GNUV201, a novel human/mouse cross-reactive and low pH-selective anti-PD-1 monoclonal antibody for cancer immunotherapy.

BACKGROUND: Several PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies. RESULTS: To overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the "FG loop" of hPD-1, distinct from those of Keytruda® ("C'D loop") and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201's binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME. CONCLUSIONS: In summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.

Biomarkers for Locally Advanced Hepatocellular Carcinoma Patients Treated with Liver-Directed Combined Radiotherapy.

Introduction: In the era of biomarker-driven cancer therapy, robust biomarkers for hepatocellular carcinoma (HCC) have not been well-defined. In this hypothesis-generating study, we investigated biomarkers that can be incorporated to predict treatment outcomes in patients with locally advanced HCC who are administered liver-directed combined radiotherapy (LDCRT). Methods: Ninety-nine patients with HCC who were treated with conventional fractionation LDCRT between July 2016 and October 2018 were enrolled in this prospective single-arm study. Clinical outcomes and possible serum biomarkers, including soluble programmed cell death ligand-1 (sPD-L1), interleukin (IL)-10, IL-6, cell-free DNA (cfDNA), inter-alpha inhibitor H4, and interferon-gamma, were analyzed. The primary endpoint was disease progression, and additional endpoints were local failure-free rate, intrahepatic failure-free rate, and lung metastasis-free rate. Results: The median follow-up period was 18.7 months. The 1-year progression-free rate was 38.2%. Increasing baseline sPD-L1 per pg/mL, previous treatment history, protein induced by vitamin K absence-II >1,629 mAU/mL, and multiple tumors were the adverse factors for progression based on multivariate analysis. Survival tree analysis revealed three prognostic groups for progression, in which patients with multiple lesions and baseline sPD-L1 ≥41.07 pg/mL showed the worst outcomes. For dynamic changes in biomarker levels, sPD-L1 fold change and cfDNA fold-change values were unfavorable factors for progression. Conclusion: Baseline sPD-L1, sPD-L1 fold change, and cfDNA fold-change values showed the highest potential as biomarkers for predicting post-treatment progression after LDCRT in HCC patients. By incorporating clinical factors, these biomarkers may be useful for devising a biomarker-driven treatment paradigm in locally advanced HCC.

Structural insight into a molecular mechanism of methenyltetrahydrofolate cyclohydrolase from Methylobacterium extorquens AM1.

Bioconversion of the C1 compounds into value-added products is one of the CO2-reducing strategies. In particular, because CO2 can be easily converted into formate, the efficient and direct bioconversion of CO2 through formate assimilation is attracting attention. The tetrahydrofolate (THF) cycle is the highly efficient reconstructed formate assimilation pathway, and 5,10-methenyltetrahydrofolate cyclohydrolase (FchA) is an essential enzyme involved in the THF cycle. In this study, a kinetic analysis of FchA from Methylobacterium extorquens AM1 (MeFchA) was performed and revealed that the enzyme has much higher cyclization than hydrolyzation activity, making it an optimal enzyme for formate assimilation. The crystal structure of MeFchA in the apo- and the THF-complexed forms was also determined, revealing that the substrate-binding site of the enzyme has three differently charged regions to stabilize the three differently charged moieties of the formyl-THF substrate. The residues involved in the substrate binding were also verified through site-directed mutagenesis. This study provides a biochemical and structural basis for the molecular mechanism underlying formate assimilation.

Circulating miR-122-5p and miR-375 as Potential Biomarkers for Bone Mass Recovery after Parathyroidectomy in Patients with Primary Hyperparathyroidism: A Proof-of-Concept Study.

Primary hyperparathyroidism (PHPT) is the leading cause of secondary osteoporosis. Although bone mineral density (BMD) tends to recover after parathyroidectomy in PHPT patients, the degree of recovery varies. Circulating microRNAs (miRNAs) profiles are known to be correlated with osteoporosis and fracture. We aimed to investigate whether osteoporotic fracture-related miRNAs are associated with postoperative BMD recovery in PHPT. Here, 16 previously identified osteoporotic fracture-related miRNAs were selected. We analyzed the association between the preoperative level of each miRNA and total hip (TH) BMD change. All 12 patients (among the 18 patients enrolled) were cured of PHPT after parathyroidectomy as parathyroid hormone (PTH) and calcium levels were restored to the normal range. Preoperative miR-19b-3p, miR-122-5p, and miR-375 showed a negative association with the percent changes in TH BMD from baseline. The association remained robust for miR-122-5p and miR-375 even after adjusting for sex, age, PTH, and procollagen type 1 N-terminal propeptide levels in a multivariable model. In conclusion, preoperative circulating miR-122-5p and miR-375 levels were negatively associated with TH BMD changes after parathyroidectomy in PHPT patients. miRNAs have the potential to serve as predictive biomarkers of treatment response in PHPT patients, which merits further investigation.

