High-throughput Quantitative Polymerase Chain Reaction Research Articles

COMPASS core subunits MpSet1 and MpSwd3 regulate Monascus pigments synthesis in Monascus purpureus.

In eukaryotes, methylation of histone H3 at lysine 4 (H3K4me) catalyzed by the complex of proteins associated with Set1 (COMPASS) is crucial for the transcriptional regulation of genes and the development of organisms. In Monascus, the functions of COMPASS in establishing H3K4me remain unclear. This study first identified the conserved COMPASS core subunits MpSet1 and MpSwd3 in Monascus purpureus and confirmed their roles in establishing H3K4me2/3. Loss of MpSet1 and MpSwd3 resulted in slower growth and development and inhibited the formation of cleistothecia, ascospores, and conidia. The loss of these core subunits also decreased the production of extracellular and intracellular Monascus pigments (MPs) by 94.2%, 93.5%, 82.7%, and 82.5%, respectively. In addition, RNA high-throughput sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) showed that the loss of MpSet1 and MpSwd3 altered the expression of 2646 and 2659 genes, respectively, and repressed the transcription of MPs synthesis-related genes. In addition, the ΔMpset1 and ΔMpswd3 strains demonstrated increased sensitivity to cell wall stress with the downregulation of chitin synthase-coding genes. These results indicated that the COMPASS core subunits MpSet1 and MpSwd3 help establish H3K4me2/3 for growth and development, spore formation, and pigment synthesis in Monascus. These core subunits also assist in maintaining cell wall integrity.

Microbial-influenced pesticide removal co-occurs with antibiotic resistance gene variation in soil-earthworm-maize system

Within human-influenced landscapes, pesticides cooccur with a variety of antibiotic stressors. However, the relationship between pesticides removal process and antibiotic resistance gene variation are not well understood. This study explored pesticide (topramezone, TPZ) and antibiotic (polymyxin E, PME) co-contamination using liquid chromatography-tandem mass spectrometry (LC-MS/MS), bacterial-16 S rRNA sequencing and high-throughput quantitative polymerase chain reaction (HT-qPCR) in a soil-earthworm-maize system. After incubating soil for 28 days with TPZ and PME (10 mg kg−1 dry weight), earthworm weight-gain, mortality rates, and maize plant weight-gain only differed slightly, but height-gain significantly decreased. PME significantly increased TPZ-removal in the soil. Accumulation of TPZ in earthworm's tissues may pose potential risks in the food chain. Combined pollution altered the microbial community structure and increased the abundance of functional microorganisms involved in aromatic compound degradation. Furthermore, maize rhizosphere can raise resistance genes, however earthworms can reduce resistance genes. Co-contamination increased absolute abundance of mobile genetic elements (MGEs) in bulk-soil samples, antibiotic resistance genes (ARGs) in skin samples and number of ARGs in bulk-soil samples, while decreased absolute abundance of transposase gene in bulk-soil samples and number of ARGs in rhizosphere-soil samples. Potential hosts harbouring ARGs may be associated with the antagonistic effect during resistance and detoxification of TPZ and PMB co-occurrence. These findings provide insights into the mechanism underlining pesticide removal regarding occurrence of ARGs in maize agroecosystem.

Establishment of Biocontrol Agents and Their Impact on Rhizosphere Microbiome and Induced Grapevine Defenses Are Highly Soil-Dependent

With a reduction in available chemical treatments, there is an increased interest in biological control of grapevine trunk diseases. Few studies have investigated the impact of introducing beneficial microorganisms in the rhizosphere on the existing indigenous soil microbiome. In this study, we explored the effect of two biocontrol agents (BCAs), Trichoderma atroviride SC1 (Ta SC1) (Vintec; Certis Belchim) and Bacillus subtilis PTA-271 (Bs PTA-271), on the grapevine rhizosphere bacterial and fungal microbiome as well as plant defense expression using high-throughput amplicon sequencing and quantitative real-time polymerase chain reaction (PCR), respectively. Additionally, we quantified both Ta SC1 and Bs PTA-271 in the rhizosphere over time using droplet digital PCR. The fungal microbiome was more affected by factors such as soil type, BCA treatment, and sampling time compared with the bacterial microbiome. Specifically, Ta SC1 application produced negative impacts on fungal diversity, whereas application of BCAs did not affect bacterial diversity. Interestingly, the survival and establishment of both BCAs showed opposite trends depending on the soil type, indicating that the physicochemical properties of soils have a role in BCA establishment. Fungal co-occurrence networks were less complex than bacterial networks but highly impacted by Ta SC1 application. Soils treated with Ta SC1 presented more complex and stable co-occurrence networks, with a higher number of positive correlations. Induced grapevine defenses also differed according to the soil, being more affected by BCA inoculation on sandy soil. The findings of this research emphasize the complex relationships among microorganisms in the rhizosphere and highlight the significance of taking into account various factors, such as soil type, sampling time, and BCA treatment, and their influence on the structure and dynamics of microbial communities.

