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Purpose Liver cancer is one of the most common tumors with the seventh-highest incidence and the third-highest mortality. Many studies have shown that small extracellular vesicles (sEVs) play an important role in liver cancer. Here, we report comprehensive signatures for sEV proteins from hepatocellular carcinoma (HCC) patient plasma, which might be valuable for the evaluation and diagnosis of HCC. Methods We extracted sEVs from the plasma of controls and HCC patients. Differentially expressed proteins in the sEVs were analyzed via label-free quantification and bioinformatic analysis. Western blotting (WB) was used to validate the abovementioned sEV proteins that differentially accumulated in the validation cohorts. Results Protein expression profiles were performed for plasma sEVs from 21 HCC patients and 15 controls. Among 335 identified proteins in our study, 27 were significantly dysregulated, including 13 increased proteins that were involved predominantly in the complement cascade (C1QB, C1QC, C4BPA, and C4BPB) and the coagulation cascade (F13B, FGA, FGB, and FGG). We verified that the protein levels of C1QB, C1QC, C4BPA, and C4BPB were increased in the plasma sEVs of HCC patients in both the discovery cohort and validation cohort. Conclusion The complement cascade in sEVs was significantly involved in HCC progression. C1QB, C1QC, C4BPA, and C4BPB were highly abundant in sEVs of HCC patients' plasma, which might be potential molecular signatures.

The accumulation of amyloid beta (Aβ) plaques in the brain is a hallmark of Alzheimer's disease (AD) pathology. Microglial activation-mediated neuroinflammation has been implicated in the pathogenesis of AD and the expression levels of interleukin-6 (IL-6) were increased in the brains of AD patients. However, the mechanisms by which IL-6 expression is regulated in human microglia are incompletely understood. Here, we show that Aβ

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. It has a poor prognosis because it is often diagnosed at the advanced stage when treatments are limited. In addition, HCC pathogenesis is not fully understood, and this has affected early diagnosis and treatment of this disease. Human alkaline ceramidase 2 (ACER2), a key enzyme that regulates hydrolysis of cellular ceramides, affects cancer cell survival, however its role in HCC has not been well characterized. Our results showed that ACER2 is overexpressed in HCC tissues and cell lines. In addition, high ACER2 protein expression was associated with tumor growth; ACER2 knockdown resulted in decreased cell growth and migration. Sphingomyelin phosphodiesterase acid‐like 3B (SMPDL3B) promoted HCC cell growth, invasion, and migration; SMPDL3B knockdown had a significant inhibitory effect on HCC tumor growth in vivo. Moreover, ACER2 positively regulated the protein level of SMPDL3B. Of note, ACER2/SMPDL3B promoted ceramide hydrolysis and S1P production. This axis induced HCC survival and could be blocked by inhibition of S1P formation. In conclusion, ACER2 promoted HCC cell survival and migration, possibly via SMPDL3B. Thus, inhibition of ACER2/SMPDL3B may be a novel therapeutic target for HCC treatment., ACER2 induced SMPDL3B resulting in S1P formation and ceramide hydrolysis, which promoted HCC cells growth, invasion and migration.

Background Whole-genome approaches are widely preferred for species delineation in prokaryotes. However, these methods require pairwise alignments and calculations at the whole-genome level and thus are computationally intensive. To address this problem, a strategy consisting of sieving (pre-selecting closely related genomes) followed by alignment and calculation has been proposed. Results Here, we initially test a published approach called “genome-wide tetranucleotide frequency correlation coefficient” (TETRA), which is specially tailored for sieving. Our results show that sieving by TETRA requires > 40% completeness for both genomes of a pair to yield > 95% sensitivity, indicating that TETRA is completeness-dependent. Accordingly, we develop a novel algorithm called “fragment tetranucleotide frequency correlation coefficient” (FRAGTE), which uses fragments rather than whole genomes for sieving. Our results show that FRAGTE achieves ~ 100% sensitivity and high specificity on simulated genomes, real genomes and metagenome-assembled genomes, demonstrating that FRAGTE is completeness-independent. Additionally, FRAGTE sieved a reduced number of total genomes for subsequent alignment and calculation to greatly improve computational efficiency for the process after sieving. Aside from this computational improvement, FRAGTE also reduces the computational cost for the sieving process. Consequently, FRAGTE extremely improves run efficiency for both the processes of sieving and after sieving (subsequent alignment and calculation) to together accelerate genome-wide species delineation. Conclusions FRAGTE is a completeness-independent algorithm for sieving. Due to its high sensitivity, high specificity, highly reduced number of sieved genomes and highly improved runtime, FRAGTE will be helpful for whole-genome approaches to facilitate taxonomic studies in prokaryotes.

Objectives AKR1B10, first cloned from liver cancer tissues, has recently been reported to be up-regulated significantly in hepatocellular carcinoma (HCC) tissues, but the relationship between serum level of AKR1B10 and the risk of HCC is not understood. Methods 170 HCC patients and 120 health donors from October 2014 to March 2017 were recruited in the affiliated hospital of Guilin Medical University. Serum AKR1B10 in all cases were detected and in 30 HCC patients were analyzed preoperatively and postoperatively by Time-resolved fluoroimmunoassay. Results The level of serum AKR1B10 was significantly higher in HCC patients (1800.24±2793.79) than in health donors (129.34±194.129), and downregulation of serum AKR1B10 in HCC patients was observed after hepatectomy. When samples were grouped according to the serum level of AKR1B10 (≥232.7pg/ml), serum AKR1B10 positively correlated to serum AFP (χ2=6.295, P=0.012), ALT (χ2=18.803, P=0.000), AST (χ2=33.421, P=0.000), tumor nodule number (χ2=6.777, P=0.009), cirrhosis (χ2=43.458, P=0.000), and tumor size (χ2=6.042, P=0.014) in the Chi-square test. Conclusions Diagnosis of HCC could be improved using the both predictors of serum AKR1B10 and AFP. AKR1B10 was thus considered to be a new serological biomarker for HCC.

