Heterozygous knockout of Synaptotagmin13 phenocopies ALS features and TP53 activation in human motor neurons.

Spinal motor neurons (MNs) represent a highly vulnerable cellular population, which is affected in fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In this study, we show that the heterozygous loss of SYT13 is sufficient to trigger a neurodegenerative phenotype resembling those observed in ALS and SMA. SYT13+/- hiPSC-derived MNs displayed a progressive manifestation of typical neurodegenerative hallmarks such as loss of synaptic contacts and accumulation of aberrant aggregates. Moreover, analysis of the SYT13+/- transcriptome revealed a significant impairment in biological mechanisms involved in motoneuron specification and spinal cord differentiation. This transcriptional portrait also strikingly correlated with ALS signatures, displaying a significant convergence toward the expression of pro-apoptotic and pro-inflammatory genes, which are controlled by the transcription factor TP53. Our data show for the first time that the heterozygous loss of a single member of the synaptotagmin family, SYT13, is sufficient to trigger a series of abnormal alterations leading to MN sufferance, thus revealing novel insights into the selective vulnerability of this cell population.

Mitochondrial H2O2 release does not directly cause damage to chromosomal DNA.

Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of chromosomal DNA mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondria can directly damage nuclear DNA is largely lacking. In this study, we investigated the effects of H2O2 released by mitochondria or produced at the nucleosomes using a titratable chemogenetic approach. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2. Nuclear H2O2 gives rise to DNA damage and mutations and a subsequent p53 dependent cell cycle arrest. Mitochondrial H2O2 release shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. We conclude that H2O2 released from mitochondria is unlikely to directly damage nuclear genomic DNA, limiting its contribution to oncogenic transformation and aging.

Identified senescence endotypes in aged cartilage are reflected in the blood metabolome.

Heterogeneous accumulation of senescent cells expressing the senescence-associated secretory phenotype (SASP) affects tissue homeostasis which leads to diseases, such as osteoarthritis (OA). In this study, we set out to characterize heterogeneity of cellular senescence within aged articular cartilage and explored the presence of corresponding metabolic profiles in blood that could function as representative biomarkers. Hereto, we set out to perform cluster analyses, using a gene-set of 131 senescence genes (N = 57) in a previously established RNA sequencing dataset of aged articular cartilage and a generated metabolic dataset in overlapping blood samples. Using unsupervised hierarchical clustering and pathway analysis, we identified two robust cellular senescent endotypes. Endotype-1 was enriched for cell proliferating pathways, expressing forkhead box protein O4 (FOXO4), RB transcriptional corepressor like 2 (RBL2), and cyclin-dependent kinase inhibitor 1B (CDKN1B); the FOXO mediated cell cycle was identified as possible target for endotype-1 patients. Endotype-2 showed enriched inflammation-associated pathways, expressed by interleukin 6 (IL6), matrix metallopeptidase (MMP)1/3, and vascular endothelial growth factor (VEGF)C and SASP pathways were identified as possible targets for endotype-2 patients. Notably, plasma-based metabolic profiles in overlapping blood samples (N = 21) showed two corresponding metabolic clusters in blood. These non-invasive metabolic profiles could function as biomarkers for patient-tailored targeting of senescence in OA.

WNT/beta-catenin signalling interrupts a senescence-induction cascade in human mesenchymal stem cells that restricts their expansion.

Senescence, the irreversible cell cycle arrest of damaged cells, is accompanied by a deleterious pro-inflammatory senescence-associated secretory phenotype (SASP). Senescence and the SASP are major factors in aging, cancer, and degenerative diseases, and interfere with the expansion of adult cells in vitro, yet little is known about how to counteract their induction and deleterious effects. Paracrine signals are increasingly recognized as important senescence triggers and understanding their regulation and mode of action may provide novel opportunities to reduce senescence-induced inflammation and improve cell-based therapies. Here, we show that the signalling protein WNT3A counteracts the induction of paracrine senescence in cultured human adult mesenchymal stem cells (MSCs). We find that entry into senescence in a small subpopulation of MSCs triggers a secretome that causes a feed-forward signalling cascade that with increasing speed induces healthy cells into senescence. WNT signals interrupt this cascade by repressing cytokines that mediate this induction of senescence. Inhibition of those mediators by interference with NF-κB or interleukin 6 signalling reduced paracrine senescence in absence of WNT3A and promoted the expansion of MSCs. Our work reveals how WNT signals can antagonize senescence and has relevance not only for expansion of adult cells but can also provide new insights into senescence-associated inflammatory and degenerative diseases.