Active pathways of anaerobic methane oxidization in deep-sea cold seeps of the South China Sea.

Anaerobic oxidation of methane (AOM) is critical for controlling methane emissions from deep-sea cold seeps. Microbial groups associated with different AOM processes have been briefly investigated, but the AOM activities associated with the utilization of different electron acceptors in the deep-sea methane seeps remain largely unknown. Here, surface sediments (0-4 cm) from two cold seeps (Site F/Haima) and the Xisha trough in the South China Sea (SCS) were incubated to measure AOM potentials driven by nitrite, nitrate, and sulfate as different electron acceptors with isotopically labeled methane gas (13CH4), and the population shifts of microbial communities along the incubation were investigated by high-throughput sequencing and quantitative polymerase chain reaction based on both the 16S rRNA gene and functional genes. Nitrite and nitrate were preferentially used prior to sulfate during the incubation, and nitrate-/nitrite-dependent anaerobic methane oxidation (Nr-/N-DAMO) contributed more to the methane flux in both the cold seeps and the trough. The highest rates of DAMO and sulfate-dependent anaerobic methane oxidation (SAMO) were detected at Site F, consistent with the transcript abundance of mcrA and dsrB genes during the incubation, and might be explained by the in situ active functional groups. The former process might be explained by Campylobacteria, which could couple sulfide oxidation and nitrate consumption to further participate in the N-DAMO process, while the latter process might be due to the higher proportions of Desulfobacterota. This study elucidates the diversity and potential activities of major microbial groups in the DAMO and SAMO processes in the deep-sea cold seeps using isotopic tracing incubations and provides direct evidence for the occurrence of Nr-/N-DAMO as a previously overlooked microbial methane sink in the deep-sea hydrate-bearing sediments in the SCS. IMPORTANCE Cold seeps occur in continental margins worldwide and are deep-sea oases. Anaerobic oxidation of methane is an important microbial process in the cold seeps and plays an important role in regulating methane content. This study elucidates the diversity and potential activities of major microbial groups in dependent anaerobic methane oxidation and sulfate-dependent anaerobic methane oxidation processes and provides direct evidence for the occurrence of nitrate-/nitrite-dependent anaerobic methane oxidation (Nr-/N-DAMO) as a previously overlooked microbial methane sink in the hydrate-bearing sediments of the South China Sea. This study provides direct evidence for occurrence of Nr-/N-DAMO as an important methane sink in the deep-sea cold seeps.

A Comparative Assessment of High-Throughput Quantitative Polymerase Chain Reaction versus Shotgun Metagenomic Sequencing in Sediment Resistome Profiling

Prolonged and excessive use of antibiotics has resulted in the development of antimicrobial resistance (AMR), which is considered an emerging global challenge that warrants a deeper understanding of the antibiotic-resistant gene elements (ARGEs/resistomes) involved in its rapid dissemination. Currently, advanced molecular methods such as high-throughput quantitative polymerase chain reaction (HT-qPCR) and shotgun metagenomic sequencing (SMS) are commonly applied for the surveillance and monitoring of AMR in the environment. Although both methods are considered complementary to each other, there are some appreciable differences that we wish to highlight in this communication. We compared both these approaches to map the ARGEs in the coastal sediments of Kuwait. The study area represents an excellent model as it receives recurrent emergency waste and other anthropogenic contaminants. The HT-qPCR identified about 100 ARGs, 5 integrons, and 18 MGEs (total—122). These ARGs coded for resistance against the drug classes of beta-lactams > aminoglycoside > tetracycline, macrolide lincosamide streptogramin B (MLSB) > phenicol > trimethoprim, quinolone, and sulfonamide. The SMS picked a greater number of ARGs (402), plasmid sequences (1567), and integrons (168). Based on the evidence, we feel the SMS is a better method to undertake ARG assessment to fulfil the WHO mandate of “One Health Approach.” This manuscript is a useful resource for environmental scientists involved in AMR monitoring.

