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Background: The gut microbiome has emerged as a clear player in health and disease, in part by mediating host response to environment and lifestyle. The urobiome (microbiota of the urinary tract) likely functions similarly. However, efforts to characterize the urobiome and assess its functional potential have been limited due to technical challenges including low microbial biomass and high host cell shedding in urine. Here, to begin addressing these challenges, we evaluate urine sample volume (100 ml - 5 mL), and host DNA depletion methods and their effects on urobiome profiles in healthy dogs, which are a robust large animal model for the human urobiome. We collected urine from seven dogs and fractionated samples into aliquots. One set of samples was spiked with host (canine) cells to model a biologically relevant host cell burden in urine. Samples then underwent DNA extraction followed by 16S rRNA gene and shotgun metagenomic sequencing. We then assembled metagenome assembled genomes (MAGs) and compared microbial composition and diversity across groups. We tested six methods of DNA extraction: QIAamp BiOstic Bacteremia (no host depletion), QIAamp DNA Microbiome, Molzym MolYsis, NEBNext Microbiome DNA Enrichment, Zymo HostZERO, and Propidium Monoazide. Results: In relation to urine sample volume, 3 3.0 mL resulted in the most consistent urobiome profiling. In relation to host depletion, individual (dog) but not extraction method drove overall differences in microbial composition. DNA Microbiome yielded the greatest microbial diversity in 16S rRNA sequencing data and shotgun metagenomic sequencing data, and maximized MAG recovery while effectively depleting host DNA in host-spiked urine samples. As proof-of-principle, we then mined MAGs for core metabolic functions and environmental chemical metabolism. We identified long chain alkane utilization in two of the urine MAGs. Long chain alkanes are common pollutants that result from industrial combustion processes and end up in urine. Conclusions: This is the first study, to our knowledge, to demonstrate environmental chemical degradation potential in urine microbes through genome-resolved metagenomics. These findings provide guidelines for studying the urobiome in relation to sample volume and host depletion, and lay the foundation for future evaluation of urobiome function in relation to health and disease.
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Since the discovery of complete ammonia oxidizers (comammox) within the genus Nitrospira, their distribution and abundance across habitats have been intensively studied to better understand their ecological significance. Many primers targeting their ammonia monooxygenase subunit A gene (amoA) have been designed to detect and quantify comammox bacteria and to describe their community structure. We identified 38 published primers, but only few had high coverage and specificity for all known comammox Nitrospira or one of the two described subclades. For each target group, we comprehensively evaluated selected primer pairs using in silico analyses, endpoint PCRs, qPCRs, and amplicon sequencing on samples from various environments. Endpoint PCRs and qPCRs showed that the most commonly used primer pairs (comaA-244F/659R, comaB-244F/659R, and Ntsp-amoA162F/359R) produced several bands, which likely inflated quantifications via qPCR. In contrast, the recently published primer combinations CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R resulted mostly in a single band. Furthermore, amplicon sequencing demonstrated that these primer combinations also captured the highest richness of comammox Nitrospira. Taken together, our results indicate that few existing comammox amoA primer combinations have both high specificity and coverage and that the choice of these high-specificity and high-coverage primer pairs substantially impacts the accurate detection, quantification, and community description of comammox bacteria. We, therefore, recommend using the CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R primer pairs.IMPORTANCEBacteria that can fully convert ammonia via nitrite to nitrate, the complete ammonia oxidizers (comammox), were recently discovered and are found in many natural and engineered environments. PCR-based tools to study their abundance and diversity were rapidly developed, resulting in a plethora of primers available, many of which are widely used. The presence of comammox bacteria in an environment can, however, only be correctly determined if the used primers detect all members of this group while not detecting any other guilds. This study assesses the coverage and specificity of existing primers targeting comammox bacteria using both computational and standard molecular techniques, revealing large differences in their performance. The uniform usage of well-performing primers across studies could aid in generating comparable and generalizable data to better understand the importance of comammox bacteria in the environment.
