Hepatitis B Virus Induces IL-23 Production in Antigen Presenting Cells and Causes Liver Damage via the IL-23/IL-17 Axis

IL-23 regulates myriad processes in the innate and adaptive immune systems, and is a critical mediator of the proinflammatory effects exerted by Th17 cells in many diseases. In this study, we investigated whether and how hepatitis B virus (HBV) causes liver damage directly through the IL-23 signaling pathway. In biopsied liver tissues from HBV-infected patients, expression of both IL-23 and IL-23R was remarkably elevated. In vivo observations also indicated that the main sources of IL-23 were myeloid dendritic cells (mDCs) and macrophages. Analysis of in vitro differentiated immature DCs and macrophages isolated from healthy donors revealed that the HBV surface antigen (HBsAg) efficiently induces IL-23 secretion in a mannose receptor (MR)-dependent manner. Culture with an endosomal acidification inhibitor and the dynamin inhibitor showed that, upon binding to the MR, the HBsAg is taken up by mDCs and macrophages through an endocytosis mechanism. In contrast, although the HBV core antigen (HBcAg) can also stimulate IL-23 secretion from mDCs, the process was MR- and endocytosis-independent. In addition, IL-23 was shown to be indispensible for HBsAg-stimulated differentiation of naïve CD4⁺ T cells into Th17 cells, which were determined to be the primary source of IL-17 in HBV-infected livers. The cognate receptor, IL-17R, was found to exist on the hepatic stellate cells and mDCs, both of which might represent the potential target cells of IL-17 in hepatitis B disease. These data provide novel insights into a yet unrecognized mechanism of HBV-induced hepatitis, by which increases in IL-23 expression, through an MR/endocytosis-dependent or -independent manner, produce liver damage through the IL-23/IL-17 axis.     

Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome - lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3⁺ structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival.     

A non-catalytic function of Rev1 in translesion DNA synthesis and mutagenesis is mediated by its stable interaction with Rad5

DNA damage tolerance consisting of template switching and translesion synthesis is a major cellular mechanism in response to unrepaired DNA lesions during replication. The Rev1 pathway constitutes the major mechanism of translesion synthesis and base damage-induced mutagenesis in model cell systems. Rev1 is a dCMP transferase, but additionally plays non-catalytic functions in translesion synthesis. Using the yeast model system, we attempted to gain further insights into the non-catalytic functions of Rev1. Rev1 stably interacts with Rad5 (a central component of the template switching pathway) via the C-terminal region of Rev1 and the N-terminal region of Rad5. Supporting functional significance of this interaction, both the Rev1 pathway and Rad5 are required for translesion synthesis and mutagenesis of 1,N⁶-ethenoadenine. Furthermore, disrupting the Rev1–Rad5 interaction by mutating Rev1 did not affect its dCMP transferase, but led to inactivation of the Rev1 non-catalytic function in translesion synthesis of UV-induced DNA damage. Deletion analysis revealed that the C-terminal 21-amino acid sequence of Rev1 is uniquely required for its interaction with Rad5 and is essential for its non-catalytic function. Deletion analysis additionally implicated a C-terminal region of Rev1 in its negative regulation. These results show that a non-catalytic function of Rev1 in translesion synthesis and mutagenesis is mediated by its interaction with Rad5.     

Identification of methylated flavonoid regioisomeric metabolites using enzymatic semisynthesis and liquid chromatography-tandem mass spectrometry

Discrimination of isomeric methylated metabolites is an important step toward identifying genes responsible for methylation, but presents substantial challenges because authentic standards are often unavailable and mass spectra of isomers have been considered indistinguishable. In this report, an approach is described for identifying methyl group positions in multiply methylated flavonoid metabolites using combinations of tandem mass spectrometry, liquid chromatography retention, and site-selective methylation by recombinant O-methyltransferases from Solanum habrochaites LA1777. The basis for observed fragment ions in tandem mass spectra of multiply methylated myricetin was further established using enzymatic incorporation of deuterium-labeled methyl groups using S-adenosylmethionine-d ₃ as precursor.     

Flow infusion electrospray ionisation mass spectrometry for high throughput,non-targeted metabolite fingerprinting: a review

Producing a comprehensive overview of the chemical content of biologically-derived material is a major challenge. Apart from ensuring adequate metabolome coverage and issues of instrument dynamic range, mass resolution and sensitivity, there are major technical difficulties associated with data pre-processing and signal identification when attempting large scale, high-throughput experimentation. To address these factors direct infusion or flow infusion electrospray mass spectrometry has been finding utility as a high throughput metabolite fingerprinting tool. With little sample pre-treatment, no chromatography and instrument cycle times of less than 5 min it is feasible to analyse more than 1,000 samples per week. Data pre-processing is limited to aligning extracted mass spectra and mass-intensity matrices are generally ready in a working day for a month’s worth of data mining and hypothesis generation. ESI-MS fingerprinting has remained rather qualitative by nature and as such ion suppression does not generally compromise data information content as originally suggested when the methodology was first introduced. This review will describe how the quality of data has improved through use of nano-flow infusion and mass-windowing approaches, particularly when using high resolution instruments. The increasingly wider availability of robust high accurate mass instruments actually promotes ESI-MS from a merely fingerprinting tool to the ranks of metabolite profiling and combined with MS/MS capabilities of hybrid instruments improved structural information is available concurrently. We summarise current applications in a wide range of fields where ESI-MS fingerprinting has proved to be an excellent tool for “first pass” metabolome analysis of complex biological samples. The final part of the review describes a typical workflow with reference to recently published data to emphasise key aspects of overall experimental design.     

