µMET: A Novel Reusable Microfluidic Chip for Precision Microbial Enumeration Tests.

This work describes µMET, a novel microfluidic device for precise microbial enumeration tests (MET), essential in pharmaceutical, cosmetic, and food industries for ensuring microbiological safety standards. The µMET chip, comprising two hydrophobic glass plates, features a 15-µm deep µMET chamber enhanced by nanopillars and air supply units, facilitating both immediate and growth-dependent MET. Experimental results, with E. coli as a model bacterium, demonstrate that µMET provides counting linearity that outperforms traditional hemocytometers. The chip's design mitigates challenges like evaporation and ensures high-resolution imaging, making it a cost-effective and reusable alternative to conventional methods. Notably, bright-field µMET eliminates the need for fluorescent staining, streamlining operations with deep-learning algorithms for bacterial counts. Furthermore, we have developed a high-parallel µMET chip featuring 16 counting chambers, enhancing throughput and accommodating immediate and growth-dependent MET approaches. Its innovative design and adaptability render the µMET chip as a valuable tool for microbiology, medicine, and industry applications.

Visual whole-process monitoring of pesticide residues: An environmental perspective using surface-enhanced Raman spectroscopy with dynamic borohydride-reduced silver nanoparticles.

Environmental monitoring of pesticide residues in crops is essential for both food safety and environmental protection. Traditional methodologies face challenges due to the interference of endogenous compounds in peel and pulp tissues, often being invasive, labor-intensive, and inadequate for real-time observation of hazardous substance distribution. In this study, dynamic borohydride-reduced nanoparticles were employed as enhanced substrates. For the first time, surface-enhanced Raman spectroscopy (SERS) imaging was harnessed to enable whole-process visual detection of pesticide residues. The developed method is both stable and sensitive, boasting a detection lower limit below 1 pg/mL, coupled with robust quantitative analytical capabilities. This technique was successfully employed to detect residue signals across various crops and fruit juices. Furthermore, SERS imaging was utilized to map the distribution of pesticide residues from the exterior to the interior of fruits and vegetables. Vertex component analysis further refined the process by mitigating interference from plant autofluorescence. Collectively, this innovative strategy facilitates comprehensive pesticide residue monitoring, offering a potent tool for controlling hazardous substances in crops. Its potential applications extend beyond food safety, holding significant promise for sustainable agricultural production and enhanced environmental safeguarding.

Accelerating effects of growing-season warming on tree seasonal activities are progressively disappearing.

The phenological changes induced by climate warming have profound effects on water, energy, and carbon cycling in forest ecosystems. In addition to pre-season warming, growing-season warming may drive tree phenology by altering photosynthetic carbon uptake. It has been reported that the effect of pre-season warming on tree phenology is decreasing. However, temporal change in the effect of growing-season warming on tree phenology is not yet clear. Combining long-term ground observations and remote-sensing data, here we show that spring and autumn phenology were advanced by growing-season warming, while the accelerating effects of growing-season warming on tree phenology were progressively disappearing, manifesting as phenological events converted from being advanced to being delayed, in the temperate deciduous broadleaved forests across the Northern Hemisphere between 1983 and 2014. We further observed that the effect of growing-season warming on photosynthetic productivity showed a synchronized decline over the same period. The responses of phenology and photosynthetic productivity had a strong linear relationship with each other, and both showed significant negative correlations with the elevated temperature and vapor pressure deficit during the growing season. These findings indicate that warming-induced water stress may drive the observed decline in the responses of tree phenology to growing-season warming by decelerating photosynthetic productivity. Our results not only demonstrate a close link between photosynthetic carbon uptake and tree seasonal activities but also provide a physiological perspective of the nonlinear phenological responses to climate warming.

Multiple Metabolism Pathways of Bentazone Potentially Regulated by Metabolic Enzymes in Rice.