Role of Interleukin-7 in the Development of and Recovery from Radiation-Induced Lymphopenia: A Post-hoc Analysis of a Prospective Cohort.

PURPOSE: Radiation-induced lymphopenia is associated with worse outcomes in solid tumors. We assessed the impact of interleukin-7 (IL-7), a key cytokine in lymphocyte homeostasis, on radiation-induced lymphopenia. MATERIALS AND METHODS: A post-hoc analysis was performed in a prospective cohort of 98 patients with hepatocellular carcinoma who were treated with radiotherapy in 2016-2018. Blood IL-7 levels were assayed before and at the end of radiotherapy. Acute severe lymphopenia (ASL) was defined as a total lymphocyte count of < 200/µL during radiotherapy. Cox and logistic regression analyses were performed to identify predictors of survival and ASL development, respectively. RESULTS: Patients with ASL (n=41) had significantly poorer overall survival than those without (12.0 months vs. 25.3 months, p=0.001). Patients with lymphocyte recovery showed significantly longer overall survival than those without (21.8 months vs. 10.3 months, p=0.042). ASL was an independent predictor of poor survival (hazard ratio, 2.07; p=0.015). Patients with ASL had significantly lower pre-radiotherapy IL-7 levels (2.07 pg/mL vs. 3.01 pg/mL, p=0.010). A high pre-radiotherapy IL-7 level was an independent predictor of a reduced risk of ASL development (hazard ratio, 0.40; p=0.004). IL-7 levels reflected a feedback response to ASL, with a higher ΔIL-7 in patients with ASL and a lower ΔIL-7 in those without ASL (0.48 pg/mL vs. -0.66 pg/mL, p < 0.001). Post-radiotherapy IL-7 levels were significantly positively correlated with the total lymphocyte counts at 2 months. CONCLUSION: IL-7 is associated with the development of and recovery from ASL, which may impact survival. To overcome radiation-induced lymphopenia, a novel strategy using IL-7 may be considered.

Effect of Interleukin-7 on Radiation-Induced Lymphopenia and Its Antitumor Effects in a Mouse Model.

PURPOSE: Local ionizing radiation (IR) can lead to systemic lymphocyte depletion, which is associated with poor survival outcomes in patients with cancer. Interleukin-7 (IL-7) plays an important role in lymphocyte homeostasis; however, its role in alleviating radiation-induced lymphopenia remains unclear. Hence, we established a radiation-induced lymphopenia animal model and evaluated the effect of exogenous IL-7 administration. METHODS: C3H/HeN mice underwent x-ray irradiation of 30 Gy in 10 fractions at the right hind limbs. Next, 10 mg/kg of IL-7 was injected subcutaneously, and the lymphocyte count in blood was measured. Murine hepatocellular carcinoma (HCa-1) cells were inoculated subcutaneously into the right thighs of tumor model mice, which underwent the same treatment. RESULTS: In the naïve mouse model, the decreased CD45+ cell count after irradiation gradually recovered to the initial level over 3 weeks in the IR group, whereas it markedly increased to 373% of the initial level in 1 week in the IR+IL-7 group. Similar trends were observed for the CD3+, CD8+, CD4+, regulatory T cells, and CD19+ B cell counts. Similar findings were observed in the tumor mouse model. CD8+ and CD4+ T cell infiltration in tumor specimens was higher in the IL-7 and IR+IL-7 groups than in the nontreated and IR groups. Tumor growth was significantly more suppressed in the IR+IL-7 group than in the IR group. The median survival time was significantly longer in the IR+IL-7 group (not reached) than in the IR (56 days; P = .0382), IL-7 (36 days; P = .0004), or nontreated groups (36 days; P < .0001). CONCLUSIONS: Administration of exogenous IL-7 after IR not only restored lymphocyte counts but also enhanced the antitumor effect. Exogenous IL-7 can be beneficial in overcoming radiation-induced lymphopenia and in enhancing the treatment outcome in combination with radiation therapy, which needs validation through future clinical studies.

Combination therapy with anti-T-cell immunoglobulin and mucin-domain containing molecule 3 and radiation improves antitumor efficacy in murine hepatocellular carcinoma.

