Identification and characterization of a phosphinothricin N-acetyltransferase from Enterobacter LSJC7.

Phosphinothricin (PPT) is a widely used and non-selective herbicide. PPT-resistance genes, especially PPT N-acetyltransferase genes, have been used in the development of transgenic PPT-resistant crops. However, there are only a limited number of available PPT-resistance genes for use in plant biotechnology. In this study, we found that Enterobacter LSJC7 is highly resistant to PPT and can acetylate PPT to N-acetyl phosphinothricin (Ac-PPT). Furthermore, a novel PPT N-acetyltransferase gene, named LsarsN, was identified from LSJC7. When LsarsN was expressed in E. coli AW3110, it confered resistance to PPT. Ac-PPT was detected in both the culture medium and cells of AW3110 expressing the LsarsN-pET22b plasmid. The purified LsArsN protein also showed strong N-acetylation ability in vitro, and its enzymatic kinetic curve was fitted with the Michaelis-Mentan equation. Compared with wild-type LsArsN, both R72A and R74A mutants showed significantly lower PPT N-acetylation ability. In summary, our results systematically characterized LsArsN with strong ability for PPT N-acetylation, which lays the groundwork for future research into the use of this novel gene, LsarsN, to create PPT-resistant crops.

Incorporating bioaccessibility and source apportionment into human health risk assessment of heavy metals in urban dust of Xiamen, China.

Heavy metals in urban dust could pose noticeable human health risks, but there are few studies focusing on comprehensive human health risk assessment with the incorporation of both bioaccessibility and source apportionment in urban dust. Thus, fifty-eight urban dust samples were collected from kindergartens in Xiamen to analyze the bioaccessibility-based, source-specific health risk of heavy metals (V, Co, Ni, As, Mo, Cr, Mn, Cu, Zn, and Pb). Most heavy metals, except for V and Mn, were significantly enriched in urban dust based on their values of geoaccumulation index (Igeo) and may be influenced by human activities. The oral bioaccessibility values of heavy metals, which were estimated by the Solubility/Bioaccessibility Research Consortium (SBRC) in vitro model, ranged from 1.563% to 76.51%. The source apportionment determined by applying the absolute principal component analysis-multiple linear regression (APCS-MLR) model indicated five main potential sources, coal combustion, traffic and industrial, natural, construction and furniture sources, and unidentified sources, with contributions of 34.09%, 20.72%, 18.72%, 7.597% and 18.87%, respectively, to the accumulation of heavy metals in urban dust. After incorporating bioaccessibility adjustments, lower non-carcinogenic and carcinogenic risks of heavy metals were observed than those based on total metal content, with the mean hazard index (HI) values being less than the threshold value (1) and the mean total carcinogenic risk (TCR) values exceeding the precautionary criterion (10-6) for both adults and children. By combining bioaccessibility-based health risk assessment and source apportionment, traffic and industrial emissions and coal combustion dominated the noncarcinogenic and carcinogenic risks induced by heavy metals in urban dust, respectively. This study is expected to promote the systematic integration of source apportionment and bioaccessibility into health risk estimation for heavy metal contamination in urban dust, thus providing useful implications for better human health protection.

Occurrence and spatiotemporal distribution of arsenic biotransformation genes in urban dust.

Microbially-mediated arsenic biotransformation plays a pivotal role in the biogeochemical cycling of arsenic; however, the presence of arsenic biotransformation genes (ABGs) in urban dust remains unclear. To investigate the occurrence and spatiotemporal distributions of ABGs, a total of one hundred and eighteen urban dust samples were collected from different districts of Xiamen city, China in summer and winter. Although inorganic arsenic species, including arsenate [As(V)] and arsenite [As(III)], were found to be predominant, the methylated arsenicals, particularly trimethylarsine oxide [TMAs(V)O] and dimethylarsenate [DMAs(V)], were detected in urban dust. Abundant ABGs were identified in urban dust via AsChip analysis (a high-throughput qPCR chip for ABGs), of which As(III) S-adenosylmethionine methyltransferase genes (arsM), As(V) reductase genes (arsC), As(III) oxidase genes (aioA), As(III) transporter genes (arsB), and arsenic-sensing regulator genes (arsR) were the most prevalent, collectively constituting more than 90 % of ABGs in urban dust. Microbes involved in arsenic methylation were assigned to bacteria (e.g., Actinomycetes and Alphaproteobacteria), archaea (e.g., Halobacteria), and eukaryotes (e.g., Chlamydomonadaceae) in urban dust via the arsM amplicon sequencing. Temperature, a season-dependent environmental factor, profoundly affected the abundance of ABGs and the composition of microbes involved in arsenic methylation. This study provides new insights into the presence of ARGs within the urban dust.