Greenhouse Gas Inventory Model for Biochar Additions to Soil.

Stabilizing the global climate within safe bounds will require greenhouse gas (GHG) emissions to reach net zero within a few decades. Achieving this is expected to require removal of CO2 from the atmosphere to offset some hard-to-eliminate emissions. There is, therefore, a clear need for GHG accounting protocols that quantify the mitigation impact of CO2 removal practices, such as biochar sequestration, that have the potential to be deployed at scale. Here, we have developed a GHG accounting methodology for biochar application to mineral soils using simple parameterizations and readily accessible activity data that can be applied at a range of scales including farm, supply chain, national, or global. The method is grounded in a comprehensive analysis of current empirical data, making it a robust method that can be used for many applications including national inventories and voluntary and compliance carbon markets, among others. We show that the carbon content of biochar varies with feedstock and production conditions from as low as 7% (gasification of biosolids) to 79% (pyrolysis of wood at above 600 °C). Of this initial carbon, 63-82% will remain unmineralized in soil after 100 years at the global mean annual cropland-temperature of 14.9 °C. With this method, researchers and managers can address the long-term sequestration of C through biochar that is blended with soils through assessments such as GHG inventories and life cycle analyses.

A short de novo synthesis of nucleoside analogs.

Nucleoside analogs are commonly used in the treatment of cancer and viral infections. Their syntheses benefit from decades of research but are often protracted, unamenable to diversification, and reliant on a limited pool of chiral carbohydrate starting materials. We present a process for rapidly constructing nucleoside analogs from simple achiral materials. Using only proline catalysis, heteroaryl-substituted acetaldehydes are fluorinated and then directly engaged in enantioselective aldol reactions in a one-pot reaction. A subsequent intramolecular fluoride displacement reaction provides a functionalized nucleoside analog. The versatility of this process is highlighted in multigram syntheses of d- or l-nucleoside analogs, locked nucleic acids, iminonucleosides, and C2'- and C4'-modified nucleoside analogs. This de novo synthesis creates opportunities for the preparation of diversity libraries and will support efforts in both drug discovery and development.

Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms.

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, and shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, which included interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltransferase and the stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associated antibiotic effects, especially the lack of resistance development, probably stem from the compound's polypharmacology.

Isolating Pediatric Mesenchymal Stem Cells with Enhanced Expansion and Differentiation Capabilities.

Mesenchymal stem cells/marrow stromal cells (MSCs) are attractive for applications ranging from research and development to use in clinical therapeutics. However, the most commonly studied MSCs, adult bone marrow MSCs (A-MSCs), are limited by significant donor variation resulting in inconsistent expansion rates and multilineage differentiation capabilities. We have recently obtained permission to isolate pediatric MSCs (P-MSCs) from surplus iliac crest bone chips. Here, we developed a simple and easily replicable isolation protocol yielding P-MSCs, which adhere to MSC defining guidelines. After confirming immunophenotypic marker expression, we compared expansion rates, senescence, morphology, and trilineage differentiation of P-MSCs to A-MSCs for multiple donors. We found P-MSCs have faster in vitro replication, consistently show significantly lower senescence, and are capable of more reproducible multilineage differentiation than A-MSCs. We, therefore, believe P-MSCs are a promising candidate for use in research applications and potentially as part of an allogeneic therapeutic treatment.

Vitamin D and Its Analogues Decrease Amyloid-ß (Aß) Formation and Increase Aß-Degradation.

