High-risk ARGs (HRA) Chip: A high-throughput qPCR-based array for assessment of high-risk ARGs from the environment.

The global surge in antibiotic resistance genes (ARGs) presents a serious public health challenge. While methods like metagenomic analysis and qPCR arrays have been instrumental in investigating ARG distributions and dynamics, the vast diversity of ARGs often complicates effective monitoring and risk assessment. Here, we developed a High-Risk ARGs (HRA) chip based on high-capacity quantitative PCR array targeting previously identified high-risk ARGs. These ARGs are known to be prevalent in human-related environments, exhibit gene mobility, and are present in ESKAPE pathogens. The HRA chip include 101 primer sets and the 16S rRNA gene as a reference. These primer sets consist of 34 obtained from previous studies, and 67 newly designed primer sets which were validated in silico and experimentally. Absolute quantification of targeted ARGs is accomplished by generating standard curves for all ARGs with serially ten-fold diluted mixed plasmids containing targeted ARG sequences. The amplification efficiencies of all ARGs exceed 99% via plasmid template dilution tests, suggesting high reliability in quantification. The performance of HRA chip is further evaluated by practical applications in environmental samples from wastewater treatment plants (WWTPs) and soils with various land use types and fertilization regimes. The results indicate the dynamics of high-risk ARGs during wastewater treatment process, and reveal the profiles of soil high-risk ARGs which is distinct from those derived via metagenomic approaches. These findings highlight the potentials of HRA Chip in the evaluation of anthropogenic impacts on the environmental resistome with a more focused spectrum of high-risk ARGs. Overall, the HRA Chip emerges as a powerful and efficient high-throughput tool for rapid detection and quantification of high-risk ARGs, facilitating comprehensive profiling of high-risk resistomes in environmental samples which is essential for human health risk assessment of ARGs.

Local environment, surface characteristics and stochastic processes shape the dynamics of urban dustbin surface microbiome.

Dustbins function as critical infrastructures for urban sanitation, creating a distinct breeding ground for microbial assemblages. However, there is no information regarding the dynamics of microbial communities and the underlying mechanism for community assembly on dustbin surfaces. Here, surface samples were collected from three sampling zones (business building, commercial street and residential community) with different types (kitchen waste, harmful waste, recyclables, and others) and materials (metallic and plastic); and distribution pattern and assembly of microbial communities were investigated by high-throughput sequencing. Bacterial and fungal communities showed the distinct community variations across sampling zones and waste sorting. Core community and biomarker species were significantly correlated with the spatial distribution of overall community. The detection of pathogens highlighted the potential risk of surface microbiome. Human skin, human feces and soil biomes were the potential source environments of the surface microbiomes. Neutral model prediction suggested that microbial community assembly was significantly driven by stochastic processes. Co-association patterns varied with sampling zones and waste types, and neutral amplicon sequence variants (ASVs) that fall within the 95 % confidence intervals of neutral model were largely involved in the stability of microbial networks. These findings improve our understanding of the distribution pattern and the underlying assembly of microbial community on the dustbin surface, thus enabling prospective prediction and assessment of urban microbiomes and their impacts on human health.

Dynamics of Microbial Community and Potential Microbial Pollutants in Shopping Malls.

Shopping malls offer various niches for microbial populations, potentially serving as sources and reservoirs for the spread of microorganisms of public health concern. However, knowledge about the microbiome and the distribution of human pathogens in malls is largely unknown. Here, we examine the microbial community dynamics and genotypes of potential pathogens from floor and escalator surfaces in shopping malls and adjacent road dusts and greenbelt soils. The distribution pattern of microbial communities is driven primarily by habitats and seasons. A significant enrichment of human-associated microbiota in the indoor environment indicates that human interactions with surfaces might be another strong driver for mall microbiomes. Neutral community models suggest that the microbial community assembly is strongly driven by stochastic processes. Distinct performances of microbial taxonomic signatures for environmental classifications indicate the consistent differences of microbial communities of different seasons/habitats and the strong anthropogenic effect on homogenizing microbial communities of shopping malls. Indoor environments harbored higher concentrations of human pathogens than outdoor samples, also carrying a high proportion of antimicrobial resistance-associated multidrug efflux genes and virulence genes. These findings enhanced the understanding of the microbiome in the built environment and the interactions between humans and the built environment, providing a basis for tracking biothreats and communicable diseases and developing sophisticated early warning systems. IMPORTANCE Shopping malls are distinct microbial environments which can facilitate a constant transmission of microorganisms of public health concern between humans and the built environment or between human and human. Despite extensive investigation of the natural environmental microbiome, no comprehensive profile of microbial ecology has been reported in malls. Characterizing microbial distribution, potential pathogens, and antimicrobial resistance will enhance our understanding of how these microbial communities are formed, maintained, and transferred and help establish a baseline for biosurveillance of potential public health threats in malls.

Uncovering the diversity and contents of gene cassettes in class 1 integrons from the endophytes of raw vegetables.

