Establishment and application of a loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for detecting Clostridium piliforme.

BACKGROUND: Clostridium piliforme (causative agent of Tyzzer disease) infects various animals, including primates, and hence a threat to animal and human health worldwide. At present, it is detected using traditional methods, such as path morphology, polymerase chain reaction and enzyme-linked immunosorbent assay. Therefore, it is necessary to develop convenient, efficient visual molecular biological methods for detecting C. piliforme. OBJECTIVES: To establish a method with good specificity, high sensitivity and simple operation for the detection of C. piliforme. METHODS: In this study, we designed internal and external primers based on the conserved 23S rRNA region of C. piliforme to develop a biotin-labelled diarrhoea-suffered loop-mediated isothermal amplification (LAMP) system for detecting of C. piliforme and assessed the specificity, sensitivity and repeatability of the LAMP system. RESULTS: The LAMP system did not exhibit cross-reactivity with 24 other common pathogenic species, indicating that it had good specificity. The minimum concentration of sensitivity was 1 × 10-7  ng/µL. Mouse models (Meriones unguiculatus) of Tyzzer disease were established and a LAMP-lateral flow dipstick (LAMP-LFD) was developed for detecting C. piliforme. The detection rate of C. piliforme was 5.08% in clean-grade animals and 9.96% in specific-pathogen-free-grade animals from Jiangsu, Zhejiang and Shanghai. In addition, the detection rates of C. piliforme were 10.1%, 8.6% and 20%, in animals from Hangzhou, Wenzhou and Shaoxing, respectively. The detection rate of C. piliforme was higher in experimental animals used in schools than in those used in companies and research institutes. CONCLUSIONS: The LAMP-LFD method established in this study can be used to detect C. piliforme in animals handled in laboratory facilities of universities, pharmaceutical enterprises and research and development institutions.

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.

Biogeographic distribution, ecotype partitioning and controlling factors of Chloroflexi in the sediments of six hadal trenches of the Pacific Ocean.

The hadal trenches are "hot spots" for mineralization of organic matter in the deep ocean. Chloroflexi are one of the most dominant and active taxa in trench sediments, serving as important drivers of carbon cycles in hadal trenches. However, current understanding on hadal Chloroflexi is largely restricted to individual trench. This study systematically analyzed the diversity, biogeographic distribution, ecotype partitioning as well as environmental drivers of Chloroflexi in the sediments of hadal trenches, by reanalyzing 16S rRNA gene libraries of 372 samples from 6 trenches around the Pacific Ocean. The results showed that Chloroflexi averagely account for 10.10 % and up to 59.95 % of total microbial communities in the trench sediments. Positive correlations between relative abundance of Chloroflexi and depths down the vertical sediment profiles were observed in all of the sediment cores analyzed, suggesting the increasing significance of Chloroflexi in deeper sediment layers. Overall, trench sediment Chloroflexi were mainly composed of the classes Dehalococcidia, Anaerolineae and JG30-KF-CM66, and four orders i.e. SAR202, Anaerolineales, norank JG30-KF-CM66 and S085, were identified as core taxa that were dominant and prevalent in the hadal trench sediments. A total of 22 subclusters were identified within these core orders, and distinct patterns of ecotype partitioning related with depths down the vertical sediment profiles were observed, suggesting the great diversification of metabolic potentials and environment preference of different Chloroflexi lineages. The spatial distribution of hadal Chloroflexi were found to be significantly related with multiple environmental factors, while depths down the vertical sediment profiles explained the highest proportion of variations. These results provide valuable information for further exploring the roles of Chloroflexi in biogeochemical cycle of the hadal zone, and lay the foundation for understanding the adaptive mechanisms and evolutionary characteristics of microorganisms in hadal trenches.

Methyl Jasmonate- and Salicylic Acid-Induced Transcription Factor ZjWRKY18 Regulates Triterpenoid Accumulation and Salt Stress Tolerance in Jujube.

Triterpenoids are important, pharmacologically active substances in jujube (Ziziphus jujuba Mill.), and play an important role in the plant's resistance to abiotic stress. However, regulation of their biosynthesis, and the underlying mechanism of their balance with stress resistance, remain poorly understood. In this study, we screened and functionally characterized the ZjWRKY18 transcription factor, which is associated with triterpenoid accumulation. The transcription factor is induced by methyl jasmonate and salicylic acid, and its activity was observed by gene overexpression and silencing experiments, combined with analyses of transcripts and metabolites. ZjWRKY18 gene silencing decreased the transcription of triterpenoid synthesis pathway genes and the corresponding triterpenoid content. Overexpression of the gene promoted the biosynthesis of jujube triterpenoids, as well as triterpenoids in tobacco and Arabidopsis thaliana. In addition, ZjWRKY18 binds to W-box sequences to activate promoters of 3-hydroxy-3-methyl glutaryl coenzyme A reductase and farnesyl pyrophosphate synthase, suggesting that ZjWRKY18 positively regulates the triterpenoid synthesis pathway. Overexpression of ZjWRKY18 also increased tolerance to salt stress in tobacco and Arabidopsis thaliana. These results highlight the potential use of ZjWRKY18 to improve triterpenoid biosynthesis and salt stress tolerance in plants, and provide a strong basis for metabolic engineering to improve the content of triterpenoids and breeding of jujube varieties that are resistant to stress.