Alzheimer's disease (AD) is characterized by extracellular plaques in the brain, mainly consisting of amyloid-ß (Aß), as derived from sequential cleavage of the amyloid precursor protein. Epidemiological studies suggest a tight link between hypovitaminosis of the secosteroid vitamin D and AD. Besides decreased vitamin D level in AD patients, an effect of vitamin D on Aß-homeostasis is discussed. However, the exact underlying mechanisms remain to be elucidated and nothing is known about the potential effect of vitamin D analogues. Here we systematically investigate the effect of vitamin D and therapeutically used analogues (maxacalcitol, calcipotriol, alfacalcidol, paricalcitol, doxercalciferol) on AD-relevant mechanisms. D2 and D3 analogues decreased Aß-production and increased Aß-degradation in neuroblastoma cells or vitamin D deficient mouse brains. Effects were mediated by affecting the Aß-producing enzymes BACE1 and γ-secretase. A reduced secretase activity was accompanied by a decreased BACE1 protein level and nicastrin expression, an essential component of the γ-secretase. Vitamin D and analogues decreased ß-secretase activity, not only in mouse brains with mild vitamin D hypovitaminosis, but also in non-deficient mouse brains. Our results further strengthen the link between AD and vitamin D, suggesting that supplementation of vitamin D or vitamin D analogues might have beneficial effects in AD prevention.

Systematic Comparison of Protocols for the Preparation of Human Articular Cartilage for Use as Scaffold Material in Cartilage Tissue Engineering.

Natural extracellular matrix-derived biomaterials from decellularized allogenic tissues are of increasing interest for tissue engineering because their structure and composition provide a complexity that is not achievable with current manufacturing techniques. The prerequisite to bring allogenic tissue from bench to bedside as a functional biomaterial is the full removal of cells while preserving most of its native characteristics such as structure and composition. The exceptionally dense structure of articular cartilage, however, poses a special challenge for decellularization, scaffold preparation, and reseeding. Therefore, we tested 24 different protocols aiming to remove cells and glycosaminoglycans (GAG) while preserving the collagen backbone and ultrastructure. The resulting matrices were analyzed for cell removal (DNA quantification, haematoxylin and eosin staining), GAG content (dimethyl methylene blue assay, Alcian blue staining and micro-computed tomography), collagen integrity (immunohistochemistry and ultrastructure), and biomechanics (compression test). Furthermore, seeding tests were conducted to evaluate cell viability and attachment to the scaffolds. Sodium dodecyl sulfate-based protocols yielded satisfactory reduction of DNA content, yet had negative effects on cell viability and attachment. Hydrochloric acid efficiently decellularized the scaffold and pepsin emerged as best option for GAG depletion. Combining these two reagents led to our final protocol, most efficient in DNA and GAG depletion while preserving the collagen architecture. The compressive modulus decreased in the absence of GAG to ∼1/3 of native cartilage, which is significantly higher than that by commercially available scaffolds tested as a reference (ranging from 1/25 to 1/100 of native cartilage). Cytocompatibility tests showed that human adipose-derived stromal cells readily adhered to the scaffold. In this study, we established a protocol combining freeze-thaw cycles, osmotic shock, and treatment with hydrochloric acid followed by a pepsin digestion step, achieving successful decellularization and GAG depletion within 1 week, resulting in a cytocompatible material with intact collagen structure. The protocol provides a basis for the generation of allogeneic scaffolds, potentially substituting manufactured scaffolds currently used in clinical articular cartilage treatment.

Differential Effects of Small Molecule WNT Agonists on the Multilineage Differentiation Capacity of Human Mesenchymal Stem Cells.

Human bone marrow-derived mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies, but loss of expansion and differentiation potential in vitro limits their applicability. Recently we showed that WNT3A protein promoted MSC proliferation and enhanced their chondrogenic potential, while simultaneously suppressing the propensity of the cartilage to undergo hypertrophic maturation. Since WNT3A protein is costly and rapidly loses its activity in culture, we investigated the possibility of replacing it with cheaper commercially available WNT agonists, specifically lithium chloride (LiCl), CHIR99021 (CHIR), SKL2001, and AMBMP. Of these, we found that only CHIR and LiCl stimulated MSC proliferation. Moreover, CHIR enhanced the chondrogenic capacity of MSCs, whereas LiCl predominantly increased the osteo- and adipogenic capacity. The different WNT agonists also differentially impacted the surface marker profile of the MSCs, possibly explaining the observed differences. Moreover, CHIR suppressed the hypertrophic propensity of the MSC-derived cartilage after in vivo implantation to an extent approaching that of WNT3A protein. These results indicate that CHIR may be a promising alternative for WNT3A protein for certain applications of human bone marrow-derived MSCs.

