Single-cell somatic copy number alteration profiling of vitreous humor seeds in retinoblastoma.

BACKGROUND: Heterogeneity can impact biomarker identification. Thus, we investigated the somatic copy number alterations (SCNAs) of individual tumor cells in the vitreous humor of a retinoblastoma patient using single-cell whole-genome profiling and explored the genomic concordance among vitreous and aqueous humor, vitreous seeds, and tumor. METHODS: Aqueous humor (AH), vitreous humor (VH), and tumor biopsy were obtained from an enucleated globe with retinoblastoma and vitreous seeding. Micromanipulation was used to manually isolate 39 live single tumor cells from vitreous seeds harvested from the VH. The SCNA profiles of these individual cells were generated via whole-genome sequencing and analyzed alongside profiles from the tumor mass and cell-free DNA (cfDNA) from AH and VH. RESULTS: Heatmap of VH single-cell SCNA profiles demonstrates heterogeneity among individual vitreous seeds with one clearly dominant subclone (23 of 37 cells). The SCNA profiles from the cells in this subclone demonstrate an average concordance of 98% with cfDNA profiles from acellular AH and VH and with the tumor profile. CONCLUSIONS: Our findings reveal some heterogeneity among single-cell SCNA profiles in individual VH seeds. Despite this heterogeneity, the dominant vitreous subclone exhibits extremely (>98%) high concordance with the SCNA profile from tumor and AH, suggesting AH cfDNA is representative of the dominant genomic subclone. This may facilitate tumoral biomarker identification via the AH. This preliminary work supports the potential of applying single-cell technology to VH seeds in retinoblastoma as a platform to study tumor subclones, which may provide insight into the genomic complexity of disease.

GGA1 interacts with the endosomal Na+/H+ Exchanger NHE6 governing localization to the endosome compartment.

Mutations in the endosomal Na+/H+ exchanger (NHE6) cause Christianson Syndrome (CS), an X-linked neurological disorder. NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl-terminus as bait, we identify Golgi-associated, Gamma adaptin ear containing, ARF binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using: co-immunoprecipitation (co-IP); over-expressed constructs in mammalian cells; and co-IP of endogenously-expressed GGA1 and NHE6 from neuroblastoma cells, as well as from mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7 and NHE9), and that there is significantly less interaction with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate that the cytoplasmic tail of NHE6 interacts most strongly with GGA1. We demonstrate the co-localization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mis-localized in GGA1 knockout cells, wherein we find less NHE6 in endosomes, but more NHE6 transport to lysosomes, and more Golgi retention of NHE6, with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mis-localization, and Golgi retention, we find the intra-luminal pH in Golgi to be alkalinized in GGA1-null cells. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intra-cellular NHE to the endosome compartment.

Sialic acids on T cells are crucial for their maintenance and survival.

Sialic acids are found as terminal sugars on glycan structures on cellular surfaces. T cells carry these sialoglycans abundantly, and they are thought to serve multiple functions in cell adhesion, cell migration, and protection from complement attack. We studied the role of sialoglycans on T cells in a mouse model with a T cell-specific deletion of cytidine monophosphate-sialic acid synthase (CMAS), the enzyme that is crucial for the synthesis of sialoglycans. These mice showed a T-cell deficiency in peripheral lymphoid organs. Many T cells with an undeleted Cmas allele were found in the periphery, suggesting that they escaped the Cre-mediated deletion. The remaining peripheral T cells of T cell-specific Cmas KO mice had a memory-like phenotype. Additional depletion of the complement factor C3 could not rescue the phenotype, showing that the T-cell defect was not caused by a host complement activity. Cmas-deficient T cells showed a high level of activated caspase 3, indicating an ongoing apoptosis. In bone marrow chimeric cellular transfer experiments, we observed a strong competitive disadvantage of Cmas-deficient T cells compared to wild-type T cells. These results show that sialoglycans on the surface of T cells are crucial for T-cell survival and maintenance. This function has not been recognized before and is similar to the function of sialoglycans on B cells.

Kavalactones support motivation to move during intensive training in males preparing for military special operations forces.