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BACKGROUND: Prokaryotic microbes have impacted marine biogeochemical cycles for billions of years. Viruses also impact these cycles, through lysis, horizontal gene transfer, and encoding and expressing genes that contribute to metabolic reprogramming of prokaryotic cells. While this impact is difficult to quantify in nature, we hypothesized that it can be examined by surveying virus-encoded auxiliary metabolic genes (AMGs) and assessing their ecological context. RESULTS: We systematically developed a global ocean AMG catalog by integrating previously described and newly identified AMGs and then placed this catalog into ecological and metabolic contexts relevant to ocean biogeochemistry. From 7.6 terabases of Tara Oceans paired prokaryote- and virus-enriched metagenomic sequence data, we increased known ocean virus populations to 579,904 (up 16%). From these virus populations, we then conservatively identified 86,913 AMGs that grouped into 22,779 sequence-based gene clusters, 7248 (~ 32%) of which were not previously reported. Using our catalog and modeled data from mock communities, we estimate that ~ 19% of ocean virus populations carry at least one AMG. To understand AMGs in their metabolic context, we identified 340 metabolic pathways encoded by ocean microbes and showed that AMGs map to 128 of them. Furthermore, we identified metabolic "hot spots" targeted by virus AMGs, including nine pathways where most steps (≥ 0.75) were AMG-targeted (involved in carbohydrate, amino acid, fatty acid, and nucleotide metabolism), as well as other pathways where virus-encoded AMGs outnumbered cellular homologs (involved in lipid A phosphates, phosphatidylethanolamine, creatine biosynthesis, phosphoribosylamine-glycine ligase, and carbamoyl-phosphate synthase pathways). CONCLUSIONS: Together, this systematically curated, global ocean AMG catalog and analyses provide a valuable resource and foundational observations to understand the role of viruses in modulating global ocean metabolisms and their biogeochemical implications. Video Abstract.
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Oceanos e Mares , Água do Mar , Água do Mar/virologia , Água do Mar/microbiologia , Metagenômica , Bactérias/genética , Bactérias/classificação , Bactérias/metabolismo , Vírus/genética , Vírus/classificação , Vírus/isolamento & purificação , Células Procarióticas/metabolismo , Células Procarióticas/virologia , Metagenoma , Redes e Vias Metabólicas/genética , Transferência Genética Horizontal , Fosfatidiletanolaminas/metabolismoRESUMO
Through a behavioural coordination game played by groups of humans and simulated with agent-based models, we investigated a social network dilemma that we call fraughtness. Seven players, connected to one another in various topologies via a computer network, each had to move a slider to the left or right along a horizontal bar on their screen. The goal was for all the players to move their slider to the same side. Players received feedback indicating the degree to which they and their neighbours agreed about the choice of side. When the topology had a hierarchical branching structure, the groups often got stuck in fraughtness: players on one branch favoured one side, while players on the other branch favoured the other; because all were receiving supportive local feedback, nobody wanted to change. Nevertheless, after being stuck in fraughtness for some time, most groups managed to escape it. Fraughtness is arguably an analog of generally negatively viewed social phenomena like polarization and echo chambers. Our analyses suggest that while fraughtness is problematic, it is closely linked to successful structure formation-it thus may be most effective to focus not on how to banish it, but on how to resolve it.
