Rhizobia-diatom symbiosis fixes missing nitrogen in the ocean.

Nitrogen (N2) fixation in oligotrophic surface waters is the main source of new nitrogen to the ocean1 and has a key role in fuelling the biological carbon pump2. Oceanic N2 fixation has been attributed almost exclusively to cyanobacteria, even though genes encoding nitrogenase, the enzyme that fixes N2 into ammonia, are widespread among marine bacteria and archaea3-5. Little is known about these non-cyanobacterial N2 fixers, and direct proof that they can fix nitrogen in the ocean has so far been lacking. Here we report the discovery of a non-cyanobacterial N2-fixing symbiont, 'Candidatus Tectiglobus diatomicola', which provides its diatom host with fixed nitrogen in return for photosynthetic carbon. The N2-fixing symbiont belongs to the order Rhizobiales and its association with a unicellular diatom expands the known hosts for this order beyond the well-known N2-fixing rhizobia-legume symbioses on land6. Our results show that the rhizobia-diatom symbioses can contribute as much fixed nitrogen as can cyanobacterial N2 fixers in the tropical North Atlantic, and that they might be responsible for N2 fixation in the vast regions of the ocean in which cyanobacteria are too rare to account for the measured rates.

Developing and validating a clinlabomics-based machine-learning model for early detection of retinal detachment in patients with high myopia.

BACKGROUND: Retinal detachment (RD) is a vision-threatening disorder of significant severity. Individuals with high myopia (HM) face a 2 to 6 times higher risk of developing RD compared to non-myopes. The timely identification of high myopia-related retinal detachment (HMRD) is crucial for effective treatment and prevention of additional vision impairment. Consequently, our objective was to streamline and validate a machine-learning model based on clinical laboratory omics (clinlabomics) for the early detection of RD in HM patients. METHODS: We extracted clinlabomics data from the electronic health records for 24,440 HM and 5607 HMRD between 2015 and 2022. Lasso regression analysis assessed fifty-nine variables, excluding collinear variables (variance inflation factor > 10). Four models based on random forest, gradient boosting machine (GBM), generalized linear model, and Deep Learning Model were trained for HMRD diagnosis and employed for internal validation. An external test of the models was done. Three random data sets were further processed to validate the performance of the diagnostic model. The primary outcomes were the area under the receiver operating characteristic curve (AUC) and the area under the precision-recall curve (AUCPR) to diagnose HMRD. RESULTS: Nine variables were selected by all models. Given the AUC and AUCPR values across the different sets, the GBM model was chosen as the final diagnostic model. The GBM model had an AUC of 0.8550 (95%CI = 0.8322-0.8967) and an AUCPR of 0.5584 (95%CI = 0.5250-0.5879) in the training set. The AUC and AUCPR in the internal validation were 0.8405 (95%CI = 0.8060-0.8966) and 0.5355 (95%CI = 0.4988-0.5732). During the external test evaluation, it reached an AUC of 0.7579 (95%CI = 0.7340-0.7840) and an AUCPR of 0.5587 (95%CI = 0.5345-0.5880). A similar discriminative capacity was observed in the three random data sets. The GBM model was well-calibrated across all the sets. The GBM-RD model was implemented into a web application that provides risk prediction for HM individuals. CONCLUSION: GBM algorithms based on nine features successfully predicted the diagnosis of RD in patients with HM, which will help ophthalmologists to establish a preliminary diagnosis and to improve diagnostic accuracy in the clinic.

Interaction of lncRNA-CR33942 with Dif/Dorsal Facilitates Antimicrobial Peptide Transcriptions and Enhances Drosophila Toll Immune Responses.