BACKGROUND: Military special operators, elite athletes, and others requiring uninterrupted optimal performance currently lack options for sleep and mood support without performance-inhibiting effects. Kavalactones, derived from the root of the kava plant (Piper methysticum Forst), have been shown to elevate mood and wellbeing by producing a feeling of relaxation without addiction or cognitive impairment. METHODS: In this placebo-controlled, crossover study (NCT05381025), we investigated the effects of 2 weeks of kavalactones use on cortisol (diurnal salivary), sleep (RSQ-W; Restorative Sleep Questionnaire, Weekly), mood (DASS-21; Depression Anxiety Stress Scale-21), and motivation state to expend (Move) or conserve (Rest) energy (CRAVE; Cravings for Rest and Volitional Energy Expenditure, Right Now) in a cohort of 15 healthy, physically fit young males engaged in a rigorous, two-a-day preparation class for special operations forces qualification. RESULTS: Cortisol, sleep, and mood were within normal, healthy parameters in this cohort at baseline. This remained unchanged with kavalactones use with no significant findings of clinical interest. However, a statistically similar, positive slope for within-group Move scores was seen in both groups during kavalactones loading (first group Move slope 2.25, second group Move slope 3.29, p = 0.299). This trend was seen regardless of order and with no apparent effects on the Rest metric (all p ≥ 0.05). Moreover, a significant between-group difference appeared after 1 week of kavalactones use in the first phase (p = 0.044) and persisted through the end of the first loading period (p = 0.022). Following the 10-day washout, this between-groups divergence remained significant (p = 0.038) but was reversed by 1 week after the crossover (p = 0.072), with Move scores once again statistically similar between groups and compared to baseline at study end. Furthermore, the group taking kavalactones first never experienced a significant decrease in Move motivation state (lowest mean score 21.0, highest 28.6, all p ≥ 0.05), while the group receiving kavalactones in the last 2 weeks of the study had Move scores that were statistically lower than baseline (lowest mean score 8.6, highest 25.9, all p ≤ 0.05) at all time points but the last (p = 0.063) after 2 weeks of kavalactones exposure. CONCLUSIONS: We report a novel finding that kavalactones may support performance by maintaining or rescuing the desire to expend energy in the context of significant physical and mental strain in well-conditioned individuals, even in a context of already normal cortisol, sleep, and mood.

The Space Omics and Medical Atlas (SOMA) and international astronaut biobank.

Spaceflight induces molecular, cellular and physiological shifts in astronauts and poses myriad biomedical challenges to the human body, which are becoming increasingly relevant as more humans venture into space1-6. Yet current frameworks for aerospace medicine are nascent and lag far behind advancements in precision medicine on Earth, underscoring the need for rapid development of space medicine databases, tools and protocols. Here we present the Space Omics and Medical Atlas (SOMA), an integrated data and sample repository for clinical, cellular and multi-omic research profiles from a diverse range of missions, including the NASA Twins Study7, JAXA CFE study8,9, SpaceX Inspiration4 crew10-12, Axiom and Polaris. The SOMA resource represents a more than tenfold increase in publicly available human space omics data, with matched samples available from the Cornell Aerospace Medicine Biobank. The Atlas includes extensive molecular and physiological profiles encompassing genomics, epigenomics, transcriptomics, proteomics, metabolomics and microbiome datasets, which reveal some consistent features across missions, including cytokine shifts, telomere elongation and gene expression changes, as well as mission-specific molecular responses and links to orthologous, tissue-specific mouse datasets. Leveraging the datasets, tools and resources in SOMA can help to accelerate precision aerospace medicine, bringing needed health monitoring, risk mitigation and countermeasure data for upcoming lunar, Mars and exploration-class missions.

Tilting and Distortion in the Multiferroic Aurivillius Phase Bi6Ti3Fe1.5Mn0.5O18.

Aurivillius structured Bi6Ti3Fe1.5Mn0.5O18 (B6TFMO) has emerged as a rare room temperature multiferroic, exhibiting reversible magnetoelectric switching of ferroelectric domains under cycled magnetic fields. This layered oxide presents exceptional avenues for advancing data storage technologies owing to its distinctive ferroelectric and ferrimagnetic characteristics. Despite its immense potential, a comprehensive understanding of the underlying mechanisms driving multiferroic behavior remains elusive. Herein, we employ atomic resolution electron microscopy to elucidate the interplay of octahedral tilting and atomic-level structural distortions within B6TFMO, associating these phenomena with functional properties. Fundamental electronic features at varying bonding environments within this complex system are scrutinized using electron energy loss spectroscopy (EELS), revealing that the electronic nature of the Ti4+ cations within perovskite BO6 octahedra is influenced by position within the Aurivillius structure. Layer-by-layer EELS analysis shows an ascending crystal field splitting (Δ) trend from outer to center perovskite layers, with an average increase in Δ of 0.13 ± 0.06 eV. Density functional theory calculations, supported by atomic resolution polarization vector mapping of B-site cations, underscore the correlation between the evolving nature of Ti4+ cations, the extent of tetragonal distortion and ferroelectric behavior. Integrated differential phase contrast imaging unveils the position of light oxygen atoms in B6TFMO for the first time, exposing an escalating degree of octahedral tilting toward the center layers, which competes with the magnitude of BO6 tetragonal distortion. The observed octahedral tilting, influenced by B-site cation arrangement, is deemed crucial for juxtaposing magnetic cations and establishing long-range ferrimagnetic order in multiferroic B6TFMO.