Oxidative stress is elevated in the recipients of allogeneic hematopoietic transplantation (allo-HCT) and likely contributes to the development of graft-versus-host disease (GVHD). GVHD is characterized by activation, expansion, cytokine production and migration of alloreactive donor T cells, and remains a major cause of morbidity and mortality after allo-HCT. Hence, strategies to limit oxidative stress in GVHD are highly desirable. Thioredoxin1 (Trx1) counteracts oxidative stress by scavenging reactive oxygen species (ROS) and regulating other enzymes that metabolize H2O2. The present study sought to elucidate the role of Trx1 in the pathophysiology of GVHD. Using murine and xenograft models of allogeneic bone marrow transplantation (allo-BMT) and genetic (human Trx1-transgenic, Trx1-Tg) as well as pharmacologic (human recombinant Trx1, RTrx1) strategies; we found that Trx1-Tg donor T cells or administration of the recipients with RTrx1 significantly reduced GVHD severity. Mechanistically, we observed RTrx1 reduced ROS accumulation and cytokine production of mouse and human T cells in response to alloantigen stimulation in vitro. In allo-BMT settings, we found that Trx1-Tg or RTrx1 decreased downstream signaling molecules including NFκB activation and T-bet expression, and reduced proliferation, IFN-γ production and ROS accumulation in donor T cells within GVHD target organs. More importantly, administration of RTrx1 did not impair the graft-versus-leukemia (GVL) effect. Taken together, the current work provides a strong rationale and demonstrates feasibility to target the ROS pathway, which can be readily translated into clinic.

Aim: The roles of S1P in acute pancreatitis (AP) or non-AP patients with pancreatic acinar cell injury (PACI) are not well understood. Materials & methods: Serum S1P, in 40 healthy individuals and 99 patients with PACI, was retrospectively analyzed. Additionally, we detected and analyzed S1P in AP mice and the AR42J acinar cell line. Results: Serum S1P was significantly decreased in PACI patients, compared with that of healthy controls. Patients with gall stones, normal serum calcium or normal blood lipids showed relative higher levels of serum S1P. Interestingly, in patients with gall or liver disease, serum S1P was positively associated with γ-GT and ALT. Additionally, S1P and SPHK2 were decreased in AP mice and AR42J cells, relative to the levels of corresponding controls. Conclusion: Serum S1P is decreased in PACI, which may be partly due to downregulation of pancreatic SPHK2.

Aldo‐keto reductase family 1 member B10 (AKR1B10) is a secretory protein overexpressed in hepatocellular carcinoma (HCC). We aimed to evaluate AKR1B10 as a serum marker for detection of HCC. Herein, we conducted a cohort study that consecutively enrolled 1,244 participants from three independent hospitals, including HCC, healthy controls (HCs), benign liver tumors (BLTs), chronic hepatitis B (CHB), and liver cirrhosis (LC). Serum AKR1B10 was tested by time‐resolved fluorescent assays. Data were plotted for receiver operating characteristic (ROC) curve analyses. Alpha‐fetoprotein (AFP) was analyzed for comparison. An exploratory discovery cohort demonstrated that serum AKR1B10 increased in patients with HCC (1,567.3 ± 292.6 pg/mL; n = 69) compared with HCs (85.7 ± 10.9 pg/mL; n = 66; P < 0.0001). A training cohort of 519 participants yielded an optimal diagnostic cutoff of serum AKR1B10 at 267.9 pg/mL. When ROC curve was plotted for HCC versus all controls (HC + BLT + CHB + LC), serum AKR1B10 had diagnostic parameters of the area under the curve (AUC) 0.896, sensitivity 72.7%, and specificity 95.7%, which were better than AFP with AUC 0.816, sensitivity 65.1%, and specificity 88.9%. Impressively, AKR1B10 showed promising diagnostic potential in early‐stage HCC and AFP‐negative HCC. Combination of AKR1B10 with AFP increased diagnostic accuracy for HCC compared with AKR1B10 or AFP alone. A validation cohort of 522 participants confirmed these findings. An independent cohort of 68 patients with HCC who were followed up showed that serum AKR1B10 dramatically decreased 1 day after operation and was nearly back to normal 3 days after operation. Conclusion: AKR1B10 is a potent serum marker for detection of HCC and early‐stage HCC, with better diagnostic performance than AFP.

