Unveiling the impact of short-term polyethylene microplastics exposure on metabolomics and gut microbiota in earthworms (Eudrilus euganiae).

Microplastics (MPs) pose a significant environmental concern, particularly for terrestrial fauna. In this study, earthworms were used as a model organism to investigate the ecotoxicological effects of short-term exposure to virgin MPs on changes in metabolome and gut microbiota. High-throughput untargeted metabolomics showed significant internal reactions in the earthworms' metabolic processes due to MPs exposure, even when no visible stress signs, such as changes in growth or mortality rates, were present. Earthworms exposed to different concentrations of polyethylene (PE) MP exhibited significant disruption in 39 and 199 molecular features related to energy and lipid metabolism, anti-inflammatory, cell signaling, and membrane integrity. The activities of enzymes and transport proteins in earthworms were dysregulated when exposed to PE. Changes in the gut microbiota's community structure and complexity were observed in response to PE MPs exposure. Despite the relative stability in alpha-diversity and relative abundance, shifts in beta-diversity and network analysis in the PE-exposed group were indicative of an adaptive response to MPs. Earthworms exhibited resilience or adaptation in response to MPs exposure, potentially maintaining their functionality. This study provides preliminary insights into the impact of MPs on soil invertebrates like earthworms and highlights the need for further exploration of long-term effects and underlying molecular mechanisms.

Different types of land use influence soil physiochemical properties, the abundance of nitrifying bacteria, and microbial interactions in tropical urban soil.

Anthropogenic activities have led to unexpected changes in microbial community composition and structure, resulting in an interruption of soil ecological roles in urban environments. We questioned the impact of the different land use (e.g., agricultural, industrial, recreational, coastal, and residential areas) on the distribution of nitrifying bacteria and microbial interaction in tropical soil. The dominant nitrifying bacteria were ammonia-oxidizing archaea (AOA) in tropical soils up to 107 copies/g of soil, while the abundance of ammonia-oxidizing bacteria (AOB) was significantly higher in agricultural soil only. Comammox (CMX) was ubiquitous up to 105 copies/g of tropical soil, indicating that CMX might share ecological niches with AOA and considerably contribute to nitrification in urban areas. The most abundant phylum is Actinobacteria, accounting for 27-34 % relative abundance among most land-use types, but Proteobacteria was observed as the most prevalent phylum in agricultural soil. The physicochemical properties (e.g., soil pH and nutrient contents) of different types of land use influenced microbial richness and diversities associated with nitrogen cycling. Multivariate analysis disclosed that agricultural soils were distinct from other land uses because of the concentrations of nutrients and heavy metals and the abundance of microorganisms associated with nitrogen cycles. Also, the microbial co-occurrence network revealed that agricultural soils were a highly interconnected network of the microbial community. In this study, C: N ratio might have a significant impact on ecological networks and the abundance of nitrogen-related taxa, which could influence microbial interactions and complexity in tropical soils. Thus, the impact of anthropogenic land use induced changes in microbial composition and diversity, co-occurrence network, and nitrifying bacteria, leading to potential transformation in ecological services of tropical soils and nitrogen cycling in urban environments.

Acute exposure to microplastics induces metabolic disturbances and gut dysbiosis in adult zebrafish (Danio rerio).

There is limited knowledge of the ecotoxicological impacts of MPs at the environmentally relevant concentration on freshwater animals, even though numerous studies have demonstrated the toxic effects of MPs on living organisms. In this study, zebrafish (Danio rerio) was used as a model organism to investigate the ecotoxicological effects of acute exposure of virgin MPs on changes in metabolome and gut microbiota. High-throughput untargeted metabolomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS) provided comprehensive insights into the metabolic responses of zebrafish exposed to PE (polyethylene) and PES (polyester) MPs. Statistical analysis of metabolomics data indicated that 39 and 27 metabolites, such as lysophosphatidylcholine, phosphocholine, phosphatidylserine, triglyceride, glycosphingolipid, psychosine, 8-amino-7-oxononanoate, cholesterol fatty acid ester, phosphatidylinositol, n-Triacontanol, were significantly altered in PE- and PES-exposed zebrafish, respectively. Furthermore, the enrichment pathway analysis unveiled the synthesis of the structural and functional lipids, signaling molecules, fatty alcohol metabolism, and amino acid metabolism, which was considerably perturbated in MPs-exposed zebrafish. In addition, high-throughput DNA sequencing was conducted to examine changes in gut microbiota in the MPs-treated zebrafish. The MPs exposure increased in the relative abundance of Fusobacteria and Proteobacteria, while the relative abundance of Firmicutes declined in MPs-treated zebrafish. Also, microbial diversity and linear discriminant analyses indicated microbiota dysbiosis, metabolomic dysregulation, and oxidative stress. Taken together, the acute exposure of MPs at environmentally relevant concentrations could disrupt the metabolic interaction via the microbiota-gut-liver-brain relationship, implying gastrointestinal and neurological/immune disorders in zebrafish.