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BACKGROUND, INTRODUCTION AND AIM: Ureteral stent-related symptoms (USRS) often result in unplanned phone calls and ER visits. We hypothesize that patient factors can be identified to predict these unplanned encounters. METHODS AND MATERIALS: Retrospective analysis of indwelling ureteral stent placements from 2014 to 2019 at a single institution by CPT code was performed. Patient demographics, discharge medications, and clinical factors were evaluated using multiple logistic regression with respect to postoperative telephone and emergency room (ER) encounters for USRS. RESULTS: Of 374 patients, 75 (20.1%) had one or more encounters for USRS: 48 (12.8%) called the clinic and 39 (10.4%) returned to the ER. Chronic opioid use was predictive of calls to clinic and ER visits (OR 3.21 [CI 1.42-6.97], p < 0.01 and OR 3.64 [CI 1.45-8.98], p < 0.01). Survival analysis stratified by history of chronic opioid use and discharge opioid prescriptions demonstrated that opioid naïve patients receiving opioids at discharge had unplanned encounters sooner and more often [Calls p = 0.025, ER p = 0.041]), whereas patients with chronic opioid use returned to the ER sooner and more frequently when prescribed additional opioids (Calls p = 0.4, ER p = 0.002). CONCLUSION: Patients with a history of chronic opioid use may experience more intense USRS or have a lower threshold to seek medical care than opioid naïve patients and tend to bypass calling the clinic for the ER. Given that none of the studied medications reduced unplanned patient contact for USRS, urologists should consider upfront definitive management of urinary obstruction when appropriate.
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Analgésicos Opioides , Visitas ao Pronto Socorro , Humanos , Estudos Retrospectivos , Alta do Paciente , StentsRESUMO
Agricultural drainage ditches are subjected to high anthropogenic nitrogen input, leading to eutrophication and greenhouse gas emissions. Nitrate-dependent anaerobic methane oxidation (N-DAMO) could be a promising remediation strategy to remove methane (CH4) and nitrate (NO3-) simultaneously. Therefore, we aimed to evaluate the potential of N-DAMO to remove excess NO3- and decrease CH4 release from agricultural drainage ditches. Microcosm experiments were conducted using sediment and surface water collected from three different sites: a sandy-clay ditch (SCD), a freshwater-fed peatland ditch (FPD), and a brackish peatland ditch (BPD). The microcosms were inoculated with an N-DAMO enrichment culture dominated by Candidatus Methanoperedens and Candidatus Methylomirabilis and supplemented with 13CH4 and 15NO3-. A significant decrease in CH4 and NO3- concentration was only observed in the BPD sediment. In freshwater sediments (FPD and SCD), the effect of N-DAMO inoculation on CH4 and NO3- removal was negligible, likely because N-DAMO microorganisms were outcompeted by heterotrophic denitrifiers consuming NO3- much faster. Overall, our results suggest that bioaugmentation with N-DAMO might be a potential strategy for decreasing NO3- concentrations and CH4 emission in brackish ecosystems with increasing agricultural activities where the native microbial community is incapable of efficient denitrification.
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Agricultura , Biodegradação Ambiental , Metano , Nitratos , Purificação da Água , Anaerobiose , Reatores Biológicos , Desnitrificação , Ecossistema , Metano/metabolismo , Nitratos/metabolismo , Oxirredução , Areia , Purificação da Água/métodos , Água Doce , Águas Salinas , Microbiologia da Água , Bactérias/metabolismoRESUMO
The drinking water quality in Southeast Asia is at risk due to arsenic (As) groundwater contamination. Intensive use of fertilizers may lead to nitrate (NO3-) leaching into aquifers, yet very little is known about its effect on iron (Fe) and As mobility in water. We ran a set of microcosm experiments using aquifer sediment from Vietnam supplemented with 15NO3- and 13CH4. To assess the effect of nitrate-dependent anaerobic methane oxidation (N-DAMO) we also inoculated the sediment with two different N-DAMO enrichment cultures. We found that native microorganisms and both N-DAMO enrichments could efficiently consume all NO3- in 5 days. However, CH4 oxidation was observed only in the inoculated microcosms, suggesting that the native microbial community did not perform N-DAMO. In uninoculated microcosms, NO3- was preferentially used over Fe(III) as an electron acceptor and consequently inhibited Fe(III) reduction and As mobilization. The addition of N-DAMO enrichment cultures led to Fe(III) reduction and stimulated As and Mn release into the water. The archaeal community in all treatments was dominated by Ca. Methanoperedens while the bacterial community consisted of various denitrifiers. Our results suggest that input of N fertilizers to the aquifer decreases As mobility and that CH4 cannot serve as an electron donor for NO3- reduction.