N6-methyladenosine (m6A) mRNA modifications play critical roles in various biological processes. However, no study addresses the role of m6A in macroautophagy/autophagy. Here, we show that m6A modifications are increased in H/R-treated cardiomyocytes and ischemia/reperfusion (I/R)-treated mice heart. We found that METTL3 (methyltransferase like 3) is the primary factor involved in aberrant m6A modification. Silencing METTL3 enhances autophagic flux and inhibits apoptosis in H/R-treated cardiomyocytes. However, overexpression of METTL3 or inhibition of the RNA demethylase ALKBH5 has an opposite effect, suggesting that METTL3 is a negative regulator of autophagy. Mechanistically, METTL3 methylates TFEB, a master regulator of lysosomal biogenesis and autophagy genes, at two m6A residues in the 3ʹ-UTR, which promotes the association of the RNA-binding protein HNRNPD with TFEB pre-mRNA and subsequently decreases the expression levels of TFEB. Further experiments show that autophagic flux enhanced by METTL3 deficiency is TFEB dependent. In turn, TFEB regulates the expression levels of METTL3 and ALKBH5 in opposite directions: it induces ALKBH5 and inhibits METTL3. TFEB binds to the ALKBH5 promoter and activates its transcription. In contrast, inhibition of METTL3 by TFEB does not involve transcriptional repression but rather downregulation of mRNA stability, thereby establishing a negative feedback loop. Together, our work uncovers a critical link between METTL3-ALKBH5 and autophagy, providing insight into the functional importance of the reversible mRNA m6A methylation and its modulators in ischemic heart disease. Abbreviations: ACTB, actin beta; ALKBH5, alkB homolog 5, RNA demethylase; ANXA5, annexin A5; ATG, autophagy-related; BafA, bafilomycin A1; CASP3, caspase 3; ELAVL1, ELAV like RNA binding protein 1; FTO, FTO, alpha-ketoglutarate dependent dioxygenase; GFP, green fluorescent protein; GST, glutathione S-transferase; HNRNPD, heterogeneous nuclear ribonucleoprotein D; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; m6A, N6-methyladenosine; MEFs, mouse embryo fibroblasts; Mer, mutated estrogen receptor domains; METTL3, methyltransferase like 3; METTL14, methyltransferase like 14; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin kinase complex 1; NMVCs, neonatal mouse ventricular cardiomyocytes; PCNA, proliferating cell nuclear antigen; PE, phosphatidylethanolamine; PI, propidium iodide; PTMs, post-translational modifications; PVDF, polyvinylidenedifluoride; RIP, RNA-immunoprecipitation; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TFEB, transcription factor EB; TUBA: tublin alpha; WTAP, WT1 associated protein; YTHDF, YTH N6-methyladenosine RNA binding protein

Background Obesity increases the risk of developing diabetes mellitus. Clinical studies suggest that risk factors like palmitic acid (PA) and lipopolysaccharide (LPS) exist simultaneously in diabetes with obesity. Combination of PA and LPS even at low concentration can induce strong inflammatory reaction. Monocyte chemoattractant protein-1 (MCP-1) is an important inflammatory chemokine related to insulin resistance and type II diabetes. Our previous study using PCR array revealed that LPS and PA synergistically induce MCP-1 mRNA expression in macrophage cells RAW264.7, while the protein expression of MCP-1 in this case was not investigated. Moreover, the underling mechanism in the synergistic effect of MCP-1 expression or production induced by treatment of LPS and PA combination remains unclear. Methods Protein secretion of MCP-1 was measured by the enzyme-linked immunosorbent assay (ELISA) and mRNA levels of MCP-1 and Toll-like receptor 4 (TLR4) were measured by real-time PCR. Statistical analysis was conducted using SPSS software. Results LPS could increase MCP-1 transcription as well as secretion in RAW264.7, and PA amplified this effect obviously. Meanwhile, combination of LPS with PA increased TLR4 mRNA expression while LPS alone or PA alone could not, TLR4 knockdown inhibited MCP-1 transcription/secretion induced by LPS plus PA. Moreover, not NF-κB inhibitor but inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK were found to block MCP-1 generation stimulated by LPS plus PA. Conclusion LPS and PA synergistically induced MCP-1 secretion in RAW264.7 macrophage cells, in which MCP-1 transcription mediated by MAPK/TLR4 signaling pathways was involved. Combined treatment of PA and LPS in RAW264.7 cells mimics the situation of diabetes with obesity that has higher level of PA and LPS, MAPK/TLR4/ MCP-1 might be potential therapeutic targets for diabetes with obesity.

UVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide in vitro and in vivo evidence that UVRAG ubiquitination at lysine residues 517 and 559 or prevention of Ser522 phosphorylation by D4476, a CSNK1A1 inhibitor, enhances the lysosomal degradation of EGFR, which significantly inhibits hepatocellular carcinoma (HCC) growth. Furthermore, UVRAG S522 phosphorylation levels correlate with ZRANB1 T35/S209 phosphorylation levels and poor prognosis in HCC patients. These findings identify a novel molecular mechanism by which ubiquitination and phosphorylation of UVRAG regulate its function in autophagosome maturation and HCC growth, encouraging further study of their potential therapeutic implications. Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; BECN1: beclin 1; CHX: cycloheximide; CSNK1A1/CK1α: casein kinase 1 alpha 1; CQ: chloroquine; DUB: deubiquitinase; EBSS: Earle’s balanced salt solution; EGF: epidermal growth factor; GFP: green fluorescent protein; GST: glutathione S-transferase; HBSS: Hanks balanced salts solution; HCC: hepatocellular carcinoma; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryo fibroblasts; mRFP: monomeric red fluorescent protein; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PTMs: post-translational modifications; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; Ub-AMC: ubiquitin-7-amido-4-methylcoumarin: a fluorogenic substrate; UVRAG: UV radiation resistance associated; ZRANB1/TRABID: zinc finger RANBP2-type containing 1

Long non-coding RNAs (lncRNAs) are non-coding RNAs longer than 200 nucleotides that function as regulatory factors in many human diseases, including cancer. However, majority of lncRNAs remain to be characterized. In this study, we characterized a novel lncRNA transcript, named UNC5B antisense RNA1 (UASR1). UASR1 is 647bp in length consisting of two exons. This lncRNA is an antisense of intron 1 of unc-5 netrin receptor B (UNC5B) gene. In breast cancer tissues, UASR1 was upregulated. Ectopic expression of UASR1 promoted proliferation and clonogenic growth of breast cancer cells MCF7 and MDA-MB-231. The migration of these cells also increased as demonstrated by wound healing and transwell assays. In contrast, silencing of UASR1 suppressed cell proliferation and migration. Further studies showed that UASR1 activated AKT and AKT-mediated mTOR signaling pathway to stimulate cell proliferation and growth. In these cells, active pAKT, pTSC2, p4EBP1 and pp70S6K were increased. Taken together, our data suggest that UASR1 plays an oncogenic role in breast cancer cells through activation of the AKT/mTOR signaling pathway, being a novel RNA oncogene.

