In Situ Remediation of TNT Red Water Contaminated Soil: Field Demonstration

In China, about 1.5 × 105 m3 soil was contaminated by TNT red water, which contained mainly dinitrotoluene sulfonates (DNTS). These nitroaromatic explosives are toxic and exhibit human mutagenic and carcinogenic potential. Biotechnology capable of decontaminating these contaminated sites and applicable to a larger scale field application was in urgent need. A two-year pilot study was conducted in the TNT red water contaminated sites (1000 m3). The sites were treated by integrated treatment technologies of “Desorption-Biostimulation and Bioaugmentation-Phytoremediation.” Soil samples were taken every six months to determine the concentration of nitro-aromatic compounds. Acute toxicity and Fluorescein diacetate (FDA) hydrolytic activity tests were conducted to evaluate the remediation effect. After 2 years of remediation, the total nitroaromatic compounds were effectively removed, with the average removal efficiency of 99.88% and 90.47% in sites planted with alfalfa and reed, respectively. The toxicity was significantly reduced. The average luminescence inhibition ratio reduced from 92.64% to 3.37% and 29.16%, respectively. The soil microbial activity was significantly improved, with the highest FDA hydrolase activity in the surface layer. The remediation costs for the treatment of 1 cubic meter of contaminated soil were estimated to approximately $126 US. The integrated technologies used have huge potential for decontaminating munitions contaminated sites.</p

Table_1_Application of third-generation sequencing to herbal genomics.docx

There is a long history of traditional medicine use. However, little genetic information is available for the plants used in traditional medicine, which limits the exploitation of these natural resources. Third-generation sequencing (TGS) techniques have made it possible to gather invaluable genetic information and develop herbal genomics. In this review, we introduce two main TGS techniques, PacBio SMRT technology and Oxford Nanopore technology, and compare the two techniques against Illumina, the predominant next-generation sequencing technique. In addition, we summarize the nuclear and organelle genome assemblies of commonly used medicinal plants, choose several examples from genomics, transcriptomics, and molecular identification studies to dissect the specific processes and summarize the advantages and disadvantages of the two TGS techniques when applied to medicinal organisms. Finally, we describe how we expect that TGS techniques will be widely utilized to assemble telomere-to-telomere (T2T) genomes and in epigenomics research involving medicinal plants.</p

DataSheet1_TaqMan Probe-Based Quantitative Real-Time PCR to Detect Panax notoginseng in Traditional Chinese Patent Medicines.docx

Background: There has been global concern about the safety and accuracy of traditional Chinese patent medicines (TCPMs). Panax notoginseng, also known as sanqi, is an important constituent of TCPMs. However, identifying the species contained in TCPMs is challenging due to the presence of multiple ingredients and the use of various preparation processes.Objective: To detect P. notoginseng in TCPMs.Methods: A TaqMan probe-based qPCR assay was constructed and validated with DNA extracted from P. notoginseng and adulterants. In total, 75 samples derived from 25 batches of TCPMs were tested using the constructed qPCR method.Results: A TaqMan probe-based qPCR assay targeting P. notoginseng was established. The constructed qPCR assay could specifically discriminate P. notoginseng from Panax ginseng, Panax quinquefolium and Curcuma aromatica Salisb. cv. Wenyujin. The sensitivity study showed that the detectable DNA template concentration of P. notoginseng for this qPCR assay was 0.001 ng/μl. All 75 samples from TCPMs were confirmed to contain P. notoginseng by the qPCR assay.Conclusions: The qPCR method can accurately identify P. notoginseng in TCPMs and is promising as a powerful tool for quality control and market regulation.</p

“Surface Browsing” May Allow “Filter-Feeding” Protozoa to Exert Top-Down Control on Colony-Forming Toxic Cyanobacterial Blooms

Blooms of the cyanobacterium Microcystis threaten aquatic ecosystems. Protozoa grazing can control unicellular Microcystis populations; however, Microcystis blooms are composed of multicellular colonies that are thought to prevent grazing. We show that this is not so: the model ciliate Paramecium has an impact on Microcystis populations through grazing, even when large colonies occur, and this leads to a corresponding decrease in toxic microcystins. Notably, as the number of large colonies increased, Paramecium exerted top-down control by altering its feeding behavior: once the colony size was >12–20 μm, Paramecium no longer acted as a “filter feeder”; instead, it became a “surface browser,” grazing around and between larger colonies, removing individual Microcystis and small colonies. However, as the proportion of large colonies increased, exponentially reducing the surface area to volume ratio, the impact of Paramecium decreased exponentially. This study provides new insights into how protozoa may affect Microcystis populations through top-down control of blooms

