Summary Tertiary lymphoid structure (TLS) provides a local and critical microenvironment for both cellular and humoral immunity and supports effective antigen presentation and lymphocyte activation. However, the gene expression profile and prognostic significance of TLS in oral cancer remain largely unrevealed. In this study, we found the presence of both intratumoral and peritumoral TLSs in a series of 65 patients with oral cancer treated by surgical resection, with positive detection rates of 33.8 and 75.4%, respectively. The presence of intratumoral TLSs, but not peritumoral TLSs, was significantly associated with decreased P53 and Ki67 scores (P = 0·027 and 0·047, respectively). The survival analyses revealed that oral cancer patients with higher grades of TLSs was associated with improved disease-free survival (DFS) and overall survival (OS) (P = 0·037 and 0·031, respectively). Gene expression profiling analysis of the cytokines and chemokines responsible for lymph-node neogenesis identified a three-up-regulated-gene set, i.e. IL7, LTB and CXCL13, which was shown to be correlated with human oral cancer-associated TLSs. This study provides a framework for better understanding of oral cancer-associated TLSs and for delineating future innovative prognostic biomarkers and immune therapeutic strategies for oral cancer.

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Chemical demulsification is widely used in the petroleum industry to remove water from crude oil all over the world. In this work, the relationship between the rheological properties of oil/water interfacial film and demulsification of crude oil emulsions was investigated. The results showed that the elastic modulus was the critical factor for the dehydration ratio, the emulsions showed high dehydration ratio when demulsifiers reduced the elastic modulus of oil–water film to a certain extent (below 5 mN/m). Correlations between dehydration ratio and equilibrium interfacial tension, dynamic interfacial tension, and loss modulus were also investigated. The results showed no correlation between the interfacial tension (IFT), the loss modulus, and the dehydration ratio. However, correlations were observed between dynamic IFT, loss modulus, and demulsification speed when the demulsifier could reduce the elastic modulus of the oil–water film to <5 mN/m. The loss modulus was directly proportional to the surface viscosity and could influence the demulsification speed, but it was not the critical factor. The dynamic interfacial tension was decisive factor to influence the demulsification speed. Dynamic Interfacial tension could be used to characterize the adsorption time (τ1) and the reorganization time (τ2) of demulsifier molecules at the interface, with the decrease of the τ (τ = τ1 + τ2), the demulsification speed increased. The rupture rate constant (k) of interfacial film increased with the decrease of τ, which accelerated the thinning and rupture of the film, thus increasing the demulsification speed.

BackgroundThe dysfunction of type I interferon (IFN) signaling is an important mechanism of immune escape and metastasis in tumors. Increased NOS1 expression has been detected in melanoma, which correlated with dysfunctional IFN signaling and poor response to immunotherapy, but the specific mechanism has not been determined. In this study, we investigated the regulation of NOS1 on the interferon response and clarified the relevant molecular mechanisms.MethodsAfter stable transfection of A375 cells with NOS1 expression plasmids, the transcription and expression of IFNα-stimulated genes (ISGs) were assessed using pISRE luciferase reporter gene analysis, RT-PCR, and western blotting, respectively. The effect of NOS1 on lung metastasis was assessed in melanoma mouse models. A biotin-switch assay was performed to detect the S-nitrosylation of HDAC2 by NOS1. ChIP-qPCR was conducted to measure the binding of HDAC2, H4K16ac, H4K5ac, H3ac, and RNA polymerase II in the promoters of ISGs after IFNα stimulation. This effect was further evaluated by altering the expression level of HDAC2 or by transfecting the HDAC2-C262A/C274A site mutant plasmids into cells. The coimmunoprecipitation assay was performed to detect the interaction of HDAC2 with STAT1 and STAT2. Loss-of-function and gain-of-function approaches were used to examine the effect of HDAC2-C262A/C274A on lung metastasis. Tumor infiltrating lymphocytes were analyzed by flow cytometry.ResultsHDAC2 is recruited to the promoter of ISGs and deacetylates H4K16 for the optimal expression of ISGs in response to IFNα treatment. Overexpression of NOS1 in melanoma cells decreases IFNα-responsiveness and induces the S-nitrosylation of HDAC2-C262/C274. This modification decreases the binding of HDAC2 with STAT1, thereby reducing the recruitment of HDAC2 to the ISG promoter and the deacetylation of H4K16. Moreover, expression of a mutant form of HDAC2, which cannot be nitrosylated, reverses the inhibition of ISG expression by NOS1 in vitro and decreases NOS1-induced lung metastasis and inhibition of tumor infiltrating lymphocytes in a melanoma mouse model.ConclusionsThis study provides evidence that NOS1 induces dysfunctional IFN signaling to promote lung metastasis in melanoma, highlighting NOS1-induced S-nitrosylation of HDAC2 in the regulation of IFN signaling via histone modification.