BACKGROUND AND AIM: T-cell immunoglobulin and mucin-domain containing molecule 3 (TIM3) has emerged as a promising immune checkpoint inhibitor target; however, immune checkpoint inhibitor monotherapy does not benefit a substantial percentage of patients. Therefore, this study investigated the antitumor effect of anti-TIM3 therapy combined with radiation in a murine hepatocellular carcinoma (HCC) model. METHODS: The effect of radiation on TIM3 expression was determined in murine and human HCC cells using western blotting, immunohistochemistry, and flow cytometry. Tumor growth and survival rate were measured to evaluate the antitumor effect of this combination therapy. Tumor immunological parameters were assessed using flow cytometry and histology. RESULTS: TIM3 was upregulated in tumor-infiltrating CD8+ and CD4+ T cells in radiation-treated HCa-1-implanted mice. Combination treatment significantly delayed tumor growth compared with monotherapy (P < 0.01). Overall survival was improved in the combination group compared with that in the anti-TIM3 or radiation monotherapy groups (median survival time: 52 days vs 26 or 38 days, respectively, P < 0.001). The antitumor effect of the combination treatment was associated with increased apoptosis and decreased proliferation of tumor cells and reinvigorated CD8+ T-cell activation. CD8+ T-cell depletion reversed the antitumor efficacy of the combination treatment. These findings suggest that CD8+ T cells play key roles in the therapeutic effect of the combination treatment. CONCLUSION: Anti-TIM3 and radiation combination therapy significantly improved the antitumor effect in a murine HCC model, as evidenced by inhibited tumor growth and increased overall survival. This approach could be a novel combined immune-radiotherapy strategy for HCC.

Clinical significance of soluble programmed cell death ligand-1 (sPD-L1) in hepatocellular carcinoma patients treated with radiotherapy.

PURPOSE: To investigate the clinical implications of the soluble programmed cell death-ligand 1 (sPD-L1) level in hepatocellular carcinoma (HCC) patients treated with radiotherapy (RT). MATERIALS/METHODS: HCC patients treated with RT between June 2011 and March 2015 were prospectively recruited and sPD-L1 levels were measured using an enzyme-linked immunosorbent assay. Blood samples were obtained at the RT start, RT end, and 1-month follow-up. The associations of the sPD-L1 level with the clinical features and outcomes were analyzed. RESULTS: Fifty-three patients with HCC were included. Thirty-four patients received conventional fractionated RT with hepatic arterial infusional chemotherapy, while 19 patients received stereotactic body radiotherapy (SBRT). The initial sPD-L1 level was significantly associated with stage, tumor size, portal vein tumor thrombosis, and venous invasion. The overall-survival was significantly poorer in patients with a higher level of initial sPD-L1 (≥1.315 pg/mL). A higher level of sPD-L1 at 1 month (≥12.9 pg/mL) was significantly related to early lung metastasis. The sPD-L1 level was significantly increased after RT and the change pattern of sPD-L1 was different between two RT schemes. CONCLUSIONS: The level of sPD-L1 was associated with tumor aggressiveness and outcomes, suggesting its role as a possible predictive biomarker. The increases in sPD-L1 after RT suggests that combined treatment with RT and immune checkpoint inhibitors may be a promising therapeutic strategy in HCC.

Inter-alpha Inhibitor H4 as a Potential Biomarker Predicting the Treatment Outcomes in Patients with Hepatocellular Carcinoma.

PURPOSE: Early prediction of treatment outcomes represents an essential step towards increased treatment efficacy and survival in patients with hepatocellular carcinoma (HCC). In this study, we performed two-dimensional electrophoresis (2-DE) followed by protein profiling to identify biomarkers predictive of therapeutic outcomes in patients with HCC who received liver-directed therapy (LDTx) involving local radiotherapy (RT), and studied the underlying mechanisms of the identified proteins. MATERIALS AND METHODS: 2-DE analysis was conducted by pooling sera from patients with a good or poor prognosis; serum proteomic profiles of the two groups were compared and analyzed using matrixassisted laser desorption/ionization time-of-flight mass spectrometry. Identified proteins were confirmed via enzyme-linked immunosorbent assay. An invasion assay was performed after overexpression and knockdown of target protein in Huh7 cells. RESULTS: Levels of inter-alpha inhibitor H4 (ITIH4), fibrinogen gamma chain, keratin 9/1 complex, carbonic anhydrase I, and carbonmonoxyhemoglobin S were changed by more than 4-fold in response to LDTx. In particular, pre-LDTx ITIH4 expression was more than 5-fold higher in patients with a good prognosis, compared to patients with a poor prognosis. The migration ability of Huh7 cells was significantly suppressed and enhanced by ITIH4 overexpression and knockdown, respectively. The tumors of patients with HCC and a good prognosis expressed high levels of ITIH4, compared to those of patients with a poor prognosis. CONCLUSION: Taken together, ITIH4 may be a potential therapeutic target that could inhibit cancer metastasis, as well as a prognostic marker for patients with HCC who are receiving LDTx.

