Phylogenetic diversity of core rumen microbiota as described by cryo-ET.

Microbial taxonomy is critical for describing ecosystem composition, yet the link between taxonomy and properties of microbes, such as their cellular architecture, remains poorly defined. We hypothesized that the cellular architecture represents microbial niche adaptation. We used cryo-electron microscopy and tomography to analyze microbial morphology in order to associate cellular architecture with phylogeny and genomic contents. As a model system, we chose the core rumen microbiome and imaged a large isolate collection covering 90% of its richness at the order level. Based on quantifications of several morphological features, we found that the visual similarity of microbiota is significantly related to their phylogenetic distance. Up to the Family level, closely related microbes have similar cellular architectures, which are highly correlated with genome similarity. However, in more distantly related bacteria, the correlation both with taxonomy and genome similarity is lost. This is the first comprehensive study of microbial cellular architecture and our results highlight that structure remains an important parameter in classification of microorganisms, along with functional parameters such as metabolomics. Furthermore, the high-quality images presented in this study represent a reference database for the identification of bacteria in anaerobic ecosystems.

Mechanistic modeling indicates rapid glyphosate dissipation and sorption-driven persistence of its metabolite AMPA in soil.

Residual concentrations of glyphosate and its main transformation product aminomethylphosphonic acid (AMPA) are often observed in soils. The factors controlling their biodegradation are currently not well understood. We analyzed sorption-limited biodegradation of glyphosate and AMPA in soil with a set of microcosm experiments. A mechanistic model that accounts for equilibrium and kinetic sorption facilitated interpretation of the experimental results. Both compounds showed a biphasic dissipation with an initial fast (up to Days 7-10) and subsequent slower transformation rate, pointing to sorption-limited degradation. Glyphosate transformation was well described by considering only equilibrium sorption. Model simulations suggested that only 0.02-0.13% of total glyphosate was present in the soil solution and thus bioavailable. Glyphosate transformation was rapid in solution (time required for 50 % dissipation of the total initially added chemical [DT50 ] = 3.9 min), and, despite strong equilibrium sorption, total glyphosate in soil dissipated quickly (DT50  = 2.4 d). Aminomethylphosphonic acid dissipation kinetics could only be described when considering both equilibrium and kinetic sorption. In comparison to glyphosate, the model simulations showed that a higher proportion of total AMPA was dissolved and directly bioavailable (0.27-3.32%), but biodegradation of dissolved AMPA was slower (DT50  = 1.9 h). The model-based data interpretation suggests that kinetic sorption strongly reduces AMPA bioavailability, leading to increased AMPA persistence in soil (DT50  = 12 d). Thus, strong sorption combined with rapid degradation points to low risks of glyphosate leaching by vertical transport through soil in the absence of preferential flow. Ecotoxicological effects on soil microorganisms might be reduced. In contrast, AMPA persists, rendering these risks more likely.

13C assimilation as well as functional gene abundance and expression elucidate the biodegradation of glyphosate in a field experiment.

Glyphosate (N-phosphonomethylglycine; GLP) and its main metabolite AMPA (aminomethylphosphonic acid), are frequently detected in relatively high concentrations in European agricultural topsoils. Glyphosate has a high sorption affinity, yet it can be detected occasionally in groundwater. We hypothesized that shrinkage cracks occurring after dry periods could facilitate GLP transport to greater depths where subsoil conditions slow further microbial degradation. To test this hypothesis, we simulated a heavy rainfall event (HRE) on a clay-rich arable soil. We applied 2.1 kg ha-1 of 100% 13C3, 15N-labeled GLP one day before the simulated rainfall event. Microbial degradation of translocated GLP over a 21-day period was assessed by quantifying 13C incorporation into phospholipid fatty acids. Microbial degradation potential and activity were determined by quantifying the abundance and expression of functional genes involved in the two known degradation pathways of GLP; to AMPA (goxA) or sarcosine (sarc). We confirmed that goxA transcripts were elevated in the range of 4.23 x 105 copy numbers g-1 soil only one day after application. The increase in AMPA associated with a rise in goxA transcripts and goxA-harboring microorganisms indicated that the degradation pathway to AMPA dominated. Based on 13C-enrichment 3 h after the HRE, fungi appeared to initiate glyphosate degradation. At later time points, Gram+-bacteria proved to be the main degraders due to their higher 13C-incorporation. Once GLP reached the subsoil, degradation continued but more slowly. By comparing GLP distribution and its microbial degradation in macropores and in the bulk soil, we demonstrated different time- and depth-dependent GLP degradation dynamics in macropores. This indicates the need for field studies in which soil properties relevant to GLP degradation are related to limiting environmental conditions, providing a realistic assessment of GLP fate in soils.