Reactor performance of static magnetic field membrane bioreactor for treating actual high-salt textile dyeing wastewater and possible mechanism on magnetically enhanced membrane fouling control

Performance of the static magnetic field membrane bioreactor (SMFMBR) for treating actual high-salt textile dyeing wastewater was evaluated. Membrane fouling behavior and possible mechanisms were investigated using multiple methods including chemical composition analysis, microbial community analysis, and four-dimensional label-free quantification metaproteomic analysis. The results showed that three SMFMBRs equipped with the SMFs of 97.2 mT (1#), 202.3 mT (2#) and 303.4 mT (3#) possessed higher removal efficiency of color, chemical oxygen demand (COD) and acute toxicity, as well as higher activity of key enzymes than the conventional MBR (0#). Treatment efficiency of 3# SMFMBR was the highest among all the four groups. Potentially effective bacterial and fungal species were selectively enriched in the suspended sludge of three SMFMBRs according to the results of both high-throughput sequencing and quantitative real-time polymerase chain reaction (QRT-PCR). Membrane fouling rate of 3# SMFMBR was the lowest among all the groups, followed by that of 2#, 0# and 1# reactors. SMF of higher intensity (in 2# and 3# SMFMBRs) mitigated membrane fouling by inhibiting the production of microbial products (mainly consisting of proteins) and the growth of potential fouling-causing microbes on the membrane surface. By contrast, SMF of lower intensity (in 1# SMFMBR) aggravated membrane fouling by improving the yield of fouling-causing microbial products and the growth of biofilm on the membrane surface. Furthermore, the down-regulated porin and the up-regulated Ca2+-binding protein in biocake EPS were also responsible for the mitigation of membrane fouling in 3# SMFMBR.

Uncovering the prevalence and drivers of antibiotic resistance genes in soils across different land-use types

The emergence and spread of antibiotic resistance genes (ARGs) in soil due to animal excreta and organic waste is a major threat to human health and ecosystems, and global efforts are required to tackle the issue. However, there is limited knowledge of the variation in ARG prevalence and diversity resulting from different land-use patterns and underlying driving factors in soils. This study aimed to comprehensively characterize the profile of ARGs and mobile genetic elements and their drivers in soil samples collected from 11 provinces across China, representing three different land-use types, using high-throughput quantitative polymerase chain reaction and 16S rRNA amplicon sequencing. Our results showed that agricultural soil had the highest abundance and diversity of ARGs, followed by tea plantation and forest land. A total of 124 unique ARGs were detected in all samples, with shared subtypes among different land-use patterns indicating a common origin or high transmission frequency. Moreover, significant differences in ARG distribution were observed among different geographical regions, with the greatest enrichment of ARGs found in southern China. Biotic and abiotic factors, including soil properties, climatic factors, and bacterial diversity, were identified as the primary drivers associated with ARG abundance, explaining 71.8% of total ARG variation. The findings of our study demonstrate that different land-use patterns are associated with variations in ARG abundance in soil, with agricultural practices posing the greatest risk to human health and ecosystems regarding ARGs. Our identification of biotic and abiotic drivers of ARG abundance provides valuable insights into strategies for mitigating the spread of these genes. This study emphasizes the need for coordinated and integrated approaches to address the global antimicrobial resistance crisis.

Potential Association of Cytochrome P450 Copy Number Alteration in Tumour with Chemotherapy Resistance in Lung Adenocarcinoma Patients.