Mis-localization of endogenous TDP-43 leads to ALS-like early-stage metabolic dysfunction and progressive motor deficits.

BACKGROUND: The key pathological signature of ALS/ FTLD is the mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm. However, TDP-43 gain of function in the cytoplasm is still poorly understood since TDP-43 animal models recapitulating mis-localization of endogenous TDP-43 from the nucleus to the cytoplasm are missing. METHODS: CRISPR/Cas9 technology was used to generate a zebrafish line (called CytoTDP), that mis-locates endogenous TDP-43 from the nucleus to the cytoplasm. Phenotypic characterization of motor neurons and the neuromuscular junction was performed by immunostaining, microglia were immunohistochemically localized by whole-mount tissue clearing and muscle ultrastructure was analyzed by scanning electron microscopy. Behavior was investigated by video tracking and quantitative analysis of swimming parameters. RNA sequencing was used to identify mis-regulated pathways with validation by molecular analysis. RESULTS: CytoTDP fish have early larval phenotypes resembling clinical features of ALS such as progressive motor defects, neurodegeneration and muscle atrophy. Taking advantage of zebrafish's embryonic development that solely relys on yolk usage until 5 days post fertilization, we demonstrated that microglia proliferation and activation in the hypothalamus is independent from food intake. By comparing CytoTDP to a previously generated TDP-43 knockout line, transcriptomic analyses revealed that mis-localization of endogenous TDP-43, rather than TDP-43 nuclear loss of function, leads to early onset metabolic dysfunction. CONCLUSIONS: The new TDP-43 model mimics the ALS/FTLD hallmark of progressive motor dysfunction. Our results suggest that functional deficits of the hypothalamus, the metabolic regulatory center, might be the primary cause of weight loss in ALS patients. Cytoplasmic gain of function of endogenous TDP-43 leads to metabolic dysfunction in vivo that are reminiscent of early ALS clinical non-motor metabolic alterations. Thus, the CytoTDP zebrafish model offers a unique opportunity to identify mis-regulated targets for therapeutic intervention early in disease progression.

Single-cell multi-ome and immune profiles of the Inspiration4 crew reveal conserved, cell-type, and sex-specific responses to spaceflight.

Spaceflight induces an immune response in astronauts. To better characterize this effect, we generated single-cell, multi-ome, cell-free RNA (cfRNA), biochemical, and hematology data for the SpaceX Inspiration4 (I4) mission crew. We found that 18 cytokines/chemokines related to inflammation, aging, and muscle homeostasis changed after spaceflight. In I4 single-cell multi-omics data, we identified a "spaceflight signature" of gene expression characterized by enrichment in oxidative phosphorylation, UV response, immune function, and TCF21 pathways. We confirmed the presence of this signature in independent datasets, including the NASA Twins Study, the I4 skin spatial transcriptomics, and 817 NASA GeneLab mouse transcriptomes. Finally, we observed that (1) T cells showed an up-regulation of FOXP3, (2) MHC class I genes exhibited long-term suppression, and (3) infection-related immune pathways were associated with microbiome shifts. In summary, this study reveals conserved and distinct immune disruptions occurring and details a roadmap for potential countermeasures to preserve astronaut health.

Collection of biospecimens from the inspiration4 mission establishes the standards for the space omics and medical atlas (SOMA).