Rapid quantification of fecal indicator bacteria in water using the most probable number - loop-mediated isothermal amplification (MPN-LAMP) approach on a polymethyl methacrylate (PMMA) microchip.

Fecal contamination of water and its associated pathogens are a major public health concern in both developing and industrialized areas. Fecal indicator bacteria (FIB) are commonly used to assess microbial water quality, but they require a relatively long period of incubation time. Currently, molecular techniques have been applied to rapidly detect FIB. However, these molecular techniques require expensive and sophisticated equipment. In this study, we developed a rapid on-chip gene quantification method based on loop-mediated isothermal amplification (LAMP) PCR. The LAMP assays can measure the target genes of the fecal indicator bacteria (FIB), including E. coli and Enterococcus spp, using the most probable number (MPN) approach. The colorimetric LAMP assay allows for naked-eye observation of the PCR reaction as few as 4 gene copies / well. When the reaction ends, MPN measurement of positive outcomes on the white-based PMMA (polymethacrylic acid) microchips provides the concentrations of the target genes of FIB with a confidence interval. We validated the feasibility of the MPN-LAMP approach by obtaining a strong correlation between the results of the MPN estimations and the qPCR analysis. Moreover, the MPN-LAMP approach was used to quantify the FIB in different environmental water collected from the freshwater reservoirs, beach, agriculture farm, and sewage. Our research demonstrates that the MPN- LAMP method enables us to easily and quickly quantifying FIB genes isolated from the environment without expensive qPCR instruments.

Rapid and in-situ detection of fecal indicator bacteria in water using simple DNA extraction and portable loop-mediated isothermal amplification (LAMP) PCR methods.

Contamination of water by fecal matter and potential human enteric pathogens is a serious health concern. Microbiological water quality has been assessed by conventional culture-based methods of fecal indicator bacteria (FIB). Recently, molecular techniques for FIB have been introduced as alternative tools for rapid detection. However, such molecular techniques require a modern laboratory setting, expensive equipment, and skilled personnel. In this study, we developed a simple and rapid DNA extraction method based on a syringe filter without any specialized equipment. Furthermore, loop-mediated isothermal amplification (LAMP) PCR for fecal indicator bacteria (FIB) (i.e. E. coli and E. faecalis) was carried out using the DNA extracts from the syringe-filter based DNA extraction method. The efficiency of the extracted DNA from the syringe-filter based method was comparable to the results of the commercial kit method. We also tested fresh and marine-water collected directly from different locations in Singapore that were spiked with E. coli or E. faecalis. The LAMP assays combined with our DNA extraction method showed higher sensitivity and more tolerance to PCR inhibitors than that of conventional PCR methods. We further developed a portable LAMP device to conduct isothermal PCR reactions for rapid on-site measurement of FIB. As the color changes in the end point of the LAMP reaction can be observed with the naked eye, the portable LAMP device was easily operated and quick, obtaining results in 30 min. The simple, portable and user-friendly platform can be used as an initial screening for the rapid detection of the presence FIB in lower-resource settings. In conclusion, the portable LAMP device coupled with a syringe-filter based DNA extraction method enables us to detect the presence of FIB for assessing microbial water quality within 1 h without any sophisticated laboratory equipment or highly trained personnel.

High-resolution metabolomics determines the mode of onset of type 2 diabetes in a 3-year prospective cohort study.

Type 2 diabetes mellitus (DM) is a progressive disease and the rate of progression from non-diabetes to DM varies considerably between individuals, ranging from a few months to many years. It is important to understand the mechanisms underlying the progression of diabetes. In the present study, a high-resolution metabolomics (HRM) analysis was performed to detect potential biomarkers and pathways regulating the mode of onset by comparing subjects who developed and did not develop type 2 DM at the second year in a 3-year prospective cohort study. Metabolic profiles correlated with progression to DM were examined. The subjects (n=98) were classified into four groups: Control (did not develop DM for 3 years), DM (diagnosed with DM at the start of the study), DM onset at the third year and DM onset at the second year. The focus was on the comparison of serum samples of the DM groups with onset at the second and third year from the first year, where these two groups had not developed DM, yet. Analyses involved sample examination using liquid chromatography-mass spectrometry-based HRM and multivariate statistical analysis of the data. Metabolic differences were identified across all analyses with the affected pathways involved in metabolism associated with steroid biosynthesis and bile acid biosynthesis. In the first year, higher levels of cholesterol {mass-to charge ratio (m/z) 369.35, (M+H-H2O)+}, 25-hydroxycholesterol [m/z 403.36, (M+H)+], 3α,7α-dihydroxy-5ß-cholestane [m/z 443.33, (M+K)+], 4α-methylzymosterol-4-carboxylate [m/z 425.34, (M+H­H2O)+], and lower levels of 24,25-dihydrolanosterol [m/z 429.40, (M+H)+] were evident in the group with DM onset at the second year compared with those in the group with DM onset at the third year. These results, with a focus on the cholesterol biosynthesis pathway, point to important aspects in the development of DM and may aid in the development of more effective means of treatment and prevention.

