Model of shrimp pond-mediated spatiotemporal dynamic distribution of antibiotic resistance genes in the mangrove habitat of a subtropical gulf.

Aquacultures are the main reason for the environmental selection of antibiotic resistance genes (ARGs), resulting in the enrichment of ARGs. As a filter, a marine mangrove ecosystem can reduce antimicrobial resistance (AMR) or eliminate ARGs; however, its elimination mechanism remains unclear. This study investigated the spatiotemporal dynamic distribution of ARGs in two different types of mangrove habitats (shrimp ponds and virgin forests), within a subtropical gulf located in the Beibu Gulf, China, during dry and wet seasons by using metagenomics and real time quantitative polymerase chain reaction (RT-qPCR) analysis. As the key environmental factors, sulfide, salinity, and mobile genetic elements significantly were found to contribute to ARGs distribution, respectively. Wet and dry seasons influenced the dispersal of ARGs but did not affect the microbial community structure. Three potential biomarkers, TEM-116, smeD, and smeE, played key roles in seasonal differences. The key different genes in the biological relevance of absolute abundance were demonstrated by RT-qPCR. Co-occurrence network analysis indicated that high-abundance ARGs were distributed in a modular manner. For the first time, a risk index weighted by risk rank (RIR) was proposed and used to quantify the human risk of ARGs in the mangrove metagenome. The shrimp ponds during the wet season showed the highest RIR detected. In addition to offering a perspective on reducing AMR in mangrove wetlands, this study constructed the first spatiotemporal dynamic model of ARGs in the Beibu Gulf, China and contributed to revealing the global spread of ARGs. Meanwhile, this study proposes a new pipeline for assessing the risk of ARGs, while also exploring the concept of "One Health."

Toll-like receptor may be involved in acquired immune response in pearl oyster Pinctada fucata.

The increasing experimental evidence suggests that there are some forms of specific acquired immunity in invertebrates, in which Toll-like receptors (TLRs) play vital roles in activating innate and adaptive immunity and have been comprehensively investigated in mammalian species. Yet, the immune mechanisms underlying TLR mediation in mollusks remain obscure. In this study, we identified a TLR13 gene in the pearl oyster Pinctada fucata for the first time and named it PfTLR13 which consists of a 5'-untranslated terminal region (5'-UTR) of 543 bp, an open reading frame (ORF) of 2667 bp, and a 3'-UTR of 729 bp. We found that PfTLR13 mRNA was expressed in all tissues examined, with the highest level in the gills. The expression of PfTLR13 in the gills of oysters exposed to Vibrio alginolyticus or pathogen-associated molecular patterns (PAMPs) (including LPS, PGN, and poly(I:C)) was significantly higher than in the control group. Interestingly, the immune response to the first stimulation was weaker than the response to the second stimulation, suggesting that the primary stimulation may lead to immune priming of TLR in pearl oysters, similar to acquired immunity in vertebrates. Furthermore, we found that PfTLR13 expression was differentially associated with allograft and xenograft in the pearl oyster P. fucata, with the highest expression levels observed at 12 h post-allograft and 24 h post-xenograft. Overall, our findings provide new insights into the immune mechanisms underlying TLR mediation in mollusks and suggest that PfTLR13 may play a crucial role in the specific acquired immunity of pearl oysters.

Melatonin attenuates lipopolysaccharide-induced immune dysfunction in dendritic cells.