Polymer hydrogels are generally insufficient biomechanics, strong resistance to cell adhesion, and weak bioactivity which limits their application in bone tissue engineering considerably. In order to develop a bone tissue engineering material with both good mechanical properties, osteogenic and angiogenic activity. Nanofibers carrying DNA plasmid (pNF) are introduced to gelatin methacryloyl (GelMA) and thiolated chitosan (TCS) system for preparing a novel GelMA/TCS/pNF composite hydrogel with dual network structure. By characterization of the compressive measurements, the resulting composite scaffold shows greatly enhanced mechanical strength (0.53 MPa) and is not damaged after 20 cycles of compression. And the fabricated composite scaffold displays sustained release of bone morphogenetic protein-2 that can induce osteogenic differentiation and angiopoietin-1 that promotes vascularization. The cell experiment shows that this system can significantly promote MC3T3-E1 cell attachment, proliferation, as well as osteogenic-related and angiogenic-related genes expression of MC3T3-E1 cells. Moreover, the in vivo results show that the composite scaffold with activated gene fibers can significantly promote osteogenesis and vascularization leading to favorable capacity of bone regeneration, meaning that the resulting biomimetic composite hydrogel scaffolds are excellent candidates for bone repair materials.

The incidence of pancreatitis (AP) is increasing and there is no specific treatment available. Intracellular digestive enzyme activation is a key event in the pathogenesis of AP downstream of cytosolic calcium overload and impaired autophagy. Siraitia grosvenorii (Swingle) was used in Traditional Chinese Medicine to reduce inflammation and facilitate bowel movement. The bioactive components of this plant show hypolipedimic, antidiabetic, antifibrotic activity and have been used against pancreatic cancer. Here, we examined whether mogroside IIE, a major bioactive component of unripe S. grosvenorii fruit, can protect against AP. We found that mogroside IIE decreased the activity of trypsin and cathepsin B induced by cerulein plus lipopolysaccharide (LPS) in the pancreatic acinar cell line AR42J and primary acinar cells in a dose- and time-dependent manner. Mogroside IIE treatment decreased the levels of serum lipase and serum amylase in mice injected with cerulein plus LPS without influencing inflammation significantly. A multi-cytokine array revealed that mogroside IIE decreased the level of interleukin 9 (IL-9) in AP mice. Exogenous IL-9 eliminated the mogroside IIE induced reduction of trypsin and cathepsin B activity and reversed the inhibition of cytosolic calcium and modulation of autophagy mediated by mogroside IIE. An IL-9 receptor antibody neutralized the effect of IL-9, restoring mogroside IIE activity. The mogroside IIE targeted IL-9 may partially arise from Th9 cells. Taken together, we provide experimental evidence that mogroside IIE ameliorates AP in cell models and mice through downregulation of the IL-9/IL-9 receptor pathway.

Additional file 1: Figure S1. This diagram illustrates the sieving process and its contribution to species delineation by whole-genome approaches. Figure S2. Illustration of genome-specific composition. Figure S3. Determination and sensitivity of the length-specific cutoffs (LSCs). Figure S4. Genome-specific cutoffs (GSCs). Figure S5. Setting of l in FRAGTE. Figure S6. Statistics for both Genomes and MAGs. Figure S7. Sieving performance comparison of FRAGTE and TETRA on MAGs. Figure S8. Sieving performance of FastANI on simulated genomes. Figure S9. Sieving performance of the 16S rRNA-based approach. Figure S10. FRAGTE pruned pairs with > 96% ANI but

Aldo-keto reductase 1B10 (AKR1B10) is upregulated in breast cancer and promotes tumor growth and metastasis. However, little is known of the molecular mechanisms of action. Herein we report that AKR1B10 activates lipid second messengers to stimulate cell proliferation. Our data showed that ectopic expression of AKR1B10 in breast cancer cells MCF-7 promoted lipogenesis and enhanced levels of lipid second messengers, including phosphatidylinositol bisphosphate (PIP2), diacylglycerol (DAG), and inositol triphosphate (IP3). In contrast, silencing of AKR1B10 in breast cancer cells BT-20 and colon cancer cells HCT-8 led to decrease of these lipid messengers. Qualitative analyses by liquid chromatography-mass spectrum (LC-MS) revealed that AKR1B10 regulated the cellular levels of total DAG and majority of subspecies. This in turn modulated the phosphorylation of protein kinase C (PKC) isoforms PKCδ (Thr505), PKCµ (Ser744/748), and PKCα/βII (Thr638/641) and activity of the PKC-mediated c-Raf/MEK/ERK signaling cascade. A pan inhibitor of PKC (Go6983) blocked ERK1/2 activation by AKR1B10. In these cells, phospho-p90RSK, phospho-MSK, and Cyclin D1 expression was increased by AKR1B10, and pharmacological inhibition of the ERK signaling cascade with MEK1/2 inhibitors U0126 and PD98059 eradicated induction of phospho-p90RSK, phospho-MSK, and Cyclin D1. In breast cancer cells, AKR1B10 promoted the clonogenic growth and proliferation of breast cancer cells in two-dimension (2D) and three-dimension (3D) cultures and tumor growth in immunodeficient female nude mice through activation of the PKC/ERK pathway. These data suggest that AKR1B10 stimulates breast cancer cell growth and proliferation through activation of DAG-mediated PKC/ERK signaling pathway.