Resistance to anticancer agents is a major obstacle to efficacious tumour therapy and responsible for high cancer-related mortality rates. Some resistance mechanisms are associated with pharmacokinetic variability in anticancer drug exposure due to genetic polymorphisms of drug-metabolizing cytochrome P450 (CYP) enzymes, whereas variations in tumoural metabolism as a consequence of CYP copy number alterations are assumed to contribute to the selection of resistant cells. A high-throughput quantitative polymerase chain reaction (qPCR)-based method was developed for detection of CYP copy number alterations in tumours, and a scoring system improved the identification of inappropriate reference genes that underwent deletion/multiplication in tumours. The copy numbers of both the target (CYP2C8, CYP3A4) and the reference genes (ALB, B2M, BCKDHA, F5, CD36, MPO, TBP, RPPH1) established in primary lung adenocarcinoma by the qPCR-based method were congruent with those determined by next-generation sequencing (for 10 genes, slope = 0.9498, r2 = 0.72). In treatment naïve adenocarcinoma samples, the copy number multiplication of paclitaxel-metabolizing CYP2C8 and/or CYP3A4 was more prevalent in non-responder patients with progressive disease/exit than in responders with complete remission. The high-throughput qPCR-based method can become an alternative approach to next-generation sequencing in routine clinical practice, and identification of altered CYP copy numbers may provide a promising biomarker for therapy-resistant tumours.

Improvement of Straw Changed Soil Microbial Flora Composition and Suppressed Chinese Cabbage (Brassica rapa L. ssp. pekinensis) Clubroot Disease

Straw incorporation is known as an environmentally friendly agricultural practice that can effectively enhance soil nutrient contents and crop yields; its potential to suppress soil-borne disease has also been reported in recent years. Here, we perform a field experiment for two consecutive years (2017–2018) to evaluate the effectiveness of maize (Zea mays), rice (Oryzae sativa L.) and wheat (Triticum aestivum L.) straws incorporation in alleviating Chinese cabbage (Brassica rapa L. ssp. pekinensis) clubroot disease caused by Plasmodiophora brassicae Woronin. Microbial composition in Chinese cabbage rhizosphere and soil P. brassicae abundance were estimated by high-throughput amplicon sequencing and quantitative polymerase chain reaction (qPCR). Results showed that, during the two-year field experimental cycle, all three straw amendments promoted Chinese cabbage plant growth, inhibited clubroot disease and increased the alpha diversity of the bacterial community in Chinese cabbage rhizosphere. Rice and wheat straws also increased the alpha diversity of the fungal community. These straws diversified the composition of the Chinese cabbage rhizosphere microbial community. All three straws promoted Cryptococcus carnescens; both rice and wheat straws stimulated Lysobacter sp.; maize straw boosted Sphingomonas sp. and wheat straw increased Talaromyces sp. These microbial taxa are either considered to have positive influences on plant growth or potential biocontrol effects. In addition, straw amendments also increased soil pH, electrical conductivity, available nitrogen and available potassium contents in both years of the field experiment. Taken together, we concluded that these three gramineous straw amendments ameliorated Chinese cabbage rhizosphere microorganisms, inhibited clubroot disease and promoted the growth of Chinese cabbage, and that rice straw worked best amongst the three. This study could potentially provide a new tactic of massive grain crop straw utilization and a direction in dealing with clubroot disease.

Antibiotic Resistance Genes in Aerosols: Baseline from Kuwait.

Antimicrobial resistance (AMR) is one of the biggest threats to human health worldwide. The World Health Organization (WHO, Geneva, Switzerland) has launched the "One-Health" approach, which encourages assessment of antibiotic-resistant genes (ARGs) within environments shared by human-animals-plants-microbes to constrain and alleviate the development of AMR. Aerosols as a medium to disseminate ARGs, have received minimal attention. In the present study, we investigated the distribution and abundance of ARGs in indoor and outdoor aerosols collected from an urban location in Kuwait and the interior of three hospitals. The high throughput quantitative polymerase chain reaction (HT-qPCR) approach was used for this purpose. The results demonstrate the presence of aminoglycoside, beta-lactam, fluoroquinolone, tetracycline, macrolide-lincosamide-streptogramin B (MLSB), multidrug-resistant (MDR) and vancomycin-resistant genes in the aerosols. The most dominant drug class was beta-lactam and the genes were IMP-2-group (0.85), Per-2 group (0.65), OXA-54 (0.57), QnrS (0.50) and OXA-55 (0.55) in the urban non-clinical settings. The indoor aerosols possessed a richer diversity (Observed, Chao1, Shannon's and Pielou's evenness) of ARGs compared to the outdoors. Seasonal variations (autumn vs. winter) in relative abundances and types of ARGs were also recorded (R2 of 0.132 at p < 0.08). The presence of ARGs was found in both the inhalable (2.1 µm, 1.1 µm, 0.7 µm and < 0.3 µm) and respirable (>9.0 µm, 5.8 µm, 4.7 µm and 3.3 µm) size fractions within hospital aerosols. All the ARGs are of pathogenic bacterial origin and are hosted by pathogenic forms. The findings present baseline data and underpin the need for detailed investigations looking at aerosol as a vehicle for ARG dissemination among human and non-human terrestrial biota.