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from four crew members longitudinally before (Launch: L-92, L-44, L-3 days), during (Flight Day: FD1, FD2, FD3), and after (Return: R + 1, R + 45, R + 82, R + 194 days) spaceflight, spanning a total of 289 days across 2021-2022. The collection process included venous whole blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies. Venous whole blood was further processed to obtain aliquots of serum, plasma, extracellular vesicles and particles, and peripheral blood mononuclear cells. In total, 2,911 sample aliquots were shipped to our central lab at Weill Cornell Medicine for downstream assays and biobanking. This paper provides an overview of the extensive biospecimen collection and highlights their processing procedures and long-term biobanking techniques, facilitating future molecular tests and evaluations.As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can aid future human spaceflight and space biology experiments.

Cutting Efficiency of Diamond Grinders on Composite and Zirconia.

This in vitro study was carried out to compare the cutting efficiency of diamond grinders on zirconia and resin-based composite materials. Grinders were employed with a special holder for the handpiece to apply a constant load (160 g) for resin-based composite (8 cuts, 40 s each) and zirconia materials (4 cuts, 5 min each; n = 10 for each material and grinder). To assess the efficiency of the grinders, weight measurements of the material were taken before and after the grinding process. Scanning electron micrographs were captured for instrument surfaces before and after testing and for the resulting surface of the materials. In the resin-based composite group, there were significant differences in weight removal between the burs for both the baseline (first cut; p = 0.009) and removal after the eighth cut (p = 0.049). Statistically significant decreases in weight removal compared to the baseline values were noted for the third, fourth, sixth, and seventh steps (p ≤ 0.046). For the zirconia group, significant differences existed in weight removal between the burs for the baseline (first cut; p < 0.001) and removal after the fourth cut (p < 0.001). A significant positive correlation was observed between removal and the number of cuts (Pearson: 0.673; p < 0.001). A statistically significant decrease in removal compared to the respective baseline value was found for the fourth step (p = 0.006). The initial wear removal and durability significantly differed between the grinders used on resin-based composite and zirconia. Achieving comparable weight removal took five times longer when grinding zirconia compared to the resin-based composite.

Cu-Catalyzed Amination of Base-Sensitive Aryl Bromides and the Chemoselective N- and O-Arylation of Amino Alcohols.

We report a general and functional-group-tolerant method for the Cu-catalyzed amination of base-sensitive aryl bromides including substrates possessing acidic functional groups and small five-membered heteroarenes. The results presented herein substantially expand the scope of Cu-catalyzed C-N coupling reactions. The combination of L8, an anionic N1,N2-diarylbenzene-1,2-diamine ligand, along with the mild base NaOTMS leads to the formation of a stable yet reactive catalyst that resists deactivation from coordination to heterocycles or charged intermediates. This system enables the use of low catalyst and ligand loadings. Exploiting the differences in nucleophile deprotonation in C-O and C-N coupling reactions catalyzed by Cu·L8 we developed a method to chemoselectively N- and O-arylate a variety of amino alcohol substrates. Employing NaOt-Bu as the base resulted exclusively in C-O coupling when the amino alcohols featured primary alcohols and more hindered amines or aniline groups. Utilizing NaOTMS enabled the ability to override the steric-based selectivity of these reactions completely and exclusively promoted C-N coupling regardless of the structure of the amino alcohol. The ability to invert the observed chemoselectivity is distinct from previously described methods that require protecting group manipulations or rely entirely on steric effects to control reactivity. These results substantially improve the scope of Cu-catalyzed C-N coupling reactions using N1,N2-diarylbenzene-1,2-diamine ligands and introduce a new chemoselective method to arylate amino alcohols.

Belowground plant allocation regulates rice methane emissions from degraded peat soils.

Carbon-rich peat soils have been drained and used extensively for agriculture throughout human history, leading to significant losses of their soil carbon. One solution for rewetting degraded peat is wet crop cultivation. Crops such as rice, which can grow in water-saturated conditions, could enable agricultural production to be maintained whilst reducing CO2 and N2O emissions from peat. However, wet rice cultivation can release considerable methane (CH4). Water table and soil management strategies may enhance rice yield and minimize CH4 emissions, but they also influence plant biomass allocation strategies. It remains unclear how water and soil management influences rice allocation strategies and how changing plant allocation and associated traits, particularly belowground, influence CH4-related processes. We examined belowground biomass (BGB), aboveground biomass (AGB), belowground:aboveground ratio (BGB:ABG), and a range of root traits (root length, root diameter, root volume, root area, and specific root length) under different soil and water treatments; and evaluated plant trait linkages to CH4. Rice (Oryza sativa L.) was grown for six months in field mesocosms under high (saturated) or low water table treatments, and in either degraded peat soil or degraded peat covered with mineral soil. We found that BGB and BGB:AGB were lowest in water saturated conditions where mineral soil had been added to the peat, and highest in low-water table peat soils. Furthermore, CH4 and BGB were positively related, with BGB explaining 60% of the variation in CH4 but only under low water table conditions. Our results suggest that a mix of low water table and mineral soil addition could minimize belowground plant allocation in rice, which could further lower CH4 likely because root-derived carbon is a key substrate for methanogenesis. Minimizing root allocation, in conjunction with water and soil management, could be explored as a strategy for lowering CH4 emissions from wet rice cultivation in degraded peatlands.