It has been well established that patients with diabetes or metabolic syndrome (MetS) have increased prevalence and severity of periodontitis, an oral infection initiated by bacteria and characterized by tissue inflammation and destruction. To understand the underlying mechanisms, we have shown that saturated fatty acid (SFA), which is increased in patients with type 2 diabetes or MetS, and LPS, an important pathogenic factor for periodontitis, synergistically stimulate expression of proinflammatory cytokines in macrophages by increasing ceramide production. However, the mechanisms by which increased ceramide enhances proinflammatory cytokine expression have not been well understood. Since sphingosine 1 phosphate (S1P) is a metabolite of ceramide and a bioactive lipid, we tested our hypothesis that stimulation of ceramide production by LPS and SFA facilitates S1P production, which contributes to proinflammatory cytokine expression. Results showed that LPS and palmitate, a major SFA, synergistically increased not only ceramide, but also S1P, and stimulated sphingosine kinase (SK) expression and membrane translocation in RAW264.7 macrophages. Results also showed that SK inhibition attenuated the stimulatory effect of LPS and palmitate on IL-6 secretion. Moreover, results showed that S1P enhanced the stimulatory effect of LPS and palmitate on IL-6 secretion. Finally, results showed that targeting S1P receptors using either S1P receptor antagonists or small interfering RNA attenuated IL-6 upregulation by LPS and palmitate. Taken together, this study demonstrated that LPS and palmitate synergistically stimulated S1P production and S1P in turn contributed to the upregulation of proinflammatory cytokine expression in macrophages by LPS and palmitate. LPS and palmitate synergistically increase S1P and, in turn, S1P contributes to upregulation of proinflammatory genes by LPS and palmitate.

Hypopituitary dwarf mice demonstrate advantages of longevity, but little is known of their colon development and intestinal immunity. Herein we found that Ames dwarf mice have shorter colon and colonic crypts, but larger ratio of mesenteric lymph nodes (MLNs) over body weight than age-matched wild type (WT) mice. In the colonic lamina propria (cLP) of juvenile Ames mice, more inflammatory neutrophils (Ā: 0.15% vs. 0.03% in WT mice) and monocytes (Ā: 7.97% vs. 5.15%) infiltrated, and antigen presenting cells CD11c+ dendritic cells (Ā: 1.39% vs. 0.87%), CD11b+ macrophages (Ā: 3.22% vs. 0.81%) and gamma delta T (γδ T) cells (Ā: 5.56% vs. 1.35%) were increased. In adult Ames dwarf mice, adaptive immune cells, such as IL-17 producing CD4+ T helper (Th17) cells (Ā: 8.3% vs. 4.7%) were augmented. In the MLNs of Ames dwarf mice, the antigen presenting and adaptive immune cells also altered when compared to WT mice, such as a decrease of T-regulatory (Treg) cells in juvenile Ames mice (Ā: 7.7% vs.10.5%), but an increase of Th17 cells (Ā: 0.627% vs.0.093%). Taken together, these data suggest that somatotropic signaling deficiency influences colon development and intestinal immunity.

Complement plays a role in both hepatic ischemia reperfusion (IR) injury (IRI) and liver regeneration, but it is not clear how complement is activated in either process. We investigated the role of self-reactive immunoglobulin M (IgM) antibodies in activating complement after hepatic IR and liver resection. Natural IgM antibodies that recognize danger-associated molecular patterns (neoepitopes) activate complement following both hepatic IR and liver resection. Antibody-deficient Rag1-/- mice were protected from hepatic IRI, but had increased hepatic injury and an impaired regenerative response after 70% partial hepatectomy (PHx). We identified two IgM monoclonal antibodies (mAbs) that specifically reversed the effect of Rag1 deficiency in both models; B4 (recognizes Annexin IV) and C2 (recognizes subset of phospholipids). Focusing on the B4-specific response, we demonstrated sinusoidal colocalization of IgM and C3d in Rag1-/- mice that were reconstituted with B4 mAb, and furthermore that the Annexin IV neoepitope is specifically and similarly expressed after both hepatic IR and PHx in wild-type (WT) mice. A single-chain antibody construct (scFv) derived from B4 mAb blocked IgM binding and reduced injury post-IR in WT mice, although, interestingly, B4scFv did not alter regeneration post-PHx, indicating that anti-Annexin IV antibodies are sufficient, but not necessary, for the regenerative response in the context of an entire natural antibody repertoire. We also demonstrated expression of the B4 neoepitope in postischemic human liver samples obtained posttransplantation and a corollary depletion in IgM recognizing the B4 and C2 neoepitopes in patient sera following liver transplantation. Conclusion: These data indicate an important role for IgM in hepatic IRI and regeneration, with a similar cross-species injury-specific recognition system that has implications for the design of neoepitope targeted therapeutics. (Hepatology 2018;67:721-735).

Neutral ceramidase (NCDase) is a class of ceramidases, a key enzyme in ceramide degradation. Recently, it was observed that NCDase activity was suppressed by saturated fatty acids to increase ceramide content in rat muscle. However, little is known about its changes in activity and roles in palmitate (Palm)-induced lipotoxicity in pancreatic β cells. Here, we demonstrated that Palm treatment significantly down-regulated NCDase activity, mRNA and protein levels in rat INS-1 cells. In addition, Palm caused a significant accumulation of ceramide, while SPH level remained unchanged, suggesting that inhibition of NCDase activity led to no change of SPH level after treatment with Palm for 24 h. Furthermore, NCDase overexpression significantly reduced Palm-induced apoptosis in INS-1 cells. Conversely, NCDase siRNA knockdown markedly exacerbated Palm-induced apoptosis. In conclusion, Palm treatment suppressed the activity of NCDase and down-regulated its mRNA and protein expression. Furthermore, NCDase inhibition was involved in Palm-induced apoptosis by blocking ceramide degradation in INS-1 cells.

