High-Throughput µPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.

The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (µPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

TiO2 nanorod arrays@PDA/Ag with biomimetic polydopamine as binary mediators for duplex SERS detection of illegal food dyes.

Based on TiO2 nanorod arrays@PDA/Ag (TNRs@PDA/Ag), a better surface-enhanced Raman scattering (SERS) sensor with effective enrichment and enhancement was investigated for duplex SERS detection of illicit food dyes. Biomimetic PDA functions as binary mediators by utilizing the structural characteristics of polydopamine (PDA), which include the conjugated structure and abundant hydrophilic groups. One PDA functioned as an electron transfer mediator to enhance the efficiency of electron transfer, and the other as an enrichment mediator to effectively enrich rhodamine B (RhB) and crystal violet (CV) through hydrogen bonding, π-π stacking, and electrostatic interactions. Individual and duplex detection of illicit food dyes (RhB and CV) was performed using TNRs@PDA/Ag to estimate SERS applications. Their linear equations and limits of detection of 1 nM for RhB and 5 nM for CV were derived. Individual and duplex food colour detection was successfully accomplished even in genuine chili meal with good results. The bifunctional TNRs@PDA/Ag-based highly sensitive and duplex SERS dye detection will have enormous potential for food safety monitoring.

Effects of selenium on biogeochemical cycles of cadmium in rice from flooded paddy soil systems in the alluvial Indus Valley of Pakistan.

This study delves into the pollution status, assesses the effects of Se on Cd biogeochemical pathways, and explores their interactions in nutrient-rich paddy soil-rice ecosystems through 500 soil-rice samples in Pakistan. The results showed that 99.6 % and 12.8 % of soil samples exceeded the World Health Organization (WHO) allowable Se and Cd levels (7 and 0.35 mg/kg). In comparison, 23 % and 6 % of the grain samples exceeded WHO's allowable Se and Cd levels (0.3 and 0.2 mg/kg), respectively. Geographically Weighted Regression (GWR) model results further revealed spatial nonstationarity, confirming diverse associations between dependent variables (Se and Cd in rice grain) and independent variables from paddy soil and plant tissues (root and shoot), such as Soil Organic Matter (SOM), pH, Se, and Cd concentrations. High Se:Cd molar ratios (>1) and a negative correlation (r = -0.16, p < 0.01) between the Cd translocation factor (Cd in rice grain/Cd in root) and Se in roots suggest that increased root Se levels inhibit the transfer of Cd from roots to grains. The inverse correlation between Se and Cd in paddy grains was further characterized as Se deficiency, no risk, high Cd risk, Se risk, Cd risk, and Se-Cd co-exposure risk. There was no apparent risk for human co-consumption in 42.6 % of grain samples with moderate Se and low Cd. The remaining categories indicate differing degrees of risk. In the study area, 31 % and 20 % of grain samples with low Se and Cd indicate Se deficiency and risk, respectively. High Se and low Cd levels in rice samples suggest a potential hazard for severe Se exposure due to frequent rice consumption. This study not only systematically evaluates the pollution status of paddy-soil systems in Pakistan but also provides a reference to thoroughly contemplate the development of a scientific approach for evaluating human risks and the potential dangers associated with paddy soils and rice, specifically in regions characterized by low Se and low Cd concentrations, as well as those with moderate Se and high Cd concentrations. SYNOPSIS: This study is significant for understanding the effects of Se on Cd geochemical cycles and their interactions in paddy soil systems in Pakistan.

Agronomic and Genetic Strategies to Enhance Selenium Accumulation in Crops and Their Influence on Quality.