Effect of composted pig manure, biochar, and their combination on antibiotic resistome dissipation in swine wastewater-treated soil

The prevalence of antibiotic resistance genes (ARGs), owing to irrigation using untreated swine wastewater, in vegetable-cultivated soils around swine farms poses severe threats to human health. Furthermore, at the field scale, the remediation of such soils is still challenging. Therefore, here, we performed field-scale experiments involving the cultivation of Brassica pekinensis in a swine wastewater-treated soil amended with composted pig manure, biochar, or their combination. Specifically, the ARG and mobile genetic element (MGE) profiles of bulk soil (BS), rhizosphere soil (RS), and root endophyte (RE) samples were examined using high-throughput quantitative polymerase chain reaction. In total, 117 ARGs and 22 MGEs were detected. Moreover, we observed that soil amendment using composted pig manure, biochar, or their combination decreased the absolute abundance of ARGs in BS and RE after 90 days of treatment. However, the decrease in the abundance of ARGs in RS was not significant. We also observed that the manure and biochar co-application showed a minimal synergistic effect. To clarify this observation, we performed network and Spearman correlation analyses and used structure equation models to explore the correlations among ARGs, MGEs, bacterial composition, and soil properties. The results revealed that the soil amendments reduced the abundances of MGEs and potential ARG-carrying bacteria. Additionally, weakened horizontal gene transfer was responsible for the dissipation of ARGs. Thus, our results indicate that composted manure application, with or without biochar, is a useful strategy for soil nutrient supplementation and alleviating farmland ARG pollution, providing a justification for using an alternative to the common agricultural practice of treating the soil using only untreated swine wastewater. Additionally, our results are important in the context of soil health for sustainable agriculture.

Structure and Functional Properties of Bacterial Communities in Surface Sediments of the Recently Declared Nutrient-Saturated Lake Villarrica in Southern Chile.

Lake Villarrica, one of Chile's main freshwater water bodies, was recently declared a nutrient-saturated lake due to increased phosphorus (P) and nitrogen (N) levels. Although a decontamination plan based on environmental parameters is being established, it does not consider microbial parameters. Here, we conducted high-throughput DNA sequencing and quantitative polymerase chain reaction (qPCR) analyses to reveal the structure and functional properties of bacterial communities in surface sediments collected from sites with contrasting anthropogenic pressures in Lake Villarrica. Alpha diversity revealed an elevated bacterial richness and diversity in the more anthropogenized sediments. The phylum Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria dominated the community. The principal coordinate analysis (PCoA) and redundancy analysis (RDA) showed significant differences in bacterial communities of sampling sites. Predicted functional analysis showed that N cycling functions (e.g., nitrification and denitrification) were significant. The microbial co-occurrence networks analysis suggested Chitinophagaceae, Caldilineaceae, Planctomycetaceae, and Phycisphaerae families as keystone taxa. Bacterial functional genes related to P (phoC, phoD, and phoX) and N (nifH and nosZ) cycling were detected in all samples by qPCR. In addition, an RDA related to N and P cycling revealed that physicochemical properties and functional genes were positively correlated with several nitrite-oxidizing, ammonia-oxidizing, and N-fixing bacterial genera. Finally, denitrifying gene (nosZ) was the most significant factor influencing the topological characteristics of co-occurrence networks and bacterial interactions. Our results represent one of a few approaches to elucidate the structure and role of bacterial communities in Chilean lake sediments, which might be helpful in conservation and decontamination plans.

