There is evidence that forage grasses such as Megathyrsus and Urochloa can suppress nitrification, with direct or indirect consequences on soil inorganic N dynamics and nitrous oxide (N2O) emissions. ...However, the influence of soil chemical properties on the dynamics of functional N-genes and losses of N in maize (Zea mays L.) intercropped with forage grasses under N fertilization is poorly understood. In this study, soil samples and N2O emissions were analyzed from a field experiment in which maize (fertilized or not with ammonium-based fertilizer) was intercropped with Guinea grass (M. maximus cv. Tanzânia), palisade grass (U. brizantha cv. Marandu), and ruzigrass (U. ruziziensis cv. Comum). Soil N-cycle microorganisms 16S rRNA of bacteria and archaea, nifH (gene encoding N2-fixing bacteria), ammonia-oxidizing bacteria (AOB) and archaea (AOA), nirS (encoding nitrite reductase), and nosZ (encoding nitrous oxide reductase) were influenced by forage grass, N fertilization, and sampling time, but no evidence of biological nitrification inhibition was found. Palisade grass was associated with a higher abundance of nifH (7.0 × 105 gene copies g−1 soil, on average) in the absence of N compared with the other grasses (4.3 × 105 gene copies g−1 soil, on average). Nitrogen fertilization increased the abundance of AOB but not AOA. Furthermore, N2O flux was influenced by AOB, water-filled pore space, and N fertilization, whereas the cumulative N2O emission and fertilizer-induced emission factor (0.36%, on average) were not affected by the grasses. In conclusion, this study reveals the strong dominance of AOB under ammonium supply, potentially stimulating N2O emissions in maize-forage grass intercropping systems.
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•We assessed the abundance of N-cycle genes and N2O emissions in forage grass-maize systems.•Ammonium-based fertilizer slightly increased the AOB abundance but did not affect AOA.•There was a strong influence of WFPS and AOB on N2O fluxes in maize intercropped with grass.•The link between N2O and AOB may imply that this microbial group increases N2O.•There is no clear evidence of biological nitrification inhibition by the forage grasses.
This study was designed to investigate the metabolic and transcriptional alterations in seminal fluid caused by asthenozoospermia (AS). To address these issues, a method of metabonomics based on ...ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) and real‐time quantitative PCR (RT‐qPCR) was performed to identify some crucial biomarkers and transcription levels of the enzymes in seminal fluid. Seminal fluid samples were collected from 87 AS patients and 73 healthy males with normozoospermia. The quantitative analysis by UPLC–MS/MS showed that 19 metabolites in seminal plasma were associated with AS, and they were involved in several metabolic pathways, such as energy metabolism, purine metabolism, methionine cycle, and branched chain amino acid metabolism. Among these metabolites, the levels of citric acid, malic acid, succinic acid, and pyruvic acid, which are related to energy metabolism, were collectively reduced in the AS group, whereas the lactic acid level was enhanced. These results indicated that lesser energy source (adenosine triphosphate) was produced through the anaerobic glycolysis pathway rather than via aerobic catabolism of suger and tricarboxylic acid cycle, resulting in reduced power of sperms. Meanwhile, partial least squares discriminant analysis showed significant differences in metabolic profiles between the AS and control groups. In addition, RT‐qPCR results revealed that the expression levels of four genes encoding fructokinase citrate synthase, succinate dehydrogenase, and spermine synthase, which were related to energy metabolism, were decreased in the AS group. The 23 descriptors with differential expression in AS may be valuable for the diagnosis and sequential study on AS. These results will help highlight the role of sperm inactivity in AS pathogenesis.