Screening and identification of neuroprotective compounds from Scrophularia buergeriana using cell extraction coupled with LC-MS.

In the cell extraction_LC-MS method, when cells are incubated with natural product extracts, bioactive compounds selectively bind to extracellular or intracellular targets. The extracts and major compounds (phenylpropanoids and iridoid glycosides) of Scrophularia buergeriana Miquel have been reported to show neuroprotective effects both in vitro and in vivo. In this study, the cell extraction_LC-MS strategy was applied to screen and identify potential neuroprotective compounds from S. buergeriana by using immortalized mouse hippocampal HT22 cells. The results showed that two known compounds from S. buergeriana selectively bound HT22 cells. Additionally, metabolomics analyses were performed using the Mass Profiler Professional and Limma differential expression package of R to identify significant differences between HT22 cells treated with S. buergeriana and untreated cells. The cell extraction approach more accurately reflects in vivo conditions compared with other methods and can be readily used for screening bioactive components from natural products.

Liquid Chromatography Mass Spectrometry-Based Metabolite Pathway Analyses of Myeloma and Non-Hodgkin's Lymphoma Patients.

OBJECTIVE: This study attempted to identify altered metabolism and pathways related to non-Hodgkin's lymphoma (NHL) and myeloma patients. MATERIALS AND METHODS: In this retrospective study, we collected plasma samples from 11 patients-6 healthy controls with no evidence of any blood cancers and 5 patients with either multiple myeloma (n=3) or NHL (n=2) during the preliminary study period. Samples were analyzed using quadrupole time-of-flight liquid chromatography mass spectrometry (LC-MS). Significant features generated after statistical analyses were used for metabolomics and pathway analysis. RESULTS: Data after false discovery rate (FDR) adjustment at q=0.05 of features showed 136 for positive and 350 significant features for negative ionization mode in NHL patients as well as 262 for positive and 98 features for negative ionization mode in myeloma patients. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis determined that pathways such as steroid hormone biosynthesis, ABC transporters, and arginine and proline metabolism were affected in NHL patients. In myeloma patients, pyrimidine metabolism, carbon metabolism, and bile secretion pathways were potentially affected by the disease. CONCLUSION: The results have shown tremendous differences in the metabolites of healthy individuals compared to myeloma and lymphoma patients. Validation through quantitative metabolomics is encouraged, especially for the metabolites with significantly expression in blood cancer patients.

High-resolution metabolomics to identify urine biomarkers in corticosteroid-resistant asthmatic children.

BACKGROUND: Corticosteroid (CS) treatment has been established as the first anti-inflammatory treatment for adults and children with asthma. However, a subset of patients fails to respond to combined systemic and inhaled CS treatment. OBJECTIVE: This study was aimed at further understanding CS resistance among children with severe asthma. METHODS: High-resolution metabolomics was performed on urine samples from CS-respondent (n = 15) and CS-nonrespondent (n = 15) children to determine possible urine biomarkers related to CS resistance. The metabolic phenotypes of CS responders and CS nonresponders were analyzed using bioinformatics including Manhattan plot with false- discovery rate, hierarchical cluster analysis, Kyoto Encyclopedia Genes and Genomes, and Mummichog pathway analysis. RESULTS: The 2-way hierarchical cluster analysis study determined 30 metabolites showing significantly different levels between CS responders and CS nonresponders. The important metabolites annotated were 3,6-dihydronicotinic acid (126.05 m/z, RT: 106, [M+H]+), 3-methoxy-4-hydroxyphenyl(ethylene)glycol (185.05 m/z, RT: 155, [M+H]+), 3,4-dihydroxy-phenylalanine (198.07 m/z, RT: 446, [M+H]+), γ-glutamylcysteine (236.06 m/z, RT: 528, [M+S(34)+H]+), Cys-Gly, (253.06 m/z, RT: 528, [M-NH3+H]+), and reduced Flavin mononucleotide (517.0794 m/z, RT: 533, [M+NaCl]+). Tyrosine metabolism, degradation of aromatic compounds, and glutathione metabolism are suggested to be significant pathways relating to CS resistance. CONCLUSIONS: High-resolution metabolomics is a promising approach in asthma research. Five candidate markers were identified to be related to CS-resistant children with severe asthma. These compounds, upon validation, may contribute further in the understanding of CS resistance among children with severe asthma through the use of urine.