The Drosophila Toll signaling pathway mainly responds to Gram-positive (G+) bacteria or fungal infection, which is highly conserved with mammalian TLR signaling pathway. Although many positive and negative regulators involved in the immune response of the Toll pathway have been identified in Drosophila, the roles of long noncoding RNAs (lncRNAs) in Drosophila Toll immune responses are poorly understood to date. In this study, our results demonstrate that lncRNA-CR33942 is mainly expressed in the nucleus and upregulated after Micrococcus luteus infection. Especially, lncRNA-CR33942 not only modulates differential expressions of multiple antimicrobial peptide genes but also affects the Drosophila survival rate during response to G+ bacterial infection based on the transiently overexpressing and the knockdown lncRNA-CR33942 assays in vivo. Mechanically, lncRNA-CR33942 interacts with the NF-κB transcription factors Dorsal-related immunity factor/Dorsal to promote the transcriptions of antimicrobial peptides drosomycin and metchnikowin, thus enhancing Drosophila Toll immune responses. Taken together, this study identifies lncRNA-CR33942 as a positive regulator of Drosophila innate immune response to G+ bacterial infection to facilitate Toll signaling via interacting with Dorsal-related immunity factor/Dorsal. It would be helpful to reveal the roles of lncRNAs in Toll immune response in Drosophila and provide insights into animal innate immunity.

Development and characterization of 40 InDel markers from the major histocompatibility complex genes of largemouth bass (Micropterus salmoides).

BACKGROUND: The genetic improvement in growth and food habit domestication of largemouth bass (Micropterus salmoides) have made breakthroughs in past decades, while the relevant work on disease resistance were rarely carried out. Major histocompatibility complex (MHC) genes, which are well known as their numbers and high polymorphisms, have been used as candidate genes to mine disease-resistant-related molecular markers in many species. METHODS AND RESULTS: In present study, we developed and characterized 40 polymorphic and biallelic InDel markers from the major histocompatibility complex genes of largemouth bass. The minor allele frequency, observed heterozygosity, expected heterozygosity and polymorphic information content of these markers ranged from 0.0556 to 0.5000, 0.1111 to 0.6389, 0.1064 to 0.5070, and 0.0994 to 0.3750, respectively. Three loci deviated significantly from Hardy-Weinberg equilibrium, while linkage disequilibrium existed at none of these loci. CONCLUSION: These InDel markers might provide references for the further correlation analysis and molecular assisted selection of disease resistance in largemouth bass.

Expression characteristics of the cyp19a1b aromatase gene and its response to 17ß-estradiol treatment in largemouth bass (Micropterus salmoides).

To investigate the regulatory role of the cyp19a1b aromatase gene in the sexual differentiation of largemouth bass (Micropterus salmoides, LMB), we obtained the full-length cDNA sequence of cyp19a1b using rapid amplification of cDNA ends technique. Tissue expression characteristics and feedback with 17-ß-estradiol (E2) were determined using quantitative real-time PCR (qRT-PCR), while gonad development was assessed through histological section observations. The cDNA sequence of LMB cyp19a1b was found to be1950 base pairs (bp) in length, including a 5' untranslated region of 145 bp, a 3' untranslated region of 278 bp, and an open reading frame encoding a protein consisting of 1527 bp that encoded 508 amino acids. The qRT-PCR results indicated that cyp19a1b abundantly expressed in the brain, followed by the gonads, and its expression in the ovaries was significantly higher than that observed in the testes (P < 0.05). After feeding fish with E2 for 30 days, the expression of cyp19a1b in the pseudo-female gonads (XY-F) was significantly higher than that in males (XY-M) (P < 0.05), whereas expression did not differ significantly between XX-F and XY-F fish (P > 0.05). Although the expression of cyp19a1b in XY-F and XX-F fish was not significantly different after 60 days (P>0.05), both exhibited significantly higher levels than that of XY-M fish (P<0.05). Histological sections analysis showed the presence of oogonia in both XY-F and XX-F fish at 30 days, while spermatogonia were observed in XY-M fish. At 60 days, primary oocytes were abundantly observed in both XY-F and XX-F fish, while a few spermatogonia were visible in XY-M fish. At 90 days, the histological sections' results showed that a large number of oocytes were visible in XY-F and XX-F fish. Additionally, the gonads of XY-M fish contained numerous spermatocytes. These results suggest that cyp19a1b plays a pivotal role in the development of ovaries and nervous system development in LMB.

Leaching kinetics of fluorine during the aluminum removal from spent Li-ion battery cathode materials.