Siberian sturgeon (Acipenser baerii) represents a high-value fish farmed for obtaining high-priced food. In this study, the occurrence of antibiotic resistance (AR) genes encoding for resistance to ...antimicrobials administered in clinical practice, as aminoglycosides, β-lactams, macrolide-lincosamide-streptogramin B, tetracyclines, and vancomycin, were studied in the gut of sturgeons fed a diet containing 50% Hermetia illucens larvae by qPCR assays. Erm(A), erm(B), erm(C), tet(K), tet(M), tet(S), and tet(W) were detected in all samples. Feed and guts of sturgeons at beginning of rearing were negative for tet(O), vanA, vanB, mecA, and aac-aph. BlaZ was exclusively detected in control diet. The highest copy numbers were observed for tet(M), tet(K) and tet(S) in control diet. The copy numbers of most of the genes were higher in control diet than in diet containing 50% insect. Despite the differences in the gene copy numbers in control or insect-based diet, the results for AR genes contained in the guts of fish highlighted that, at 50% substitution level, no effect of the insect-based diet on the AR genes seems to occur in the experimental sturgeons at the end of rearing.
•Antibiotic resistances in sturgeons fed Hermetia illucens-based feed were studied.•The detected genes were higher in control diet than in diet containing insect.•At 50% substitution no effect of the insect diet on the AR genes was seen in sturgeons.•The most detected genes were those for resistance to tetracycline and erythromycin.
Due to the high value of Persian angelica (Heracleum persicum L.) and the limited genetic information regarding this plant, the investigation of the main secondary metabolites and corresponding ...biosynthesis pathway is of great importance. In this study, sequencing and transcriptome analysis were performed for both leaf and fruit tissues of H. persicum. Functional annotation and gene ontology analysis for 165,597 assembled transcripts was carried out. The most abundant categories belonged to proteins involved in cellular, metabolic, binding, and biosynthetic processes class. After comprehensive transcript annotation, the genes involved in the important metabolites biosynthesis of this plant were identified and the possible pathway for the biosynthesis of furanocoumarins and terpenoids were proposed. Finally, the relative expression of the genes involved in the biosynthesis of furanocoumarin metabolites as important compounds of this plant and their amount were investigated using real-time PCR and HPLC methods, respectively. In general, according to the results of the study, the expression of most genes in fruit tissue was higher than in leaf tissue. Angelicin synthase, bergaptol methyltransferase, and psoralen synthase in fruit tissue expressed 3.3, 2.4, and 1.9 times more than that in leaf tissue, respectively. The amount of bergapten, psoralen, xanthotoxol, and isopimpinellin in fruit was measured as 94.06, 4.6, 17.9, and 0.1 mg per gram of dry weight, respectively. Our findings will be valuable in designing metabolite engineering strategies based on genetic engineering and classical plant breeding with the aim of genetic manipulation and metabolite engineering in this plant.
•In this study, sequencing and transcriptome analysis of Heracleum persicum L. were performed.•Functional annotation and gene ontology analysis for 165,597 assembled transcripts was carried out.•The most abundant categories belonged to proteins involved in biosynthetic processes and metabolic activity class.•The expression of most genes in fruit tissue was higher than in leaf tissue.•The amount of bergapten, psoralen and xanthotoxol in fruit was measured as 94.06, 4.6 and 17.9 mg/ g DW, respectively.
The chip‐based digital polymerase chain reaction (PCR) is an indispensable technique for amplifying and quantifying nucleic acids, which has been widely employed in molecular diagnostics at both ...fundamental and clinical levels. However, the previous designs have yet to achieve widespread application due to limitations in complex chip fabrication, pretreatment procedures, special surface properties, and low throughput. This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Interestingly, regardless of the hydrophilicity or hydrophobicity of the inner chip surface, an efficient digitization process can be achieved. PCR reagents introduced into the inlet can be allocated to 9600 microchambers and subsequently isolated by the immiscible phase (silicone oil). The centrifugal priming approach offers a facile means to achieve high‐throughput analysis. The design was further employed for the quantification of nucleic acids using digital PCR. The calculated result exhibited a strong correlation with the measured value at the concentrations from 1 copy/μL to 1000 copies/μL (R2 = 0.99). Additionally, the chip also allowed digital multiplexed analysis, thereby indicating its potential for multi‐target detection applications.
