High-Throughput μPAD with Cascade Signal Amplification through Dual Enzymes for <i>arsM</i> 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

DataSheet1_Nonnegligible pathogenic exposure risk of coarse part of PM10 in non-open environments.docx

In non-open environments, pathogenic microorganisms are more likely to invade the human respiratory tract due to their limited diffusion in the environment, which has received little attention. In this study, we explored the distribution characteristics of particulate matter (PM) in non-open environments, and included sewage treatment plants and farms, which are occupational exposure risks, and G-series high-speed trains and waiting rooms, which are crowded. The results showed orders of magnitude differences in PM and microbial concentrations and the DNA/PM values of adsorption in the different non-open spaces. The concentration of PM with a size in the 4.7–10.0 μm range was higher than those of PM in the 1.1–4.7 μm and 0.43–1.1 μm ranges in all three types of places, accounting for 74.64%, 46.59%, and 51.49%, respectively. The DNA/PM value for the 1.1–4.7 μm range was higher than those for PM in the other two ranges in all three types of places at 0.175, 3.78 × 10−3, and 9.98 ng/μg, respectively. Although the relative abundances of Class II potentially pathogenic bacteria with sizes ranging from 1.1 to 4.7 μm were higher in all three types of places, the total abundance and the relative abundance of identified pathogenic microorganisms with sizes ranging from 4.7 to 10.0 μm were higher in all three types of places. Here, in non-open spaces, the pathogen exposure risk associated with PM10, particularly the coarse fraction of PM10, deserves special attention. Infectious diseases caused by aerosol transmission of pathogens in non-open environments should receive more attention and require further investigation in the future.</p

Table_2_Microbial and metabolomic insights into the bovine lipometabolic responses of rumen and mammary gland to zymolytic small peptide supplementation.XLSX

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.</p

Table_3_Microbial and metabolomic insights into the bovine lipometabolic responses of rumen and mammary gland to zymolytic small peptide supplementation.XLSX

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.</p

Table_1_Microbial and metabolomic insights into the bovine lipometabolic responses of rumen and mammary gland to zymolytic small peptide supplementation.XLSX

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.</p

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

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

Additional file 1 of Revealing the developmental characterization of rumen microbiome and its host in newly received cattle during receiving period contributes to formulating precise nutritional strategies

Additional file 1: Table S1. Composition and nutrient levels of experimental diet (air-dry basis, %)

Table_2_Rumen Microbial Metabolic Responses of Dairy Cows to the Honeycomb Flavonoids Supplement Under Heat-Stress Conditions.XLSX

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.</p