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Arsênio , Compostos Férricos , Água Subterrânea , Anaerobiose , Fertilizantes , Água Subterrânea/química , Metano/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , OxirreduçãoRESUMO
OBJECTIVE: To determine factors predicting if a radiologists... report of a .. stone... on ultrasound (US) was not actually a clinically significant stone, based on subsequent computed tomogram (CT). US often overestimates stone size and various pathologic entities are also hyperechoic;.ßthus, a subsequent CT without a clinically significant stone may represent unnecessary radiation exposure. A decision-tree and nomogram were developed to predict when stones are unlikely on subsequent CT. METHODS: Retrospective analysis of patients, of any age, receiving CT within 24.ßhours of a sonographic report documenting a single renal stone, during 2019...2020, in any phase of care, at one institution. Novel stone-likelihood-systems for US and CT (US-SLS, CT-SLS) were devised and validated to classify stones as clinically significant or insignificant, with CT as the gold standard. Binomial logistic regression predicting clinically significant stones was performed with sonographic and patient characteristics. RESULTS: Eight hundred twenty patients had US followed by CT, 228 (27.8%) reported documented stones, 140 (17.1%) reported a single stone. Clinically significant stones were associated with larger stone size (P: .002), location (P: .002), hydronephrosis (P: .04), shadowing-artifact (P: .02) depth.ßto.ßstone (P: .008), and Body mass Index (BMI) (P: .01). US-SLS had higher sensitivity (95.4%) and negative-predictive-value (81.8%) compared to a multivariate model of significant variables. CONCLUSION: US-SLS appears to exclude clinically irrelevant .. stones... better than established criteria including twinkle or shadow in this retrospective analysis. A diagnostic algorithm and nomogram are presented. US-SLS and the associated decision tree can assist providers in avoiding unnecessary radiation when clinically significant stones are unlikely.
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Cálculos Renais , Tomografia Computadorizada por Raios X , Humanos , Estudos Retrospectivos , Cálculos Renais/diagnóstico por imagem , Valor Preditivo dos Testes , UltrassonografiaRESUMO
Axonal sprouting of dentate gyrus (DG) afferents after entorhinal cortex (EC) lesion is a model preparation to assess lesion-induced functional reorganization in a denervated target structure. Following a unilateral EC lesion, the surviving contralateral entorhinal projection, termed the crossed temporodentate pathway (CTD), and the heterotypic septal input to the DG, the septodentate pathway (SD), undergo extensive axonal sprouting. We explored whether EC lesion alters the capacity of the SD pathway to influence CTD-evoked granule cell excitability in the DG. We recorded extracellular field excitatory postsynaptic potentials (fEPSPs) after CTD stimulation alone and paired SD-CTD stimulation. Male rats were given unilateral EC lesions or sham operations; evoked fEPSPs in the DG were recorded at 4-, 15-, and 90-days post-entorhinal lesion to assess functional reorganization of the CTD and SD pathways. We found significantly increased fEPSP amplitudes in cases with unilateral lesions compared to sham-operates at 15- and 90-days post lesion. Within each time point, paired SD-CTD stimulation resulted in significantly depressed fEPSP amplitudes compared to amplitudes evoked after CTD stimulation alone and this effect was solely seen in cases with EC lesion. In cases where granule cell discharge was observed, SD stimulation increased discharge amplitude elicited by the CTD stimulation at 90-days postlesion. These findings demonstrate that synaptic remodeling following unilateral cortical lesion results in a synergistic interaction between two established hippocampal afferents that is not seen in uninjured brains. This work may be important for models of neurodegenerative disease and neural injury that target these structures and associated hippocampal circuitry.