Bentazone (BNTZ) is a selective and efficient herbicide used in crop production worldwide. However, the persistence of BNTZ residues in the environment has led to their increasing accumulation in farmland and crops, posing a high risk to human health. To evaluate its impact on crop growth and environmental safety, a comprehensive study was conducted on BNTZ toxicity, metabolic mechanism, and resultant pathways in rice. The rice growth was compromised to the treatment with BNTZ at 0.2-0.8 mg/L (529.95-1060.05 g a.i./ha), while the activities of enzymes including SOD, POD, CAT, GST, GT, and CYP450 were elevated under BNTZ stress. A genome-wide RNA-sequencing (RNA-Seq) was performed to dissect the variation of transcriptomes and metabolic mechanisms in rice exposed to BNTZ. The degradative pathways of BNTZ in rice are involved in glycosylation, hydrolysis, acetylation, and conjugation processes catalyzed by the enzymes. Our data provided evidence that helps understand the BNTZ metabolic and detoxic mechanisms.

An ABCG-type transporter intensifies ametryn catabolism by Phase III reaction mechanism in rice.

Pesticide residues in food crops are one of the seriously environmental contaminants that risk food safety and human health. Understanding the mechanism for pesticide catabolism is critical to develop effective biotechniques for rapid eliminating the residues in food crops. In this study we characterized a novel ABC transporter family gene ABCG52 (PDR18) in regulating rice response to pesticide ametryn (AME) widely used in the farmland. Efficient biodegradation of AME was evaluated by measuring its biotoxicity, accumulation, and metabolites in rice plants. OsPDR18 was localized to the plasma membrane and strongly induced under AME exposure. Transgenic rice overexpressing OsPDR18 (OE) conferred rice resistance and detoxification to AME by increasing chlorophyll contents, improving growth phenotypes, and reducing AME accumulation in plants. The AME concentrations in OE plants were only 71.8-78.1% (shoots) and 75.0-83.3% (roots) of the wild type. Mutation of OsPDR18 by CRISPR/Cas9 protocol led to the compromised growth and enhanced AME accumulation in rice. Five AME metabolites for Phase I and 13 conjugates for Phase II reactions in rice were characterized by HPLC/Q-TOF-HRMS/MS. Relative content analysis revealed that the AME metabolic products in OE plants were significantly reduced compared with wild-type. Importantly, the OE plants accumulated less AME metabolites and conjugates in rice grains, suggesting that OsPDR18 expression may actively facilitate the transport of AME for catabolism. These data unveil a AME catabolic mechanism by which OsPDR18 contributes to the AME detoxification and degradation in rice crops.

Acetyltransferase OsACE2 acts as a regulator to reduce the environmental risk of oxyfluorfen to rice production.

The constant use of the pesticide oxyfluorfen (OFF) in farmland contaminates the soil, posing threats to crop growth and human health. To avoid the contamination of food crops with OFF, it is critically important to understand its absorption and degradation mechanisms. In this study, we characterized a new functional locus encoding an acetyltransferase (OsACE2) that can facilitate OFF degradation in rice. OsACE2 was drastically induced by OFF at 0.04-0.2 mg L-1 for 6 days and the rice growth was significantly inhibited. To demonstrate the regulatory role of OsACE2 in resistance to OFF toxicity, we generated OsACE2 overexpression (OE) and knockout mutant using genetic transformation and gene-editing technologies (CRISPR/Cas9). The OE plants grown in the hydroponic medium showed improved growth (plant elongation and biomass), increased chlorophyll content, and reduced OFF-induced oxidative stress. The OsACE2-improved growth phenotypes of rice were attributed to the significantly lower OFF accumulation in OE plants. Conversely, knocking out OsACE2 resulted in compromised growth phenotypes compared to the wild-type (WT). Using LC-LTQ-HRMS/MS, five mono-metabolites and eleven conjugates of OFF were characterized through various canonical pathways, such as hydrolysis, oxidation, reduction, glycosylation, acetylation, malonylation, and interaction with amino acids. These metabolites increased in the OE plants, and five acetylated conjugates were reported for the first time. Collectively, OsACE2 plays a primary role in catabolizing OFF residues in rice through multiple degradation pathways and reducing OFF in its growth environment.

Interpretation of ametryn biodegradation in rice based on joint analyses of transcriptome, metabolome and chemo-characterization.