Radiation improves antitumor effect of immune checkpoint inhibitor in murine hepatocellular carcinoma model.

BACKGROUND & AIMS: Although immunotherapy has emerged as an attractive therapy for refractory cancers, its limited efficacy in hepatocellular carcinoma (HCC) suggests the need for a combination strategy that can either enhance or complement therapeutic effect. We investigated whether combination of immune checkpoint blockade (ICB) and radiation could enhance antitumor effect in a murine HCC model. METHODS: Using murine HCC, HCa-1, the effect of radiation on programmed death-ligand1 (PD-L1) expression was determined by real-time PCR, flow cytometry, and western blotting. Signaling pathways involved in altered PD-L1 expression were examined. Tumor growth and survival rate were evaluated for a combination of anti-PD-L1 and radiation. Immunological parameters in the tumor were assessed using flow cytometry and histological study. RESULTS: Radiation upregulated PD-L1 expression in tumor cells through IFN-γ/STAT3 signaling, which could facilitate therapeutic action of anti-PD-L1. Combination of anti-PD-L1 and radiation significantly suppressed tumor growth compared to treatment with anti-PD-L1 alone or radiation alone group (P<0.01). Survival was significantly improved in the combination group compared to anti-PD-L1 alone or radiation alone group (7-week survival rate; 90% vs. 0% or 30%, respectively, P<0.001). The underlying mechanism involved increasing apoptosis, decreasing tumor cell proliferation, as well as restoration of CD8+ T cell functions. CONCLUSIONS: The combination of anti-PD-L1 and radiation significantly improved the antitumor effect shown in tumor growth delay as well as in survival, supporting a novel combination strategy of immunoradiotherapy in HCC.

A MALDI-MS-based quantitative analytical method for endogenous estrone in human breast cancer cells.

The level of endogenous estrone, one of the three major naturally occurring estrogens, has a significant correlation with the incidence of post-menopausal breast cancer. However, it is challenging to quantitatively monitor it owing to its low abundance. Here, we develop a robust and highly sensitive mass-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based quantitative platform to identify the absolute quantities of endogenous estrones in a variety of clinical specimens. The one-step modification of endogenous estrone provided good linearity (R(2) > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol). In addition, we could identify the absolute amount of endogenous estrones in cells of the breast cancer cell line MCF-7 (34 fmol/10(6) cells) by using a deuterated estrone as an internal standard. Finally, by applying the MALDI-MS-based quantitative method to endogenous estrones, we successfully monitored changes in the metabolic expression level of estrones (17.7 fmol/10(6) letrozole-treated cells) in MCF-7 cells resulting from treatment with an aromatase inhibitor. Taken together, these results suggest that this MALDI-MS-based quantitative approach may be a general method for the targeted metabolomics of ketone-containing metabolites, which can reflect clinical conditions and pathogenic mechanisms.

Radiation Inhibits Interleukin-12 Production via Inhibition of C-Rel through the Interleukin-6/ Signal Transducer and Activator of Transcription 3 Signaling Pathway in Dendritic Cells.

Radiotherapy (RT) is a potent anti-tumor modality. However, unwanted effects including increased recurrence and metastasis that involve factors such as cytokines, which induce complex molecular mechanisms, have also been reported. In a previous study, we showed that interleukin (IL)-12 and radiotherapy combination treatment suppressed tumor growth and metastasis in a hepatoma mouse model. In this study, we investigated the mechanism underlying the IL-12 anti-tumor effect during radiotherapy. In tumor-bearing mice, irradiation decreased IL-12 expression in the tumors and spleens. However, a number of dendritic cells infiltrated into the tumors in which IL-12 expression did not decrease. To further study the underlying detailed mechanism for this decrease in IL-12, LPS-stimulated bone marrow-derived dendritic cells (BMDCs) were irradiated, and then IL-12- and IL-6-associated molecules were examined in irradiated tumors and BMDCs. Irradiation resulted in IL-12 suppression and IL-6 increase. IL-6 and signal transducer and activator of transcription 3 (STAT3) inhibitors restored the irradiation-induced IL-12 decrease via suppression of C-Rel activation. Taken together, our study suggests that irradiation-induced IL-6 can decrease IL-12 production through the inhibition of C-Rel phosphorylation by the IL-6/STAT3 signaling pathway.

Expression of Cryptosporidium parvum thioredoxin peroxidase in COS-7 cells confers radioprotection.