Comprehensive analysis of metal(loid)s and associated metal(loid) resistance genes in atmospheric particulate matter.

Despite global concerns about metal(loid)s in atmospheric particulate matter (PM), the presence of metal(loid) resistance genes (MRGs) in PM remains unknown. Therefore, we conducted a comprehensive investigation of the metal(loid)s and associated MRGs in PMs in two seasons (summer and winter) in Xiamen, China. According to the geoaccumulation index (Igeo), most metal(loid)s, except for V and Mn, exhibited enrichment in PM, suggesting potential anthropogenic sources. By employing Positive Matrix Factorization (PMF) model, utilizing a dataset encompassing both total and bioaccessible metal(loid)s, along with backward trajectory simulations, traffic emissions were determined to be the primary potential contributor of metal(loid)s in summer, whereas coal combustion was observed to have a dominant contribution in winter. The major contributor to the carcinogenic risk of metal(loid)s in both summer and winter was predominantly attributed to coal combustion, which serves as the main source of bioaccessible Cr. Bacterial communities within PMs showed lower diversity and network complexity in summer than in winter, with Pseudomonadales being the dominant order. Abundant MRGs, including the As(III) S-adenosylmethionine methyltransferase gene (arsM), Cu(I)-translocating P-type ATPase gene (copA), Zn(II)/Cd(II)/Pb(II)-translocating P-type ATPase gene (zntA), and Zn(II)-translocating P-type ATPase gene (ziaA), were detected within the PMs. Seasonal variations were observed for the metal(loid) concentration, bacterial community structure, and MRG abundance. The bacterial community composition and MRG abundance within PMs were primarily influenced by temperature, rather than metal(loid)s. This research offers novel perspectives on the occurrence of metal(loid)s and MRGs in PMs, thereby contributing to the control of air pollution.

Roxarsone biotransformation by a nitroreductase and an acetyltransferase in Pseudomonas chlororaphis, a bacterium isolated from soil.

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox), a widely used organoarsenical feed additive, can enter soils and be further biotransformed into various arsenic species that pose human health and ecological risks. However, the pathway and molecular mechanism of Rox biotransformation by soil microbes are not well studied. Therefore, in this study, we isolated a Rox-transforming bacterium from manure-fertilized soil and identified it as Pseudomonas chlororaphis through morphological analysis and 16S rRNA gene sequencing. Pseudomonas chlororaphis was able to biotransform Rox to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), arsenate [As(V)], arsenite [As(III)], and dimethylarsenate [DMAs(V)]. The complete genome of Pseudomonas chlororaphis was sequenced. PcmdaB, encoding a nitroreductase, and PcnhoA, encoding an acetyltransferase, were identified in the genome of Pseudomonas chlororaphis. Expression of PcmdaB and PcnhoA in E. coli Rosetta was shown to confer Rox(III) and 3-AHPAA(III) resistance through Rox nitroreduction and 3-AHPAA acetylation, respectively. The PcMdaB and PcNhoA enzymes were further purified and functionally characterized in vitro. The kinetic data of both PcMdaB and PcNhoA were well fit to the Michaelis-Menten equation, and nitroreduction catalyzed by PcMdaB is the rate-limiting step for Rox transformation. Our results provide new insights into the environmental risk assessment and bioremediation of Rox(V)-contaminated soils.

Inhibitory effect of polyethylene microplastics on roxarsone degradation in soils.