Agrochemicals such as pesticide residues become environmental contaminants due to their ecotoxic risks to plant, animal and human health. Ametryn (AME) is a widely used farmland pesticide and its residues are widespread in soils, surface stream and groundwater. However, its toxicological and degradative mechanisms in plants and food crops are largely unknown. This study comprehensively investigated AME toxicology and degradation mechanisms in a paddy crop. AME was freely absorbed by rice roots, translocated to the above-ground and thus repressed plant elongation, and reduced dry weight and chlorophyll concentration, but increased oxidative injury and subcellular electrolyte permeability. Analysis of the transcriptome and metabolome revealed that exposure to AME evoked global AME-responsive genes and step-wise catabolism of AME. We detected 995 (roots) and 136 (shoots) upregulated and differentially expressed genes (DEGs) in response to AME. Metabolomic profiling revealed that many basal metabolites such as carbohydrates, amino acids, glutathione, hormones and phenylpropanoids involved in AME catabolism were accordingly accumulated in rice. Eight metabolites and twelve conjugates of AME were characterized by HPLC-Q-TOF-HRMS/MS. These AME metabolites and conjugates are closely related to DEGs, differentially accumulated metabolites (DAMs) and activities of antioxidative enzymes. Collectively, our work highlights the specific mechanisms for AME degradative metabolism through Phase I and II reactive pathways (e.g. hydroxylation and dealkylation), with will help develop genetically engineered rice used to bioremediate AME-contaminated paddy soils and minimize AME accumulation rice crops.

Online prediction of total sugar content and optimal control of glucose feed rate during chlortetracycline fermentation based on soft sensor modeling.

In the process of chlortetracycline (CTC) fermentation, no instrument can be used to measure the total sugar content of the fermentation broth online due to its high viscosity and large amount of impurities, so it is difficult to realize the optimal control of glucose feed rate in the fermentation process. In order to solve this intractable problem, the relationship between on-line measurable parameters and total sugar content (One of the parameters that are difficult to measure online) in fermentation tank is deeply analyzed, and a new soft sensor model of total sugar content in fermentation tank and a new optimal control method of glucose feed rate are proposed in this paper. By selecting measurable variables of fermentation tank, determining different fermentation stages, constructing recursive fuzzy neural network (RFNN) and applying network rolling training method, an online soft sensor model of total sugar content is established. Based on the field multi-batch data, the change trend of the amount of glucose feed required at each fermentation stage is divided, and the online prediction of total sugar content and the optimal control strategy of glucose feed rate are realized by using the inference algorithm of expert experience regulation rules and soft sensor model of total sugar content. The experiment results in the real field demonstrate that the proposed scheme can effectively predict the total sugar content of fermentation broth online, optimize the control of glucose feed rate during fermentation process, reduce production cost and meet the requirements of production technology.

Warming-induced increase in carbon uptake is linked to earlier spring phenology in temperate and boreal forests.

Under global warming, advances in spring phenology due to rising temperatures have been widely reported. However, the physiological mechanisms underlying the advancement in spring phenology still remain poorly understood. Here, we investigated the effect of temperature during the previous growing season on spring phenology of current year based on the start of season extracted from multiple long-term and large-scale phenological datasets between 1951 and 2018. Our findings indicate that warmer temperatures during previous growing season are linked to earlier spring phenology of current year in temperate and boreal forests. Correspondingly, we observed an earlier spring phenology with the increase in photosynthesis of the previous growing season. These findings suggest that the observed warming-induced earlier spring phenology is driven by increased photosynthetic carbon assimilation in the previous growing season. Therefore, the vital role of warming-induced changes in carbon assimilation should be considered to accurately project spring phenology and carbon cycling in forest ecosystems under future climate warming.

OsBR6ox, a member in the brassinosteroid synthetic pathway facilitates degradation of pesticides in rice through a specific DNA demethylation mechanism.