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Doenças Neurodegenerativas , Ratos , Masculino , Animais , Neurônios/fisiologia , Hipocampo/fisiologia , Córtex Entorrinal/fisiologia , Giro DenteadoRESUMO
BACKGROUND AND PURPOSE: Previous studies have proposed multiple parallel channels for waste clearance from the brain, though many gaps remain in our understanding of these systems. In this study, we examined periarterial fluid drainage around intracranial and extracranial segments of the internal carotid arteries (ICAs) from the brain into the cervical lymph nodes using a noninvasive and clinical-based method. METHODS: Eighty-one subjects (45 females, aged 15-80 years old) with nonlesioned epilepsy underwent high-resolution 3-dimensional T2-weighted fluid-attenuated inversion recovery (FLAIR) MRI. We utilized a noninvasive and clinical-based method with a manual semiautomated approach to characterize the periarterial lymphatic system's maximum thickness and signal intensities along the ICAs using high-resolution 3-dimensional FLAIR imaging. We conducted group comparisons and correlation analyses to investigate sex- and age-based trends. Results were corrected with Bonferroni's test for multiple comparisons, and we performed power analysis for sample size calculations. RESULTS: Using high-resolution FLAIR images, we show evidence that fluid drainage emerges around the ICA petrous segment and joins lymphatic flow from cranial nerves in the upper neck, with this flow ultimately draining into the cervical lymph nodes bilaterally. Lymphatic signal at the petrous segment level was significantly thinner in females compared to males bilaterally (w = 413, p = .0001 on the right, w = 356, p < .0001 on the left). Lymphatic drainage around the petrous segments of the ICAs bilaterally was thicker with age in males but not in females. CONCLUSIONS: We describe the in vivo high-resolution imaging characteristics of periarterial fluid drainage along the vessel walls of ICAs. This represents a potentially major channel for brain waste clearance. We also report interesting sex- and age-based trends in these structures within our cohort.
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Linfonodos , Imageamento por Ressonância Magnética , Masculino , Feminino , Humanos , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Imageamento por Ressonância Magnética/métodos , Artéria Carótida Primitiva , EncéfaloRESUMO
Cue-based retrieval theories of sentence processing assume that syntactic dependencies are resolved through a content-addressable search process. An important recent claim is that in certain dependency types, the retrieval cues are weighted such that one cue dominates. This cue-weighting proposal aims to explain the observed average behavior, but here we show that there is systematic individual-level variation in cue weighting. Using the Lewis and Vasishth cue-based retrieval model, we estimated individual-level parameters for reading speed and cue weighting using 13 published datasets; hierarchical approximate Bayesian computation (ABC) was used to estimate the parameters. The modeling reveals a nuanced picture of cue weighting: we find support for the idea that some participants weight cues differentially, but not all participants do. Only fast readers tend to have the predicted higher weighting for structural cues, suggesting that reading proficiency (approximated here by reading speed) might be associated with cue weighting. A broader achievement of the work is to demonstrate how individual differences can be investigated in computational models of sentence processing without compromising the complexity of the model.
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Pharmaceuticals are relatively new to nature and often not completely removed in wastewater treatment plants (WWTPs). Consequently, these micropollutants end up in water bodies all around the world posing a great environmental risk. One exception to this recalcitrant conversion is paracetamol, whose full degradation has been linked to several microorganisms. However, the genes and corresponding proteins involved in microbial paracetamol degradation are still elusive. In order to improve our knowledge of the microbial paracetamol degradation pathway, we inoculated a bioreactor with sludge of a hospital WWTP (Pharmafilter, Delft, NL) and fed it with paracetamol as the sole carbon source. Paracetamol was fully degraded without any lag phase and the enriched microbial community was investigated by metagenomic and metatranscriptomic analyses, which demonstrated that the microbial community was very diverse. Dilution and plating on paracetamol-amended agar plates yielded two Pseudomonas sp. isolates: a fast-growing Pseudomonas sp. that degraded 200 mg/L of paracetamol in approximately 10 h while excreting 4-aminophenol, and a slow-growing Pseudomonas sp. that degraded paracetamol without obvious intermediates in more than 90 days. Each Pseudomonas sp. contained a different highly-expressed amidase (31% identity to each other). These amidase genes were not detected in the bioreactor metagenome suggesting that other as-yet uncharacterized amidases may be responsible for the first biodegradation step of paracetamol. Uncharacterized deaminase genes and genes encoding dioxygenase enzymes involved in the catabolism of aromatic compounds and amino acids were the most likely candidates responsible for the degradation of paracetamol intermediates based on their high expression levels in the bioreactor metagenome and the Pseudomonas spp. genomes. Furthermore, cross-feeding between different community members might have occurred to efficiently degrade paracetamol and its intermediates in the bioreactor. This study increases our knowledge about the ongoing microbial evolution towards biodegradation of pharmaceuticals and points to a large diversity of (amidase) enzymes that are likely involved in paracetamol metabolism in WWTPs.