Melatonin, a ubiquitous hormone, is principally secreted from pineal gland in mammals and possesses strong antioxidant and anti-inflammatory properties. However, its specific roles in the immune functions of dendritic cells (DCs) during acute lung injury (ALI) remain unknown. In this study, we found that melatonin restored the body weight, decreased the lung weight/body weight ratio, alleviated the histopathological lung injury, and decreased the levels of cytokines (tumor necrosis factor-α (TNF-α), interleukin (IL)-12p70, IL-17, and IL-10) in bronchoalveolar lavage fluid of the lipopolysaccharide (LPS)-induced ALI murine model. Moreover, melatonin inhibited the major histocompatibility complex II (MHCII) expression of lung CD11b+ DCs after LPS challenge in vivo. In vitro, melatonin reversed the shape index, promoted the endocytosis, and inhibited phenotypic expression of MHCII, CD40, CD80, and CD86 in LPS-activated DCs. Furthermore, melatonin decreased the expression of an activated marker, CD69, and the secretion of pro-inflammatory cytokines (TNF-α, IL-12p70, and IL-17) after LPS challenge. It hampered the LPS-activated DCs migration by downregulating the C-C chemokine receptor 7 (CCR7) expression, and then weakened the ability of LPS-induced DCs to stimulate allogeneic CD4+ T cell proliferation. Melatonin shaped the immune function of DCs in a nuclear factor erythroid-2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) axis-dependent manner. These findings indicate that melatonin protects DCs from ALI-induced immunological stress and may be used to develop novel DC-targeting strategies for ALI therapy.

Sulfur metabolism in subtropical marine mangrove sediments fundamentally differs from other habitats as revealed by SMDB.

Shotgun metagenome sequencing provides the opportunity to recover underexplored rare populations and identify difficult-to-elucidate biochemical pathways. However, information on sulfur genes, including their sequences, is scattered in public databases. Here, we introduce SMDB ( https://smdb.gxu.edu.cn/ )-a manually curated database of sulfur genes based on an in-depth review of the scientific literature and orthology database. The SMDB contained a total of 175 genes and covered 11 sulfur metabolism processes with 395,737 representative sequences affiliated with 110 phyla and 2340 genera of bacteria/archaea. The SMDB was applied to characterize the sulfur cycle from five habitats and compared the microbial diversity of mangrove sediments with that of other habitats. The structure and composition of microorganism communities and sulfur genes were significantly different among the five habitats. Our results show that microorganism alpha diversity in mangrove sediments was significantly higher than in other habitats. Genes involved in dissimilatory sulfate reduction were abundant in subtropical marine mangroves and deep-sea sediments. The neutral community model results showed that microbial dispersal was higher in the marine mangrove ecosystem than in others habitats. The Flavilitoribacter of sulfur-metabolizing microorganism becomes a reliable biomarker in the five habitats. SMDB will assist researchers to analyze genes of sulfur cycle from the metagenomic efficiently.

Melatonin Suppresses LPS-Induced Oxidative Stress in Dendritic Cells for Inflammatory Regulation via the Nrf2/HO-1 Axis.

Melatonin, an indoleamine synthesized in the pineal gland of mammals, is a natural bioactive compound with powerful antioxidant and anti-inflammatory properties. Here, we evaluated whether melatonin has the capacity to moderate the oxidative stress of dendritic cells (DCs) for inflammatory control in an acute lung injury (ALI) model. Our findings showed that melatonin remarkably inhibited total nitric oxide synthase (T-NOS) activity, nitric oxide (NO) production, intracellular reactive oxygen species (ROS) levels, and lipid peroxidation (MDA detection) levels in both an LPS-induced murine ALI model and LPS-induced DCs. Meanwhile, the reduced glutathione (GSH) level and the GSH/GSSG ratio were recovered. In addition, antioxidant enzymes, such as glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), were increased in these processes. Moreover, melatonin also inhibited the LPS-induced secretions of IL-1ß, IL-6, and TGF-ß in vivo and in vitro. Finally, we found that the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) axis was required in the inhibition of LPS-induced oxidative stress in DCs by melatonin. Altogether, these data indicate that melatonin strongly suppresses the LPS-induced oxidative stress in DCs, which is a promising DC-targeted strategy via inflammatory control for ALI treatment.

Identification and Characterization of miRNAs and Their Predicted mRNAs in the Larval Development of Pearl Oyster Pinctada fucata.

