Large-scale map of RNA-binding protein interactomes across the mRNA life cycle.

mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress.

Nuclear mRNPs are compact particles packaged with a network of proteins promoting RNA-RNA interactions.

Messenger RNAs (mRNAs) are at the center of the central dogma of molecular biology. In eukaryotic cells, these long ribonucleic acid polymers do not exist as naked transcripts; rather, they associate with mRNA-binding proteins to form messenger ribonucleoprotein (mRNP) complexes. Recently, global proteomic and transcriptomic studies have provided comprehensive inventories of mRNP components. However, knowledge of the molecular features of distinct mRNP populations has remained elusive. We purified endogenous nuclear mRNPs from Saccharomyces cerevisiae by harnessing the mRNP biogenesis factors THO and Sub2 in biochemical procedures optimized to preserve the integrity of these transient ribonucleoprotein assemblies. We found that these mRNPs are compact particles that contain multiple copies of Yra1, an essential protein with RNA-annealing properties. To investigate their molecular and architectural organization, we used a combination of proteomics, RNA sequencing, cryo-electron microscopy, cross-linking mass spectrometry, structural models, and biochemical assays. Our findings indicate that yeast nuclear mRNPs are packaged around an intricate network of interconnected proteins capable of promoting RNA-RNA interactions via their positively charged intrinsically disordered regions. The evolutionary conservation of the major mRNA-packaging factor (yeast Yra1 and Aly/REF in metazoans) points toward a general paradigm governing nuclear mRNP packaging.

Structural basis of PETISCO complex assembly during piRNA biogenesis in C. elegans.

Piwi-interacting RNAs (piRNAs) constitute a class of small RNAs that bind PIWI proteins and are essential to repress transposable elements in the animal germline, thereby promoting genome stability and maintaining fertility. C. elegans piRNAs (21U RNAs) are transcribed individually from minigenes as precursors that require 5' and 3' processing. This process depends on the PETISCO complex, consisting of four proteins: IFE-3, TOFU-6, PID-3, and ERH-2. We used biochemical and structural biology approaches to characterize the PETISCO architecture and its interaction with RNA, together with its effector proteins TOST-1 and PID-1. These two proteins define different PETISCO functions: PID-1 governs 21U processing, whereas TOST-1 links PETISCO to an unknown process essential for early embryogenesis. Here, we show that PETISCO forms an octameric assembly with each subunit present in two copies. Determination of structures of the TOFU-6/PID-3 and PID-3/ERH-2 subcomplexes, supported by in vivo studies of subunit interaction mutants, allows us to propose a model for the formation of the TOFU-6/PID-3/ERH-2 core complex and its functionality in germ cells and early embryos. Using NMR spectroscopy, we demonstrate that TOST-1 and PID-1 bind to a common surface on ERH-2, located opposite its PID-3 binding site, explaining how PETISCO can mediate different cellular roles.

MicroRNA Clustering Assists Processing of Suboptimal MicroRNA Hairpins through the Action of the ERH Protein.

Microprocessor initiates the processing of microRNAs (miRNAs) from the hairpin regions of primary transcripts (pri-miRNAs). Pri-miRNAs often contain multiple miRNA hairpins, and this clustered arrangement can assist in the processing of otherwise defective hairpins. We find that miR-451, which derives from a hairpin with a suboptimal terminal loop and a suboptimal stem length, accumulates to 40-fold higher levels when clustered with a helper hairpin. This phenomenon tolerates changes in hairpin order, linker lengths, and the identities of the helper hairpin, the recipient hairpin, the linker-sequence, and the RNA polymerase that transcribes the hairpins. It can act reciprocally and need not occur co-transcriptionally. It requires Microprocessor recognition of the helper hairpin and linkage of the two hairpins, yet predominantly manifests after helper-hairpin processing. It also requires enhancer of rudimentary homolog (ERH), which copurifies with Microprocessor and can dimerize and interact with other proteins that can dimerize, suggesting a model in which one Microprocessor recruits another Microprocessor.

SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

Heat and mass transfer induced by alternating current during desorption of PAHs from soil using electrical resistance heating.

The transfer of heat and contaminants by alternating current (AC) and the removal mechanism of polycyclic aromatic hydrocarbons (PAHs) in electrical resistance heating (ERH) need further study. The main factors affecting heat transfer and water evaporation in the ERH experiment were studied, and the desorption efficiency, temporal and spatial distribution and kinetic behavior under various conditions were analyzed. The results suggested that moisture content was a necessary condition to ensure effective heating of soil, and soil moisture content above 30% was recommended. Higher voltage intensity and/or ion concentration meant stronger input power, resulting in the rapider heating process and the shorter the boiling time. At a low desorption temperature (about 100°C), the Phe desorption mainly depended on the volatilization of surface Phe and the co-boiling of Phe-water. In ERH, the participation of AC would accelerate the diffusion of pollutants from the internal pores of soil particles and their redistribution with water phase, thus improving the Phe removed by co-boiling. It was noteworthy that AC just greatly promoted solid-liquid mass transfer, but it hardly promoted desorption directly, and the removal still depended on Phe-water co-boiling. The Phe desorption efficiency could be significantly improved from 14.0~18.4% to 59.6~70.8% under the combined action of current strengthening Phe diffusion and co-boiling. Thermogravimetric and product analysis confirmed that no new organic matter was generated, but only Phe entered the gas phase through phase change.

Mycotoxins along the tea supply chain: A dark side of an ancient and high valued aromatic beverage.

ABSTRACTSTea (Camellia sinensis L.) is a high valued beverage worldwide since ancient times; more than three billion cups of tea are consumed each day. Leaf extracts of the plant are used for food preservation, cosmetics, and medicinal purposes. Nevertheless, tea contaminated with mycotoxins poses a serious health threat to humans. Mycotoxin production by tea fungi is induced by a variety of factors, including poor processing methods and environmental factors such as high temperature and humidity. This review summarizes the studies published to date on mycotoxin prevalence, toxicity, the effects of climate change on mycotoxin production, and the methods used to detect and decontaminate tea mycotoxins. While many investigations in this domain have been carried out on the prevalence of aflatoxins and ochratoxins in black, green, pu-erh, and herbal teas, much less information is available on zearalenone, fumonisins, and Alternaria toxins. Mycotoxins in teas were detected using several methods; the most commonly used being the High-Performance Liquid Chromatography (HPLC) with fluorescence detection, followed by HPLC with tandem mass spectrometry, gas chromatography and enzyme-linked immunosorbent assay. Further, mycotoxins decontamination methods for teas included physical, chemical, and biological methods, with physical methods being most prevalent. Finally, research gaps and future directions have also been discussed.

Theabrownin of raw and ripened pu-erh tea varies in the alleviation of HFD-induced obesity via the regulation of gut microbiota.

PURPOSE: Pu-erh tea can be classified into raw pu-erh tea and ripened pu-erh tea. Theabrownin (TB) is one of the major components of pu-erh tea. The difference of the anti-obesity activity between raw pu-erh tea TB (R-TB) and ripened pu-erh tea TB (F-TB) has not been comprehensively investigated yet. Therefore, this article aimed to systemically study the anti-obesity activity and the underlying mechanism of R-TB and F-TB. METHOD: High-fat diet (HFD)-induced C57BL/6J mice with obesity were gavaged with R-TB or F-TB to assess the effect of R-TB and F-TB on the amelioration of obesity, the expression of lipid metabolism-related genes, and the regulation of gut flora imbalance. RESULTS: Administration of both R-TB and F-TB could suppress body weight gain, improve insulin sensitivity and glucose homeostasis, regulate the lipid level and reduce the chronic inflammation in obese mice. The underlying anti-obesity mechanism of R-TB and F-TB might involve the regulation of lipogenesis and lipolysis, amelioration of the gut microbiota disorder and promotion of microbial metabolism. Interestingly, R-TB was more efficient in the regulation of blood glucose, reduction of inflammation and suppression of partial adipogenesis-related genes and protein, while F-TB was more effective in the inhibition of lipolysis-related genes and protein. In addition, F-TB might be more effective in adjusting the dysbacteria caused by HFD back to normal by promoting the proliferation of the beneficial microbiota, such as Lactobacillus and Lachnospiraceae_NK4A136_group. CONCLUSION: Taken together, both R-TB and F-TB had the potential to be developed as beneficial dietary supplements or functional foods for ameliorating obesity and obesity-related metabolic disorders, but their effects and the ability to regulate the intestinal flora varied.