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Rivers have a significant role in global carbon and nitrogen cycles, serving as a nexus for nutrient transport between terrestrial and marine ecosystems. Although rivers have a small global surface area, they contribute substantially to worldwide greenhouse gas emissions through microbially mediated processes within the river hyporheic zone. Despite this importance, research linking microbial and viral communities to specific biogeochemical reactions is still nascent in these sediment environments. To survey the metabolic potential and gene expression underpinning carbon and nitrogen biogeochemical cycling in river sediments, we collected an integrated data set of 33 metagenomes, metaproteomes, and paired metabolomes. We reconstructed over 500 microbial metagenome-assembled genomes (MAGs), which we dereplicated into 55 unique, nearly complete medium- and high-quality MAGs spanning 12 bacterial and archaeal phyla. We also reconstructed 2,482 viral genomic contigs, which were dereplicated into 111 viral MAGs (vMAGs) of >10 kb in size. As a result of integrating gene expression data with geochemical and metabolite data, we created a conceptual model that uncovered new roles for microorganisms in organic matter decomposition, carbon sequestration, nitrogen mineralization, nitrification, and denitrification. We show how these metabolic pathways, integrated through shared resource pools of ammonium, carbon dioxide, and inorganic nitrogen, could ultimately contribute to carbon dioxide and nitrous oxide fluxes from hyporheic sediments. Further, by linking viral MAGs to these active microbial hosts, we provide some of the first insights into viral modulation of river sediment carbon and nitrogen cycling. IMPORTANCE Here we created HUM-V (hyporheic uncultured microbial and viral), an annotated microbial and viral MAG catalog that captures strain and functional diversity encoded in these Columbia River sediment samples. Demonstrating its utility, this genomic inventory encompasses multiple representatives of dominant microbial and archaeal phyla reported in other river sediments and provides novel viral MAGs that can putatively infect these. Furthermore, we used HUM-V to recruit gene expression data to decipher the functional activities of these MAGs and reconstruct their active roles in Columbia River sediment biogeochemical cycling. Ultimately, we show the power of MAG-resolved multi-omics to uncover interactions and chemical handoffs in river sediments that shape an intertwined carbon and nitrogen metabolic network. The accessible microbial and viral MAGs in HUM-V will serve as a community resource to further advance more untargeted, activity-based measurements in these, and related, freshwater terrestrial-aquatic ecosystems.
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Ecossistema , Rios , Dióxido de Carbono/metabolismo , Archaea/genética , Ciclo do Nitrogênio , Nitrogênio/metabolismoRESUMO
The treatment of advanced and metastatic kidney cancer has entered a golden era with the addition of more therapeutic options, improved survival and new targeted therapies. Tyrosine kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors and immune checkpoint blockade have all been shown to be promising strategies in the treatment of renal cell carcinoma (RCC). However, little is known about the best therapeutic approach for individual patients with RCC and how to combat therapeutic resistance. Cancers, including RCC, rely on sustained replicative potential. The cyclin-dependent kinases CDK4 and CDK6 are involved in cell-cycle regulation with additional roles in metabolism, immunogenicity and antitumour immune response. Inhibitors of CDK4 and CDK6 are now commonly used as approved and investigative treatments in breast cancer, as well as several other tumours. Furthermore, CDK4/6 inhibitors have been shown to work synergistically with other kinase inhibitors, including mTOR inhibitors, as well as with immune checkpoint inhibitors in preclinical cancer models. The effect of CDK4/6 inhibitors in kidney cancer is relatively understudied compared with other cancers, but the preclinical studies available are promising. Collectively, growing evidence suggests that targeting CDK4 and CDK6 in kidney cancer, alone and in combination with current therapeutics including mTOR and immune checkpoint inhibitors, might have therapeutic benefit and should be further explored.