MicroRNAs (miRNAs) can regulate gene expression at post-transcriptional levels, thereby influence cancer risk. The aim of the current study is to investigate association between miR-199a rs74723057 and MET rs1621 and HCC risk in 1032 HCC patients and 1060 cancer-free controls. These two SNPs were genotyped by using the Agena MassARRAY genotyping system. Odds ratio (OR) and 95% confidence interval (95%CI) were calculated to assess the strength of the associations. We found that compared with the wild-type AA genotype of MET rs1621, the variant GG genotype was associated with a decreased risk for HCC (OR = 0.24, 95% CI = 0.06-0.96, P = 0.043). No association between miR-199a rs74723057 and HCC risk was observed. In addition, an interaction effect on HCC risk between the selected two SNPs was found. Among those who carried the CG/GG genotypes of miR-199a rs74723057, those who carried the GG genotype of MET rs1621 had a reduced risk of HCC, when compared with those who carried the AG/AA genotypes of MET rs1621 (OR = 0.15, 95% CI = 0.03~0.73, P for interaction = 0.018). Our results suggest that MET rs1621 polymorphism, alone and combined with miR-199a rs74723057, may influence susceptibility to HCC. Further large-scale association studies and functional studies are needed to validate our findings.

BackgroundGenomic composition has been found to be species specific and is used to differentiate bacterial species. To date, almost no published composition-based approaches are able to distinguish between most closely related organisms, including intra-genus species and intra-species strains. Thus, it is necessary to develop a novel approach to address this problem.ResultsHere, we initially determine that the “tetranucleotide-derived z-value Pearson correlation coefficient” (TETRA) approach is representative of other published statistical methods. Then, we devise a novel method called “Tetranucleotide-derivedZ-valueManhattanDistance” (TZMD) and compare it with the TETRA approach. Our results show that TZMD reflects the maximal genome difference, while TETRA does not in most conditions, demonstrating in theory that TZMD provides improved resolution. Additionally, our analysis of real data shows that TZMD improves species differentiation and clearly differentiates similar organisms, including similar species belonging to the same genospecies, subspecies and intraspecific strains, most of which cannot be distinguished by TETRA. Furthermore, TZMD is able to determine clonal strains with the TZMD = 0 criterion, which intrinsically encompasses identical composition, high average nucleotide identity and high percentage of shared genomes.ConclusionsOur extensive assessment demonstrates that TZMD has high resolution. This study is the first to propose a composition-based method for differentiating bacteria at the strain level and to demonstrate that composition is also strain specific. TZMD is a powerful tool and the first easy-to-use approach for differentiating clonal and non-clonal strains. Therefore, as the first composition-based algorithm for strain typing, TZMD will facilitate bacterial studies in the future.

Zhou et al. characterize a critical role of CLK3 on purine metabolism in cholangiocarcinoma (CCA). Overexpressed or mutated CLK3 promotes CCA progression in vitro and in vivo. Therefore, CLK3 may serve as a biomarker and potential drug target for CCA., CDC-like kinase 3 (CLK3) is a dual specificity kinase that functions on substrates containing serine/threonine and tyrosine. But its role in human cancer remains unknown. Herein, we demonstrated that CLK3 was significantly up-regulated in cholangiocarcinoma (CCA) and identified a recurrent Q607R somatic substitution that represented a gain-of-function mutation in the CLK3 kinase domain. Gene ontology term enrichment suggested that high CLK3 expression in CCA patients mainly was associated with nucleotide metabolism reprogramming, which was further confirmed by comparing metabolic profiling of CCA cells. CLK3 directly phosphorylated USP13 at Y708, which promoted its binding to c-Myc, thereby preventing Fbxl14-mediated c-Myc ubiquitination and activating the transcription of purine metabolic genes. Notably, the CCA-associated CLK3-Q607R mutant induced USP13-Y708 phosphorylation and enhanced the activity of c-Myc. In turn, c-Myc transcriptionally up-regulated CLK3. Finally, we identified tacrine hydrochloride as a potential drug to inhibit aberrant CLK3-induced CCA. These findings demonstrate that CLK3 plays a crucial role in CCA purine metabolism, suggesting a potential therapeutic utility., Graphical Abstract

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is a newly identified cytosolic DNA sensor, but its function in lung epithelial cells is relatively unknown. In the present study, the effects of lipopolysaccharide (LPS) on the expression and function of cGAS in the A549 lung epithelial cell line was investigated. The cells were treated with LPS at different concentrations (e.g., 100, 200, 400 and 800 ng/ml), and the cGAS expression levels were examined via western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The cells were pretreated with LPS, followed by E. coli DNA transfection using Lipofectamine(®) 3000. After 24 h, interferon (IFN)-β production was measured using ELISA and the expression of the autophagic markers, microtubule-associated proteins 1A/1B light chain 3 and sequestosome-1, were determined using western blot analysis. The cells were also pretreated with either a toll-like receptor (TLR) 4 inhibitor, a serine/threonine-protein kinase TBK1 (TBK1) inhibitor or an nuclear factor (NF)-κB inhibitor, followed by LPS treatment, and the cGAS expression levels were examined via western blot analysis and RT-qPCR. The result showed that LPS treatment upregulated cGAS expression in a dose-dependent manner. E. coli DNA treatment could induce IFN-β production and autophagy via cGAS, which was enhanced by LPS pretreatment. The effect of LPS on cGAS expression was suppressed by treatment with a TLR4 inhibitor, a TBK1 inhibitor and an NF-κB inhibitor. In conclusion, LPS enhances DNA-induced IFN-β production and autophagy by upregulating cGAS expression through the myeloid differentiation primary response protein MyD88-independent TLR4 signaling pathway in A549 cells.