Heavy rainfall following a summer drought stimulates soil redox dynamics and facilitates rapid and deep translocation of glyphosate in floodplain soils.

We present field data on the effects of heavy rainfall after drought on the mobility of glyphosate and redox conditions in a clayey floodplain soil. By applying glyphosate together with deuterated water as conservative tracer in combination with time resolved in situ redox potential measurements, the spatial and temporal patterns of water infiltration and pesticide transport as well as the concomitant changes of the redox conditions were revealed. Our findings demonstrate that shrinkage cracks in dry soils can serve as effective transport paths for atmospheric oxygen, water and glyphosate. The rain intensity of a typical summer storm event (approx. 25 mm within one hour) was sufficient to translocate deuterated water and glyphosate to the subsoil (50 cm) within 2 hours. Soil wetting induced partial closure of the shrinkage cracks and stimulated microbial activity resulting in pronounced dynamics of in situ soil redox conditions. Redox potentials in 40 to 50 cm depth dropped permanently to strongly reducing conditions within hours to days but fluctuated between reducing and oxidizing conditions in 10 to 30 cm depth. Our findings highlight the close link between the presence of macropores (shrinkage cracks), heavy rainfall after drought, redox dynamics and pesticide translocation to the subsoil and thus call for further studies addressing the effects of dynamic redox conditions as a limiting factor for glyphosate degradation.

Phosphate addition enhances alkaline extraction of glyphosate from highly sorptive soils and aquatic sediments.

BACKGROUND: Analytical constraints complicate environmental monitoring campaigns of the herbicide glyphosate and its major degradation product aminomethylphosphonic acid (AMPA): their strong sorption to soil minerals requires harsh extraction conditions. Coextracted matrix compounds impair downstream analysis and must be removed before analysis. RESULTS: A new extraction method combined with subsequent capillary electrophoresis-mass spectrometry for derivatization-free analysis of glyphosate and AMPA in soil and sediment was developed and applied to a suite of environmental samples. It was compared to three extraction methods from literature. We show that no extraction medium reaches 100% recovery. The new phosphate-supported alkaline extraction method revealed (1) high recoveries of 70-90% for soils and aquatic sediments, (2) limits of detections below 20 µg kg-1 , and (3) a high robustness, because impairing matrix components (trivalent cations and humic acids) were precipitated prior to the analysis. Soil and sediment samples collected around Tübingen, Germany, revealed maximum glyphosate and AMPA residues of 80 and 2100 µg kg-1 , respectively, with residues observed along a core of lake sediments. Glyphosate and/or AMPA were found in 40% of arable soils and 57% of aquatic sediment samples. CONCLUSION: In this work, we discuss soil parameters that influence (de)sorption and thus extraction. From our results we conclude that residues of glyphosate in environmental samples are easily underestimated. With its possible high throughput, the method presented here can resolve current limitations in monitoring campaigns of glyphosate by addressing soil and aquatic sediment samples with critical sorption characteristics.

From properties to toxicity: Comparing microplastics to other airborne microparticles.