Integrated microRNA and transcriptome profiling reveals the regulatory network of embryo abortion in jujube.

Hybridization is an important approach to the production of new varieties with exceptional traits. Although the kernel rate of wild jujube (Ziziphus jujuba Mill. var. spinosa Hu.) is generally high, that of cultivated jujube (Z. jujuba Mill.) is low, greatly hampering the jujube breeding process. However, the mechanism by which this trait changed during jujube domestication remains unclear. Here, we explored the potential regulatory network that governs jujube embryo abortion using correlation analysis of population traits, artificial pollination, sugar content measurements and multi-omics analysis. The results showed that embryo abortion was an important reason for the low kernel rate of cultivated jujube, and kernel rate was negatively correlated with edible rate. Twenty-one days after pollination was a critical period for embryo abortion. At this time, the sugar content of cultivated 'Junzao' kernels decreased significantly compared with that of the pulp, but sugar content remained relatively stable in kernels of wild 'Suanzao'. A total of 1142 differentially expressed genes targeted by 93 microRNAs (miRNAs) were identified by transcriptome, miRNA and degradome sequencing, and may be involved in the regulation of embryo abortion during kernel development. Among them, DELLA protein, TCP14 and bHLH93 transcription factors have been shown to participate in the regulation of embryonic development. Our findings suggest that carbohydrate flow between different tissues of cultivated jujube exhibits a bias toward the pulp at 21 days after pollination, thereby restricting the process of kernel development. This information enhances our understanding of the embryo abortion process and reveals miRNA-target gene pairs that may be useful for molecular-assisted breeding.

Pedestrians' psychological preferences for urban street lighting with different color temperatures.

White LEDs, which have been widely used in the urban street lighting, are increasingly applied to replace traditional HPS lamps with a lower CCT (correlated color temperature). Generally, studies on the CCT of street lighting focus on providing safe functional lighting for vehicle drivers. However, it is still unknown how the street light color can affect pedestrians' perception and preferences with respect to lighting levels and ambient temperature. In this study, a wide range of CCTs (1,600-5,400 K) was measured for urban street lighting in Beijing, China, for example. And the transition from traditional HPS lamps to LEDs lacks a reference street lighting standard for CCT. The study aims to conduct a cross-sensory test to evaluate urban street lighting with multiple combinations of CCT values and illuminance levels according to pedestrians' visual perception and psychological preferences. A total of 18 night street lighting scenes with six CCT values and three illuminance levels were first selected in Beijing city, and then HDR videos of these scenes were taken from the view of pedestrians to conduct psychological experiments in an indoor environment with three ambient temperatures. A total of 77 university students (24 males) were invited to assess videos of the 18 lighting scenes in terms of seven factors, such as lighting brightness, color temperature sensation, light color preference, sense of safety, recognition, comfort, and overall preference. Several key findings were achieved as follows. (1) The CCT of urban street lighting can have significant effects on the visual psychological perceptions of participants. (2) There was a significant interaction between CCT, illuminance, and ambient temperature on the visual psychological performances of participants. (3) The higher ambient temperature will deliver the higher level of overall preference for the street lighting with medium and high CCT, and the perception of warmer light color. (4) There was a strong correlation found between participants' light color preference, comfort, and overall preferences.

Active compound analysis of Ziziphus jujuba cv. Jinsixiaozao in different developmental stages using metabolomic and transcriptomic approaches.

Jujube (Ziziphus jujuba Mill.) is a popular fruit with health benefits ascribed to its various metabolites. These metabolites determine the flavors and bioactivities of the fruit, as well as their desirability. However, the dynamics of the metabolite composition and the underlying gene expression that modulate the overall flavor and accumulation of active ingredients during fruit development remain largely unknown. Therefore, we conducted an integrated metabolomic and transcriptomic investigation covering various developmental stages in the jujube cultivar Z. jujuba cv. Jinsixiaozao, which is famous for its nutritional and bioactive properties. A total of 407 metabolites were detected by non-targeted metabolomics. Metabolite accumulation during different jujube developmental stages was examined. Most nucleotides and amino acids and their derivatives accumulated during development, with cAMP increasing notably during ripening. Triterpenes gradually accumulated and were maintained at high concentrations during ripening. Many flavonoids were maintained at relatively high levels in early development, but then rapidly decreased later. Transcriptomic and metabolomic analyses revealed that chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS), and dihydroflavonol 4-reductase (DFR) were mainly responsible for regulating the accumulation of flavonoids. Therefore, the extensive downregulation of these genes was probably responsible for the decreases in flavonoid content during fruit ripening. This study provide an overview of changes of active components in 'Jinsixiaozao' during development and ripening. These findings enhance our understanding of flavor formation and will facilitate jujube breeding for improving both nutrition and function.