Selenium (Se) is an essential trace element that plays a crucial role in maintaining the health of humans, animals, and certain plants. It is extensively present throughout the Earth's crust and is absorbed by crops in the form of selenates and selenite, eventually entering the food chain. Se biofortification is an agricultural process that employs agronomic and genetic strategies. Its goal is to enhance the mechanisms of crop uptake and the accumulation of exogenous Se, resulting in the production of crops enriched with Se. This process ultimately contributes to promoting human health. Agronomic strategies in Se biofortification aim to enhance the availability of exogenous Se in crops. Concurrently, genetic strategies focus on improving a crop's capacity to uptake, transport, and accumulate Se. Early research primarily concentrated on optimizing Se biofortification methods, improving Se fertilizer efficiency, and enhancing Se content in crops. In recent years, there has been a growing realization that Se can effectively enhance crop growth and increase crop yield, thereby contributing to alleviating food shortages. Additionally, Se has been found to promote the accumulation of macro-nutrients, antioxidants, and beneficial mineral elements in crops. The supplementation of Se biofortified foods is gradually emerging as an effective approach for promoting human dietary health and alleviating hidden hunger. Therefore, in this paper, we provide a comprehensive summary of the Se biofortification conducted over the past decade, mainly focusing on Se accumulation in crops and its impact on crop quality. We discuss various Se biofortification strategies, with an emphasis on the impact of Se fertilizer strategies on crop Se accumulation and their underlying mechanisms. Furthermore, we highlight Se's role in enhancing crop quality and offer perspective on Se biofortification in crop improvement, guiding future mechanistic explorations and applications of Se biofortification.

Revealing the developmental characterization of rumen microbiome and its host in newly received cattle during receiving period contributes to formulating precise nutritional strategies.

BACKGROUND: Minimizing mortality losses due to multiple stress and obtaining maximum performance are the production goals for newly received cattle. In recent years, vaccination and metaphylaxis treatment significantly decreased the mortality rate of newly received cattle, while the growth block induced by treatment is still obvious. Assessment of blood metabolites and behavior monitoring offer potential for early identification of morbid animals. Moreover, the ruminal microorganisms' homeostasis is a guarantee of beef steers' growth and health. The most critical period for newly received cattle is the first-month post-transport. Therefore, analyzing rumen metagenomics, rumen metabolomics, host metabolomics, and their interaction during receiving period (1 day before transport and at days 1/4, 16, and 30 after transport) is key to revealing the mechanism of growth retardation, and then to formulating management and nutritional practices for newly received cattle. RESULTS: The levels of serum hormones (COR and ACTH), and pro-inflammatory factors (IL-1ß, TNF-α, and IL-6) were highest at day 16, and lowest at day 30 after arrival. Meanwhile, the antioxidant capacity (SOD, GSH-Px, and T-AOC) was significantly decreased at day 16 and increased at day 30 after arrival. Metagenomics analysis revealed that rumen microbes, bacteria, archaea, and eukaryota had different trends among the four different time points. At day 16 post-transport, cattle had a higher abundance of ruminal bacteria and archaea than those before transport, but the eukaryote abundance was highest at day 30 post-transport. Before transport, most bacteria were mainly involved in polysaccharides digestion. At day 4 post-transport, the most significantly enriched KEGG pathways were nucleotide metabolism (pyrimidine metabolism and purine metabolism). At day 16 post-transport, the energy metabolism (glycolysis/gluconeogenesis, pyruvate metabolism) and ruminal contents of MCP and VFAs were significantly increased, but at the same time, energy loss induced by methane yields (Methanobrevibacter) together with pathogenic bacteria (Saccharopolyspora rectivirgula) were also significantly increased. At this time, the most upregulated ruminal L-ornithine produces more catabolite polyamines, which cause oxidative stress to rumen microbes and their host; the most downregulated ruminal 2',3'-cAMP provided favorable growth conditions for pathogenic bacteria, and the downregulated ruminal vitamin B6 metabolism and serum PC/LysoPC disrupt immune function and inflammation reaction. At day 30 post-transport, the ruminal L-ornithine and its catabolites (mainly spermidine and 1,3-propanediamine) were decreased, and the serum PC/LysoPC and 2',3'-cNMPs pools were increased. This is also consistent with the changes in redox, inflammation, and immune status of the host. CONCLUSIONS: This study provides new ideas for regulating the health and performance of newly received cattle during the receiving period. The key point is to manage the newly received cattle about day 16 post-transport, specifically to inhibit the production of methane and polyamines, and the reproduction of harmful bacteria in the rumen, therefore improving the immunity and performance of newly received cattle. Video Abstract.

Coupling of different antioxidative systems in rice under the simultaneous influence of selenium and cadmium.