Effects of carbamazepine on gut microbiota, ARGs and intestinal health in zebrafish

Carbamazepine (CBZ) in the aquatic environment is recognized as a potential threat to aquatic organisms and public health. However, the response of organism intestinal health, resistome, microbiota, and their relationship after CBZ exposure has been rarely reported. This study aimed to explore the impacts of CBZ on gut microbiota, antibiotic resistance genes (ARGs) and the expression of intestinal health related genes as well as their interaction using the zebrafish model. 16 S ribosomal RNA sequencing indicated CBZ altered the composition of gut microbiota. Using high-throughput quantitative polymerase chain reaction (HT-qPCR), we found the number and abundance of ARGs were impacted by CBZ levels and exposure duration. We also observed the upregulated expression of the pro-inflammatory gene IL6 and downregulated expression of toll-like receptor gene TLR2 and intestinal barrier gene TJP2a at different exposure times. Correlation analyses revealed that Geobacillus, Rhodococcus, Ralstonia, Delftia, Luteolibacter and Escherichia-Shigella might be the main bacterial genera carrying ARGs. Meanwhile, Cetobacterium and Aeromonas could be the dominant bacteria affecting intestinal health related genes. Our results could contribute to understanding the health risks of CBZ to the intestinal microecology of aquatic animals.

Sediment diffusion is feasible to simultaneously reduce nitrate discharge from recirculating aquaculture system and ammonium release from sediments in receiving intensive aquaculture pond

Nitrogen accumulation has become one of the greatest unresolved challenges restricting the development of aquaculture worldwide. In recirculating aquaculture system (RAS), lack of organic matter (OM) and sensitive organisms makes it difficult to apply efficient denitrifying technology, thus leading to a high nitrate‑nitrogen (NO3−-N) accumulation. In contrast, excess OM accumulation in intensive aquaculture pond sediments is associated with dissolved oxygen depletion and ammonium‑nitrogen (NH4+-N) accumulation in the sediments. Based on the opposing effects of OM on the nitrogen accumulation in RAS and intensive aquaculture ponds, this study assessed the feasibility of simultaneously reducing NO3−-N discharge from RAS and controlling NH4+-N accumulation in intensive aquaculture ponds by in situ diffusing RAS tailwater containing NO3−-N into intensive aquaculture pond sediments. The results showed that NO3−-N diffusion strategy improved the native sediment denitrification capacity, thus increasing NO3−-N removal efficiency from RAS tailwater and significantly decreasing the NH4+-N concentration in interstitial water and the total organic carbon content in intensive aquaculture pond sediments. High-throughput sequencing and quantitative real-time polymerase chain reaction (qPCR) results revealed that NO3−-N addition significantly increased both nitrifying bacteria and denitrifying bacteria abundance. These results implied that NO3−-N diffusion strategy could effectively stimulate microbial decomposition of OM, thus relieving the hypoxia limitation of sediment nitrification. Overall, this study offers a feasible method for simultaneous reduction of NO3−-N from RAS tailwater and NH4+-N in intensive aquaculture ponds with low cost and high efficiency.

Iron-based passivator mitigates the coupling process of anaerobic methane oxidation and arsenate reduction in paddy soils

Arsenic (As) is a toxic metalloid that is ubiquitous in paddy soils, where passivation is the most widely used method for remediating As contamination. Recently, anaerobic methane oxidation coupled with arsenate (As(V)) reduction (AOM-AsR) has been shown to act as a critical driver for As release in paddy fields. However, the effect and mechanism of the passivators on the AOM-AsR process remain unclear. In this study, we incubated arsenate-contaminated paddy soils under anaerobic conditions. Using isotopically labelled methane and different passivators, we found that an iron-based passivator containing calcium sulfate and iron oxide (9:1, m/m) named IBP showed a much better performance than the other passivators. Adding IBP decreased the arsenite (As(III)) concentration in the soil solution by 78% and increased the AOM rate by 55%. Furthermore, we employed high-throughput sequencing and real-time quantitative polymerase chain reaction (qPCR) to investigate the ability of IBP to control As release mediated by AOM-AsR in paddy fields, as well as its underlying mechanism. Our results showed that IBP addition significantly increased anaerobic methanotrophic (ANME) archaea (ANME-2a-c, ANME-2d, and ANME-3) by 91%, and increased the methane-oxidizing bacterium Methylobacter by 262%. Similarly, IBP addition significantly increased the Fe(III) concentration in soil solution by 39% and increased the absolute abundance of Fe(III)-reducing bacteria (Geobacteraceae) by 21 times in soil. Adding IBP may significantly promote AOM coupled with Fe(III) reduction, significantly reducing electron transfer from AOM to As(V) reduction. Hence, IBP may be used as an efficient passivator to remediate As-contaminated soil using an active AOM-AsR process. These results provide a novel insight into controlling soil As release by regulating an active and critical As mobilization pathway in the environment.