Objective: To overcome the disadvantages of cisplatin, numerous platinum (Pt) complexes have been prepared. However, the anticancer activity and mechanism of Pt(II) complexed with 2-benzoylpyridine [Pt(II)- Bpy]: [PtCl2(DMSO)L] (DMSO = dimethyl sulfoxide, L = 2-benzoylpyridine) in cancer cells remain unknown. Methods: Pt(II)-Bpy was synthesized and characterized by spectrum analysis. Its anticancer activity and underlying mechanisms were demonstrated at the cellular, molecular, and in vivo levels. Results: Pt(II)-Bpy inhibited tumor cell growth, especially HepG2 human liver cancer cells, with a halfmaximal inhibitory concentration of 9.8±0.5μM, but with low toxicity in HL-7702 normal liver cells. Pt(II)- Bpy induced DNA damage, which was demonstrated through a marked increase in the expression of cleavedpoly (ADP ribose) polymerase (PARP) and gamma-H2A histone family member X and a decrease in PARP expression. The interaction of Pt(II)-Bpy with DNA at the molecular level was most likely through an intercalation mechanism, which might be evidence of DNA damage. Pt(II)-Bpy initiated cell cycle arrest at the S phase in HepG2 cells. It also caused severe loss of the mitochondrial membrane potential; a decrease in the expression of caspase-9 and caspase-3; an increase in reactive oxygen species levels; the release of cytochrome c and apoptotic protease activation factor; and the activation of caspase-9 and caspase-3 in HepG2 cells, which in turn resulted in apoptosis. Meanwhile, changes in p53 and related proteins were observed including the upregulation of p53, the phosphorylation of p53, p21, B-cell lymphoma-2-associated X protein, and NOXA; and the downregulation of B-cell lymphoma 2. Moreover, Pt(II)-Bpy displayed marked inhibitory effects on tumor growth in the HepG2 nude mouse model. Conclusion: Pt(II)-Bpy is a potential candidate for cancer chemotherapy.

Premature trypsinogen activation is the early event of acute pancreatitis. Therefore, the studies on the processes of trypsinogen activation induced by compounds are important to understand mechanism underly acute pancreatitis under various conditions. Calcium overload in the early stage of acute pancreatitis was previously found to cause intracellular trypsinogen activation; however, treatment of acute pancreatitis using calcium channel blockers did not produced consistent results. Proteasome activity that could be inhibited by some calcium channel blocker has recently been reported to affect the development of acute pancreatitis; however, the associated mechanism were not fully understood. Here, the roles of nicardipine were investigated in trypsinogen activation in pancreatic acinar cells. The results showed that nicardipine could increase cathepsin B activity that caused trypsinogen activation, but higher concentration of nicardipine or prolonged treatment had an opposite effect. The effects of short time treatment of nicardipine at low concentration were studied here. Proteasome inhibition was observed under nicardipine treatment that contributed to the up-regulation in cytosolic calcium. Increased cytosolic calcium from ER induced by nicardipine resulted in the release and activation of cathepsin B. Meanwhile, calcium chelator inhibited cathepsin B as well as trypsinogen activation. Consistently, proteasome activator protected acinar cells from injury induced by nicardipine. Moreover, proteasome inhibition caused by nicardipine depended on CaMKII. In conclusion, CaMKII down-regulation/proteasome inhibition/cytosolic calcium up-regulation/cathepsin B activation/trypsinogen activation axis was present in pancreatic acinar cells injury under nicardipine treatment.

Phosphatidylinositol 3-kinase (PI3K)/AKT pathway regulates cell growth, proliferation, survival, mobility and invasion. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway is also an important mitogenic signaling pathway involved in various cellular progresses. AKT, also named protein kinase B (PKB), is a primary mediator of the PI3K signaling pathway; and ERK at the end of MAPK signaling is the unique substrate and downstream effector of mitogen-activated protein/extracellular signal-regulated kinase (MEK). The AKT and ERK signaling are both aberrantly activated in a wide range of human cancers and have long been targeted for cancer therapy, but the clinical benefits of these targeted therapies have been limited due to complex cross-talk. Novel strategies, such as AKT/ERK dual inhibitors, may be needed.

Serum predictors for early diagnosis of hepatocellular carcinoma (HCC) have been investigated. Sphingosine-1-phosphate (S1P) has been widely reported to promote the survival of many types of cancer cells. However, the potential of serum S1P as a diagnostic marker in HCC has not been well characterized. The aim of this study was to identify the relationship between serum S1P and the risk of HCC.We retrospectively reviewed serum S1P in 63 HCC patients and 39 normal people. Receiver operating characteristic (ROC) curve analysis was performed to define the cut-off value of S1P in the serum. Chi-square test, t-test and multivariate regression analysis were used to investigate the association between serum S1P and individual clinicopathologic parameters.S1P showed significantly higher level in healthy subjects (1.372±0.116 μM) than that in patients (1.372±0.116 μM). Serum S1P in HCC patients was positively correlated to globulin (t = -3.122, p=0.003), hepatitis B virus (HBV) DNA copies (x2=4.386, p=0.036) and negatively related to AST (x2=2.870, p=0.09). Besides, part of the amount of serum S1P was negatively correlated to albumin (correlation coefficient (β) = -0.056) and positively correlated to alanine aminotransferase (ALT) (β=0.016) according to the regression analysis.These results suggested that serum S1P could be used as an auxiliary marker for HCC diagnosis, and used to monitor HBV infection in patients with HCC.