Association of Bitter Metabolites and Flavonoid Synthesis Pathway in Jujube Fruit.

Jujube is rich in nutrients and can be eaten fresh or made into dried fruit, candied fruit, and preserved fruit. Its slightly bitter peel affects nutritional value and commercial value, but the mechanism of the formation of bitter substances is still unclear. We dynamically analyzed the biosynthesis of jujube peel bitterness and related nutrient metabolites through the transcriptome and metabolome. The results demonstrated that flavonoids were the main bitter substances in 'Junzao' jujube fruit skins and a total of 11,106 differentially expressed genes and 94 differentially abundant flavonoid metabolites were identified. Expression patterns of genes in the flavonoid synthesis pathway showed that flavonol synthase (FLS) expression was significantly correlated with quercetin content. Transient overexpression and virus induced gene silencing (VIGS) of ZjFLS1 and ZjFLS2 in jujube fruits and sour jujube seedlings significantly affected flavonol accumulation, especially the content of quercetin-3-O-rutinoside. Moreover, in vitro enzymatic reactions showed that ZjFLS1 and ZjFLS2 could catalyze the formation of quercetin from dihydroquercetin. These findings indicate that ZjFLS gene is the key gene in the biosynthesis of bitter substances in jujube fruit skins and provide basis for the research on the development of functional nutrients in jujube and the synthesis mechanism of bitter compounds.

Novel Chloroflexi genomes from the deepest ocean reveal metabolic strategies for the adaptation to deep-sea habitats.

BACKGROUND: The deep sea harbors the majority of the microbial biomass in the ocean and is a key site for organic matter (OM) remineralization and storage in the biosphere. Microbial metabolism in the deep ocean is greatly controlled by the generally depleted but periodically fluctuating supply of OM. Currently, little is known about metabolic potentials of dominant deep-sea microbes to cope with the variable OM inputs, especially for those living in the hadal trenches-the deepest part of the ocean. RESULTS: In this study, we report the first extensive examination of the metabolic potentials of hadal sediment Chloroflexi, a dominant phylum in hadal trenches and the global deep ocean. In total, 62 metagenome-assembled-genomes (MAGs) were reconstructed from nine metagenomic datasets derived from sediments of the Mariana Trench. These MAGs represent six novel species, four novel genera, one novel family, and one novel order within the classes Anaerolineae and Dehalococcoidia. Fragment recruitment showed that these MAGs are globally distributed in deep-sea waters and surface sediments, and transcriptomic analysis indicated their in situ activities. Metabolic reconstruction showed that hadal Chloroflexi mainly had a heterotrophic lifestyle, with the potential to degrade a wide range of organic carbon, sulfur, and halogenated compounds. Our results revealed for the first time that hadal Chloroflexi harbor pathways for the complete hydrolytic or oxidative degradation of various recalcitrant OM, including aromatic compounds (e.g., benzoate), polyaromatic hydrocarbons (e.g., fluorene), polychlorobiphenyl (e.g., 4-chlorobiphenyl), and organochlorine compounds (e.g., chloroalkanes, chlorocyclohexane). Moreover, these organisms showed the potential to synthesize energy storage compounds (e.g., trehalose) and had regulatory modules to respond to changes in nutrient conditions. These metabolic traits suggest that Chloroflexi may follow a "feast-or-famine" metabolic strategy, i.e., preferentially consume labile OM and store the energy intracellularly under OM-rich conditions, and utilize the stored energy or degrade recalcitrant OM for survival under OM-limited condition. CONCLUSION: This study expands the current knowledge on metabolic strategies in deep-ocean Chlorolfexi and highlights their significance in deep-sea carbon, sulfur, and halogen cycles. The metabolic plasticity likely provides Chloroflexi with advantages for survival under variable and heterogenic OM inputs in the deep ocean. Video Abstract.

A symbiotic bacterium of Antarctic fish reveals environmental adaptability mechanisms and biosynthetic potential towards antibacterial and cytotoxic activities.