As an important economic shellfish, the pearl oyster, Pinctada fucata, and its larvae are an ideal model for studying molecular mechanisms of larval development in invertebrates. Larval development directly affects the quantity and quality of pearl oysters. MicroRNAs (miRNAs) may play important roles in development, but the effects of miRNA expression on P. fucata early development remain unknown. In this study, miRNA and mRNA transcriptomics of seven different P. fucata developmental stages were analyzed using Illumina RNA sequencing. A total of 329 miRNAs, including 87 known miRNAs and 242 novel miRNAs, and 33,550 unigenes, including 26,333 known genes and 7217 predicted new genes, were identified in these stages. A cluster analysis showed that the difference in the numbers of miRNAs was greatest between fertilized eggs and trochophores. In addition, the integrated mRNA transcriptome was used to predict target genes for differentially expressed miRNAs between adjacent developmental stages, and the target genes were subjected to a gene ontology enrichment analysis. Using the gene ontology annotation, 100 different expressed genes and 95 differentially expressed miRNAs were identified as part of larval development regulation. Real-time PCR was used to identify eight mRNAs and three miRNAs related to larval development. The present findings will be helpful for further clarifying the regulatory mechanisms of miRNA in invertebrate larval development.

Real-world PM2.5 exposure induces pathological injury and DNA damage associated with miRNAs and DNA methylation alteration in rat lungs.

Fine particulate matter (PM2.5) has been demonstrated to threaten public health and increase lung cancer risk. DNA damage is involved in the pathogenesis of lung cancer. However, the mechanisms of epigenetic modification of lung DNA damage are still unclear. This study developed a real-world air PM2.5 inhalation system and exposed rats for 1 and 2 months, respectively, and investigated rat lungs pathological changes, inflammation, oxidative stress, and DNA damage effects. OGG1 and MTH1 expression was measured, along with their DNA methylation status and related miRNAs expression. The results showed that PM2.5 exposure led to pathological injury, influenced levels of inflammatory cytokines and oxidative stress factors in rat lungs. Of note, 2-month PM2.5 exposure aggravated pathological injury. Besides, PM2.5 significantly elevated OGG1 expression and suppressed MTH1 expression, which was correlated to oxidative stress and partially mediated by reducing OGG1 DNA methylation status and increasing miRNAs expression related to MTH1 in DNA damage with increases of γ-H2AX, 8-OHdG and GADD153. PM2.5 also activated c-fos and c-jun levels and inactivated PTEN levels in rat lungs. These suggested that epigenetic modification was probably a potential mechanism by which PM2.5-induced genotoxicity in rat lungs.

Study on antibiotics, antibiotic resistance genes, bacterial community characteristics and their correlation in the landfill leachates.

AIM: This study aimed to investigate the contamination levels of antibiotics and antibiotic resistance genes (ARGs) in the landfill leachates and their correlations with the bacteria. METHODS AND RESULTS: Using HPLC-MS, quantitative PCR and high-throughput sequencing, we measured the pollution levels of 14 antibiotics and 10 ARGs in the leachates of the landfill in Taiyuan, China, and analysed changes in the bacterial community and the correlations of bacteria with antibiotics and ARGs. The main results showed high levels of antibiotics (like enrofloxacin, pefloxacin and oxytetracycline) and ARGs (like sulfonamides, tetracycline, macrolides, quinolones and ß-lactam-resistance genes) in the landfill leachates, along with higher diversity and richness of the bacteria. Some types of antibiotics had positive correlations with their corresponding ARGs. The dominant bacteria in the landfill leachates were Pseudomonas, Defluviitoga and Sulfurimonas, which correlated with the antibiotics and ARGs and might have potential effects on degrading them. CONCLUSIONS: Antibiotics and ARG pollution existed in the landfill leachates, while bacteria were closely associated with them. SIGNIFICANCE AND IMPACT OF THE STUDY: It will provide helpful information for the potential application of the bacteria in antibiotics and ARGs pollution control and landfill leachate management.

Ambient air PM2.5 exposure induces heart injury and cardiac hypertrophy in rats through regulation of miR-208a/b, α/ß-MHC, and GATA4.