The impact of cholesterol, DHA, and sphingolipids on Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder currently affecting over 35 million people worldwide. Pathological hallmarks of AD are massive amyloidosis, extracellular senile plaques, and intracellular neurofibrillary tangles accompanied by an excessive loss of synapses. Major constituents of senile plaques are 40-42 amino acid long peptides termed ß -amyloid (A ß ). A ß is produced by sequential proteolytic processing of the amyloid precursor protein (APP). APP processing and A ß production have been one of the central scopes in AD research in the past. In the last years, lipids and lipid-related issues are more frequently discussed to contribute to the AD pathogenesis. This review summarizes lipid alterations found in AD postmortem brains, AD transgenic mouse models, and the current understanding of how lipids influence the molecular mechanisms leading to AD and A ß generation, focusing especially on cholesterol, docosahexaenoic acid (DHA), and sphingolipids/glycosphingolipids.

Micro- and nano-environments of C sequestration in soil: a multi-elemental STXM-NEXAFS assessment of black C and organomineral associations.

Black C is an essential component of the terrestrial C pool and its formation is often credited as a CO(2) sink by transferring the fast-cycling C from the atmosphere-biosphere system into slower cycling C in the geosphere. This study is the first multi-element K- (C, N, Ca, Fe, Al and Si) soft-X-ray STXM-NEXAFS investigation conducted at a submicron-scale spatial resolution specifically targeting black C and its interaction with the mineral and non-black C organic matter in the organomineral assemblage. The STXM-NEXAFS micrographs and spectra demonstrated that pyrogenic C was dominated by quinoide, aromatic, phenol, ketone, alcohol, carboxylic and hydroxylated- and ether-linked C species. There was also evidence for the presence of pyridinic, pyridonic, pyrrolic, amine and nitril N functionalities. The non-black C organic matter contained amino acids, amino sugars, nucleic acids and polysaccharides known to exhibit negatively charged carboxylic, phenolic, enolic, thiolate and phosphate functionalities highly reactive towards metal ions and black C. The metal-rich mineral matrix was composed of phyllosilicate clay minerals, Fe and Al hydroxypolycations, oxides, hydroxides and oxyhydroxide that can attract and bind organic biopolymers. STXM-NEXAFS provided evidence for interactive association between pyrogenic C, non-black C organic matter and the mineral oxide and oxyhydroxide communities in the organomineral interface. These intimate associations occurred through a "two-way" direct linkage between black C and the mineral or non-black C organic matter or via a "three-way" indirect association where non-black C organic matter could serve as a molecular cross-linking agent binding black C with the mineral matrix or vice versa where inorganic oxides, hydroxides and polycations could act as a bridge to bind black C with non-black C organic matter. The binding and sequestration of black C in the investigated micro- and nano-C repository environments seem to be the combined action of physical entrapment in seemingly terminal biotic exclusion zone through the action of metal oxides and organic matter induced microaggregation and through molecular-level association ranging from ligand exchange, polyvalent cation bridging to weak hydrophobic interactions including van der Waals and H-bonding.

Temperature sensitivity of black carbon decomposition and oxidation.

Global warming accelerates decomposition of soil organic carbon (SOC) pools with varying rates and temperature sensitivities. Black carbon (BC) materials are among the slowest decomposing components of the SOC pool. Although BC is a large component of SOC in many systems, the influence of temperature on decomposition of BC bearing different chemical and physical structures remains poorly understood. Four BC materials, produced by carbonizing corn residue and oak wood at 350 and 600 degrees C (corn-350-BC, corn-600-BC, oak-350-BC, and oak-600-BC), were mixed with pure sand and incubated at 4, 10, 20, 30, 45, and 60 degrees C for 1 year. Corn-BC was more porous than oak-BC as determined by scanning electron microscopy (SEM). Increasing the charring temperature from 350 to 600 degrees C led to greater aromaticity with 5-15% more C in aromatic rings and a 39-57% increase in both nonprotonated aromatic C and aromatic bridgehead C quantified by nuclear magnetic resonance (NMR) spectroscopy and a greater degree of order and development of C layers as observed by transmission electron microscopy (TEM). With a temperature increase from 4 to 60 degrees C, C loss of corn-350-BC increased from 10 to 20%, corn-600-BC, from 4 to 20%, oak-350-BC, from 2.3 to 15%, and oak-600-BC from 1.5 to 14% of initial C content, respectively. Temperature sensitivity (Q(10)) decreased with increasing incubation temperature and was highest in oak-600-BC, followed by oak-350-BC, corn-600-BC, and corn-350-BC, indicating that decomposition of more stable BC was more sensitive to increased temperature than less stable materials. Carbon loss and potential cation exchange capacity (CECp) significantly (p < 0.05) correlated with O/C ratios and change in O/C ratios, suggesting that oxidative processes were the most important mechanism controlling BC decomposition in this study.