Selenium (Se) elevates the antioxidant ability of rice against cadmium (Cd) stress, but previous studies only focused on the variation in antioxidant enzymes or nonenzymatic substances induced by Se under Cd stress and ignored the relationships between different antioxidant parameters during the interaction. Here, hydroponic experiments with rice were performed by adding both Cd and Se at doses in the range of 0-50 µM to explore the physiological responses of rice and their relationships in the presence of different levels of Se and Cd. Exogenous Cd markedly promoted the activity of antioxidant enzymes with the exception of catalase (CAT) and the concentration of nonenzymatic substances in aerial parts. Se enhanced the antioxidant capacity by improving the activities of all the enzymes tested in this study and increasing the concentrations of nonenzymatic compounds. The couplings among different antioxidant substances within paddy rice were then determined based on cluster and linear fitting results and their metabolic process and physiological functions. The findings specifically highlight that couplings among the ascorbic acid (AsA)-glutathione (GSH) cycle, glutathione synthase (GS)-phytochelatin synthetase (PCS) coupling system and glutathione peroxidase (GPX)-superoxide dismutase (SOD) coupling system in aerial parts helps protect plants from Cd stress. These coupling systems form likely due to the fact that one enzyme generated a product that could be the substrate for another enzyme. Noticeably, such coupling systems do not emerge in roots because the stronger damage to roots than other organs activates the ascorbate peroxidase (APX)-GPX-CAT and PCS-GS-SOD systems with distinct functions and structures. This study provides new insights into the detoxification mechanisms of rice caused by the combined effect of Se and Cd.

Transcriptomic and Functional Analyses of Two Cadmium Hyper-Enriched Duckweed Strains Reveal Putative Cadmium Tolerance Mechanisms.

Cadmium (Cd) is one of the most toxic metals in the environment and exerts deleterious effects on plant growth and production. Duckweed has been reported as a promising candidate for Cd phytoremediation. In this study, the growth, Cd enrichment, and antioxidant enzyme activity of duckweed were investigated. We found that both high-Cd-tolerance duckweed (HCD) and low-Cd-tolerance duckweed (LCD) strains exposed to Cd were hyper-enriched with Cd. To further explore the underlying molecular mechanisms, a genome-wide transcriptome analysis was performed. The results showed that the growth rate, chlorophyll content, and antioxidant enzyme activities of duckweed were significantly affected by Cd stress and differed between the two strains. In the genome-wide transcriptome analysis, the RNA-seq library generated 544,347,670 clean reads, and 1608 and 2045 differentially expressed genes were identified between HCD and LCD, respectively. The antioxidant system was significantly expressed during ribosomal biosynthesis in HCD but not in LCD. Fatty acid metabolism and ethanol production were significantly increased in LCD. Alpha-linolenic acid metabolism likely plays an important role in Cd detoxification in duckweed. These findings contribute to the understanding of Cd tolerance mechanisms in hyperaccumulator plants and lay the foundation for future phytoremediation studies.

Physiological response, microbial diversity characterization, and endophytic bacteria isolation of duckweed under cadmium stress.

Duckweed is a cadmium (Cd) hyperaccumulator. However, its enrichment characteristics and physiological responses to Cd have not been systematically studied. The physiological responses, enrichment characteristics, diversity of endophytic bacterial communities, and isolation of Cd-resistant endophytes in duckweed (Lemna minor 0014) were studied for different durations and Cd concentrations. The results indicated that peroxidase (POD) and catalase (CAT) activities decreased while superoxide dismutase activity first increased and then decreased with increasing Cd stress duration. POD activities, CAT activities, and O2- increased as Cd concentrations increased. Malondialdehyde content and Cd accumulation in duckweed increased with increasing concentrations and time. This endophytic diversity study identified 488 operational taxonomic units, with the dominant groups being Proteobacteria, Firmicutes, and Actinobacteria. Paenibacillus sp. Y11, a strain tolerant to high concentrations of Cd and capable of significantly promoting duckweed growth, was isolated from the plant. Our study revealed the effects of heavy metals on aquatic plants, providing a theoretical basis for the application of duckweed in water pollution.

Impacts of household PM2.5 pollution on blood pressure of rural residents: Implication for clean energy transition.