This manuscript described a comprehensive study on a pesticide degradation factor OsBR6ox that promoted the degradation of pesticides atrazine (ATZ) and acetochlor (ACT) in rice tissues and grains through an epigenetic mechanism. OsBR6ox was transcriptionally induced under ATZ and ACT stress. Genetic disruption of OsBR6ox increased rice sensitivity and led to more accumulation of ATZ and ACT, whereas transgenic rice overexpressing OsBR6ox lines (OEs) showed opposite effects with improved growth and lower ATZ and ACT accumulation in various tissues, including grains. OsBR6ox-mediated detoxification of ATZ and ACT was associated with the increased abundance of brassinolide (one of the brassinosteroids, BRs), a plant growth regulator for stress responses. Some Phase I-II reaction protein genes for pesticide detoxification such as genes encoding laccase, O-methyltransferase and glycosyltransferases were transcriptionally upregulated in OE lines under ATZ and ACT stress. HPLC-Q-TOF-MS/MS analysis revealed an enhanced ATZ/ATC metabolism in OE plants, which removed 1.21-1.49 fold ATZ and 1.31-1.44 fold ACT from the growth medium but accumulated only 83.1-87.1 % (shoot) and 71.7-84.1 % (root) of ATZ and 69.4-83.4 % of ACT of the wild-type. Importantly, an ATZ-responsive demethylated region in the upstream of OsBR6ox was detected. Such an epigenetic modification marker was responsible for the increased OsBR6ox expression and consequent detoxification of ATZ/ACT in rice and environment. Overall, this work uncovered a new model showing that plants utilize two mechanisms to co-regulate the detoxification and metabolism of pesticides in rice and provided a new approach for building up cleaner crops and eliminating residual pesticides in environments.

Acute Alcohol Intake Affects Internal Additive Noise and the Perceptual Template in Visual Perception.

A reduction in visual performance is among the serious consequences of acute alcohol intake. Contrast sensitivity (CS) is a fundamental feature of visual function. Here, we investigated the negative effect of a moderate dose of alcohol on CS across a wide range of spatial frequencies and with multiple levels of external noise and clarified the corresponding mechanisms in the context of a perceptual template model (PTM). To avoid the effect of alcohol washout, a quick contrast sensitivity function (qCSF) method was used to assess the visual performance of subjects before and 30 min after a moderate dose of alcohol intake. We found that (1) CS was significantly disrupted by acute alcohol intake; (2) alcohol-induced CS loss was dependent on spatial frequency and external noise; and (3) increased internal additive noise and an impaired perceptual template accounted for alcohol-induced CS loss. These results help us better understand the negative effect of alcohol consumption and provide guidance for driver safety studies.

Genome-wide identification of Oryza sativa: A new insight for advanced analysis of ABC transporter genes associated with the degradation of four pesticides.

ATP-binding cassette (ABC) transporter is a large genes superfamily. It involves transportation of diverse substrates (e.g., heavy metal, amino acids, pesticides, metabolites). The ABC transporters can be strongly induced by environmental stress and responsible for the phase III metabolic process of toxic compounds in plants. To investigate the potential molecular and biochemical function of ABC transporters in response to pesticides, we used bioinformatics and high-throughput sequencing to identify 107 loci from rice (Oryza sativa) exposed to different pesticides (ametryn, AME; bentazone, BNTZ; fomesafen, FSA; mesotrione, MTR) and annotated as ABC transporter genes. ABC transporter genes were categorized to eight subfamilies including ABCA-G and ABCI. ABCG subfamily was the largest group in rice genome followed by ABCC subfamily and ABCB subfamily. The distribution of each ABC transporter on twelve chromosomes was identified. The result showed that a large number of genes were scattered around chromosome. Differentially expressed genes (DEGs) were selected for cis-acting analysis under pesticide stress. Multiple cis-elements for biological functions such as hormone-sensitive elements and defense-related elements were found to involve the initiation and regulation of transcription. Comprehensive phylogenetic analysis and domain prediction of all ABC DEGs from rice and Arabidopsis were carried out. The docking analysis of ABC transporters and pesticides provided insights into the key amino acid residues involved in the binding of the pesticides. Consequently, the results provided applicable information and reference for a more functional analysis of ABC transporter genes on regulation of pesticide metabolism and transport in plants.