Ambient air fine particulate matter (PM2.5) may increase cardiovascular disease risks. In this study, we investigated the miR-208/GATA4/myosin heavy chain (MHC) regulation mechanisms on cardiac injury in rats after PM2.5 exposure via an animal inhalation device. The results showed that PM2.5 exposure for 2 months caused pathological heart injury, reduced nucleus-cytoplasm ratio, and increased the levels of CK-MB and cTnI, showing cardiac hypertrophy. Oxidative stress and inflammatory responses were also observed in rats' hearts exposed to PM2.5. Of note, PM2.5 exposure for 2-month significantly elevated GATA4 and ß-MHC mRNA and protein expression compared with the corresponding controls, along with the high-expression of miR-208b. The ratios of ß-MHC/α-MHC expression induced by PM2.5 were remarkably raised in comparison to their controls. It suggested that the up-regulation of miR-208b/ß-MHC and GATA4 and the conversion from α-MHC to ß-MHC may be the important causes of cardiac hypertrophy in rats incurred by PM2.5.

The mitochondrial genome of Erronea caurica (Cypraeidae).

The mitochondrial genome of Erronea caurica from the South China Sea has been determined (GenBank Accession No. MT522622), which was the second report of mitochondrial genome in the superfamily Cypraeoidea. It is 16,053 bp long and consists of 21 tRNA genes, 2 rRNA genes, 13 protein-coding genes, and 1 control region. As previously reported mitochondrial genome in Cypraeoidea, all protein-coding genes of E. caurica use a typical start codon (ATN) and a complete stop codon (TAA or TAG). Phylogenetic tree demonstrated that E. caurica belongs to the family Cypraeoidea and closer to the superfamily Tonnoidea.

Multiple functions of thymosin ß4 in the pearl oyster Pinctada fucata suggest its multiple potential roles in artificial pearl culture.

Thymosin ß4 is a multifunctional protein in vertebrates that participates in physiological processes, such as wound healing, immune response, cell proliferation and migration. We assessed the multifarious roles of this small peptide in Pinctada fucata, an oyster commonly used in pearl culture in China. Our results showed that when P. fucata was challenged by bacterial pathogens or LPS, the relative expression level of Pfthymosin ß4 mRNA was significantly up-regulated, suggesting its involvement in immune response of the animal. Recombinant Pfthymosin ß4 (rPfthymosin ß4) was produced and showed in vitro different antibacterial activities against different pathogenic bacteria; the inhibitory effect of rPfthymosin ß4 on bacterial growth was relatively stronger in the broth culture than agar culture. The overexpression of Pfthymosin ß4 in Escherichia coli BL21(DE3) cells could improve their resistance to Cu2+, Zn2+, Cd2+, and H2O2, suggesting that Pfthymosin ß4 is likely involved with antioxidant. rPfthymosin ß4 also significantly promoted the proliferation and migration of mouse aortic vascular smooth muscle cells as indicated by MTT assay and cell scratch assay, respectively. In addition, chemically synthesized or recombinant Pfthymosin ß4 could transiently increase the circulating total hemocytes counts but down-regulated by RNAi in P. fucata. Taking together above results and previous studies suggested that Pfthymosin ß4 is potentially able to promote wound healing through enhancing antibacterial activity and antioxidant capacity, promotion of cell proliferation and migration, and increase of circulating hemocytes in P. fucata due to nucleus implantation injury. Thus, the future of recombinant Pfthymosin ß4 should be promising in the culture of pearls in P. fucata.

Fine particulate matter aggravates intestinal and brain injury and affects bacterial community structure of intestine and feces in Alzheimer's disease transgenic mice.