Graphical and Lay Summary
This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Accurate, quantitative, and multiplexed digital RT‐qPCR tests can be achieved regardless of the hydrophilicity or hydrophobicity of the inner chip surface.
•SWSSs were important reservoirs for opportunistic and enteric pathogens.•SWSSs could promote the microbial regrowth and colonization of pathogens.•Potential pathogenic Legionella had a higher ...detection frequency in local SWSSs.•Water temperature and chlorine residuals drove the changes of microbiomes.
Secondary water supply systems (SWSSs) are characterized by long water stagnation and low levels of chlorine residuals, which may pose a high microbial risk to terminal users. In this study, the SWSSs of 12 residential neighborhoods in a metropolitan area of 5 million people in southeastern China were seasonally investigated to assess their microbial risks by determining more than 30 physicochemical and biological parameters. Although the microbiological quality of SWSS water met the requirements of the standards for drinking water quality of China, it did deteriorate in various aspects. The heterotrophic plate counts with R2A media were high (> 100 CFU/mL) in some SWSS tank and tap water samples. Propidium monoazide (PMA)-qPCR revealed a one magnitude higher abundance of viable bacteria in the tank and tap water samples (average 103.63±1.10 and 103.65±1.25 gene copies/mL, respectively) compared with the input water samples, and Enterococcus, Acanthamoeba, and Hartmannella vermiformis were only detected in the tanks. In particular, the high detection frequency of Legionella in 35% tank and 21% tap water samples suggested it is a supplementary microbial safety indicator in SWSSs. The microbial regrowth potential was more obvious in summer, and Illumina sequencing also demonstrated distinct seasonal changes in the relative abundance of bacterial gene sequences at the genus level. Turbidity and residual chlorine were closely connected with total bacterial biomass, and the latter seemed responsible for microbial community structure alteration. The extremely low chlorine residuals associated with a high abundance of total bacteria (as high as 106.48 gene copies/mL) and Legionella (as high as 106.71 gene copies/100 mL) in the closed valve tanks highlighted the high microbial risk increased by mishandling the operation of SWSSs. This study found that SWSSs possessed a higher microbial risk than the drinking water network, which suggested that the frequency and scope of monitoring the microbial risk of SWSSs in megacities should be strengthened for the purpose of waterborne epidemic disease prevention and control.
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Faecal pollution in aquatic environments is a worldwide public health concern, yet the reliability and comprehensiveness of the methods used to assess faecal contamination are still debated. We ...compared three approaches, namely a culture-based method to enumerate Faecal Indicator Bacteria (FIB), a FIB-targeting qPCR assay, and High-Throughput Sequencing (HTS) to detect faeces- and sewage-associated taxa in water and sediment samples of an impacted model lagoon and its adjacent sea across one year. Despite at different levels, all approaches agreed in showing a higher contamination in the lagoon than in the sea, and higher in sediments than water. FIB significantly correlated when considering separately sediment and water, and when using both cultivation and qPCR. Similarly, FIB correlated between cultivation and qPCR, but qPCR provided consistently higher estimates of FIB. Faeces-associated bacteria positively correlated with cultivated FIB in both compartments, whereas sewage-associated bacteria did only in water. Considering their benefits and limitations, we conclude that, in our study site, improved quali-quantitative information on contamination is provided when at least two approaches are combined (e.g., cultivation and qPCR or HTS data). Our results provide insights to move beyond the use of FIB to improve faecal pollution management in aquatic environments and to incorporate HTS analysis into routine monitoring.
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•Three approaches to assess faecal pollution were compared.•Each approach exhibited benefits and limitations.•Combining at least two approaches provides exhaustive information on contamination.
Ready-to-eat products, such as leafy greens, must be carefully controlled as they are directly consumed without any treatment to reduce the presence of potential pathogens. Food industries, ...especially those that process products with short shelf-life, demand rapid detection of foodborne pathogens such as Shiga Toxin-producing Escherichia coli (STEC). In this sense, molecular methods can fulfill both requirements of turnaround time and consumer safety. The most popular rapid methods are those based on real-time PCR (qPCR) however, vegetables contain inhibitory compounds that may inhibit the amplification reaction thus, there is a need for novel sample preparation protocols.