Simultaneous Determination of Pesticide Residues and Mycotoxins in Storage Pu-erh Tea Using Ultra-High-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Mycotoxins and pesticides are the most concerning chemical contaminants that can affect the quality of Pu-erh tea during its production and storage. This study presents a method that can simultaneously determine 31 pesticide residues and six mycotoxins in Pu-erh tea within 11 min using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) after QuEChERS extraction. The lower limit of quantification (LOQ) for all analytes ranged between 0.06 and 50 ppb. Recoveries for each pesticide and mycotoxin ranged between 62.0 and 130.3%, with intra- and inter-day precisions lower than 15%. Good linear relationships were obtained, with correlation coefficients of r2 > 0.991 for all analytes. The established method was applied to 31 Pu-erh tea samples, including raw and ripened Pu-erh tea with different storage times. As a result, pesticide residues were not detected in any of the collected samples, and the mycotoxins detected in the samples were well below the official maximum residue limits (MRLs). Notably, the levels of aflatoxin B1 (AFB1), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) were lower than 1 ppb in the samples stored for more than 30 years.

Inactivation of fission yeast Erh1 de-represses pho1 expression: evidence that Erh1 is a negative regulator of prt lncRNA termination.

Fission yeast Erh1 exists in a complex with RNA-binding protein Mmi1. Deletion of erh1 up-regulates the phosphate homeostasis gene pho1, which is normally repressed by transcription in cis of a 5' flanking prt lncRNA. Here we present evidence that de-repression of pho1 by erh1Δ is achieved through precocious 3'-processing/termination of prt lncRNA synthesis, to wit: (i) erh1Δ does not affect the activity of the prt or pho1 promoters per se; (ii) de-repression by erh1Δ depends on CPF (cleavage and polyadenylation factor) subunits Ctf1, Dis2, Ssu72, Swd22, and Ppn1 and on termination factor Rhn1; (iii) de-repression requires synthesis by the Asp1 IPP kinase of inositol 1-pyrophosphates (1-IPPs); (iv) de-repression is effaced by mutating Thr4 of the RNA polymerase II CTD to alanine; and (v) erh1Δ exerts an additive effect on pho1 de-repression in combination with mutating CTD Ser7 to alanine and with deletion of the IPP pyrophosphatase Aps1. These findings point to Erh1 as an antagonist of lncRNA termination in the prt-pho1 axis. In contrast, in mmi1Δ cells there is a reduction in pho1 mRNA and increase in the formation of a prt-pho1 read-through transcript, consistent with Mmi1 being an agonist of prt termination. We envision that Erh1 acts as a brake on Mmi1's ability to promote CPF-dependent termination during prt lncRNA synthesis. Consistent with this idea, erh1Δ de-repression of pho1 was eliminated by mutating the Mmi1-binding sites in the prt lncRNA.

Role of enemy release and hybridization in the invasiveness of Impatiens balfourii and I. glandulifera.