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox(V)), an extensively used organoarsenical feed additive, enters soils through the application of Rox(V)-containing manure and further degrades to highly toxic arsenicals. Microplastics, as emerging contaminants, are also frequently detected in soils. However, the effects of microplastics on soil Rox(V) degradation are unknown. A microcosm experiment was conducted to investigate soil Rox(V) degradation responses to polyethylene (PE) microplastics and the underlying mechanisms. PE microplastics inhibited soil Rox(V) degradation, with the main products being 3-amino-4-hydroxyphenylarsonic acid [3-AHPAA(V)], N-acetyl-4-hydroxy-m-arsanilic acid [N-AHPAA(V)], arsenate [As(V)], and arsenite [As(III)]. This inhibition was likely driven by the decline in soil pH by PE microplastic addition, which may directly enhance Rox(V) sorption in soils. The decreased soil pH further suppressed the nfnB gene related to nitroreduction of Rox(V) to 3-AHPAA(V) and nhoA gene associated with acetylation of 3-AHPAA(V) to N-AHPAA(V), accompanied by a decrease in the relative abundance of possible Rox(V)-degrading bacteria (e.g., Pseudomonadales), although the diversity, composition, network complexity, and assembly of soil bacterial communities were largely influenced by Rox(V) rather than PE microplastics. Our study emphasizes microplastic-induced inhibition of Rox(V) degradation in soils and the need to consider the role of microplastics in better risk assessment and remediation of Rox(V)-contaminated soils.

Characteristics and provenances of rare earth elements and Nd isotopes in surface sediments of mangrove wetlands in the Jiulong River Estuary, China.

Rare earth elements (REEs) and Nd isotopes are frequently employed to determine provenance, although their characteristics and provenances in the surface sediments of mangrove wetlands are rarely analyzed. In this study, a thorough analysis of the characteristics and provenances of REEs and Nd isotopes in the surface sediments of mangrove wetland in the Jiulong River Estuary was carried out. According to the results, the mean concentration of REEs in the surface sediments was 290.9 mg·kg-1, which was greater than the background value. Unpolluted to moderately polluted for La and Ce, as well as a moderate ecological risk for Lu, were indicated by the geoaccumulation index (Igeo) and potential ecological risk of individual factors ([Formula: see text]), respectively. The surface sediments showed substantial negative Eu anomalies but no significant Ce anomalies. The enrichments in LREE and flat HREE patterns are visible in the chondrite-normalized REE patterns. REEs in the surface sediments might be attributed to both natural sources (granite and magmatic rocks) and anthropogenic activities, including coal combustion, vehicle exhaust, steel smelting, and fertilizer, based on the (La/Yb)N-∑REE and ternary (La/Yb)N-(La/Sm)N-(Gd/Yb)N plots. The three-dimensional ∑LREE/∑HREE-Eu/Eu*-εNd(0) plot, when combined with the Nd isotope, further demonstrated that the REEs in the surface sediments appeared to have come from additional nonlocal potential sources.

Randomized Trial of First-Line Tyrosine Kinase Inhibitor With or Without Radiotherapy for Synchronous Oligometastatic EGFR-Mutated Non-Small Cell Lung Cancer.

BACKGROUND: Adding radiotherapy (RT) to systemic therapy improves progression-free survival (PFS) and overall survival (OS) in oligometastatic non-small cell lung cancer (NSCLC). Whether these findings translate to epidermal growth factor receptor (EGFR)-mutated NSCLC remains unknown. The SINDAS trial (NCT02893332) evaluated first-line tyrosine kinase inhibitor (TKI) therapy for EGFR-mutated synchronous oligometastatic NSCLC and randomized to upfront RT vs no RT; we now report the prespecified interim analysis at 68% accrual. METHODS: Inclusion criteria were biopsy-proven EGFR-mutated adenocarcinoma (per amplification refractory mutation system or next generation sequencing), with synchronous (newly diagnosed, treatment naïve) oligometastatic (≤5 metastases; ≤2 lesions in any one organ) NSCLC without brain metastases. All patients received a first-generation TKI (gefitinib, erlotinib, or icotinib), and randomization was between no RT vs RT (25-40 Gy in 5 fractions depending on tumor size and location) to all metastases and the primary tumor/involved regional lymphatics. The primary endpoint (intention to treat) was PFS. Secondary endpoints included OS and toxicities. All statistical tests were 2-sided. RESULTS: A total of 133 patients (n = 65 TKI only, n = 68 TKI with RT) were enrolled (2016-2019). The median follow-up was 23.6 months. The respective median PFS was 12.5 months vs 20.2 months (P < .001), and the median OS was 17.4 months vs 25.5 months (P < .001) for TKI only vs TKI with RT. Treatment yielded no grade 5 events and a 6% rate of symptomatic grade 3-4 pneumonitis in the TKI with RT arm. Based on the efficacy results of this prespecified interim analysis, the ethics committee recommended premature cessation of this trial. CONCLUSIONS: As compared with a first-line TKI alone, addition of upfront local therapy using RT statistically significantly improved PFS and OS for EGFR-mutated NSCLC.