Responses of diversity and arsenic-transforming functional genes of soil microorganisms to arsenic hyperaccumulator (Pteris vittata L.)/pomegranate (Punica granatum L.) intercropping

Intercropping of arsenic (As) hyperaccumulator (Pteris vittata L.) with crops can reduce the As concentration in soil and the resulting ecological and health risks, while maintaining certain economic benefits. However, it is still unclear how As-transforming functional bacteria and dominant bacteria in the rhizosphere of P. vittata affect the microbial properties of crop rhizosphere soil, as well as how As concentration and speciation change in crop rhizosphere soil under intercropping. This is of great significance for understanding the biogeochemical cycle of As in soil and crops. This study aimed to use high-throughput sequencing and quantitative polymerase chain reaction (qPCR) to analyze the effects of different rhizosphere isolation patterns on the bacterial diversity and the copy number of As-transforming functional genes in pomegranate (Punica granatum L.) rhizosphere soil. The results showed that the abundance of bacteria in the rhizosphere soil of pomegranate increased by 16.3 %, and the soil bacterial community structure significantly changed. C_Alphaproteobacteria and o_Rhizobiales bacteria significantly accumulated in the rhizosphere of pomegranate. The copy number of As methylation (arsM) gene in pomegranate rhizosphere soil significantly increased by 63.37 %. The concentrations of nonspecifically sorbed As (F1), As associated with amorphous Fe (hydr)oxides (F3), and the total As (FT) decreased; the proportion of As (III) in pomegranate rhizosphere soil decreased; and the proportion of As (V) increased in pomegranate rhizosphere soil. c_Alphaproteobacteria and o_Rhizobiales accumulated in crop rhizosphere soil under the intercropping of P. vittata with crops. Also, the copy number of As methylation functional genes in crop rhizosphere soil significantly increased, which could reduce As (III) proportion in crop rhizosphere soil. These changes favored simultaneous agricultural production and soil remediation. The results provided the theoretical basis and practical guidance for the safe utilization of As-contaminated soil in the intercropping of As-hyperaccumulator and cash crops.

MRG Chip: A High-Throughput qPCR-Based Tool for Assessment of the Heavy Metal(loid) Resistome.

Bacterial metal detoxification mechanisms have been well studied for centuries in pure culture systems. However, profiling metal resistance determinants at the community level is still a challenge due to the lack of comprehensive and reliable quantification tools. Here, a novel high-throughput quantitative polymerase chain reaction (HT-qPCR) chip, termed the metal resistance gene (MRG) chip, has been developed for the quantification of genes involved in the homeostasis of 9 metals. The MRG chip contains 77 newly designed degenerate primer sets and 9 published primer sets covering 56 metal resistance genes. Computational evaluation of the taxonomic coverage indicated that the MRG chip had a broad coverage matching 2 kingdoms, 29 phyla, 64 classes, 130 orders, 226 families, and 382 genera. Temperature gradient PCR and HT-qPCR verified that 57 °C was the optimal annealing temperature, with amplification efficiencies of over 94% primer sets achieving 80-110%, with R2 > 0.993. Both computational evaluation and the melting curve analysis of HT-qPCR validated a high specificity. The MRG chip has been successfully applied to characterize the distribution of diverse metal resistance determinants in natural and human-related environments, confirming its wide scope of application. Collectively, the MRG chip is a powerful and efficient high-throughput quantification tool for exploring the microbial metal resistome.

Exosomal circWDR62 promotes temozolomide resistance and malignant progression through regulation of the miR-370-3p/MGMT axis in glioma