The high content of aluminum (Al) impurity in the recycled cathode powder seriously affects the extraction efficiency of Nickel, Cobalt, Manganese, and Lithium resources and the actual commercial value of recycled materials, so Al removal is crucially important to conform to the industrial standard of spent Li-ion battery cathode materials. In this work, we systematically investigated the leaching process and optimum conditions associated with Al removal from the cathode powder materials collected in a wet cathode-powder peeling and recycling production line of spent Li-ion batteries (LIBs). Moreover, we specifically studied the leaching of fluorine (F) synergistically happened along with the removal process of Al, which was not concerned about in other studies, but one of the key factors affecting pollution prevention in the recovery process. The mechanism of the whole process including the leaching of Al and F from the cathode powder was indicated by using NMR, FTIR, and XPS, and a defluoridation process was preliminarily investigated in this study. The leaching kinetics of Al could be successfully described by the shrinking core model, controlled by the diffusion process and the activation energy was 11.14 kJ/mol. While, the leaching of F was attributed to the dissolution of LiPF6 and decomposition of PVDF, and the kinetics associated was described by Avrami model. The interaction of Al and F is advantageous to realize the defluoridation to some degree. It is expected that our investigation will provide theoretical support for the large-scale recycling of spent LIBs.

[Effects of Personalized Exercise Prescriptions on Sarcopenia in Patients Undergoing Hemodialysis].

Sarcopenia is a major factor affecting the health and quality of life of the patients undergoing hemodialysis.Exercise can effectively ameliorate sarcopenia in these patients.However,the type,intensity,time,and frequency of exercise influence the effect of exercise.This review describes the effects of different exercise prescriptions on sarcopenia in the patients undergoing hemodialysis.It aims to assist medical staff in developing personalized exercise prescriptions,guiding patients to engage in exercise,and provide effective strategies for the prevention and treatment of sarcopenia in the patients undergoing hemodialysis.

circTOP2A functions as a ceRNA to promote glioma progression by upregulating RPN2.

Competing endogenous RNA (ceRNA)-mediated signaling pathway dysregulation provides great insight into comprehensively understanding the molecular mechanism and combined targeted therapy for glioblastoma. circRNA is characterized by high stability, tissue/developmental stage-specific expression and abundance in brain and plays significant roles in the initiation and progression of cancer. Our previous published data have demonstrated that RPN2 was significantly upregulated in glioma and promoted tumor progression via the activation of the Wnt/ß-catenin pathway. Furthermore, we proved that miR-422a regulated the Wnt/ß-catenin signaling pathway by directly targeting RPN2. In this study, based on the glioblastoma microarray profiles, we identified the upstream circTOP2A, which completely bound to miR-422a and was co-expressed with the RPN2. circTOP2A was significantly overexpressed in glioma and conferred a poor prognosis. circTOP2A could regulate RPN2 expression by sponging miR-422a, verified by western blot, dual-luciferase reporter gene assay, and RNA pull-down assay. Functional assays including CCK8, transwell and FITC-annexin V were performed to explore the RPN2-mediated role of the circTOP2A effect on the glioma malignant phenotype. Additionally, TOP/FOP and immunofluorescence analysis were used to confirm that sh-circTOP2A could suppress the Wnt/ß-catenin pathway partly through RPN2. Finally, a tumor xenograft model was applied to validate the biological function of circTOP2A in vivo. Taken together, our findings reveal the critical role of circTOP2A in promoting glioma proliferation and invasion via a ceRNA mechanism and provide an exploitable biomarker and therapeutic target for glioma patients.

Life strategies and metabolic interactions of core microbes during thiosulphate-based denitrification.