Extraction of soil nitrate nitrogen (NO 3 − -N) and ammonium nitrogen (NH 4 + -N) by chemical reagents and their determinations by continuous flow analysis were used to ascertain factors affecting analysis of soil mineral N. In this study, six factors affecting extraction of soil NO 3− -N and NH 4 + -N were investigated in 10 soils sampled from five arable fields in autumn and spring in northwestern China, with three replications for each soil sample. The six factors were air drying, sieve size (1, 3, and 5 mm), extracting solution [0.01 mol L −1 calcium chloride (CaCl 2 ), 1 mol L −1 potassium chloride (KCl), and 0.5 mol L −1 potassium sulfate (K 2 SO 4 )] and concentration (0.5, 1, and 2 mol L −1 KCl), solution-to-soil ratio (5:1, 10:1, and 20:1), shaking time (30, 60, and 120 min), storage time (2, 4, and 6 weeks), and storage temperature (−18 o C, 4 o C, and 25 o C) of extracted solution. The recovery of soil NO 3 − -N and NH 4 + -N was also measured to compare the differences of three extracting reagents (CaCl 2 , KCl, and K 2 SO 4 ) for NO 3 − -N and NH 4 + -N extraction. Air drying decreased NO 3 − -N but increased NH 4 + -N concentration in soil. Soil passed through a 3-mm sieve and shaken for 60 min yielded greater NO 3 − -N and NH 4 + -N concentrations compared to other treatments. The concentrations of extracted NO 3 − -N and NH 4 + -N in soil were significantly (P < 0.05) affected by extracting reagents. KCl was found to be most suitable for NO 3 − -N and NH 4 + -N extraction, as it had better recovery for soil mineral N extraction, which averaged 113.3% for NO 3 − -N and 94.9% for NH 4 + -N. K 2 SO 4 was not found suitable for NO 3 − -N extraction in soil, with an average recovery as high as 137.0%, and the average recovery of CaCl 2 was only 57.3% for NH 4 + -N. For KCl, the concentration of extracting solution played an important role, and 0.5 mol L −1 KCl could fully extract NO 3 − -N. A ratio of 10:1 of solution to soil was adequate for NO 3 − -N extraction, whereas the NH 4 + -N concentration was almost doubled when the solution-to-soil ratio was increased from 5:1 to 20:1. Storage of extracted solution at −18 • C, 4 • C, and 25 • C had no significant effect (P < 0.05) on NO 3 − -N concentration, whereas the NH 4 + -N concentration varied greatly with storage temperature. Storing the extracted solution at −18 o C obtained significantly (P < 0.05) similar results with that determined immediately for both NO 3 − -N and NH 4 + -N concentrations. Compared with the immediate extraction, the averaged NO 3 − -N concentration significantly (P < 0.05) increased after storing 2, 4, and 6 weeks, respectively, whereas NH 4 + -N varied in the two seasons. In conclusion, using fresh soil passed through a 3-mm sieve and extracted by 0.5 mol 571 Downloaded by [University of New Hampshire] at 11:25 18 February 2015 572 K.-Y. Li et al. L −1 KCl at a solution-to-soil ratio of 10:1 was suitable for extracting NO 3 − -N, whereas the concentration of extracted NH 4 + -N varied with KCl concentration and increased wi...

Biomaterials regulate macrophages and promote regeneration function, which is a new hot pot in tissue engineering and regenerative medicine. The research based on macrophage materials biology has appeared happy future, but related research on regulating macrophages and promoting tissue regeneration is still in its infancy. The surface roughness of biomaterials is one of the important factors affecting macrophage behavior. Previous study also found that the surface roughness of many biomaterials regulating macrophage polarization, but not including mineralized collagen (MC). In this study, we designed and fabricated MC with different roughness and investigated the influence of MC with different roughness on macrophages. In the study, we found that on the rough surface of MC, macrophages exhibited M1 phenotype-amoeboid morphology and high-level secretory of inflammatory factor (tumor necrosis factor-α and interleukin-6), while smoother surface exhibited M2 phenotype. These data will be beneficial to understand the mechanism deeply and enrich biomaterials tissue regeneration theory, provide a new train of thought biomaterials inducing tissue regeneration and repair and guide the optimum design of new biomaterials, development and reasonable applications.

Tempol (4-hydroxy-2,2,6,6-Tetramethylpiperidine-1-oxyl, TPL), a nitroxide compound, inhibits proliferation and increases the vulnerability of cancer cells to apoptosis induced by cytotoxic agents. However, the molecular mechanism of TPL inhibiting cancer cell proliferation has not been fully understood. In this study, we evaluated the metabolic effect of TPL on cancer cells and explored its cancer therapeutic potential. Extracellular flow assays showed that TPL inhibited cellular basal and maximal oxygen consumption rates of mitochondrial. 13C metabolic flux analysis showed that TPL treatment had minimal effect on glycolysis. However, we found that TPL inhibits glutamine metabolism by interfering with the oxidative tricarboxylic acid cycle (TCA) process and reductive glutamine process. We found that the inhibitory effect of TPL on metabolism occurs mainly on the step from citrate to α-ketoglutarate or vice versa. We also found that activity of isocitrate dehydrogenase IDH1 and IDH2, the key enzymes in TCA, were inhibited by TPL treatment. In xenograft mouse model, TPL treatment reduced tumor growth by inhibiting cellular proliferation of xenograft tumors. Thus, we provided a mechanism of TPL inhibiting cancer cell proliferation by interfering with glutamine utilization that is important for survival and proliferation of cancer cells. The study may help the development of a therapeutic strategy of TPL combined with other anticancer medicines.