Rapid spread of antibiotic resistance genes (ARGs) in pathogens is threatening human health. Integrons allow bacteria to integrate and express foreign genes, facilitating horizontal transfer of ARGs in environments. Consumption of raw vegetables represents a pathway for human exposure to environmental ARGs. However, few studies have focused on integron-associated ARGs in the endophytes of raw vegetables. Here, based on the approach of qPCR and clone library, we quantified the abundance of integrase genes and analyzed the diversity and contents of resistance gene cassettes in class 1 integrons from the endophytes of six common raw vegetables. The results revealed that integrase genes for class 1 integron were most prevalent compared with class 2 and class 3 integron integrase genes (1-2 order magnitude, P < 0.05). The cucumber endophytes harbored a higher absolute abundance of integrase genes than other vegetables, while the highest bacterial abundance was detected in cabbage and cucumber endophytes. Thirty-two unique resistance gene cassettes were detected, the majority of which were associated with the genes encoding resistance to beta-lactam and aminoglycoside. Antibiotic resistance gene cassettes accounted for 52.5 % of the functionally annotated gene cassettes, and blaTEM-157 and aadA2 were the most frequently detected resistance cassettes. Additionally, carrot endophytes harbored the highest proportion of antibiotic resistance gene cassettes in the class 1 integrons. Collectively, these results provide an in-depth view of acquired resistance genes by integrons in the raw vegetable endophytes and highlight the potential health risk of the transmission of ARGs via the food chain.

Nanopore sequencing analysis of integron gene cassettes in sewages and soils.

Integrons are genetic elements that can facilitate rapid spread of antibiotic resistance by insertion and removal of genes. However, knowledge about the diversity and distribution of gene cassettes embedded in class 1 integron is still limited. In this study, we sequenced integron gene cassettes using nanopore sequencing and quantified antibiotic resistance genes (ARGs) and integrase genes in the manured soils and sewages of a bioreactor. The results showed that class 1 integron integrase genes were the most abundant in soils and sewages compared with class 2 and class 3 integrase genes. Long-term manure application exacerbated the enrichment of total ARGs, integrase genes and antibiotic resistance-associated gene cassettes, while antibiotics and heavy metals showed no impact on the overall resistome profile. Sewage treatment could efficiently remove the absolute abundance of integrase genes (~3 orders of magnitude, copies/L) and antibiotic resistance gene cassettes. The resistance gene cassettes mainly carried the ARGs conferring resistance to aminoglycoside and beta-lactams in soils and sewages, some of which were persistent during the sewage treatment. This study underlined that soil and sewage were potential reservoirs for integron-mediated ARGs transfer, indicating that anthropogenic activity played a vital role in the prevalence and diversity of resistance gene cassettes in integrons.

Harnessing the hidden genetic diversity for improving multiple abiotic stress tolerance in rice (Oryza sativa L.).

To develop superior rice varieties with improved yield in most rainfed areas of Asia/Africa, we started an introgression-breeding program for simultaneously improving yield and tolerances of multiple abiotic stresses. Using eight BC1 populations derived from a widely adaptable recipient and eight donors plus three rounds of phenotypic selection, we developed 496 introgression lines (ILs) with significantly higher yield under drought, salt and/or non-stress conditions in 5 years. Six new varieties were released in the Philippines and Pakistan and many more are being evaluated in multi-location yield trials for releasing in several countries. Marker-facilitated genetic characterization revealed three interesting aspects of the breeding procedure: (1) the donor introgression pattern in specific BC populations was characteristic; (2) introgression frequency in different genomic regions varied considerably, resulting primarily from strong selection for the target traits; and (3) significantly lower heterozygosity was observed in BC progenies selected for drought and salinity tolerance. Applying strong phenotypic selection under abiotic stresses in early segregating generations has major advantages for not only improving multiple abiotic stress tolerance but also achieving quicker homozygosity in early generations. This breeding procedure can be easily adopted by small breeding programs in developing countries to develop high-yielding varieties tolerant of abiotic stresses. The large set of trait-specific ILs can be used for genetic mapping of genes/QTL that affect target and non-target traits and for efficient varietal development by designed QTL pyramiding and genomics-based recurrent selection in our Green Super Rice breeding technology.

MTSS1 overexpression correlates with poor prognosis in colorectal cancer.