In the current study, a low-cost sample treatment based on sequential filtration steps was developed. This protocol was combined with covalent organic frameworks (COFs), and compared against a chelating resin, to evaluate their performance by multiplex qPCR targeting the major virulence genes of STEC, namely stx1, stx2, and eae, along with the rfbE for the specific identification of serogroup O157 due to its particularly high incidence, and an Internal Amplification Control to assess reaction inhibition. The optimized sample treatment effectively removed vegetable qPCR inhibitory compounds, and it was possible to detect STEC in spiked ready-to-eat salad samples in one working day, roughly 5 h, with an LOD50 of 8.7 CFU/25 g with high diagnostic sensitivity and specificity. The method was also assessed in samples with cold-stressed bacteria with good results, further demonstrating its applicability.
It was demonstrated for the first time that COFs are suitable for DNA extraction and purification. In addition to this, due to the tunable nature of these materials, it is envisioned that future modifications in terms of pore size or combination with magnetic materials, will allow to further improve their performance. In addition to this, the rapid and low-cost sample treatment protocol developed demonstrated suitable for the rapid screening of STEC vegetable samples.
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•Covalent Organic Frameworks have been applied for the first time for DNA extraction and purification.•Short pre-enrichment method allowed to detect STEC in only 5 h.•A simple filtration-based sample treatment allowed to efficiently remove vegetable tissues qPCR inhibiting compounds.•The multiplex qPCR method targeted stx1, stx2, eae, rfbE and a NC-IAC.•The LOD50 of the method was 8.7 CFU/25 g.
•The technology was tested at a site contaminated by chlorinated ethenes.•A groundwater circulation system was used for heating the aquifers.•Hydrochemical and molecular genetic methods were used to ...analyse the processes.•The application of heat and whey resulted in a fast removal of chlorinated ethenes.•Temperature alone did not create favourable conditions for dechlorination.
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In situ bioremediation (ISB) using reductive dechlorination is a widely accepted but relatively slow approach compared to other technologies for the treatment of groundwater contaminated by chlorinated ethenes (CVOCs). Due to the known positive kinetic effect on microbial metabolism, thermal enhancement may be a viable means of accelerating ISB.
We tested thermally enhanced ISB in aquifers situated in sandy saprolite and underlying fractured granite. The system comprised pumping, heating and subsequent injection of contaminated groundwater aiming at an aquifer temperature of 20–30°C. A fermentable substrate (whey) was injected in separate batches.
The test was monitored using hydrochemical and molecular tools (qPCR and NGS). The addition of the substrate and increase in temperature resulted in a rapid increase in the abundance of reductive dechlorinators (e.g., Dehalococcoides mccartyi, Dehalobacter sp. and functional genes vcrA and bvcA) and a strong increase in CVOC degradation. On day 34, the CVOC concentrations decreased by 87% to 96% in groundwater from the wells most affected by the heating and substrate. On day 103, the CVOC concentrations were below the LOQ resulting in degradation half-lives of 5 to 6days. Neither an increase in biomarkers nor a distinct decrease in the CVOC concentrations was observed in a deep well affected by the heating but not by the substrate.
NGS analysis detected Chloroflexi dechlorinating genera (Dehalogenimonas and GIF9 and MSBL5 clades) and other genera capable of anaerobic metabolic degradation of CVOCs. Of these, bacteria of the genera Acetobacterium, Desulfomonile, Geobacter, Sulfurospirillum, Methanosarcina and Methanobacterium were stimulated by the substrate and heating. In contrast, groundwater from the deep well (affected by heating only) hosted representatives of aerobic metabolic and aerobic cometabolic CVOC degraders.
The test results document that heating of the treated aquifer significantly accelerated the treatment process but only in the case of an abundant substrate.
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