Daytime warming triggers tree growth decline in the Northern Hemisphere.

Global warming has been linked to declines in tree growth. However, it is unclear how the asymmetry in daytime and nighttime warming influences this response. Here, we use 2947 residual tree-ring width chronologies covering 32 species at 2493 sites, between 1901 and 2018, across the Northern Hemisphere, to analyze the effects of daytime and nighttime temperatures, precipitation, and drought stress on the radial growth of trees. We show that drought stress was primarily triggered by daytime rather than nighttime warming. The radial growth of trees was more sensitive to drought stress in warm regions than in cold regions, especially for angiosperms. Our study provides robust evidence that daytime warming is the primary driver of the observed declines in forest productivity related to drought stress and that daytime and nighttime warming should be considered separately when modelling forest-climate interactions and feedbacks in a future, warmer world.

Fluorescence-activated droplet sorting of PET degrading microorganisms.

Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. Still, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PET-degrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new plastic-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of degrading enzymes using synthetic biology.

Tunable and Contamination-Free Injection with Microfluidics by Stepinjection.

Droplet microfluidics with picoinjection provides significant advantages to multistep reactions and screenings. The T-junction design for picoinjection is convenient in adding picoliter reagents into passing droplets to initiate reactions. However, conventional picoinjectors face difficulties in eliminating cross-contamination between droplets, preventing them from widespread use in sensitive biological and molecular assays. Here, we introduce stepinjection, which uses a T-junction with a stepped channel design to elevate the diffusional buffer zone into the main channel and consequently increases the pressure difference between droplets and the inlet of the injection channel. To demonstrate the stepinjector's ability to perform contamination-sensitive enzymatic assays, we inject casein fluorescein isothiocyanate (FITC-casein) into a mixture of savinase and savinase-free (labeled with a red fluorescent dye) droplets. We observe no cross-contamination using stepinjection but find a severe cross-talk using an optimal picoinjection design. We envision that the simple, tunable, and reliable stepinjector can be easily integrated in various droplet processing devices, and facilitate various biomedical and biochemical applications including multiplex digital PCR, single-cell sequencing, and enzymatic screening.

Characterization of diacylglycerol acyltransferase 2 from Idesia polycarpa and function analysis.

Idesia polycarpa is an oil-producing tree native to China and Northeast Asia. The fruits of I. polycarpa which are named oil grape are unique in that they contain large amounts saturated and unsaturated lipids. Diacylglycerol acyltransferase 2 (DGAT2) is a key enzyme catalyzing the final step of triacylglyceride (TAG) synthesis. However, expression and bioinformatics of DGAT2 in I. polycarpa are still blank. In order to further understand the lipogenesis of oil grape, we contrasted seven various growth periods fruits from seed formation to seed maturation. Lipid accumulation rates and final lipid content were significantly different among the different periods. We cloned and characterized the DGAT2 gene from fruits of I. polycarpa. A partial fragment of 239 bp of IpDGAT2 was amplified by PCR. We cloned the open-reading frame (ORF) of IpDGAT2 by RACE technique. The ORF of IpDGAT2 contains 984 bp and encodes 327 amino acids. The qPCR analysis manifested that IpDGAT2 was expressed in all oil grape growing periods and expression was highest on September 20 (seed maturation). In I. polycarpa fruits the expression of IpDGAT2 was positively correlated with the lipid accumulation rates. Rhodotorula glutinis expression analysis showed that IpDGAT2 have a diacylglycerol acyltransferase bio-functional. Heterologous expression of the 35S::IpDGAT2 in Arabidopsis thaliana confirmed that the isolated IpDGAT2 could catalyze lipid synthesis. The lipid content increased by 40 % in transgenic plants relative to the control. which suggests that high lipid content fruits can be created by the overexpression of IpDGAT2 in I. polycarpa.

Dissipation and dietary risk assessment of kasugamycin and saisentong in Chinese cabbage.