BACKGROUND: The aims of this study were to investigate metastasis suppressor 1 (MTSS1) expression in benign and malignant colorectal tissues and to explore its significance in the prognosis of colorectal cancer (CRC) patients. METHODS: MTSS1 expression was detected by immunohistochemistry in CRC, colorectal adenomatous polyp (precancerous lesion) and normal colorectal tissues. The relationship between MTSS1 expression in CRC tissues and clinicopathologic factors was analyzed with Mann-Whitney U test. MTSS1 protein expression was observed by Western blot in CRC tissues and adjacent nontumor colorectal tissues. Two factors between MTSS1 expression and CRC patient tumor node metastasis (TNM) stage were analyzed by Spearman rank correlation analysis. The Kaplan-Meier method and log-rank test were employed to compare the overall survival between MTSS1 negative/weak positive expression group and MTSS1 strong positive expression group. RESULTS: MTSS1 expression rates were significantly higher in CRC tissues (99 out of 135, 73.30%) than that in normal colorectal tissues (one out of seven, 14.29%), nontumor colorectal tissues (six out of 32, 18.75%), and adenomatous polyp tissues (four out of 15, 26.67%; P = 0.003, P < 0.001, P = 0.001, respectively). The upregulated MTSS1 expression in CRC tissues was significantly correlated to poor differentiation (P = 0.005), tissue invasion (P = 0.018), high preoperative CEA level (P = 0.022), present lymph node metastasis (P = 0.003), and high TNM stage (P = 0.002). MTSS1 expression was positively correlated with clinical TNM stage, that suggested the more advanced clinical TNM stage corresponding to the higher expression level of MTSS1 (r(s) = 0.327, P < 0.05). Western blotting demonstrated that MTSS1 expression was upregulated in 25 of 32 CRC tissues (75.0%) compared to corresponding adjacent nontumor colorectal tissues. The overall 5-year survival of MTSS1 strong positive expression CRC patients was significantly shorter than that of MTSS1 negative and weakly positive expression group. In multivariate analysis, MTSS1 expression maintained independent prognostic influence on overall survival (P = 0.004). CONCLUSION: MTSS1 may be a good biomarker to be applied in the clinical setting to predict the prognosis of CRC patients with completely resected.

Genome-wide gene responses in a transgenic rice line carrying the maize resistance gene Rxo1 to the rice bacterial streak pathogen, Xanthomonas oryzae pv. oryzicola.

BACKGROUND: Non-host resistance in rice to its bacterial pathogen, Xanthomonas oryzae pv. oryzicola (Xoc), mediated by a maize NBS-LRR type R gene, Rxo1 shows a typical hypersensitive reaction (HR) phenotype, but the molecular mechanism(s) underlying this type of non-host resistance remain largely unknown. RESULTS: A microarray experiment was performed to reveal the molecular mechanisms underlying HR of rice to Xoc mediated by Rxo1 using a pair of transgenic and non-transgenic rice lines. Our results indicated that Rxo1 appeared to function in the very early step of the interaction between rice and Xoc, and could specifically activate large numbers of genes involved in signaling pathways leading to HR and some basal defensive pathways such as SA and ET pathways. In the former case, Rxo1 appeared to differ from the typical host R genes in that it could lead to HR without activating NDR1. In the latter cases, Rxo1 was able to induce a unique group of WRKY TF genes and a large set of genes encoding PPR and RRM proteins that share the same G-box in their promoter regions with possible functions in post-transcriptional regulation. CONCLUSIONS: In conclusion, Rxo1, like most host R genes, was able to trigger HR against Xoc in the heterologous rice plants by activating multiple defensive pathways related to HR, providing useful information on the evolution of plant resistance genes. Maize non-host resistance gene Rxo1 could trigger the pathogen-specific HR in heterologous rice, and ultimately leading to a localized programmed cell death which exhibits the characteristics consistent with those mediated by host resistance genes, but a number of genes encoding pentatricopeptide repeat and RNA recognition motif protein were found specifically up-regulated in the Rxo1 mediated disease resistance. These results add to our understanding the evolution of plant resistance genes.

[Expressions of RASSF1A, Galectin-3 and TPO mRNA in papillary thyroid carcinoma and their clinical significance].

OBJECTIVE: To investigate the mRNA expressions of RASSF1A, Galectin-3 and TPO in papillary thyroid carcinoma and some other thyroid benign lesions, and evaluate their diagnostic significance. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of RASSF1A, galectin-3 and TPO in the samples from 73 cases, including 23 cases with papillary thyroid cancer, 16 with nodular goiter, 29 with thyroid adenoma and 5 with Hashimoto's disease. RESULTS: A statistically significant difference in the mRNA expression of RASSF1A, Galectin-3 and TPO was observed between papillary thyroid carcinoma and follicular benign lesions (P<0.05). However, there was no significant difference among various kinds of benign lesions (P>0.05). A negative correlation of the expression of RASSF1A and Galectin-3 mRNA was found between thyroid benign lesions and malignant ones (P = 0.000). While the mRNA expression of RASSF1A and TPO was positively correlated between benign and malignant lesions (P = 0.028). CONCLUSION: Loss of expression of RASSF1A and TPO mRNA but high expression of Galectin-3 mRNA in papillary thyroid carcinoma are common. Therefore, the products of these three genes may be closely related to the development of thyroid papillary carcinoma, and may be used as useful markers in differential diagnosis of papillary thyroid carcinoma from the benign lesions. The results are more reliable if this detection method is used in combination with other techniques.