High blood pressure associated with PM2.5 exposure is of great concern, especially for rural residents exposed to high PM2.5 levels. However, the impact of short-term exposure to high PM2.5 on blood pressure (BP) has not been well elucidated. Thus, this study aims to focus on the association between short-term PM2.5 exposure with BP of rural residents and its variation between summer and winter. Our results showed that the summertime PM2.5 exposure concentration was 49.3 ± 20.6 µg/m3, among which, mosquito coil users had 1.5-folds higher PM2.5 exposure than non-mosquito coil users (63.6 ± 21.7 vs 43.0 ± 16.7 µg/m3, p < 0.05). The mean systolic and diastolic BP (SBP and DBP, respectively) of rural participants were 122 ± 18.2 and 76.2 ± 11.2 mmHg in summer, respectively. The PM2.5 exposure, SBP, and DBP in summer were 70.7 µg/m3, 9.0 mmHg, and 2.8 mmHg lower than that in winter, respectively. Furthermore, the correlation between PM2.5 exposure and SBP was stronger in winter than that in summer, possibly due to higher PM2.5 exposure levels in winter. The transition of household energy from solid fuels in winter to clean fuels in summer would be benefit to the decline of PM2.5 exposure as well as BP. Results from this study suggested that the reduction of PM2.5 exposure would have positive effect on human health.

Wind-dispersed seeds blur phylogeographic breaks: The complex evolutionary history of Populus lasiocarpa around the Sichuan Basin.

The strength of phylogeographic breaks can vary among species in the same area despite being subject to the same geological and climate history due to differences in biological traits. Several important phylogeographic breaks exist around the Sichuan Basin in Southwest China but few studies have focused on wind-dispersed plants. Here, we investigated the phylogeographic patterns and the evolutionary history of Populus lasiocarpa, a wind-pollinated and wind-dispersed tree species with a circum-Sichuan Basin distribution in southwest China. We sequenced and analyzed three plastid DNA fragments (ptDNA) and eight nuclear microsatellites (nSSRs) of 265 individuals of P. lasiocarpa from 21 populations spanning the entire distribution range. Distribution patterns based on nSSR data revealed that there are three genetic groups in P. lasiocarpa. This is consistent with the three phylogeographic breaks (Sichuan Basin, the Kaiyong Line and the 105°E line), where the Sichuan basin acts as the main barrier to gene flow between western and eastern groups. However, the distribution pattern based on ptDNA haplotypes poorly matched the phylogeographic breaks, and wind-dispersed seeds may be one of the main contributing factors. Species distribution modelling suggested a larger potential distribution in the last glacial maximum with a severe bottleneck during the last interglacial. A DIYABC model also suggested a population contraction and expansion for both western and eastern lineages. These results indicate that biological traits are likely to affect the evolutionary history of plants, and that nuclear molecular markers, which experience higher levels of gene flow, might be better indicators of phylogeographic breaks.

Quantifying the dynamic characteristics of indoor air pollution using real-time sensors: Current status and future implication.

People generally spend most of their time indoors, making indoor air quality be of great significance to human health. Large spatiotemporal heterogeneity of indoor air pollution can be hardly captured by conventional filter-based monitoring but real-time monitoring. Real-time monitoring is conducive to change air assessment mode from static and sparse analysis to dynamic and massive analysis, and has made remarkable strides in indoor air evaluation. In this review, the state of art, strengths, challenges, and further development of real-time sensors used in indoor air evaluation are focused on. Researches using real-time sensors for indoor air evaluation have increased rapidly since 2018, and are mainly conducted in China and the USA, with the most frequently investigated air pollutants of PM2.5. In addition to high spatiotemporal resolution, real-time sensors for indoor air evaluation have prominent advantages in 3-dimensional monitoring, pollution peak and source identification, and short-term health effect evaluation. Huge amounts of data from real-time sensors also facilitate the modeling and prediction of indoor air pollution. However, challenges still remain in extensive deployment of real-time sensors indoors, including the selection, performance, stability, as well as calibration of sensors. In future, sensors with high performance, long-term stability, low price, and low energy consumption are welcomed. Furthermore, more target air pollutants are also expected to be detected simultaneously by real-time sensors in indoor air monitoring.