Exosome-mediated delivery of circular RNAs (circRNAs) is implicated in cancer progression. However, the role of exosomal circRNAs in the chemotherapy resistance of tumours remains poorly understood. Here we identified a novel circRNA, circWDR62. It was found that circWDR62 expression was upregulated in TMZ-resistant glioma cells and TMZ-resistant glioma cell-derived exosomes compared with their controls by using high-throughput microarray analysis and quantitative real-time polymerase chain reaction, and high circWDR62 expression was associated with poor prognosis of glioma. Functionally, downregulation of circWDR62 expression could significantly inhibit the TMZ resistance and malignant progression of glioma. Further mechanistic studies showed that circWDR62 plays a role by sponging miR-370-3p as a competing endogenous RNA. Rescue experiments confirmed that MGMT is the downstream target of the circWDR62/miR-370-3p axis in glioma. In addition, circWDR62 could be transported between TMZ-resistant and TMZ-sensitive glioma cells via exosomes. Exosomal circWDR62 from TMZ-resistant cells conferred TMZ resistance in recipient sensitive cells while also enhancing the proliferation, migration and invasion of these cells. A series of clinical and in vivo trials corroborated that exosomal circWDR62 could promote TMZ chemoresistance and malignant progression of glioma. Our results demonstrate for the first time that exosome-mediated delivery of circWDR62 can promote TMZ resistance and malignant progression via targeting of the miR-370-3p/MGMT axis in vitro and in vivo in glioma, providing a new therapeutic strategy. Moreover, exosomal circWDR62 in human serum may serve as a promising therapeutic target and prognostic marker for glioma therapy.

Limited Impacts of Cover Cropping on Soil N-Cycling Microbial Communities of Long-Term Corn Monocultures.

Cover cropping (CC) is a promising in-field practice to mitigate soil health degradation and nitrogen (N) losses from excessive N fertilization. Soil N-cycling microbial communities are the fundamental drivers of these processes, but how they respond to CC under field conditions is poorly documented for typical agricultural systems. Our objective was to investigate this relationship for a long-term (36 years) corn [Zea mays L.] monocultures under three N fertilizer rates (N0, N202, and N269; kg N/ha), where a mixture of cereal rye [Secale cereale L.] and hairy vetch [Vicia villosa Roth.] was introduced for two consecutive years, using winter fallows as controls (BF). A 3 × 2 split-plot arrangement of N rates and CC treatments in a randomized complete block design with three replications was deployed. Soil chemical and physical properties and potential nitrification (PNR) and denitrification (PDR) rates were measured along with functional genes, including nifH, archaeal and bacterial amoA, nirK, nirS, and nosZ-I, sequenced in Illumina MiSeq system and quantified in high-throughput quantitative polymerase chain reaction (qPCR). The abundances of nifH, archaeal amoA, and nirS decreased with N fertilization (by 7.9, 4.8, and 38.9 times, respectively), and correlated positively with soil pH. Bacterial amoA increased by 2.4 times with CC within N269 and correlated positively with soil nitrate. CC increased the abundance of nirK by 1.5 times when fertilized. For both bacterial amoA and nirK, N202 and N269 did not differ from N0 within BF. Treatments had no significant effects on nosZ-I. The reported changes did not translate into differences in functionality as PNR and PDR did not respond to treatments. These results suggested that N fertilization disrupts the soil N-cycling communities of this system primarily through soil acidification and high nutrient availability. Two years of CC may not be enough to change the N-cycling communities that adapted to decades of disruption from N fertilization in corn monoculture. This is valuable primary information to understand the potentials and limitations of CC when introduced into long-term agricultural systems.

Impact of surface-applied liquid manure on the drainage resistance profile of an agricultural tile-drained clay till field.

Dissemination of antibiotic resistance genes (ARGs) in aquatic environments is a concern due to human and animal health. Application of liquid manure on agricultural land is an important source of ARGs, where pathogens, antibiotic-resistant bacteria, and selective agents are released. To improve our understanding of ARGs spreading through soils, our main objective was to evaluate the effectiveness of the soil as a barrier protecting water resources. Over the course of a year, profiles and abundances of ARGs and mobile genetic elements in soil and drainage from an agricultural tile-drained clay till field were investigated upon liquid pig manure application by applying high-throughput quantitative polymerase chain reaction targeting 143 genes. The findings were as follows: (a) 97 genes were detected, where only the transposon gene tnpA-03/ IS6 was shared between the genes detected in drainage and those in acidified liquid manure or fertilized soils, indicating that liquid manure application had a limited impact on the drainage resistance profile; (b) intI1 gene was present in ∼60% of drainage samples in concentrations up to 1,634intI1 ml-1 ; and (c) evapotranspiration from barley (Hordeum vulgare L., 'KWS Irina') and a low groundwater table appeared to reduce preferential transport to drainage during the first 3 mo of liquid manure application. Interestingly, the first preferential transport to drainage was observed immediately after the harvest of spring barley. Overall, during the monitoring year we found the soil to be an effective barrier against the spread of fecal ARGs even though the occurrence of the intI1 gene questions the barrier effect from previous years.