Food waste compost and digestate as novel fertilizers: Impacts on antibiotic resistome and potential risks in a soil-vegetable system.

As a novel agricultural practice, the reuse of food waste compost and digestate as fertilizers leads to a circular economy, but inevitably introduces bio-contaminants such as antibiotic resistance genes (ARGs) into the agroecosystem. Moreover, heavy metal and antibiotic contamination in farmland soil may exert selective pressures on the evolution of ARGs, posing threats to human health. This study investigated the fate, influencing mechanisms and potential risks of ARGs in a soil-vegetable system under different food waste fertilization and remediation treatments and soil contamination conditions. Application of food waste fertilizers significantly promoted the pakchoi growth, but resulted in the spread of ARGs from fertilizers to pakchoi. A total of 56, 80, 84, 41, and 73 ARGs, mobile genetic elements (MGEs) and metal resistance genes (MRGs) were detected in the rhizosphere soil (RS), bulk soil (BS), control soil (CS), root endophytes (RE), and leaf endophytes (LE), respectively. Notably, 7 genes were shared in the above five subgroups, indicating a specific soil-root-endophytes transmission pathway. 36 genes were uniquely detected in the LE, which may originate from airborne ARGs. The combined application of biochar and fertilizers reduced the occurrence of ARGs and MGEs to some extent, showing the remediation effect of biochar. The average abundance of ARGs in the RS, BS and CS was 3.15 × 10-2, 1.31 × 10-2 and 2.35 × 10-1, respectively. Rhizosphere effects may reduce the abundance of ARGs in soil. The distribution pattern of ARGs was influenced by the types of soil, endophyte and contaminant. MGEs is the key driver shaping ARGs dynamics. Soil properties and pakchoi growth status may affect the bacterial composition, and consequently regulate ARGs fate, while endophytic ARGs were more impacted by biotic factors. Moreover, the average daily doses of ARGs from pakchoi consumption is 107-109 copies/d/kg, and its potential health risks should be emphasized.

Antibiotic resistome associated with influencing factors in industrial-scale organic waste aerobic composting plants.

This study investigated the fate of antibiotic resistance genes (ARGs) and bacterial evolution in six industrial-scale organic wastes aerobic composting plants and identified key factors driving ARGs dynamics. A total of 226 ARGs and 46 mobile genetic elements (MGEs), mainly resistant to aminoglycoside and MLSB, were detected by high-throughput qPCR. Briefly, aerobic composting showed good performance in reducing the diversity and abundance of ARGs, where the total absolute abundance was reduced by 88.34%-97.08% except for cattle manures. Rapid composting may lead to a rebound of ARGs due to long-term storage compared to traditional composting. Hub ARGs and bacterial genera were screened out by co-occurrence patterns. As the dominant phyla in composting, the main potential hosts of ARGs were Firmicutes, Bacteroidota and Proteobacteria. Structural equation model indicated that MGEs and heavy metals were key factors affecting ARGs dynamics. In addition, nutrients and bacterial α-diversity can indirectly influence ARGs by affecting MGEs.

Removal behavior and key drivers of antibiotic resistance genes in two full-scale leachate treatment plants.

Leachate is a critical reservoir of antibiotic resistance genes (ARGs) and its proper treatment is closely related to human health and ecosystem safety. Here, we used high-throughput qPCR to explore the removal behavior of ARGs in two full-scale leachate treatment plants (LTPs) where biological treatment and membrane filtration processes were integrated. A total of 286 ARGs and 55 mobile genetic elements (MGEs) were detected, with aminoglycoside, multidrug and MLSB resistance genes being the most prevalent and abundant. Anaerobic digestion was found to be an important pretreatment process for leachate, while anoxic/aerobic tanks in membrane bioreactor (MBR) acted as incubators for ARGs due to their significant proliferation effect on ARGs. Integrated membrane filtration (UF-NF-RO) excelled in ARGs removal with absolute abundances reduced by 3 to 6 orders of magnitude, from about 109 copies/mL in raw leachate to 103-105 copies/mL in effluents. Our results also showed that leachate treatment processes significantly altered the composition of ARGs and bacterial communities. Procrustes analysis and network analysis revealed strong associations between microbes and ARGs, with several hub genes and bacterial genera identified. Structural equation models (SEMs) indicated that bacterial composition, MGEs and basic water properties were the key drivers shaping ARGs dynamics in the raw leachate, biological system and filtration system, respectively. Notably, several pathogens (e.g., Klebsiella, Vibrio, Aeromonas) were closely correlated with ARGs in raw leachate and may amplify the dissemination risks of ARGs. Moreover, insertion sequences in biological systems would accelerate the horizontal gene transfer of ARGs. In short, this study provides new insights into the mechanisms of ARGs removal and dissemination behavior in industrial-scale LTPs.