Microplastic (MP) debris is considered as a potentially hazardous material. It is omnipresent in our environment, and evidence that MP is also abundant in the atmosphere is increasing. Consequently, the inhalation of these particles is a significant exposure route to humans. Concerns about potential effects of airborne MP on human health are rising. However, currently, there are not enough studies on the putative toxicity of airborne MP to adequately assess its impact on human health. Therefore, we examined potential drivers of airborne MP toxicity. Physicochemical properties like size, shape, ζ-potential, adsorbed molecules and pathogens, and the MP's bio-persistence have been proposed as possible drivers of MP toxicity. Since their role in MP toxicity is largely unknown, we reviewed the literature on toxicologically well-studied non-plastic airborne microparticles (asbestos, silica, soot, wood, cotton, hay). We aimed to link the observed health effects and toxicology of these microparticles to the abovementioned properties. By comparing this information with studies on the effects of airborne MP, we analyzed possible mechanisms of airborne MP toxicity. Thus, we provide a basis for a mechanistic understanding of airborne MP toxicity. This may enable the assessment of risks associated with airborne MP pollution, facilitating effective policymaking and product design.

Role of NKG2a/c+CD8+ T cells in pathogenic versus non-pathogenic SIV infections.

Some viruses have established an equilibrium with their host. African green monkeys (AGM) display persistent high viral replication in the blood and intestine during Simian immunodeficiency virus (SIV) infection but resolve systemic inflammation after acute infection and lack intestinal immune or tissue damage during chronic infection. We show that NKG2a/c +CD8+ T cells increase in the blood and intestine of AGM in response to SIVagm infection in contrast to SIVmac infection in macaques, the latter modeling HIV infection. NKG2a/c +CD8+ T cells were not expanded in lymph nodes, and CXCR5+NKG2a/c +CD8+ T cell frequencies further decreased after SIV infection. Genome-wide transcriptome analysis of NKG2a/c +CD8+ T cells from AGM revealed the expression of NK cell receptors, and of molecules with cytotoxic effector, gut homing, and immunoregulatory and gut barrier function, including CD73. NKG2a/c +CD8+ T cells correlated negatively with IL-23 in the intestine during SIVmac infection. The data suggest a potential regulatory role of NKG2a/c +CD8+ T cells in intestinal inflammation during SIV/HIV infections.

Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies.

Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through "T"-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.

Capillary electrophoresis-mass spectrometry for the direct analysis of glyphosate: method development and application to beer beverages and environmental studies.

In this study, we developed and validated a CE-TOF-MS method for the quantification of glyphosate (N-(phosphonomethyl)glycine) and its major degradation product aminomethylphosphonic acid (AMPA) in different samples including beer, media from toxicological analysis with Daphnia magna, and sorption experiments. Using a background electrolyte (BGE) of very low pH, where glyphosate is still negatively charged but many matrix components become neutral or protonated, a very high separation selectivity was reached. The presence of inorganic salts in the sample was advantageous with regard to preconcentration via transient isotachophoresis. The advantages of our new method are the following: no derivatization is needed, high separation selectivity and thus matrix tolerance, speed of analysis, limits of detection suitable for many applications in food and environmental science, negligible disturbance by metal chelation. LODs for glyphosate were < 5 µg/L for both aqueous and beer samples, the linear range in aqueous samples was 5-3000 µg/L, for beer samples 10-3000 µg/L. For AMPA, LODs were 3.3 and 30.6 µg/L, and the linear range 10-3000 µg/L and 50-3000 µg/L, for aqueous and beer samples, respectively. Recoveries in beer samples for glyphosate were 94.3-110.7% and for AMPA 80.2-100.4%. We analyzed 12 German and 2 Danish beer samples. Quantification of glyphosate and AMPA was possible using isotopically labeled standards without enrichment, purification, or dilution, only degassing and filtration were required for sample preparation. Finally, we demonstrate the applicability of the method for other strong acids, relevant in food and environmental sciences such as N-acetyl glyphosate, N-acetyl AMPA (present in some glyphosate resistant crop), trifluoroacetic acid, 2-methyl-4-chlorophenoxyacetic acid, glufosinate and its degradation product 3-(methylphosphinico)propionic acid, oxamic acid, and others.

The mitochondrial peroxiredoxin displays distinct roles in different developmental stages of African trypanosomes.