Comparative studies with taxonomically and geographically paired alien species that exhibit different degrees of success in their invasions may help to identify the factors that determine invasiveness. Examples of such species in Europe include the noninvasive Impatiens balfourii and invasive I. glandulifera. We tested whether the low invasiveness of I. balfourii in Europe may be explained by strong pressure from local enemies. Earlier studies of these two species provided support for their hybridization. We tested this phenomenon as the potential occurrence of I. glandulifera × I. balfourii hybrids might promote the evolution of the invasiveness of I. balfourii. Both species were germinated from seeds collected in 2015 on the Swiss-Italian border in Insubria and utilized in three experiments: (1) a common garden enemy release test (leaf damage or pest pressure), (2) a test of the pressure exerted by a generalist enemy and (3) hybridization test. In the first test, the effect of enemies was assessed by the level of leaf damage and the number of pests. In the second test, a food choice experiment with a generalist herbivore (Cepaea snails) was performed. In the hybridization test, the plants were placed in a climatic chamber for self-pollination and hand cross-pollination. Analyses of enemy release and Cepaea snail preference revealed that I. balfourii experienced higher enemy pressure than I. glandulifera; however, this was not reflected in the performance of the plants. Although I. glandulifera was larger, I. balfourii had greater fecundity. Thus, the invasion success of I. glandulifera could not be unambiguously attributed to its greater degree of release from enemies compared with the noninvasive I. balfourii. Additionally, we did not obtain any evidence of hybridization between the two species. Thus, we obtained no support for the hypothesis that the evolution of the invasiveness of I. balfourii could be enhanced through hybridization with I. glandulifera.

Regulation of fungal community and the quality formation and safety control of Pu-erh tea.

Pu-erh tea belongs to dark tea among six major teas in China. As an important kind of post-fermented tea with complex microbial composition, Pu-erh tea is highly praised by many consumers owing to its unique and rich flavor and taste. In recent years, Pu-erh tea has exhibited various physiological activities to prevent and treat metabolic diseases. This review focuses on the fungi in Pu-erh tea and introduces the sources, types, and functions of fungi in Pu-erh tea, as well as the influence on the quality of Pu-erh tea and potential safety risks. During the process of fermentation and aging of Pu-erh tea, fungi contribute to complex chemical changes in bioactive components of tea. Therefore, we examine the important role that fungi play in the quality formation of Pu-erh tea. The associations among the microbial composition, chemicals excreted, and potential food hazards are discussed during the pile-fermentation of Pu-erh tea. The quality of Pu-erh tea has exhibited profound changes during the process of pile-fermentation, including color, aroma, taste, and the bottom of the leaves, which are inseparable from the fungus in the pile-fermentation of Pu-erh tea. Specifically, the application prospects of various detection methods of mycotoxins in assessing the safety of Pu-erh tea are proposed. This review aims to fully understand the importance of fungi in the production of Pu-erh tea and further provides new insights into subtly regulating the piling process to improve the nutritional properties and guarantee the safety of Pu-erh tea.

Proteogenomics Study of Blastobotrys adeninivorans TMCC 70007-A Dominant Yeast in the Fermentation Process of Pu-erh Tea.

Blastobotrys adeninivorans plays an essential role in pile-fermenting of Pu-erh tea. Its ability to assimilate various carbon and nitrogen sources makes it available for application in a wide range of industry sectors. The genome of B. adeninivorans TMCC 70007 isolated from pile-fermented Pu-erh tea was sequenced and assembled. Proteomics analysis indicated that 4900 proteins in TMCC 70007 were expressed under various culture conditions. Proteogenomics mapping revealed 48 previously unknown genes and corrected 118 gene models predicted by GeneMark-ES. Ortho-proteogenomics analysis identified 17 previously unidentified genes in B. adeninivorans LS3, the first strain with a sequenced genome among the genus Blastobotrys as well. More importantly, five species specific genes were identified from TMCC 70007, which could serve as a barcode for strain typing and were applicable for fermentation process protection of this industrial species. The datasets generated from tea aqueous extract culture not only increased the proteome coverage and accuracy but also contributed to the identification of proteins related to polyphenols and caffeine, which were considered to change greatly during the microbial fermentation of Pu-erh tea. This study provides a proteome perspective on TMCC 70007, which was considered to be an important strain in the production of Pu-erh tea. The systematic proteogenomics analysis not only made a better annotation on the genome of B. adeninivorans TMCC 70007 as previous proteogenomics study but also provided solution for fermentation process protection on valuable industrial species with species specific genes uniquely identified from proteogenomics study.