Fine particulate matter (PM2.5) was a risk factor for neurological disorders when emerging studies revealed that PM2.5 affected the bacterial community structure of gut in Alzheimer's disease (AD) patients. The purpose of this study was to explore the effects of PM2.5 on intestinal and brain injury and on bacterial community structure in the intestine and feces of APP/PS1 transgenic mice exposed to PM2.5 for eight weeks with a real-world whole-body inhalation exposure system in Taiyuan, China. The brain and intestinal tissues were collected to evaluate histopathological changes by HE staining. TNF-α and IL-6 levels in intestines, brains, and serums, and Aß-42 levels in brains were detected. Intestinal and fecal samples were subjected to 16S rRNA gene sequencing. Results showed that PM2.5 significantly aggravated the pathological injury in intestines and brains in AD mice with elevated pro-inflammatory cytokine levels. The estimators of Shannon, Simpson, Chao1, and ACE indexes reflected the diversity and richness of the bacterial community. Compared with the FA-WT group, the FA-AD group had lower diversity and richness when the PM2.5-AD group had the highest ones. PCA and NMDS revealed the specific influence of PM2.5 on the bacterial community of intestine and feces because that the PM2.5-FA and PM2.5-AD group clumped visibly closer than the other groups in both bacterial communities of intestine and feces. The KEGG pathway analysis predicted the vital functional genes and metabolic pathways in the bacterial community of PM2.5-AD mice. This study indicated the histopathological changes and inflammation in the intestine and brain were seriously caused in PM2.5-AD mice when the α-diversity of the bacterial community in intestine and feces was visibly changed.

SEM observation of early shell formation and expression of biomineralization-related genes during larval development in the pearl oyster Pinctada fucata.

Shell formation of Pinctada fucata in larval development stages plays a crucial role in their survival. Scanning electron microscopy (SEM) was used to observe the morphological changes during larval development. We found that the early shell forms soon after enlargement of the blastopore at the anterior end of the trochophore stage and the complete shell forms in the spats stage, required for metamorphosis of P. fucata. Based on our transcriptome data of trochophore, D-shaped, umbonal, eyespots and spats stages, including the whole process of shell formation, 93 differentially expressed biomineralization-related genes were identified, of which 25 genes were unique to P. fucata, 30 were identical to genes in pacific oyster, and the remaining genes were annotated to other species. Two-dimensional and three-dimensional principal components analysis (PCA) showed that different developmental stages were significantly different, with the early two stages exhibiting a larger difference compared with the next stages. The 93 genes were sorted into 20 trends with three trends being significantly enriched: an initial increase and then a decrease, a monotonic decrease, and a monotonic increase. Gene expression patterns changed with regulatory function during shell formation. Almost all the biomineralization-related genes were up-regulated in the D-shaped stage, but only five genes were up-regulated in that stage but down-regulated in the remaining stages. There were also 11 genes up-regulated in the last three stages, and a total of 24 genes showed high expression level during the last four stages. The 55 genes selected for shell incision experiment sorted into five trends and most genes presented differences in expression between 24 h and other time points. Considering all these results, there is a correlation with the morphological change and the expression of biomineralization-related genes during larval developmental stages, especially of differently expressed genes.

[Effects of salt stress on the contents of chlorophyll and organic solutes in Aeluropus littoralis var. sinensis Debeaux].

After seedlings of Aeluropus sinensis var. sinensis Debeaux were treated with different NaCl concentrations (0-200 mmol/L) for 14 days, some physiological indexes were measured. The higher the NaCl concentration, the more the growth of A. sinensis was inhibited. The increase in root/shoot ratio suggests that the shoots are more sensitive to salinity than the roots. The diminished leaf area may reduce the transpiration rate, and the root mainly grew longitudinally, which may help the root to reach the water source under the high salinity conditions. Chl a contents increased, so did Chl b, but the Chl a/Chl b ratio declined, which implies the stimulation of Chl a accepted from NaCl is smaller than that of Chl b. After salt treatment, the organic solute contents increased (P<0.05), the proline, amino acids and soluble sugar contents increased more than organic acids, the increase in soluble carbohydrate may inhibit photosynthesis in feedback. The percentage of sucrose in soluble carbohydrates increased too. Although the organic dry weight of whole plant declined, the proportion of organic dry weight in total dry weight increased, and the osmotic potential of plant cell juice declined, which implies that the contribution of organic matter to osmotic adjustment increased with salinity. That is, A. sinensis had ability to tolerate salinity to a certain degree.