Ageing of black carbon along a temperature gradient.

Black carbon (BC) is regarded as a chemically and biologically stable form of carbon and the changes of BC properties in nature are generally assumed to be minute. However, more and more observations have argued the inertness of BC. The objectives of this study were to characterize the changes of BC properties through ageing processes and to identify if these changes are associated with temperature. Our results showed that ageing of BC occurs over a temperature range from -22 degrees C to 70 degrees C within a short period of 12 months. The main changes of BC properties through ageing were found in elemental composition, surface chemistry, and adsorption properties, where the aged BCs were shown to have higher oxygen concentrations, surface acidity, and negative surface charge but lower C concentrations, pH, surface basicity, point of zero net charge, and also a lower adsorption capacity of hydroquinone, an allelopathic compound, than fresh BC. These ageing processes of BC were affected by temperature and changed over time, with higher temperature and longer incubation time enhancing BC ageing. Our results from a wide temperature range suggest that ageing of BC is likely to occur in any terrestrial regime and that the changes of BC properties through ageing should not be overlooked.

Soil mineral N dynamics beneath mixtures of leaves from legume and fruit trees in Central Amazonian multi-strata agroforests

Long term applications of leguminous green mulch could increase mineralizable nitrogen (N) beneath cupuaçu trees produced on the infertile acidic Ultisols and Oxisols of the Amazon Basin. However, low quality standing cupuaçu litter could interfere with green mulch N release and soil N mineralization. This study compared mineral N, total N, and microbial biomass N beneath cupuaçu trees grown in two different agroforestry systems, north of Manaus, Brazil, following seven years of different green mulch application rates. To test for net interactions between green mulch and cupuaçu litter, dried gliricidia and inga leaves were mixed with senescent cupuaçu leaves, surface applied to an Oxisol soil, and incubated in a greenhouse for 162 days. Leaf decomposition, N release and soil N mineralization were periodically measured in the mixed species litter treatments and compared to single species applications. The effect of legume biomass and cupuaçu litter on soil mineral N was additive implying that recommendations for green mulch applications to cupuaçu trees can be based on N dynamics of individual green mulch species. Results demonstrated that residue quality, not quantity, was the dominant factor affecting the rate of N release from leaves and soil N mineralization in a controlled environment. In the field, complex N cycling and other factors, including soil fauna, roots, and microclimatic effects, had a stronger influence on available soil N than residue quality.

Aplicações a longo prazo de leguminosas como adubo verde podem aumentar o nitrogênio (N) mineralizável sob árvores de cupuaçu em solos pouco férteis e ácidos (Ultisols e Oxisols) da Bacia Amazônica. Entretanto, a baixa qualidade da liteira de cupuaçu pode influênciara liberação de N do adubo verde e a mineralização deste no solo. Neste estudo foram comparados o N mineral, N total, e o N da biomassa microbiana sob árvores de cupuaçu cultivadas em dois sistemas agroflorestais, ao norte de Manaus, Brasil, as quais receberam diferentes aplicações de adubo verde sob sua liteira natural durante sete anos. Para testar as interações entre o adubo verde e a liteira de cupuaçu, folhas secas de gliricídia e ingá foram misturadas com as folhas senescentes de cupuaçu, distribuídas na superfície de um solo Oxisol, e incubadas em casa de vegetação durante 162 dias. A decomposição das folhas, a liberação de N e mineralização do N no solo foram periodicamente mensurados nos tratamentos de mistura de liteira de diferentes espécies e comparados com as aplicações de liteira de apenas uma espécie. O efeito da biomassa de leguminosas e da liteira de cupuaçu no N mineral do solo foi aditivo, indicando que o uso de adubação verde em plantas de cupuaçu pode ser baseado na dinâmica do N em cada espécie usada como adubo verde. Os resultados demonstraram que a qualidade do resíduo, e não a quantidade, foi o principal fator que influenciou a taxa de liberação de N das folhas e a mineralização deste no solo, em ambiente controlado. No campo, o complexo ciclo do N e outros fatores tais como a fauna do solo, raízes e os efeitos do microclima, tiveram uma influência mais forte na disponibilidade de N no solo do que a qualidade d o resíduo vegetal.