Hydroperoxide reduction in African trypanosomes relies on 2-Cys-peroxiredoxins (Prxs) and glutathione peroxidase-type enzymes (Pxs) which both obtain their reducing equivalents from the trypanothione/tryparedoxin couple and thus act as tryparedoxin peroxidases. While the cytosolic forms of the peroxidases are essential, the mitochondrial mPrx and Px III appear dispensable in bloodstream Trypanosoma brucei. This led to the suggestion that in this developmental stage which is characterized by a mitochondrion that lacks an active respiratory chain, only one of the two peroxidases might be required. Here we show that bloodstream cells in which the Px III gene is deleted and mPrx is down-regulated by RNA interference, proliferate as the parental cells indicating that both mitochondrial peroxidases are dispensable. However, when we raised the culture temperature to 39 °C, mPrx-depleted cells died indicating that under conditions mimicking a fever situation in the mammalian host, the protein becomes essential. In contrast, depletion of mPrx in insect stage procyclic T. brucei causes a proliferation defect under standard conditions at 27 °C, in the absence of any stress. In the absence of mPrx, a tryparedoxin-coupled roGFP2 biosensor expressed in the mitochondrial matrix is unable to respond to antimycin A treatment. Thus mPrx reduces mitochondrial H2O2 with the generation of trypanothione disulfide and acts as peroxidase. However, mPrx-depleted procyclic cells neither display any alteration in the cytosolic or mitochondrial trypanothione redox state nor increased sensitivity towards exogenous oxidative stressors suggesting that the peroxidase activity is not the crucial physiological function. After prolonged mPrx-depletion, the cells almost stop proliferation and display a highly elongated shape and diminished MitoTracker Red staining. In contrast to the situation in the mammalian bloodstream T. brucei and Leishmania, mPrx appears to play a constitutive role for the morphology, mitochondrial function and proliferation of the insect stage of African trypanosomes.

Challenges and applications of isotachophoresis coupled to mass spectrometry: A review.

Electrophoretic separations are of growing interest to tackle complex analytical challenges. Nevertheless, capillary electrophoresis, as the most common mode, still suffers from insufficient detection limits due to low capillary loadability. ITP is of growing interest as preconcentration method for capillary electrophoresis and is also interesting to be applied as an independent analytical method. While mass spectrometric detection is common for capillary electrophoresis, the combination of ITP with MS is still a niche technique. In this work, we want to give an overview on isotachophoretic effects in CE-MS and ITP-MS methods, as well as coupling techniques of ITP with CE-MS. The challenges and possibilities associated with mass spectrometric detection in ITP and its coupling to capillary electrophoresis are critically discussed.

Glyphosate analysis using sensors and electromigration separation techniques as alternatives to gas or liquid chromatography.

Since its introduction in 1974, the herbicide glyphosate has experienced a tremendous increase in use, with about one million tons used annually today. This review focuses on sensors and electromigration separation techniques as alternatives to chromatographic methods for the analysis of glyphosate and its metabolite aminomethyl phosphonic acid. Even with the large number of studies published, glyphosate analysis remains challenging. With its polar and depending on pH even ionic functional groups lacking a chromophore, it is difficult to analyze with chromatographic techniques. Its analysis is mostly achieved after derivatization. Its purification from food and environmental samples inevitably results incoextraction of ionic matrix components, with a further impact on analysis derivatization. Its purification from food and environmental samples inevitably results in coextraction of ionic matrix components, with a further impact on analysis and also derivatization reactions. Its ability to form chelates with metal cations is another obstacle for precise quantification. Lastly, the low limits of detection required by legislation have to be met. These challenges preclude glyphosate from being analyzed together with many other pesticides in common multiresidue (chromatographic) methods. For better monitoring of glyphosate in environmental and food samples, further fast and robust methods are required. In this review, analytical methods are summarized and discussed from the perspective of biosensors and various formats of electromigration separation techniques, including modes such as capillary electrophoresis and micellar electrokinetic chromatography, combined with various detection techniques. These methods are critically discussed with regard to matrix tolerance, limits of detection reached, and selectivity.