Sulphur-driven denitrification is a low-cost process for the treatment of nitrate-contaminated water. However, a comprehensive understanding of core populations and microbial interactions of a sulphur-based denitrifying system is lacking. This study presents results from three replicated denitrifying systems amended with thiosulphate and operated under a low C/N ratio. Amplicon sequencing revealed gradual enrichments of a few abundant denitrifiers. Based on genome-centred metagenomics and metatranscriptomics, a core set of microbes was identified in the systems, with Pseudomonas 1 and Thauera 2 being the most abundant ones. Although the replicates showed different enrichments, generalized observations were summarized. Most core populations conserved energy from denitrification coupled with sulphur. Pseudomonas 1 and Thauera 2 were able to finish complete denitrification. Surprisingly, they were also able to synthesize almost all amino acids and vitamins. In contrast, less abundant members, including Pseudomonas 2, were relatively auxotrophic and required an exogenous supply of amino acids and vitamins. The high expression of enzymes involved in biosynthesis and transport systems indicated their syntrophic relationships. The genomic findings suggested life strategies and interactions of the core thiosulphate-based denitrifying microbiome, with implications for nitrate-polluted water remediation.

Denitrification fractionates N and O isotopes of nitrate following a ratio independent of carbon sources in freshwaters.

The stable isotope technique has been used in tracking nitrogen cycling processes, but the isotopic characteristics are influenced by environmental conditions. To better understand the variability of nitrate isotopes in nature, we investigated the influence of organic carbon sources on isotope fractionation characteristics during microbial denitrification. Denitrifying cultures were inoculated with freshwater samples and enriched with five forms of organic compounds, that is, acetate, citrate, glucose, cellobiose, and leucine. Though the isotope enrichment factors of nitrogen and oxygen (15 ε and 18 ε) changed with carbon sources, 18 ε/15 ε always followed a proportionality near 1. Genome-centred metagenomics revealed the enrichment of a few populations, such as Pseudomonas, Enterobacter, and Atlantibacter, most of which contained both NapA- and NarG-type nitrate reductases. Metatranscriptome showed that both NapA and NarG were expressed but to different extents in the enrichments. Furthermore, isotopic data collected from a deep reservoir was analysed. The results showed δ18 O- and δ15 N-nitrate did not correlate in the surface water where nitrification was active, but 18 ε/15 ε followed a proportionality of 1.05 ± 011 in deeper waters (≥ 12 m) where denitrification controlled the nitrate isotope. The independence of 18 ε/15 ε from carbon sources provides an opportunity to determine heterotrophic denitrification and helps the interpretation of nitrate isotopes in freshwaters.

Modeling and insights into the structural characteristics of endocrine-disrupting chemicals.

Endocrine-disrupting chemicals (EDCs) can cause serious harm to human health and the environment; therefore, it is important to rapidly and correctly identify EDCs. Different computational models have been proposed for the prediction of EDCs over the past few decades, but the reported models are not always easily available, and few studies have investigated the structural characteristics of EDCs. In the present study, we have developed a series of artificial intelligence models targeting EDC receptors: the androgen receptor (AR); estrogen receptor (ER); and pregnane X receptor (PXR). The consensus models achieved good predictive results for validation sets with balanced accuracy values of 87.37%, 90.13%, and 79.21% for AR, ER, and PXR binding assays, respectively. Analysis of the physical-chemical properties suggested that several chemical properties were significantly (p < 0.05) different between EDCs and non-EDCs. We also identified structural alerts that can indicate an EDC, which were integrated into the web server SApredictor. These models and structural characteristics can provide useful tools and information in the discrimination and mechanistic understanding of EDCs in drug discovery and environmental risk assessment.

Novel iontophoretic drug delivery of estradiol with alendronate for osteoporosis treatment.

Alendronate and estradiol are potential molecules for the treatment of osteoporosis but their use is limited by lower skin permeation and first-pass metabolism respectively. To develop a combinatorial dosage regimen of alendronate and estradiol and enhance skin permeation through iontophoresis for the treatment of osteoporosis. The alendronate and estradiol-containing gel system were developed using carbopol-940 and triethanolamine as independent variables while viscosity and ex vivo permeation as dependent variables. The formulated gel was evaluated for viscosity, pH and ex vivo permeation study with and without iontophoresis. A permeation study was performed on rat abdominal skin. The viscosities for the developed formulations were found to be in the range of 945 cp-1298 cp, The pH of the formulation was found to be 7.4 which is ideal for the ionization of most drugs. Ex vivo permeation ranged from 79.99 to 99.89%. The permeation of both drugs was found to be increased upon application of DC (0.25, 0.50, 0.75 mA/cm2). The maximum percentage of permeation was found to be 99.89% (F3 batch). The skin permeation of alendronate and estradiol was enhanced by the novel formulation using iontophoresis.