“Surface Browsing” May Allow “Filter-Feeding” Protozoa to Exert Top-Down Control on Colony-Forming Toxic Cyanobacterial Blooms

Blooms of the cyanobacterium Microcystis threaten aquatic ecosystems. Protozoa grazing can control unicellular Microcystis populations; however, Microcystis blooms are composed of multicellular colonies that are thought to prevent grazing. We show that this is not so: the model ciliate Paramecium has an impact on Microcystis populations through grazing, even when large colonies occur, and this leads to a corresponding decrease in toxic microcystins. Notably, as the number of large colonies increased, Paramecium exerted top-down control by altering its feeding behavior: once the colony size was >12–20 μm, Paramecium no longer acted as a “filter feeder”; instead, it became a “surface browser,” grazing around and between larger colonies, removing individual Microcystis and small colonies. However, as the proportion of large colonies increased, exponentially reducing the surface area to volume ratio, the impact of Paramecium decreased exponentially. This study provides new insights into how protozoa may affect Microcystis populations through top-down control of blooms

“Surface Browsing” May Allow “Filter-Feeding” Protozoa to Exert Top-Down Control on Colony-Forming Toxic Cyanobacterial Blooms

Blooms of the cyanobacterium Microcystis threaten aquatic ecosystems. Protozoa grazing can control unicellular Microcystis populations; however, Microcystis blooms are composed of multicellular colonies that are thought to prevent grazing. We show that this is not so: the model ciliate Paramecium has an impact on Microcystis populations through grazing, even when large colonies occur, and this leads to a corresponding decrease in toxic microcystins. Notably, as the number of large colonies increased, Paramecium exerted top-down control by altering its feeding behavior: once the colony size was >12–20 μm, Paramecium no longer acted as a “filter feeder”; instead, it became a “surface browser,” grazing around and between larger colonies, removing individual Microcystis and small colonies. However, as the proportion of large colonies increased, exponentially reducing the surface area to volume ratio, the impact of Paramecium decreased exponentially. This study provides new insights into how protozoa may affect Microcystis populations through top-down control of blooms

Data_Sheet_1_Analysis of Whole-Genome facilitates rapid and precise identification of fungal species.docx

Fungal identification is a cornerstone of fungal research, yet traditional molecular methods struggle with rapid and accurate onsite identification, especially for closely related species. To tackle this challenge, we introduce a universal identification method called Analysis of whole GEnome (AGE). AGE includes two key steps: bioinformatics analysis and experimental practice. Bioinformatics analysis screens candidate target sequences named Targets within the genome of the fungal species and determines specific Targets by comparing them with the genomes of other species. Then, experimental practice using sequencing or non-sequencing technologies would confirm the results of bioinformatics analysis. Accordingly, AGE obtained more than 1,000,000 qualified Targets for each of the 13 fungal species within the phyla Ascomycota and Basidiomycota. Next, the sequencing and genome editing system validated the ultra-specific performance of the specific Targets; especially noteworthy is the first-time demonstration of the identification potential of sequences from unannotated genomic regions. Furthermore, by combining rapid isothermal amplification and phosphorothioate-modified primers with the option of an instrument-free visual fluorescence method, AGE can achieve qualitative species identification within 30 min using a single-tube test. More importantly, AGE holds significant potential for identifying closely related species and differentiating traditional Chinese medicines from their adulterants, especially in the precise detection of contaminants. In summary, AGE opens the door for the development of whole-genome-based fungal species identification while also providing guidance for its application in plant and animal kingdoms.</p

“Surface Browsing” May Allow “Filter-Feeding” Protozoa to Exert Top-Down Control on Colony-Forming Toxic Cyanobacterial Blooms