Puerhibacterium puerhi gen. nov., sp. nov., a novel member of the family Promicromonosporaceae, isolated from Pu-erh tea pile-fermentation.

A Gram-staining positive aerobic bacterium, designated TLY-12T, was isolated from the Pu-erh tea pile-fermentation process in Pu'er city, Yunnan, China. Strain TLY-12T grew at 15-37 °C (optimum, 30 °C), pH 6.0-11.0 (optimum, pH 9.0) and 0-9.0% (w/v) NaCl (optimum, 3.0%). The major cellular fatty acids were anteiso-C15:0, C16:0 and iso-C16:0. The respiratory quinone were menaquinones MK-9 (H2) and MK-9 (H4). The polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), phosphoglycolipid (PGL), glycolipid (GL) and an unidentified phospholipid (PL). The peptidoglycan contained glutamic acid, aspartic acid, alanine and lysine, with the last named being the diagnostic diamino acid. Whole-cell sugars of the isolate were ribose, galactose and glucose. Phylogenetic analyses of 16S rRNA gene showed that this strain belonged to the family Promicromonosporaceae, and was most closely related to Isoptericola cucumis DSM 101603 T, which gave sequence similarity of 97.9%. Genome sequencing revealed a genome size of 3.91 Mbp and a G + C content of 75.0%. Average nucleotide identity and digital DNA-DNA hybridization values were all below the species threshold of described Promicromonosporaceae species. Genome phylogenetic analysis showed that strain TLY-12T formed a separate evolutionary branch, and was parallel to other related genera of Promicromonosporaceae. Based on the phylogenetic, phenotypic, chemotaxonomic and genome pairwise data, strain TLY-12T is considered to represent a novel species in a new genus in the family Promicromonosporaceae, for which the name Puerhibacterium puerhi gen. nov, sp. nov. is proposed. The type strain is TLY-12T (= CGMCC 1.17157T = KCTC 49467T).

Massilia puerhi sp. nov., isolated from soil of Pu-erh tea cellar.

A Gram-reaction-negative, yellow-pigmented, non-spore-forming rod, aerobic, motile bacterium, designated SJY3T, was isolated from soil samples collected from a Pu-erh tea cellar in Bolian Pu-erh tea estate Co. Ltd. in Pu'er city, Yunnan, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Massilia. The closest phylogenetic relative was Massilia arenae CICC 24458T (99.5 %), followed by M. timonae CCUG45783T (97.9 %), M. oculi CCUG43427AT (97.8 %), and M. aurea DSM 18055T (97.8 %). The major fatty acids were C16 : 0 and C16 : 1 ω7c and/or C16 : 1 ω6c. The major respiratory quinone was ubiquinone Q-8 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Genome sequencing revealed a genome size of 5.97 M bp and a G+C content of 65.4 mol%. Pairwise determined whole genome average nucleotide identity (gANI) values and digital DNA-DNA hybridization (dDDH) values were all below the threshold. Although the 16S rRNA gene similarity of stain SJY3T and Massilia arenae CICC 24458T was more than 99 %, the gANI, dDDH values and genomic tree clearly indicated that they were not of the same species. In summary, strain SJY3T represents a new species, for which we propose the name Massilia puerhi sp. nov. with the type strain SJY3T (=CGMCC 1.17158T=KCTC 82193T).

Theabrownin from Pu-erh tea together with swinging exercise synergistically ameliorates obesity and insulin resistance in rats.