Integration of leave-one-out method and real-time live cell reporter array system to assess the toxicity of mixtures.

The ever-increasing number of chemicals and complex mixtures demands a time-saving and cost-effective platform for environmental risk assessment. However, there is limit promising tool for evaluating the contribution of each component to the total toxicity effects of the mixture. Here, four widely distributed environmental pollutants with different mode-of-actions, i.e., cadmium chloride (Cd), nitrofurazone (NFZ), triclosan (TCS), and tris(2-chloroethyl) phosphate (TCEP), were selected as components of artificial mixture. Integration of leave-one-out method and high-dimensional live cell array system was used to explore relative contribution of each component from the mixture. A quaternary mixture (All_4_chems) and four ternary mixtures (Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP) were investigated by Escherichia coli (E. coli) live cell array system with 90 environmental stress genes modified by green fluorescent protein (GFP) expressing reporter vectors. E. coli cytotoxicity tests demonstrated that TCS has antagonism effect with other three chemicals (Cd, NFZ and TCEP), while it was additive effect in other three binary combinations. A total of 26, 23, 13, 31 and 23 genes were significantly altered with fold-change greater than 2 over the 4 h exposure by All_4_chems, Leave_Cd, Leave_NFZ, Leave_TCS and Leave_TCEP, respectively. Clustering analysis based on time-series gene expression patterns and transcriptional effect level index (TELI) showed that Leave_TCEP has similar profiles with All_4_chems, demonstrating TCEP has the least contribution among four components to the quaternary mixture. Leave_NFZ has the least number of significantly altered genes, implying NFZ has the largest toxicity effect contribution to the quaternary mixture. The relative contribution in different pathways indicated that Cd has the most contribution to the mixture in redox stress, while TCS has the least contribution in DNA stress pathway. Collectively, our results demonstrated the utility of high-dimensional toxicogenomics data and leave-one-out method in prioritizing the relative contribution of each component in mixture.

Identification of a protective Bacteroides strain of alcoholic liver disease and its synergistic effect with pectin.

The depletion of Bacteroides in the gut is closely correlated with the progression of alcoholic liver disease (ALD). This study aimed to identify Bacteroides strains with protective effects against ALD and evaluate the synergistic effects of Bacteroides and pectin in this disease. Mice were fed Lieber-DeCarli alcohol diet to establish an experimental ALD model and pre-treated with 4 Bacteroides strains. The severity of the liver injury, hepatic steatosis, and inflammation was evaluated through histological and biochemical assays. We found that Bacteroides fragilis ATCC25285 had the best protective effects against ALD strains by alleviating both ethanol-induced liver injury and steatosis. B. fragilis ATCC25285 could counteract inflammatory reactions in ALD by producing short-chain fat acids (SCFAs) and enhancing the intestinal barrier. In the subsequent experiment, the synbiotic combination of B. fragilis ATCC25285 and pectin was evaluated and the underlying mechanisms were investigated by metabolomic and microbiome analyses. The combination elicited superior anti-ALD effects than the individual agents used alone. The synergistic effects of B. fragilis ATCC25285 and pectin were driven by modulating gut microbiota, improving tryptophan metabolism, and regulating intestinal immune function. Based on our findings, the combination of B. fragilis ATCC25285 and pectin can be considered a potential treatment for ALD. KEY POINTS: • B. fragilis ATCC25285 was identified as a protective Bacteroides strain against ALD. • The synbiotic combination of B. fragilis and pectin has better anti-ALD effects. • The synbiotic combination modulates gut microbiota and tryptophan metabolism.

Timing of early gonadal differentiation and effects of estradiol-17ß treatments on the sex differentiation in Largemouth bass (Micropterus salmoides).