Fertilization and cover crop effects on soil nitrogen and plant nutrition in a young guarana plantation / Efeitos da fertilização mineral e da cobertura do solo sobre a dinâmica do Nitrogênio e na nutrição de plantas jovens de guaraná

Fruit tree production is gaining an increasing importance in the central Amazon and elsewhere in the humid tropics, but very little is known about the nutrient dynamics in the soil-plant system. The present study quantified the effects of fertilization and cover cropping with a legume (Pueraria phaseoloides (Roxb.) Benth.) on soil nitrogen (N) dynamics and plant nutrition in a young guarana plantation (Paullinia cupana Kunth. (H.B. and K.) var. sorbilis (Mart.) Ducke) on a highly weathered Xanthic Ferralsol. Large subsoil nitrate (NO3-) accumulation at 0.3-3 m below the guarana plantation indicated N leaching from the topsoil. The NO3- contents to a depth of 2 m were 2.4 times greater between the trees than underneath unfertilized trees (P<0.05). The legume cover crop between the trees increased soil N availability as shown by elevated aerobic N mineralization and lower N immobilization in microbial biomass. The guarana N nutrition and yield did not benefit from the N input by biological fixation of atmospheric N2 by the legume cover (P>0.05). Even without a legume intercrop, large amounts of NO3- were found in the subsoil between unfertilized trees. Subsoil NO3- between the trees could be utilized, however, by fertilized guarana. This can be explained by a more vigorous growth of fertilized trees which had a larger nutrient demand and exploited a larger soil volume. With a legume cover crop, however, more mineral N was available at the topsoil which was leached into the subsoil and consequently accumulated at 0.3-3 m depth. Fertilizer additions of P and K were needed to increase subsoil NO3- use between trees.

A produção de fruteiras está ganhando grande importância na Amazônia Central e em outras partes dos trópicos úmidos mas, muito pouco ainda é conhecido sobre a dinâmica de nutrientes no sistema solo-planta. O presente estudo quantificou os efeitos da fertilização mineral e da cobertura do solo com uma leguminosa (Pueraria phaseoloides (Roxb) Benth.) sobre a dinâmica do N no solo e sobre a nutrição de plantas jovens de guaraná (Paullinia cupana Kunth. (H.B. and K.) var. sorbilis (Mart.) Ducke), em um Latossolo Amarelo muito argiloso. Grande acúmulo de nitrato (NO3-) encontrado na profundidade de 0,3 - 3,0m abaixo do plantio de guaraná é um indicativo da lixiviação de N da camada superficial. Os teores de NO3- na profundidade de 2m era 2,4 vezes maior entre as plantas do que na entrelinha que não recebeu fertilização (P<0,05). A leguminosa de cobertura, entre as plantas de guaraná, aumentou a disponibilidade de N, conforme é indicado pela elevada mineralização aeróbica e baixa imobilização de N na massa microbiana. A nutrição nitrogenada e a produção do guaraná não foram beneficiados pela adição de N, via fixação biológica do N2 da atmosfera pela leguminosa de cobertura (P<0,05). Mesmo sem leguminosa nas entrelinhas de plantio, grandes quantidades de NO3- foram encontradas no subsolo, entre plantas não adubadas. O NO3- do subsolo entre as plantas pode, entretanto, ter sido utilizado pelo guaraná fertilizado. Isso pode ser explicado pelo crescimento mais vigoroso das plantas fertilizadas as quais têm uma grande demanda por nutrientes e exploraram maior volume de solo. Com uma leguminosa de cobertura, contudo, mais N mineral foi disponibilizado na camada superficial, o qual foi lixiviado para o subsolo e, consequentemente, acumulado na camada de 0,3 a 3,0m de profundidade. Adições suplementares de P e K foram necessárias para aumentar a utilização do NO3- entre as plantas.