Chromosome-level de novo genome assembly of two conifer-parasitic wasps, Megastigmus duclouxiana and Megastigmus sabinae, reveals genomic imprints of adaptation to hosts.

Conifers make up about one third of global forests but are threatened by seed parasitoid wasp species. Many of these wasps belong to the genus Megastigmus, yet little is known about their genomic background. In this study, we provide chromosome-level genome assemblies for two oligophagous conifer parasitoid species of Megastigmus, which represent the first two chromosome-level genomes of the genus. The assembled genomes of Megastigmus duclouxiana and M. sabinae are 878.48 Mb (scaffold N50 of 215.60 Mb) and 812.98 Mb (scaffold N50 of 139.16 Mb), respectively, which are larger than the genome size of most hymenopterans due to the expansion of transposable elements. Expanded gene families highlight the difference in sensory-related genes between the two species, reflecting the difference in their hosts. We further found that these two species have fewer family members but more single-gene duplications than polyphagous congeners in the gene families of ATP-binding cassette transporter (ABC), cytochrome P450 (P450) and olfactory receptors (OR). These findings shed light on the pattern of adaptation to a narrow spectrum of hosts in oligophagous parasitoids. Our findings suggest potential drivers underlying genome evolution and parasitism adaptation, and provide valuable resources for understanding the ecology, genetics and evolution of Megastigmus, as well as for the research and biological control of global conifer forest pests.

Binary Double Network-like Structure: An Effective Energy-Dissipation System for Strong Tough Hydrogel Design.

Currently, hydrogels simultaneously featuring high strength, high toughness, superior recoverability, and benign anti-fatigue properties have demonstrated great application potential in broad fields; thus, great efforts have been made by researchers to develop satisfactory hydrogels. Inspired by the double network (DN)-like theory, we previously reported a novel high-strength/high-toughness hydrogel which had two consecutive energy-dissipation systems, namely, the unzipping of coordinate bonds and the dissociation of the crystalline network. However, this structural design greatly damaged its stretchability, toughness recoverability, shape recoverability, and anti-fatigue capability. Thus, we realized that a soft/ductile matrix is indispensable for an advanced strong tough hydrogel. On basis of our previous work, we herein reported a modified energy-dissipation model, namely, a "binary DN-like structure" for strong tough hydrogel design for the first time. This structural model comprises three interpenetrated polymer networks: a covalent/ionic dually crosslinked tightened polymer network (stiff, first order network), a constrictive crystalline polymer network (sub-stiff, second order network), and a ductile/flexible polymer network (soft, third order network). We hypothesized that under low tension, the first order network served as the sacrificing phase through decoordination of ionic crosslinks, while the second order and third order networks together functioned as the elastic matrix phase; under high tension, the second order network worked as the energy dissipation phase (ionic crosslinks have been destroyed at the time), while the third order network played the role of the elastic matrix phase. Owing to the "binary DN-like" structure, the as-prepared hydrogel, in principle, should demonstrate enhanced energy dissipation capability, toughness/shape recoverability, and anti-fatigue/anti-tearing capability. Finally, through a series of characterizations, the unique "binary DN-like" structure was proved to fit well with our initial theoretical assumption. Moreover, compared to other energy-dissipation models, this structural design showed a significant advantage regarding comprehensive properties. Therefore, we think this design philosophy would inspire the development of advanced strong tough hydrogel in the future.

Microbial and metabolomic insights into the bovine lipometabolic responses of rumen and mammary gland to zymolytic small peptide supplementation.