Dynamic distribution and driving mechanisms of antibiotic resistance genes in a human-intensive watershed.

Accelerated urbanization has promoted urban watersheds as important reservoirs of antibiotic resistance genes (ARGs); yet the biogeographical patterns and driving mechanisms of ARGs at the watershed scale remain unclear. Here, we examined the dynamic distribution of ARGs in a human-intensive watershed (including city, river and lake systems) over different seasons in a temperate region, as well as revealed the key factors shaping ARGs dynamics through structural equation models (SEMs). High diversity and abundance of ARGs were detected in sediments and surface water, with aminoglycoside, beta-lactamase and multidrug resistance genes dominating. PCoA showed distinct ARGs variations between the two phases. Seasonal changes and regional functions had significant impacts on the distribution patterns of ARGs. More diverse ARGs were detected in winter, while higher ARGs abundances were observed in spring and summer. The city system showed the highest level of ARGs contamination and was mainly derived from wastewater and human/animal feces based on SourceTracker analysis and ARGs indicators. Notably, watershed restoration could significantly mitigate the ARGs pollution status and improve biodiversity in the aquatic environment. Network analysis identified several hub ARGs and bacterial genera, which helped to infer potential bacterial hosts carrying ARGs. Furthermore, ARGs indicators provided insights to trace ARGs sources. SEMs indicated that bioavailable heavy metals and nutrients can greatly shape ARGs dynamics in regions with high-intensity human activities, while the microbial community and MGEs dominate the fate of ARGs in less human-impacted regions. More attention should be given to control heavy metals and nutrients to curb the spread of ARGs. Overall, this study highlights the environmental fate of ARGs and provides novel strategies to mitigate ARGs pollution in the human-intensive watershed.

A Balance of Yki/Sd Activator and E2F1/Sd Repressor Complexes Controls Cell Survival and Affects Organ Size.

The Hippo/Yki and RB/E2F pathways both regulate tissue growth by affecting cell proliferation and survival, but interactions between these parallel control systems are poorly defined. In this study, we demonstrate that interaction between Drosophila E2F1 and Sd disrupts Yki/Sd complex formation and thereby suppresses Yki target gene expression. RBF modifies these effects by reducing E2F1/Sd interaction. This regulation has significant effects on apoptosis, organ size, and progenitor cell proliferation. Using a combination of DamID-seq and RNA-seq, we identified a set of Yki targets that play a diversity of roles during development and are suppressed by E2F1. Further, we found that human E2F1 competes with YAP for TEAD1 binding, affecting YAP activity, indicating that this mode of cross-regulation is conserved. In sum, our study uncovers a previously unknown mechanism in which RBF and E2F1 modify Hippo signaling responses to modulate apoptosis, organ growth, and homeostasis.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration.

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair.

EGFR/Ras Signaling Controls Drosophila Intestinal Stem Cell Proliferation via Capicua-Regulated Genes.

Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.

Regional Cell Specific RNA Expression Profiling of FACS Isolated Drosophila Intestinal Cell Populations.

The adult Drosophila midgut is built of five distinct cell types, including stem cells, enteroblasts, enterocytes, enteroendocrine cells, and visceral muscles, and is divided into five major regions (R1 to R5), which are morphologically and functionally distinct from each other. This unit describes a protocol for the isolation of Drosophila intestinal cell populations for the purpose of cell type-specific transcriptome profiling from the five different regions. A method to select a cell type of interest labeled with green or yellow fluorescent protein (GFP, YFP) by making use of the GAL4-UAS bipartite system and fluorescent-activated cell sorting (FACS) is presented. Total RNA is isolated from the sorted cells of each region, and linear RNA amplification is used to obtain sufficient amounts of high-quality RNA for analysis by microarray, RT-PCR, or RNA sequencing. This method will be useful for quantitative transcriptome comparison across intestinal cell types in the different regions under normal and various experimental conditions.