In this study, an efficient estradiol-17ß (E2)-induced feminization method was established based on the timing of early gonadal differentiation in Largemouth bass (Micropterus salmoides). Histological section results showed that from 20 days post-hatch (dph) to 30 dph, the germ cells gradually differentiated into oogonium and spermatic deferent, respectively. Moreover, female-biased genes Foxl2 and Cyp19a1a were up-regulated to the first peak at 20 dph, while the male-biased genes Dmrt1 were up-regulated to the first peak at 30 dph. These results indicated that the timing of early gonadal differentiation in Largemouth bass was between 20 and 30 dph. Therefore, 15 dph Largemouth bass with a body length of 15.10 ± 0.09 mm were chosen, and four E2-treated diets were set as 0 (E0, control), 50 mg/kg E2 (E50), 100 mg/kg E2 (E100), and 200 mg/kg E2 (E200). After feeding with E2-treated diets for 60 days, female ratios were 55%, 100%, 100%, and 100% in E0, E50, E100, and E200 groups, respectively. No intersex fish were observed in all the groups. However, 30% of females in the E200 group possessed thinner ovaries, with smaller ovary cavity structures and a decreased number of primary oocyte cells than those in other groups. Besides, the Largemouth bass in the E0 group grew more than those in E50, E100, and E200 groups during the E2 treatments period (P < 0.05). In conclusion, our study suggested that 50-100 mg/kg E2-treated diets could effectively induce the feminization of 15 dph Largemouth bass within 60 days duration time, which provided valuable information for the breeding of the all-male Largemouth bass population.

Characteristics of the intestinal bacterial microbiota profiles in Bifidobacterium pseudocatenulatum LI09 pre-treated rats with D-galactosamine-induced liver injury.

Bifidobacterium pseudocatenulatum LI09 could prevent D-galactosamine-induced liver injury. Our previous study has preliminarily determined that different intestinal microbiota profiles existed in the LI09-treated rats. Due to the sample size limitation, some subsequent analyses could not be achieved. In the current study, we conducted different experiments and bioinformatic analyses to characterise the distinct intestinal bacterial microbiota profiles in the LI09-treated rats with liver injury (i.e., LI09 group). Partition around medoids clustering analysis determined two intestinal microbiota profiles (i.e., Cluster_1_LI09 and Cluster_2_LI09) in LI09 group. Compared with Cluster_2_LI09, Cluster_1_LI09 group was determined at less dysbiotic microbial status and with lower level of liver injury. The two microbiota profiles were determined with distinct representative amplicon sequence variants (ASVs), among which, ASV1_Akkermansia and ASV3_Bacteroides were most associated with Cluster_1_LI09 and Cluster_2_LI09, respectively. Multiple representative phylotypes in Cluster_1_LI09 negatively correlating with liver function variables were assigned to Parabacteroides, suggesting Parabacteroides could benefit LI09 on modulating the liver function. In addition, ASV310_Lachnospiraceae, ASV501_Muribaculaceae and ASV484_Lachnospiraceae were determined as network gatekeepers in Cluster_1_LI09 network. The relevant results suggest that some intestinal bacteria could assist LI09 in lowering the intestinal microbial dysbiosis in the rats with liver injury, and their clinical application deserves further investigation.

In vitro elimination of autoreactive B cells from rheumatoid arthritis patients by universal chimeric antigen receptor T cells.