Blooms of the cyanobacterium Microcystis threaten aquatic ecosystems. Protozoa grazing can control unicellular Microcystis populations; however, Microcystis blooms are composed of multicellular colonies that are thought to prevent grazing. We show that this is not so: the model ciliate Paramecium has an impact on Microcystis populations through grazing, even when large colonies occur, and this leads to a corresponding decrease in toxic microcystins. Notably, as the number of large colonies increased, Paramecium exerted top-down control by altering its feeding behavior: once the colony size was >12–20 μm, Paramecium no longer acted as a “filter feeder”; instead, it became a “surface browser,” grazing around and between larger colonies, removing individual Microcystis and small colonies. However, as the proportion of large colonies increased, exponentially reducing the surface area to volume ratio, the impact of Paramecium decreased exponentially. This study provides new insights into how protozoa may affect Microcystis populations through top-down control of blooms

Data_Sheet_1_Molecular markers associated with drug resistance in Plasmodium falciparum parasites in central Africa between 2016 and 2021.DOCX

ObjectivesThe widespread occurrence of anti-malarial drug resistance threatens the current efforts to control malaria in African regions. Molecular marker surveillance helps to track the emergence and spread of drug-resistant malaria cases.MethodsA total of 237 Plasmodium falciparum infections imported from central Africa to Zhejiang Province, China, between 2016 and 2021, were investigated. Genomic DNA was extracted from blood samples of each patient and nested PCRs was used to detect molecular markers in k13, Pfcrt, and Pfmdr1 genes. The spatial and temporal distributions of the molecular markers were analyzed.ResultsA limited polymorphism of k13 was observed, including two nonsynonymous (D464E and K503E) and five synonymous mutations. Wild-type CVMNK of Pfcrt predominated (78.5%), whereas 19.5% of the samples harbored the mutant haplotype, CVIET. The point mutation Y184F and the single mutant haplotype NF of Pfmdr1 were the most frequently observed. The geographical distributions of the Pfcrt and Pfmdr1 haplotypes displayed distinct patterns, with the mutant haplotype of Pfcrt more common in Gabon (53.9%) and Congo (50.0%), and wild haplotypes of Pfmdr1 more frequently found in Cameroon, Angola, and Congo. The prevalence of wild-type CVMNK of Pfcrt increased from 68.5–74.6% in 2016–2017 to 81.8–87.5% in 2018–2021. The proportion of wild-type Pfmdr1 also increased from 27.1% in 2016 to 38.5% in 2019.ConclusionThe geographical and temporal distribution of k13, Pfcrt, and Pfmdr1 polymorphisms in P. falciparum parasites imported from central Africa between 2016 and 2021 are demonstrated. Our data provide updated evidence that can be used to adjust anti-malarial drug policies in central Africa and China.</p

Data_Sheet_1_Identification of closely related species in Aspergillus through Analysis of Whole-Genome.docx

The challenge of discriminating closely related species persists, notably within clinical diagnostic laboratories for invasive aspergillosis (IA)-related species and food contamination microorganisms with toxin-producing potential. We employed Analysis of the whole-GEnome (AGE) to address the challenges of closely related species within the genus Aspergillus and developed a rapid detection method. First, reliable whole genome data for 77 Aspergillus species were downloaded from the database, and through bioinformatic analysis, specific targets for each species were identified. Subsequently, sequencing was employed to validate these specific targets. Additionally, we developed an on-site detection method targeting a specific target using a genome editing system. Our results indicate that AGE has successfully achieved reliable identification of all IA-related species (Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans, Aspergillus flavus, and Aspergillus terreus) and three well-known species (A. flavus, Aspergillus parasiticus, and Aspergillus oryzae) within the Aspergillus section. Flavi and AGE have provided species-level-specific targets for 77 species within the genus Aspergillus. Based on these reference targets, the sequencing results targeting specific targets substantiate the efficacy of distinguishing the focal species from its closely related species. Notably, the amalgamation of room-temperature amplification and genome editing techniques demonstrates the capacity for rapid and accurate identification of genomic DNA samples at a concentration as low as 0.1 ng/μl within a concise 30-min timeframe. Importantly, this methodology circumvents the reliance on large specialized instrumentation by presenting a singular tube operational modality and allowing for visualized result assessment. These advancements aptly meet the exigencies of on-site detection requirements for the specified species, facilitating prompt diagnosis and food quality monitoring. Moreover, as an identification method based on species-specific genomic sequences, AGE shows promising potential as an effective tool for epidemiological research and species classification.</p