Additional file 2: Figure S1. Determination of the optimal species-level cutoffs for the four published methods based on F-score. Figure S2. F-scores for 10 samplings for each method. Figure S3. Determination of the species-level cutoffs for the four published methods based on Rand index. Figure S4. Rand indexes for 10 samplings for each method. Figure S5. Instraspecific Pearson correlation coefficient distribution for each method. Figure S6. Normalization of tetranucleotide-derived z-values. Figure S7. Histrogram showing the TZMD distributions for both intraspecific and interspecific pairs. Figure S8. Comparison base on Rand index showed TZMD slightly improved species differentiation. Figure S9. TETRA cannot differentiate Brucella species belonging to one single genospecies. Figure S10. TETRA cannot differentiate Y. pseudotuberculosis and Y. pestis belonging to one single genospecies. Figure S11. TZMD differentiates Y. pestis and Y. pseudotuberculosis belonging to one single genospecies. Figure S12. TETRA cannot differentiate B. mallei and B. pseudomallei belonging to one single genospecies. Figure S13. TZMD differentiates B. mallei and B. pesudomallei belonging to one single genospecies. Figure S14. TETRA cannot differentiates sub species and intraspecific strains of Campylobacter jejuni. Figure S15. TZMD differentiates all subspecies of Franscisella tularensis. Figure S16. TETRA cannot differentiate two subspecies of Francisella tularensis. Figure S17. TZMD distinguishes intraspecific strains of Streptococcus pyogenes. Figure S18. TETRA cannot differentiate intraspecific strains of Streptococcus pyogenes. Figure S19. TZMD distinguishes intraspecific strains of Bacillus cereus. Figure S20. TETRA cannot differentiate intraspecific strains of Bacillus cereus. Figure S21. Impact of genomic completeness on TZMD and TETRA for species differentiation. Figure S22. Binning performance of TZMD and TETRA. Table S1. TZMD and TETRA values for differently-sized genomes.

Aldo-keto reductase 1B10 (AKR1B10) is not expressed in normal breast, but upregulated in primary and metastatic breast cancers, being a negative prognostic factor. This study characterized the molecular mechanisms of AKR1B10-promoted breast cancer metastasis. Ectopic expression of AKR1B10 in breast cancer cells MCF-7 and MDA-MB-231 or siRNA-mediated silencing in BT-20 cells affected cell adhesion, migration and invasion in cell culture, and metastasis to the lung in the nude mice through upregulation of integrin α5 and δ-catenin. Silencing of integrin α5 or δ-catenin eradicated the cell adhesion and migration enhanced by AKR1B10, both of which acted synergistically. In these cells, the integrin α5 mediated focal adhesion kinase (FAK) signaling pathway was activated by AKR1B10, which, along with δ-catenin, stimulated Rac1-mediated cell migration and movement. In human primary and lymph node metastatic breast cancer, AKR1B10, integrin α5 and δ-catenin were correlatively upregulated with r=0.645 (p

Triptolide, the predominant biologically active component of the Chinese herb Tripterygium wilfordii Hook f., possesses numerous pharmacological activities, including anti-inflammatory, anti-fertility, anti-neoplastic, and immunosuppressive effects. However, toxicity and severe adverse effects, particularly hepatotoxicity, limit the clinical application of triptolide. Licorice root extract contains various bioactive compounds and is potent hepatoprotective. Magnesium isoglycyrrhizinate, a magnesium salt of the 18α-glycyrrhizic acid stereoisomer of glycyrrhizic acid, is used clinically in China to treat chronic viral hepatitis and acute drug-induced liver injury. The aim of this study was to investigate the role of the factor erythroid 2-related factor 2 pathway in the protective effects of LE and MIG against triptolide-induced hepatotoxicity. Hepatotoxicity models were established in L-02 cells and rats using triptolide, and the protective effects of LE and MIG were investigated in vitro and in vivo, respectively. LE and MIG significantly protected against triptolide-induced cytotoxicity. Additionally, triptolide decreased the mRNA and protein levels of Nrf2 and down-regulated Nrf2 target genes, including UGT1A, BSEP, and MRP2, while pretreatment with LE and MIG reversed these effects. Finally, Nrf2-involved antioxidant responses were activated in the presence of LE and MIG.

Neutral sphingomyelinase 1 (NSMase1) mediates caspase-3 activation and apoptosis. However, the role of NSMase1, especially exosome-borne NSMase1 in hepatocellular carcinoma (HCC), remains unclear. We report that NSMase1, which converts sphingomyelin (SM) to ceramide, was significantly downregulated in HCC tissues. Low NSMase1 expression predicted poor long-term survival of HCC patients. NSMase1 downregulation in HCC resulted in increased SM and reduced ceramide (Cer) that led to an increased SM/Cer ratio. Interestingly, NSMase1 and NSMase activity were also decreased in exosomes isolated from HCC tissues and cell lines. Furthermore, NSMase activity increased in exosomes isolated from the culture medium of L02 cells transfected with pEGFP-C3-NSMase1 (NSMase1-Exo). NSMase1-Exo suppressed HCC cell growth and induced apoptosis via reduction of the SM/Cer ratio. Thus, NSMase1 in exosomes inhibits HCC growth by decreasing the SM/Cer ratio.