High-resolution metabolomics to discover potential parasite-specific biomarkers in a Plasmodium falciparum erythrocytic stage culture system.

BACKGROUND: Current available malaria diagnostic methods each have some limitations to meet the need for real-time and large-scale screening of asymptomatic and low density malaria infection at community level. It was proposed that malaria parasite-specific low molecular-weight metabolites could be used as biomarkers for the development of a malaria diagnostic tool aimed to address this diagnostic challenge. In this study, high resolution metabolomics (HRM) was employed to identify malaria parasite-specific metabolites in Plasmodium falciparum in vitro culture samples. METHODS: Supernatants were collected at 12 hours interval from 3% haematocrit in vitro 48-hour time-course asynchronized culture system of P. falciparum. Liquid chromatography coupled with high resolution mass spectrometry was applied to discover potential parasite-specific metabolites in the cell culture supernatant. A metabolome-wide association study was performed to extract metabolites using Manhattan plot with false discovery rate (FDR) and hierarchical cluster analysis. The significant metabolites based on FDR cutoff were annotated using Metlin database. Standard curves were created using corresponding chemical compounds to accurately quantify potential Plasmodium-specific metabolites in culture supernatants. RESULTS: The number of significant metabolite features was 1025 in the supernatant of the Plasmodium infected culture based on Manhattan plot with FDR q=0.05. A two way hierarchical cluster analysis showed a clear segregation of the metabolic profile of parasite infected supernatant from non-infected supernatant at four time points during the 48 hour culture. Among the 1025 annotated metabolites, the intensities of four molecules were significantly increased with culture time suggesting a positive association between the quantity of these molecules and level of parasitaemia: i) 3-methylindole, a mosquito attractant, ii) succinylacetone, a haem biosynthesis inhibitor, iii) S-methyl-L-thiocitrulline, a nitric oxide synthase inhibitor, and iv) O-arachidonoyl glycidol, a fatty acid amide hydrolase inhibitor, The highest concentrations of 3-methylindole and succinylacetone were 178 ± 18.7 pmoles at 36 hours and 157±30.5 pmoles at 48 hours respectively in parasite infected supernatant. CONCLUSION: HRM with bioinformatics identified four potential parasite-specific metabolite biomarkers using in vitro culture supernatants. Further study in malaria infected human is needed to determine presence of the molecules and its relationship with parasite densities.

Comparison of different disinfection processes in the effective removal of antibiotic-resistant bacteria and genes.

This study compared three different disinfection processes (chlorination, E-beam, and ozone) and the efficacy of three oxidants (H2O2, S2O(-)8, and peroxymonosulfate (MPS)) in removing antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in a synthetic wastewater. More than 30 mg/L of chlorine was needed to remove over 90% of ARB and ARG. For the E-beam method, only 1 dose (kGy) was needed to remove ARB and ARG, and ozone could reduce ARB and ARG by more than 90% even at 3 mg/L ozone concentration. In the ozone process, CT values (concentration × time) were compared for ozone alone and combined with different catalysts based on the 2-log removal of ARB and ARG. Ozone treatment yielded a value of 31 and 33 (mg·min)/L for ARB and ARGs respectively. On the other hand, ozone with persulfate yielded 15.9 and 18.5 (mg·min)/L while ozone with monopersulfate yielded a value of 12 and 14.5 (mg·min)/L. This implies that the addition of these catalysts significantly reduces the contact time to achieve a 2-log removal, thus enhancing the process in terms of its kinetics.

Transfer of antibiotic resistance plasmids in pure and activated sludge cultures in the presence of environmentally representative micro-contaminant concentrations.

The presence of antibiotics in the natural environment has been a growing issue. This presence could also account for the influence that affects microorganisms in such a way that they develop resistance against these antibiotics. The aim of this study was to evaluate whether the antibiotic resistant gene (ARG) plasmid transfer can be facilitated by the impact of 1) environmentally representative micro-contaminant concentrations in ppb (part per billion) levels and 2) donor-recipient microbial complexity (pure vs. mixed). For this purpose, the multidrug resistant plasmid, pB10, and Escherichia coli DH5α were used as a model plasmid and a model donor, respectively. Based on conjugation experiments with pure (Pseudomonas aeruginosa PAKexoT) and mixed (activated sludge) cultures as recipients, increased relative plasmid transfer frequencies were observed at ppb (µg/L) levels of tetracycline and sulfamethoxazole micro-contaminant exposure. When sludge, a more complex community, was used as a recipient, the increases of the plasmid transfer rate were always statistically significant but not always in P. aeruginosa. The low concentration (10 ppb) of tetracycline exposure led to the pB10 transfer to enteric bacteria, which are clinically important pathogens.