxidant transcription factor Nrf2 (Nfe2l2) develop age-related retinopathy relevant to human AMD. Here we evaluated the effect of consuming high glycemic (HG) or low glycemic (LG) diets until 18-months of age on development of features relevant to AMD in Nrf2-null mice. Nrf2-null mice that consumed HG diets developed atrophic AMD, characterized by photoreceptor degeneration, retinal pigment epithelium (RPE) atrophy and pigmentary abnormalities, basal laminar deposits, and loss of the choriocapillaris. In contrast, Nrf2-null-mice that consumed LG diets did not develop retinal disease phenotypes. Consumption of HG diets was associated with accumulation of advanced glycation end-products in the RPE and systemically, whereas consumption of the LG diet was associated with increased levels of anti-glycative and anti-oxidative detoxification machinery. Together our data indicate that the Nrf2-null HG mouse is a good model for atrophic AMD studies and that the LG diet can activate protective pathways to prevent AMD, even in a genetically predisposed animal.

Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes.

Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys" modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.

Soil sedimentation and quality within the roadside ditches of an agricultural watershed.

Roadside ditches are an integral component to the >6.3 million km of roadsides in the U.S. and act as drainageways for millions of hectares of watershed runoff. Our study of six roadside ditches in Lime Creek watershed characterized soil nutrients and heavy metal patterns as well as quantified the physical and hydrological properties of ditch soils. At all ditch sites, we identified significant sedimentation of silt-sized particles, total nitrogen, and soil carbon in shallow roadside ditch soils. A post-settlement surface soil horizon significantly higher in silt content was observed compared to the underlying subsoil and parent material. Although accumulation of several heavy metals was measured in ditch soils, significant variability was not observed within the ditch environment. Most of the heavy metal concentrations were found to be either similar to or lower than state-wide averages. Higher levels of calcium near the roads were likely due to annual use of road deicers. Overall, we estimated that 42 Mg/ha of total carbon and 5 Mg/ha of total nitrogen are being stored in agricultural ditch soils, which is similar to that of surrounding agricultural land in terms of total carbon storage, but much higher than estimates of total nitrogen storage. Our study of six roadside ditches in an eastern Iowa watershed documented the soil chemistry, morphology, and sediment accumulation that occurred since ditch construction. Further research is needed to develop a better understanding of how the soil and water conditions in the ditches related to the watershed areas that feed them.