OBJECTIVES: Autoreactive B cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA), and B cell-depleting therapies using an antibodies, such as rituximab, have been suggested to be effective in RA treatment. However, transient B cell depletion with rituximab is associated with significant safety challenges related to global suppression of the immune system and thus increases the risks of infection and cancer development. To address selective and persistent issues associated with RA therapy, we developed a customised therapeutic strategy employing universal antifluorescein isothiocyanate (FITC) chimeric antigen receptor T cells (CAR-T cells) combined with FITC-labelled antigenic peptide epitopes to eliminate autoreactive B cell subsets recognising these antigens in RA. METHODS: For a proof-of-concept study, four citrullinated peptide epitopes derived from citrullinated autoantigens, namely, citrullinated vimentin, citrullinated type II collagen, citrullinated fibrinogen and tenascin-C, and a cyclocitrulline peptide-1 were selected as ligands for targeting autoreactive B cells; Engineered T cells expressing a fixed anti-FITC CAR were constructed and applied as a universal CAR-T cell system to specifically eliminate these protein-specific autoreactive B cells via recognition of the aforementioned FITC-labelled autoantigenic peptide epitopes. RESULTS: We demonstrated that anti-FITC CAR-T cells could be specifically redirected and kill hybridoma cells generated by immunisation with antigenic peptides, and autoreactive B cell subsets from RA patients via recognition of corresponding FITC-labelled citrullinated peptide epitopes. Additionally, the cytotoxicity of the CAR-T cells was dependent on the presence of the peptides and occurred in a dose-dependent manner. CONCLUSIONS: The approach described here provides a direction for precise, customised approaches to treat RA and can likely be applied to other systemic autoimmune diseases.

Analysis of carotenoid profile changes and carotenogenic genes transcript levels in Rhodosporidium toruloides mutants from an optimized Agrobacterium tumefaciens-mediated transformation method.

Rhodosporidium toruloides has been reported as a potential biotechnological microorganism to produce carotenoids. The most commonly used molecular and genetic manipulation methods based on Agrobacterium-mediated transformation (ATMT). However, this method was of relatively lower transformation efficiency. In this study, we optimized the ATMT method for R. toruloides on account of the promoter on T-DNA, the ratio of A. tumefaciens to R. toruloides NP11, acetosyringone concentration, cocultivation temperature and time, and a transformation efficiency of 2,369 cells per 105 recipient cells was obtained and was 24 times as that of the previous report. With this optimized method, four redder mutants and four yellower mutants were selected out with torularhodin and ß-carotene production preference, respectively. The highest torularhodin production was 1,638.15 µg/g dry cell weight in A1-13. The yellower mutants were found to divert the metabolic flux from torularhodin and torulene to γ-carotene and ß-carotene, and the proportion of γ-carotene and ß-carotene were all over 92%. TAIL-PCR was carried out to found T-DNA insertion in these mutants, and insertion hotspot was found. RT-qPCR results showed that CTA1 genes in these mutants were closely related to the synthesis of total carotenoids, especially torularhodin, and was a potenial metabolic engineering site in the future.

Chemical profiling of Zilong Jin tablets using ultra high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry.

Zilongjin tablets as a traditional Chinese medicine are widely used for primary lung cancer patients with deficiency of "qi " and "blood " syndrome undergoing chemotherapy. It is a compound preparation that consists of eight herbs. To clarify the chemical profiling of Zilong Jin tablets rapidly, a feasible and accurate strategy was developed by the ultra high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry. According to the accurate mass and fragment ion information provided by high resolution mass spectrometry, the compounds were reasonably identified. In total, 74 compounds were characterized, including 20 flavonoids, 14 quinones, 15 organic acids, 6 phthalide compounds, and 19 other compounds. Among them, 34 major compounds were unambiguously confirmed by comparing with reference standards. This study could provide an important scientific basis for further research on quality control, pharmacokinetics and pharmacodynamics, and clinical application of Zilong Jin tablets.

Structure and DNA damage-dependent derepression mechanism for the XRE family member DG-DdrO.

DdrO is an XRE family transcription repressor that, in coordination with the metalloprotease PprI, is critical in the DNA damage response of Deinococcus species. Here, we report the crystal structure of Deinococcus geothermalis DdrO. Biochemical and structural studies revealed the conserved recognizing α-helix and extended dimeric interaction of the DdrO protein, which are essential for promoter DNA binding. Two conserved oppositely charged residues in the HTH motif of XRE family proteins form salt bridge interactions that are essential for promoter DNA binding. Notably, the C-terminal domain is stabilized by hydrophobic interactions of leucine/isoleucine-rich helices, which is critical for DdrO dimerization. Our findings suggest that DdrO is a novel XRE family transcriptional regulator that forms a distinctive dimer. The structure also provides insight into the mechanism of DdrO-PprI-mediated DNA damage response in Deinococcus.