Virtual fitness buddy ecosystem: a mixed reality precision health physical activity intervention for children.

6-11-year-old children provide a critical window for physical activity (PA) interventions. The Virtual Fitness Buddy ecosystem is a precision health PA intervention for children integrating mixed reality technology to connect people and devices. A cluster randomized, controlled trial was conducted across 19 afterschool sites over two 6-month cohorts to test its efficacy in increasing PA and decreasing sedentary behavior. In the treatment group, a custom virtual dog via a mixed reality kiosk helped children set PA goals while sharing progress with parents to receive feedback and support. Children in the control group set PA goals using a computer without support from the virtual dog or parents. 303 children had 8+ hours of PA data on at least one day of each of the 3 intervention time intervals. Conversion of sedentary time was primarily to light-intensity PA and was strongest for children with low baseline moderate-to-vigorous PA than children above 45 min of baseline moderate-to-vigorous PA. Findings suggest that the VFB ecosystem can promote sustainable PA in children and may be rapidly diffused for widespread public health impact.

Dissemination of antimicrobial resistance in agricultural ecosystems following irrigation with treated municipal wastewater.

The spread of antimicrobial resistance (AMR) in agricultural systems via irrigation water is a serious public health issue as it can be transmitted to humans through the food chain. Therefore, understanding the dissemination routes of antibiotic resistance genes (ARGs) in agricultural systems is crucial for the assessment of health risks associated with eating fresh vegetables such as spinach and radish irrigated with treated municipal wastewater (TMW). In this study, we investigated the bacterial community structure and resistome in the soil-plant-earthworm continuum after irrigation of spinach and radish with TMW containing the antibiotics trimethoprim (TMP), sulfamethoxazole (SMZ), and sulfapyridine (SPD) using 16S rRNA gene sequencing and high throughput quantitative PCR (HT-qPCR). The study was conducted in two phases: Phase I involved eight weeks of spinach and radish production using TMW for irrigation, whereas Phase II entailed three weeks of earthworm exposure to contaminated plant material obtained in Phase I. The 16S data indicated that the rhizosphere bacterial community composition and structure were more resilient to antibiotic residuals in the irrigated water, with radish showing less susceptibility than spinach than those of bulk soils. The HT-qPCR analysis revealed that a total of 271 ARGs (out of 285) and 9 mobile genetic elements (MGEs) (out of 10) were detected in all samples. Higher diversity and abundance of ARGs were observed for samples irrigated with higher concentrations of antibiotics in both spinach and radish treatments. However, compared to spinach, radish ARG dynamics in the soil biome were more stable due to the change of antibiotic introduction to the soil. At the class level, multi-drug resistance (MDR) class was altered significantly by the presence of antibiotics in irrigation water. Compared to earthworm fecal samples, their corresponding soil environments showed a higher number of detected ARGs, suggesting that earthworms could play a role in reducing ARG dissemination in the soil environments. These findings will not only provide insight into the dissemination of ARGs in agricultural environments due to antibiotic residuals in irrigated water but could help understand the potential human health risks associated with ARGs.

West Nile Virus Emergence in Germany 2019: Looking for Hidden Human West Nile Virus Infections.