Antarctic microbes are important agents for evolutionary adaptation and natural resource of bioactive compounds, harboring the particular metabolic pathways to biosynthesize natural products. However, not much is known on symbiotic microbiomes of fish in the Antarctic zone. In the present study, the culture method and whole-genome sequencing were performed. Natural product analyses were carried out to determine the biosynthetic potential. We report the isolation and identification of a symbiotic bacterium Serratia myotis L7-1, that is highly adaptive and resides within Antarctic fish, Trematomus bernacchii. As revealed by genomic analyses, Antarctic strain S. myotis L7-1 possesses carbohydrate-active enzymes (CAZymes), biosynthetic gene clusters (BGCs), stress response genes, antibiotic resistant genes (ARGs), and a complete type IV secretion system which could facilitate competition and colonization in the extreme Antarctic environment. The identification of microbiome gene clusters indicates the biosynthetic potential of bioactive compounds. Based on bioactivity-guided fractionation, serranticin was purified and identified as the bioactive compound, showing significant antibacterial and antitumor activity. The serranticin gene cluster was identified and located on the chrome. Furthermore, the multidrug resistance and strong bacterial antagonism contribute competitive advantages in ecological niches. Our results highlight the existence of a symbiotic bacterium in Antarctic fish largely represented by bioactive natural products and the adaptability to survive in the fish living in Antarctic oceans.

Metabolomic and Transcriptomic Analyses of Anthocyanin Biosynthesis Mechanisms in the Color Mutant Ziziphus jujuba cv. Tailihong.

The purplish-red color of "Tailihong" jujube fruit skins is caused primarily by anthocyanin accumulation, but the mechanisms that underlie anthocyanin biosynthesis in jujube fruit have rarely been studied. We performed metabolomic and transcriptomic analyses of jujube fruit skins at different developmental stages and identified a total of 158 flavonoids, among which cyanidin-3-O-rutinoside and peonidin-3,5-O-diglucoside were the primary anthocyanins. During fruit development and maturation, the anthocyanin content was strongly correlated with the expression of ZjANS and ZjUGT79B1, suggesting that these are key genes in the anthocyanin biosynthesis process. Transcriptomic analysis indicated that the transcription factors ZjMYB5, ZjTT8, and ZjWDR3 regulated anthocyanin biosynthesis in jujube fruit skins. Subcellular localization experiments confirmed that ZjANS and ZjUGT79B1 were localized to the nucleus and the endoplasmic reticulum. ZjMYB5 and ZjTT8 were found only in the nucleus, whereas strong fluorescence signals from ZjWDR3 were observed in the nucleus and cytoplasm. Prokaryotic expression and in vitro enzyme activity assays showed that the recombinant ZjANS protein catalyzed the formation of cyanidin from (+)-catechin. Secondary glycosylation by ZjUFGT79B1 modified cyanidin-3-O-glucoside to produce cyanidin-3-O-rutinoside, and ZjCCoAOMT readily catalyzed the production of the methylated anthocyanin peonidin-3,5-O-diglucoside from cyanidin 3,5-O-glucoside. Dual-Luciferase and GUS activity assays showed that the ZjANS and ZjUGT79B1 promoters were activated by ZjMYB5, ZjTT8, and ZjWDR3. All data indicated that these three transcription factors played important roles in anthocyanin biosynthesis in the color mutant Ziziphus jujuba cv. Tailihong, contributing to anthocyanin accumulation by enhancing the expression of ZjANS and ZjUGT79B1.

Early Detection of Toxoplasma gondii Infection In Mongolian Gerbil By Quantitative Real-Time PCR.

Toxoplasmosis, caused by Toxoplasma gondii, is associated with several clinical syndromes, including encephalitis, chorioretinitis, and congenital infection. Toxoplasma gondii is a ubiquitous apicomplexan parasite found in both humans and animals. Mongolian gerbils, which are more susceptible to both high- and low-virulence Toxoplasma strains compared with mice, are considered useful models for assessing diagnosis and treatment methods for toxoplasmosis, as well as infection by and host defense to this organism. Here we established a quantitative real-time polymerase chain reaction (qPCR) method targeting the B1 gene for early and specific detection of T. gondii infection in Mongolian gerbil. The detection limit of the developed qPCR was approximately 1 T. gondii tachyzoite. This method was also applied to detect T. gondii genomic DNA in experimentally infected Mongolian gerbils, with positive results in blood (66.7%), liver (73.3%), lung (80.0%), spleen (80.0%), and peritoneal fluid (66.7%) samples as early as 1 day postinfection. Specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum, Cryptosporidium parvum, Eimeria tenella, Trypanosoma evansi, Schistosoma japonicum, Angiostrongylus cantonensis, and Strongyloides stercoralis. This study first reports the use of Mongolian gerbils as an animal model for early diagnosis of toxoplasmosis by qPCR.