Small peptides provide the easily utilized nitrogen for rumen microbial and promote acetate generation for milk fat synthesis. However, the impacts of peptide supplements on lipometabolic processes were still unclear. Therefore, a total of 800 multiparous dairy herds (with an average live weight of 667.6 ± 39.4 kg, an average lactation of 89.3 ± 18.8 days, and an average calving parity of 2.76 ± 0.47) were randomly allocated to the control (CON) and the small peptide (SP) supplement (100 g/day for each cow) treatments, respectively. A 35-day-long feeding procedure that includes a 7-day-long pretreatment test and a 28-day-long treatment test was followed for all cows. Dry matter intake (DMI) was recorded every day and calculated by the deviation between the supply and residue, while the daily milk production was automatically recorded through the rotary milking facilities. Milk samples were collected from each replicate on the last day, followed by the milk quality and milk lipid composition measurement. Rumen fluid samples were collected on the last day through esophageal tubing 3 h after morning feeding for the determination of the underlying mechanism of the small peptide on lipid metabolism through the measurement of rumen lipometabolic-related metabolites and rumen bacterial communities. Results indicated that dry matter intake showed an increasing trend, while milk production and the milk fat content remarkably increased after SP supplement (P < 0.05). Further detailed detection showed the mainly increased milk composition focused on monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA). Acetate-producing microbes, such as Acetitomaculum, Bifidobacterium, Succiniclasticum, and Succinivibrio, and butyrate-producing microbes, such as Shuttleworthia and Saccharofermentans, significantly proliferated, which causatively brought the increased ruminal content of acetate, isobutyrate, and butyrate after SP supplement (P < 0.05) compared with CON. Lipometabolic metabolites such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), triacylglycerol (TG), and Acetyl-CoA also significantly increased after SP supplement. In summary, SP supplements help to increase milk fat content through the proliferation of rumen bacterial communities, which provided more acetate and butyrate for milk fat synthesis combined with the promotion of ruminal lipometabolism.

Recent advances on phylogenomics of gymnosperms and a new classification.

Living gymnosperms comprise four major groups: cycads, Ginkgo, conifers, and gnetophytes. Relationships among/within these lineages have not been fully resolved. Next generation sequencing has made available a large number of sequences, including both plastomes and single-copy nuclear genes, for reconstruction of solid phylogenetic trees. Recent advances in gymnosperm phylogenomic studies have updated our knowledge of gymnosperm systematics. Here, we review major advances of gymnosperm phylogeny over the past 10 years and propose an updated classification of extant gymnosperms. This new classification includes three classes (Cycadopsida, Ginkgoopsida, and Pinopsida), five subclasses (Cycadidae, Ginkgoidae, Cupressidae, Pinidae, and Gnetidae), eight orders (Cycadales, Ginkgoales, Araucariales, Cupressales, Pinales, Ephedrales, Gnetales, and Welwitschiales), 13 families, and 86 genera. We also described six new tribes including Acmopyleae Y. Yang, Austrocedreae Y. Yang, Chamaecyparideae Y. Yang, Microcachrydeae Y. Yang, Papuacedreae Y. Yang, and Prumnopityeae Y. Yang, and made 27 new combinations in the genus Sabina.

Reproduction and genetic diversity of Juniperus squamata along an elevational gradient in the Hengduan Mountains.

Elevation plays a crucial factor in the distribution of plants, as environmental conditions become increasingly harsh at higher elevations. Previous studies have mainly focused on the effects of large-scale elevational gradients on plants, with little attention on the impact of smaller-scale gradients. In this study we used 14 microsatellite loci to survey the genetic structure of 332 Juniperus squamata plants along elevation gradient from two sites in the Hengduan Mountains. We found that the genetic structure (single, clonal, mosaic) of J. squamata shrubs is affected by differences in elevational gradients of only 150 m. Shrubs in the mid-elevation plots rarely have a clonal or mosaic structure compared to shrubs in lower- or higher-elevation plots. Human activity can significantly affect genetic structure, as well as reproductive strategy and genetic diversity. Sub-populations at mid-elevations had the highest yield of seed cones, lower levels of asexual reproduction and higher levels of genetic diversity. This may be due to the trade-off between elevational stress and anthropogenic disturbance at mid-elevation since there is greater elevational stress at higher-elevations and greater intensity of anthropogenic disturbance at lower-elevations. Our findings provide new insights into the finer scale genetic structure of alpine shrubs, which may improve the conservation and management of shrublands, a major vegetation type on the Hengduan Mountains and the Qinghai-Tibet Plateau.

Paper Device Combining CRISPR/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2 Detection in Wastewater.

Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.

Optimization of Vocal Singing Training Methods Using Intelligent Big Data Technology.