PURPOSE: Theabrownin (TB)-containing Pu-erh tea has been shown to be hypolipidemic in rats fed a high-fat diet. Physical exercise such as swinging is also known to reduce obesity. We hypothesized that TB in combination with swinging can synergistically ameliorate obesity and insulin resistance in rats with metabolic syndrome. METHODS: TB, rosiglitazone, or lovastatin (controls) was administered by gavage to rats fed a diet high in fat, sugar, and salt. A subgroup of the rats was subjected to a 30-min daily swinging exercise regimen, whereas the other rats did not exercise. RESULTS: Theabrownin in combination with swinging was found to significantly improve serum lipid status and prevent development of obesity and insulin resistance in rats. Liver transcriptomics data suggested that theabrownin activated circadian rhythm, protein kinase A, the adenosine monophosphate-activated protein kinase, and insulin signaling pathways by enhancing cyclic adenosine monophosphate levels and, hence, accelerating nutrient metabolism and the consumption of sugar and fat. The serum dopamine levels in rats increased significantly after exercise. In parallel work, intraperitoneal dopamine injections were shown to significantly reduce weight gain and prevent the elevation in triglyceride levels that would otherwise be induced by the high fat-sugar-salt diet. Theabrownin prevented obesity and insulin resistance mainly by affecting the circadian rhythm, while swinging exercise stimulated the overproduction of dopamine to accelerate metabolism of glucose and lipid. CONCLUSIONS: Theabrownin and exercise synergistically ameliorated metabolic syndrome in rats and effectively prevented obesity.

Pu-erh Tea Extract Treatment Could Be an Efficient Way to Enhance the Yield and Nutritional Value of Soybean Sprout.

Soybean sprouts are one of the most inexpensive and nutritious food items that can be easily grown year-round. Several studies have been conducted to increase their yield and nutritional values. This study was carried out to examine the effects of Pu-erh tea extracts on the production and nutrients content of soybean sprouts. Soybean seeds were soaked in 1%, 2%, or 3% (w/v) tea extracts, or tap water, before keeping for sprout cultivation; the sprout samples were named PE-1, PE-2, PE-3, and the control, respectively. The sprout yields were increased by up to 17% in PE-2 and PE-3 than in the control. The vitamin C, total free amino acid, total mineral, total isoflavone, total polyphenol, and flavonoid contents as well as the antioxidant potentials of the tea extract-treated sprouts were higher than those of the control. The results indicated that pre-soaking soybean seeds in 2% Pu-erh tea extracts could offer an easy, inexpensive, and efficient way to improve the yield and nutritional value of soybean sprouts.

Production of theophylline via aerobic fermentation of pu-erh tea using tea-derived fungi.

BACKGROUND: Caffeine is one of the most abundant methylxanthines in tea, and it remains stable in processing of general teas. In the secondary metabolism of microorganism, theophylline is the main conversion product in caffeine catabolism through demethylation. Microorganisms, involved in the solid-state fermentation of pu-erh tea, have a certain impact on caffeine level. Inoculating an appropriate starter strain that is able to convert caffeine to theophylline would be an alternative way to obtain theophylline in tea. The purpose of this study was to isolate and identify the effective strain converting caffeine to theophylline in pu-erh tea, and discuss the optimal conditions for theophylline production. RESULTS: Caffeine content was decreased significantly (p < 0.05) and theophylline content was increased significantly (p < 0.05) during the aerobic fermentation of pu-erh tea. Five dominant fungi were isolated from the aerobic fermentation and identified as Aspergillus niger, Aspergillus sydowii, Aspergillus pallidofulvus, Aspergillus sesamicola and Penicillium mangini, respectively. Especially, A. pallidofulvus, A. sesamicola and P. mangini were detected in pu-erh tea for the first time. All isolates except A. sydowii TET-2, enhanced caffeine content and had no significant influence on theophylline content. In the aerobic fermentation of A. sydowii TET-2, 28.8 mg/g of caffeine was degraded, 93.18% of degraded caffeine was converted to theophylline, and 24.60 mg/g of theophylline was produced. A. sydowii PET-2 could convert caffeine to theophylline significantly, and had application potential in the production of theophylline. The optimum conditions of theophylline production in the aerobic fermentation were 1) initial moisture content of 35% (w/w), 2) inoculation quantity of 8%, and 3) incubation temperature at 35 °C. CONCLUSIONS: For the first time, we find that A. sydowii PET-2 could convert caffeine to theophylline, and has the potential value in theophylline production through aerobic fermentation.