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Carcinoma de Células Renais , Neoplasias Renais , Carcinoma de Células Renais/tratamento farmacológico , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/uso terapêutico , Feminino , Humanos , Inibidores de Checkpoint Imunológico , Neoplasias Renais/tratamento farmacológico , Masculino , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR/uso terapêuticoRESUMO
Microbial ammonia oxidation is the initial nitrification step used in biological nitrogen-removal during water treatment processes, and the discovery of complete ammonia-oxidizing (comammox) bacteria added a novel member to this functional group. It is important to identify and understand the predominant microorganisms responsible for ammonium removal in biotechnological process design and optimization. In this study, we used a full-scale bioreactor to treat ammonium in groundwater (9.3 ± 0.5 mg NH4+-N/L) and investigated the key ammonia-oxidizing prokaryotes present. The groundwater ammonium was stably and efficiently oxidized throughout â¼700 days of bioreactor operation. 16S rRNA gene amplicon sequencing of the bioreactor community showed a high abundance of Nitrospira (12.5-45.9%), with the dominant sequence variant (3.5-37.8%) most closely related to Candidatus Nitrospira nitrosa. Furthermore, analyses of amoA, the marker gene for ammonia oxidation, indicated the presence of two distinct comammox Nitrospira populations, however, the relative abundance of only one of these populations was strongly correlated to ammonia oxidation rates and was robustly expressed. After 380 days of operation copper wires were immersed into the reactor at 0.04-0.06 m2/m3 tank, which caused a gradual abundance increase of one discrete comammox Nitrospira population. However, further increase of the copper dosing (0.08 m2/m3 tank) inverted the most abundant ammonia-oxidizing population to Nitrosomonas sp. These results indicate that comammox Nitrospira were capable of efficient ammonium removal in groundwater without exogenous nutrients, but copper addition can stimulate comammox Nitrospira or lead to dominance of Nitrosomonas depending on dosage.
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Amônia , Água Subterrânea , Bactérias/genética , Reatores Biológicos , Cobre , RNA Ribossômico 16S/genéticaRESUMO
Meningeal lymphatic vessels have been described in animal studies, but limited comparable data is available in human studies. Here we show dural lymphatic structures along the dural venous sinuses in dorsal regions and along cranial nerves in the ventral regions in the human brain. 3D T2-Fluid Attenuated Inversion Recovery magnetic resonance imaging relies on internal signals of protein rich lymphatic fluid rather than contrast media and is used in the present study to visualize the major human dural lymphatic structures. Moreover we detect direct connections between lymphatic fluid channels along the cranial nerves and vascular structures and the cervical lymph nodes. We also identify age-related cervical lymph node atrophy and thickening of lymphatics channels in both dorsal and ventral regions, findings which reflect the reduced lymphatic output of the aged brain.
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Cavidades Cranianas/diagnóstico por imagem , Epilepsia/diagnóstico por imagem , Sistema Glinfático/diagnóstico por imagem , Linfonodos/diagnóstico por imagem , Meninges/diagnóstico por imagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento , Estudos de Casos e Controles , Cavidades Cranianas/fisiopatologia , Epilepsia/fisiopatologia , Feminino , Sistema Glinfático/fisiopatologia , Humanos , Linfonodos/irrigação sanguínea , Linfonodos/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Meninges/fisiopatologia , Pessoa de Meia-Idade , Imagens de Fantasmas , Estudos Retrospectivos , Fatores SexuaisRESUMO
The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.