Bioaccessibility of microplastic-associated heavy metals using an in vitro digestion model and its implications for human health risk assessment.

Microplastics can act as carriers of heavy metals and may enter humans through ingestion and threaten human health. However, the bioaccessibility of heavy metals associated with microplastics and its implications for human health risk assessments are poorly understood. Therefore, in this study, four typical heavy metals (As(V), Cr(VI), Cd(II), and Pb(II)) and one typical microplastic (polyvinyl chloride, PVC) were chosen to estimate the human health risk of microplastic-associated heavy metals by incorporating bioaccessibility. Significant adsorption of heavy metals was observed with the following order for adsorption capacity: Pb(II) > Cr(VI) > Cd(II) > As(V); the efficiencies for desorption of these four heavy metals from PVC microplastics were all below 10%. The Fourier transform infrared spectroscopy results indicated that the functional groups on the surface of the virgin PVC microplastics did not play an important role in the capture process. Heavy metals in both gastric and small intestinal phases were prone to release from PVC microplastics when bioaccessibility was evaluated with the in vitro SBRC (Soluble Bioavailability Research Consortium) digestion model. In addition, Pb(II) bioaccessibility in the gastric phase was significantly higher than those in the other phases, while As(V), Cr(VI), and Cd(II) bioaccessibilities showed the opposite trend. After incorporating bioaccessibility adjustments, the noncarcinogenic hazards and carcinogenic risks determined were lower than those based on total metal contents. The individual hazard quotients (HQ) and carcinogenic risks (CR) for ingestion of these four heavy metals from PVC microplastics were all lower than the threshold values for adults and children. In summary, this study will provide a new view of the human health risks of heavy metals associated with microplastics.

[Speciation and Pollution Assessment of Heavy Metals in Mangrove Surface Sediments in Jiulong River Estuary].

The speciation of heavy metals was analyzed using modified BCR four-step extraction methods to analyze the pollution of heavy metals in surface sediments collected from the mangrove wetland in Jiulong River Estuary. Subsequently, the pollution degree and the ecological risk of heavy metals were evaluated by using the ratio of secondary phase to primary phase (RSP), risk assessment code (RAC), and modified potential ecological risk index (MRI) assessment methods. The results of BCR four-step extraction showed that Cd (52.55%) and Mn (47.71%) mainly existed in weak-acid extractable fractions. Pb, Y, and Cu mainly existed in reducible and oxidizable fractions. Ba, Tl, V, Th, Cr, As, U, Hg, Ni, Zn, and Co mainly existed in residue fractions. The results of RSP showed that the sediments were heavily polluted by Cd and Mn and moderately polluted by Pb. Cu, Y, and Co were slightly polluted, whereas Zn, Hg, As, U, Ni, Cr, Th, V, Ba, and Tl were not polluted. The results of RAC showed that Cd and Mn were high risk, whereas Co and Zn were moderate risk. Ni, Cu, Hg, and Y were slight risk, and the other elements (U, As, Pb, Cr, V, Tl, Ba, and Th) presented no risk. The MRI results showed that the comprehensive potential ecological risk of heavy metals was serious in the surface sediments, whereas Hg and Cd were the main contribution factors. Hg was a serious potential hazard, followed by Cd. Tl was a medium potential hazard, and the other elements were low potential hazards. These results demonstrated that the mangroves were polluted by heavy metals in Jiulong River Estuary, and effective strategies should be employed to remediate the mangrove sediment in the future.