Background: Autochthonous human West Nile virus (WNV) infections were notified in the infectious disease surveillance system in Germany in 2018 for the first time and every year since then. Since clinically apparent infections are infrequent, we conducted two studies to investigate subclinical infections of this emerging disease in Germany in 2019 to detect infections not visible to surveillance based on symptomatic infections: limited-scope blood donor testing and a serosurvey among employees at two Berlin zoos with a history of demonstrated WNV infections in animals. Methods: For the zoo study, employees of the two zoos in Berlin were invited to participate in the study in late 2019. Blood samples were drawn and tested for the presence of antibodies (immunoglobulin M [IgM] and immunoglobulin G [IgG]) against WNV, and two other flaviviruses present in Germany: Usutu virus and Tick-borne encephalitis virus (TBEV). For the study in blood donors, four blood establishments with collection sites in regions with documented WNV-infected animals in 2018 and 2019 participated in the study. All donations in these regions were tested for WNV genome from July to November 2019. Results: In the enzyme-linked immunosorbent assay, none of the 70 tested zoo employees were WNV IgM-positive, 8 were WNV IgG-positive, additional 2 participants had equivocal results. All 10 were negative in the virus neutralization test (VNT) for WNV, but positive in the VNT for TBEV. None of the 4273 samples from blood donors tested in areas with WNV-infected animals was positive for WNV-RNA. Conclusion: Our results indicate that WNV circulation in Germany, though clearly documented in animals in 2019, apparently affected very few humans. Still areas with WNV-positive animals remain risk areas for human infection as well.

Critical evaluation of the reliability of DNA methylation probes on the Illumina MethylationEPIC v1.0 BeadChip microarrays.

DNA methylation (DNAm) plays a crucial role in a number of complex diseases. However, the reliability of DNAm levels measured using Illumina arrays varies across different probes. Previous research primarily assessed probe reliability by comparing duplicate samples between the 450k-450k or 450k-EPIC platforms, with limited investigations on Illumina EPIC v1.0 arrays. We conducted a comprehensive assessment of the EPIC v1.0 array probe reliability using 69 blood DNA samples, each measured twice, generated by the Alzheimer's Disease Neuroimaging Initiative study. We observed higher reliability in probes with average methylation beta values of 0.2 to 0.8, and lower reliability in type I probes or those within the promoter and CpG island regions. Importantly, we found that probe reliability has significant implications in the analyses of Epigenome-wide Association Studies (EWAS). Higher reliability is associated with more consistent effect sizes in different studies, the identification of differentially methylated regions (DMRs) and methylation quantitative trait locus (mQTLs), and significant correlations with downstream gene expression. Moreover, blood DNAm measurements obtained from probes with higher reliability are more likely to show concordance with brain DNAm measurements. Our findings, which provide crucial reliability information for probes on the EPIC v1.0 array, will serve as a valuable resource for future DNAm studies.

Proteomic profiling of antibiotic-resistant Escherichia coli GW-AmxH19 isolated from hospital wastewater treated with physical plasma.

Microorganisms which are resistant to antibiotics are a global threat to the health of humans and animals. Wastewater treatment plants are known hotspots for the dissemination of antibiotic resistances. Therefore, novel methods for the inactivation of pathogens, and in particular antibiotic-resistant microorganisms (ARM), are of increasing interest. An especially promising method could be a water treatment by physical plasma which provides charged particles, electric fields, UV-radiation, and reactive species. The latter are foremost responsible for the antimicrobial properties of plasma. Thus, with plasma it might be possible to reduce the amount of ARM and to establish this technology as additional treatment stage for wastewater remediation. However, the impact of plasma on microorganisms beyond a mere inactivation was analyzed in more detail by a proteomic approach. Therefore, Escherichia coli GW-AmxH19, isolated from hospital wastewater in Germany, was used. The bacterial solution was treated by a plasma discharge ignited between each of four pins and the liquid surface. The growth of E. coli and the pH-value decreased during plasma treatment in comparison with the untreated control. Proteome and antibiotic resistance profile were analyzed. Concentrations of nitrite and nitrate were determined as long-lived indicative products of a transient chemistry associated with reactive nitrogen species (RNS). Conversely, hydrogen peroxide served as indicator for reactive oxygen species (ROS). Proteome analyses revealed an oxidative stress response as a result of plasma-generated RNS and ROS as well as a pH-balancing reaction as key responses to plasma treatment. Both, the generation of reactive species and a decreased pH-value is characteristic for plasma-treated solutions. The plasma-mediated changes of the proteome are discussed also in comparison with the Gram-positive bacterium Bacillus subtilis. Furthermore, no effect of the plasma treatment, on the antibiotic resistance of E. coli, was determined under the chosen conditions. The knowledge about the physiological changes of ARM in response to plasma is of fundamental interest to understand the molecular basis for the inactivation. This will be important for the further development and implementation of plasma in wastewater remediation.