A quick, easy, cheap, effective, rugged, and safe pretreatment method using dispersive solid-phase extraction was developed to quantify kasugamycin in Chinese cabbage samples by using ultra-performance liquid chromatography/tandem mass spectrometry. A pretreatment method involving precolumn transformation was utilized to determine the residue of saisentong in Chinese cabbage through high-performance liquid chromatography/ultraviolet detection. These methods were successfully applied through field trials to determine the contents of kasugamycin and saisentong in Chinese cabbage. The dissipation of kasugamycin and saisentong in Chinese cabbage followed first-order kinetics with a linear correlation coefficient of 0.9066-0.9731 at the 95% confidence level. The half-lives of kasugamycin and saisentong in Chinese cabbage were 1.8-2.0 and 2.2-3.8 days, respectively. Terminal residual levels of kasugamycin in Chinese cabbage were not detected 14 days after application. The dietary risk assessment of kasugamycin and saisentong in Chinese cabbage showed that their risk quotients were 0.93 and 2.58%, respectively, in the preharvest interval (PHI) of 14 days. Kasugamycin and saisentong in Chinese cabbage did not pose potential health hazards at PHI of 14 days. The maximum residue limits of kasugamycin and saisentong in Chinese cabbage were 0.02 and 0.36 mg/kg, respectively, and 14 days was the safe PHI.

Effects of fertilization on the triafamone photodegradation in aqueous solution: Kinetic, identification of photoproducts and degradation pathway.

Triafamone is a highly effective, low toxicity sulfonamide herbicide widely used for weeding paddy fields. The triafamone photodegradation in water environment must be explored for its ecological risk assessment. In this work, the effects of chemical fertilizer (urea, diammonium phosphate, potassium chloride, and potassium sulfate), urea metabolites (CO32- and HCO3-), and organic fertilizers (unfermented organic fertilizer [UOF] and fermented organic fertilizer [FOF]) on the triafamone photodegradation in aqueous solution under simulated sunlight were evaluated. Results showed that the triafamone photodegradation rate was unaffected by urea. The half-life of triafamone decreased from 106.8 h to 68.4 h with increasing diammonium phosphate concentration. Potassium chloride, potassium sulfate, CO32-, and HCO3- could accelerate the triafamone photodegradation at all concentrations, whereas the degradation rate of triafamone decreased when the concentration of potassium sulfate or CO32- was 2000 mg/L. Triafamone photodegradation was promoted by 20-200 mg/L UOF and FOF but decreased to 236.6 and 142.3 h when the concentration reached 2000 mg/L. Twenty-three transformation products were isolated and identified from triafamone by using ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry under simulated sunlight irradiation, and the kinetic evolution of these products was explored. Five possible degradation pathways were inferred, including the cleavage of C-N, C-C, and C-O bonds; CO bond hydrogenation; the cleavage of triazine ring; the cleavage of the sulfonamide bridge; hydroxylation; hydroxyl substitution; methylation; demethylation; amination; and rearrangement. In summary, these results are important for elucidating the environmental fate of triafamone in aquatic systems and further assessing environmental risks.

Identification and Characterization of Herbicide Penoxsulam Transformation Products in Aqueous Media by UPLC-QTOF-MS.

Photodegradation is an important non-biodegradation process of pesticide degradation in aquatic environments. In this study, the effect of different forms of nitrogen on the photodegradation kinetics of penoxsulam was investigated. The photodegradation of penoxsulam was accelerated by NO3- and NO2- but was not affected by NH4+. Ultra-high-performance liquid chromatography coupled with time-of-flight mass spectrometry was used to separate and identify the transformation products (TPs)converted by photodegradation of penoxsulam in an aqueous solution under UV-Vis (290-800 nm) irradiation. Seven major transformation products were identified based on mass spectral data. The structure was determined by elemental composition calculations, comparison of structural analogs, and existing literature. The main pathways of photodegradation were found to be sulfonamide bond cleavage, rearrangement, triazole ring cleavage, and hydroxylation. These findings are critical to elucidate the environmental fate of penoxsulam in aquatic ecosystems and provide a basis for further environmental risk assessment.