Cancer cachexia’s metabolic signature in a murine model confirms a distinct entity

Despite recent consensus definitions, lack of specific biomarkers remains a hurdle towards a more accurate and efficient diagnosis of cancer cachexia, distinguishing cachexia as a separate entity from other wasting syndromes. In a previous pilot study, we have shown that cancer-cachectic mice have a unique metabolic fingerprint with distinct glucose and lipid alterations compared to healthy controls. Further metabolomics studies were carried out to investigate differences in metabolic profiles of cancer-cachectic mice to tumor-bearing non-cachectic mice, calorie-restricted mice, and surgically treated cancer-cachectic mice. CD2F1 mice were divided into: (1) Cachexia Group received cachexia-inducing C26 undifferentiated colon carcinoma cells; (2) Tumor-Burden Group received, non-cachectic, P388 lymphoma cells; (3) Caloric-Restriction Group, remaining cancer-free, but subjected to caloric-restriction; (4) Surgery Group, similar to Cachexia Group, but tumors resected mid-experiment; and (5) Control Group aged intact. Baseline, mid-experiment and final serum samples were collected for ¹H NMR spectroscopic analysis. After data reduction, unsupervised principal component analysis and orthogonal projections to latent structures analyses demonstrate that the unique metabolic fingerprint is independent of tumor-burden and distinct from profiles of caloric-restriction and aging. Hyperlipidemia, hyperglycemia, and reduced branched-chain amino acids distinguish cachexia from other groups. Furthermore, the profile of surgically treated mice differs from that of cachectic mice, reverting to a profile more congruent with healthy controls indicating cachexia is amenable to correction where surgical cure is possible. That metabolomic analysis of murine serum is able to differentiate cachexia from tumor-burden and caloric-restriction warrants similar translational investigations in patients to explore cancer cachexia’s unique biomarkers.     

Metabolic responses of HeLa cells to silica nanoparticles by NMR-based metabolomic analyses

Silica nanoparticles are increasingly used in the biomedical fields due to their excellent solubility, high stability and favorable biocompatibility. However, despite being considered of low genotoxicity, their bio-related adverse effects have attracted particular concern from both the scientific field and the public. In this study, human cervical adenocarcinoma cells (HeLa line) were exposed to 0.01 or 1.0 mg/mL of hydrophilic silica nanoparticles. The ¹H NMR spectroscopy coupled with multivariate statistical analysis were used to characterize the metabolic variations of intracellular metabolites and the compositional changes of the corresponding culture media. At the early stage of silica nanoparticles-exposure, no obvious dose–effect of HeLa cell metabolome was observed, which implied that cellular stress-response regulated the metabolic variations of HeLa cell. Silica nanoparticles induced the increases of lipids including triglyceride, LDL, VLDL and lactate/alanine ratio and the decreases of alanine, ATP, choline, creatine, glycine, glycerol, isoleucine, leucine, phenylalanine, tyrosine, and valine, which involved in membrane modification, catabolism of carbohydrate and protein, and stress-response. Subsequently, a complicated synergistic effect of stress-response and toxicological-effect dominated the biochemical process and metabolic response, which was demonstrated in the reverse changes of some metabolites including acetate, ADP, ATP, choline, creatine, glutamine, glycine, lysine, methionine, phenylalanine and valine between 6 and 48 h post-treatment of silica nanoparticles. The toxicological-effects induced by high-dosage silica nanoparticles could be derived from the elevated levels of ATP and ADP, the utilization of glucose and amino acids and the production of metabolic end-products such as glutamate, glycine, lysine, methionine, phenylalanine, and valine. The results indicated that it is important and necessary to pursue further the physiological responses of silica nanoparticles in animal models and human before their practical use. NMR-based metabolomic analysis helps to understand the biological mechanisms of silica nanoparticles and their metabolic fate, and further, it offers an ideal platform for establishing the bio-safety of existing and new nanomaterials.     

不明原因肝功能异常患者的临床诊断思维

目的：建立不明原因肝功能异常患者的临床诊断思维以提高疑难肝病的诊治水平。方法：回顾性分析我院收治的4例不明原因肝功能异常患者的临床资料及诊治经过,并复习相关文献。结果：导致肝功能异常的病因虽极为复杂但通过详细询问病史,进行细致全面的体格检查以及必要的实验室和辅助检查,慎重采取诊断性治疗措施,提高少见病例对诊断影响的认识,绝大多数的病因可以查明。结论：不明原因肝功能异常患者的临床表现多样,病因复杂,建立相应的临床诊断思维可减少误诊和漏诊。