The development of art education and information technology has led to the importance of computer technology and multimedia technology in the development of students' independent inquiry and research skills. In the context of "Internet+," new modes of teaching phonics have emerged, reconfiguring the spatial and temporal relationship of phonics education. The use of Internet resources is not only a simple collection and sharing of educational resources, but also a new way of teaching voice, which has the magic charm of becoming one of the resources for the majority of voice enthusiasts. However, in practice, there are very few assistive software suitable for music classroom teaching. It is important to research and implement teaching aids suitable for music classroom teaching. Based on intelligent big data technology to optimize the phonetic training methods, the teaching methods are more specific, scientific, and diverse, and improve the self-learning ability and learning interest of Chinese phonetic learners. The experimental results show that the weight of the phonetic teaching optimization process is 0.154 higher than the weight before processing, which is expressed as the value of the control reliability fuzzy quantifier in this test. In other words, the reliability is "absolutely reliable." Therefore, this study is expected to promote the modernization and scientific process of Chinese vocal education and propose a new way of thinking for Chinese vocal education.

Recent progress in Fenton/Fenton-like reactions for the removal of antibiotics in aqueous environments.

The frequent use of antibiotics allows them to enter aqueous environments via wastewater, and many types of antibiotics accumulate in the environment due to difficult degradation, causing a threat to environmental health. It is crucial to adopt effective technical means to remove antibiotics in aqueous environments. The Fenton reaction, as an effective organic pollution treatment technology, is particularly suitable for the treatment of antibiotics, and at present, it is one of the most promising advanced oxidation technologies. Specifically, rapid Fenton oxidation, which features high removal efficiency, thorough reactions, negligible secondary pollution, etc., has led to many studies on using the Fenton reaction to degrade antibiotics. This paper summarizes recent progress on the removal of antibiotics in aqueous environments by Fenton and Fenton-like reactions. First, the applications of various Fenton and Fenton-like oxidation technologies to the removal of antibiotics are summarized; then, the advantages and disadvantages of these technologies are further summarized. Compared with Fenton oxidation, Fenton-like oxidations exhibit milder reaction conditions, wider application ranges, great reduction in economic costs, and great improved cycle times, in addition to simple and easy recycling of the catalyst. Finally, based on the above analysis, we discuss the potential for the removal of antibiotics under different application scenarios. This review will enable the selection of a suitable Fenton system to treat antibiotics according to practical conditions and will also aid the development of more advanced Fenton technologies for removing antibiotics and other organic pollutants.

Rumen Microbial Metabolic Responses of Dairy Cows to the Honeycomb Flavonoids Supplement Under Heat-Stress Conditions.

Flavonoids played critical roles in stabilizing microbial homoeostasis when animals suffered exoteric stresses. However, whether flavonoids attenuated heat stress of dairy cows is still not clear. Therefore, in the present article, flavonoids extracted from honeycomb were supplemented to investigate the production, digestibility, and rumen microbial metabolism responses of cows under heat stress conditions. A total of 600 multiparous dairy herds were randomly allotted into the control treatment (CON), the heat stress (HS) treatment, and the honeycomb flavonoids supplement under heat stress conditions (HF) treatment for a 30-day-long trial. Each treatment contains 4 replicates, with 50 cows in each replicate. Production performances including dry matter intake (DMI), milk production, and milk quality were measured on the basis of replicate. Furthermore, two cows of each replicate were selected for the measurement of the nutrient digestibility, the ruminal fermentable parameters including ruminal pH, volatile fatty acids, and ammonia-N, and the rumen microbial communities and metabolism. Results showed that HF effectively increased DMI, milk yield, milk fat, and ruminal acetate content (p < 0.05) compared with HS. Likewise, digestibility of NDF was promoted after HF supplement compared with HS. Furthermore, relative abundances of rumen microbial diversities especially Succiniclasticum, Pseudobutyrivibrio, Acetitomaculum, Streptococcus, and Succinivibrio, which mainly participated in energy metabolism, significantly improved after HF supplement. Metabolomic investigation showed that HF supplement significantly upregulated relative content of lipometabolic-related metabolites such as phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine, while it downregulated biogenic amines. In summary, HF supplement helps proliferate microbial abundances, which further promoted fiber digestibility and energy provision, and ultimately enhances the production performances of dairy cows under heat stress conditions.