Fly-FUCCI: A versatile tool for studying cell proliferation in complex tissues.

One promising approach for in vivo studies of cell proliferation is the FUCCI system (fluorescent ubiquitination-based cell cycle indicator). Here, we report the development of a Drosophila-specific FUCCI system (Fly-FUCCI) that allows one to distinguish G1, S, and G2 phases of interphase. Fly-FUCCI relies on fluorochrome-tagged degrons from the Cyclin B and E2F1 proteins, which are degraded by the ubiquitin E3-ligases APC/C and CRL4(Cdt2), during mitosis or the onset of S phase, respectively. These probes can track cell-cycle patterns in cultured Drosophila cells, eye and wing imaginal discs, salivary glands, the adult midgut, and probably other tissues. To support a broad range of experimental applications, we have generated a toolkit of transgenic Drosophila lines that express the Fly-FUCCI probes under control of the UASt, UASp, QUAS, and ubiquitin promoters. The Fly-FUCCI system should be a valuable tool for visualizing cell-cycle activity during development, tissue homeostasis, and neoplastic growth.

RNA expression profiling from FACS-isolated cells of the Drosophila intestine.

This unit describes a protocol for the isolation of Drosophila intestinal cell populations for the purpose of cell type-specific transcriptome profiling. A method to select a cell type of interest labeled with green or yellow fluorescent protein (GFP, YFP) by making use of the GAL4-UAS bipartite system and fluorescent-activated cell sorting (FACS) is presented. Total RNA is isolated from the sorted cells and linear RNA amplification is used to obtain sufficient amounts of high-quality RNA for analysis by microarray, RT-PCR, or RNA sequencing. This method will be useful for quantitative transcriptome comparison across intestinal cell types under normal and various experimental conditions.

Effects of ursolic acid on the proliferation and apoptosis of human ovarian cancer cells.

This study examined the effects of ursolic acid (UA) on the proliferation and apoptosis of a human ovarian cancer cell line, CAOV3. The CAOV3 cells were cultured in the RPMI 1640 media and treated with different concentrations of UA (0, 10, 20, 40 micromol/L). The proliferation rate of the CAOV3 cells was determined by MTT assay. The apoptosis rate was measured by flow cytometry. ERK activity was detected by immunoprecipitation and the expressions of p-ERK1/2, MKP-1, Bax and Bcl-2 by Western blotting. The results showed that the proliferation rate was significantly decreased in the cells treated with UA as compared with that in the non-treated cells (P<0.05). The intracellular ERK activity and p-ERK1/2 expression were also reduced in the UA-treated cells, while the MKP-1 expression was elevated. Moreover, the apoptosis was found in the CAOV3 cells exposed to UA; the Bax expression was increased and the Bcl-2 expression decreased. The apoptosis rate in the UA-treated cells was much higher than that in the non-treated cells (P<0.05). It is concluded that UA can inhibit the proliferation of CAOV3 cells by suppressing the ERK activity and the expression of p-ERK1/2. And it can also induce the apoptosis of the CAOV3 cells by up-regulating the Bax expression and down-regulating the Bcl-2 expression.

High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production.

Agal-fermentation-based microbio-diesel production was realized through high-cell-density fermentation of Chlorella protothecoides and efficient transesterification process. Cell density achieved was 16.8 g l(-1) in 184 h and 51.2 g l(-1) in 167 h in a 5-l bioreactor by performing preliminary and improved fed-batch culture strategy, respectively. The lipid content was 57.8, 55.2, and 50.3% of cell dry weight from batch, primary, and improved fed-batch culture in 5-l bioreactor. Transesterification was catalyzed by immobilized lipase, and the conversion rate reached up to 98%. The properties of biodiesel from Chlorella were comparable to conventional diesel fuel and comply with US standard for Biodiesel. In a word, the approach including high-density fermentation of Chlorella and enzymatic transesterification process were set up and proved to be a promising alternative for biodiesel production.