The ERH gene regulates migration and invasion in 5637 and T24 bladder cancer cells.

BACKGROUND: This study aimed to determine whether the enhancer of the rudimentary homolog (ERH) gene regulates cell migration and invasion in human bladder urothelial carcinoma (BUC) T24 cells and the underlying mechanism. METHODS: First, we knocked down ERH in BUC T24 and 5637 cells by shRNA and then used wound healing cell scratch migration assays, transwell cell migration assays, transwell cell invasion chamber experiments and nude mouse tail vein transfer assays to determine the migration and invasion ability after ERH was knocked down. Moreover, we used gene expression profiling chip analysis and further functional experiments to explore the possible mechanism through which ERH knockdown downregulated metastasis ability in T24 cells. RESULTS: Wound healing cell scratch migration assays, transwell cell migration assays, transwell cell invasion chamber experiments and nude mouse tail vein transfer assays all showed that the metastasis ability was significantly inhibited in human BUC T24 and 5637 cells with ERH knockdown. A gene expression profiling chip analysis in T24 cells showed that the MYC gene may be an important downstream target of the ERH gene, and the functional experiments showed that MYC is a functional target of ERH in BUC T24 cells. CONCLUSION: ERH knockdown could inhibit the metastasis of BUC T24 cells in vitro and in vivo. This study further explored the mechanism of the ERH gene in the metastasis of the T24 human bladder cancer cell line and found that ERH may regulate MYC gene expression. The results of this research provide a basis for the clinical application of ERH as a potential target for BUC treatment.

Aqueous raw and ripe Pu-erh tea extracts alleviate obesity and alter cecal microbiota composition and function in diet-induced obese rats.

Pu-erh tea is attracting increased attention worldwide because of its unique flavor and health effects, but its impact on the composition and function of the gut microbiota remains unclear. The aim of this study was to investigate the effects of aqueous extracts of fermented (ripe) and non-fermented (raw) Pu-erh teas on the composition and function of the intestinal microbiota of rats with diet-induced obesity. We conducted a comparative metagenomic and meta-proteomic investigation of the microbial communities in cecal samples taken from obese rats treated with or without extracts of raw or ripe Pu-erh teas. By analyzing the composition and diversity of 16S rRNA amplicons and expression profiles of 814 distinct proteins, we found that despite differences in the chemical compositions of raw and ripe Pu-erh teas, administration of either tea at two doses (0.15- and 0.40-g/kg body weight) significantly (P < 0.05) increased microbial diversity and changed the composition of cecal microbiota by increasing the relative abundances of Firmicutes and decreasing those of Bacteroidetes. Community metabolic processes, including sucrose metabolism, glycolysis, and syntheses of proteins, rRNAs, and antibiotics were significantly (P < 0.05) promoted or had a tendency (0.10 < P < 0.05) to be promoted due to the enrichment of relevant enzymes. Furthermore, evidence at population, molecular, and metabolic levels indicated that polyphenols of raw Pu-erh tea and their metabolites potentially promote Akkermansia muciniphila growth by stimulating a type II and III secretion system protein, the elongation factor Tu, and a glyceraldehyde-3-phosphate dehydrogenase. This study provides new evidence for the prebiotic effects of Pu-erh tea.