Cryptosporidium parvum is one of the most radioresistant organisms identified to date. In a previous study, we found that thioredoxin peroxidase (CpTPx) was significantly upregulated in this species following exposure to high dose (10 kGy) of γ-irradiation. To assess the potential of CpTPx to confer radioprotection in mammalian cells, it was expressed in COS-7 African green monkey kidney cells (CpTPx-COS7). For comparison, the thioredoxin peroxidase of Cryptosporidium muris (CmTPx) was also expressed in these cells (CmTPx-COS7 cells), which has been confirmed to have lesser antioxidant activity than CpTPx in the previous study. Notably, the survival rates of CpTPx-COS7 cells were significantly higher (12-22%) at 72 h after 8 Gy irradiation than CmTPx-COS7 or non-transfected COS-7 (ntCOS-7) counterparts. In addition, CpTPx revealed a 50% of ROS reduction in irradiated CpTPx-COS7 cells, while γ-H2AX DNA damage marker expression was not significantly changed. Furthermore, the amount of apoptosis only increased to about 120% after 2-8 Gy irradiation compared to 200-300% increase observed in ntCOS-7 cells. CmTPx was shown to have antioxidant and DNA damage protection activities; however, these activities were always lower than those of CpTPx. These results suggest that the potent antioxidant and protective activities of CpTPx are well conserved in this cell-based system and that CpTPx contributed to the radioprotection of mammalian cells through its exceptional antioxidant activity.

Molar root-incisor malformation: considerations of diverse developmental and etiologic factors.

OBJECTIVE: The objective of this study was to evaluate the variation in the condition referred to as molar root-incisor malformation (MRIM) and elucidate the distribution of affected teeth. This study further aimed to identify associated environmental stressors. STUDY DESIGN: Individuals were identified through retrospective review of dental radiographs and through referral to the investigators. Histologic evaluation included examination of mineralized and decalcified sections of affected first permanent molar teeth. RESULTS: Thirty cases of MRIM were identified, with all having affected first permanent molars with dysplastic root formation. The primary second molars were affected in 57% of the cases, with permanent anterior teeth being involved in 40% of the cases. A variety of medical conditions were associated with MRIM, the most common being neurologic. Several affected individuals reported no significant past medical history or environmental stressors. CONCLUSIONS: The etiology of MRIM remains unclear, and this unique developmental defect of the first permanent molar roots appears to occur in populations throughout the world. Clinicians identifying the MRIM phenotype should carefully evaluate the permanent incisors for associated developmental defects that could result in pulpal necrosis.

A MALDI-MS-based quantitative targeted glycomics (MALDI-QTaG) for total N-glycan analysis.

OBJECTIVES: To develop a sensitive and quantitative method for monitoring the abnormal glycosylation of clinical and biopharmaceutical products. RESULTS: MALDI-MS-based quantitative targeted glycomics (MALDI-QTaG) was proposed for sensitive and quantitative analysis of total N-glycans. The derivatization reactions (i.e., amidation of sialic acid and incorporation of a positive charge moiety into the reducing end) dramatically increased the linearity (R(2) > 0.99) and sensitivity (limit of detection is 0.5 pmol/glycoprotein) relative to underivatized glycans. In addition, the analytical strategy was chromatographic purification-free and non-laborious process accessible to the high-throughput analyses. We used MALDI-QTaG to analyze the N-glycans of α-fetoprotein (AFP) purified from normal cord blood and HCC cell line (Huh7 cells). The total percentages of core-fucosylated AFP N-glycans from Huh7 cells and normal cord blood were 98 and 18%, respectively. CONCLUSIONS: This MALDI-MS-based glycomics technology has wide applications in many clinical and bioengineering fields requiring sensitive, quantitative and fast N-glycosylation validation.

Mass spectrometry-based N-linked glycomic profiling as a means for tracking pancreatic cancer metastasis.

The aberrant glycosylation profile on the surface of cancer cells has been recognized for its potential diagnostic value towards assessing tumor progression. In this study, we initially investigate N-glycan profiles on the surface of normal (HPDE) and cancerous (Capan-1, Panc-1, and MIA PaCa-2) pancreatic cell lines, which are from different sites of pancreatic tumor. The enzymatically deglycosylated total N-glycans are permethylated via a quantitative solid-phase method and then analyzed by using MALDI-TOF MS and MALDI-QIT-TOF MS. We demonstrate that the level of high-mannose type glycans is higher among Capan-1 cells-pancreatic cancer cells that have metastasized to the liver-than that observed among Panc-1 and MIA PaCa-2 cells-pancreatic cancer cells from the pancreas duct head and tail regions, respectively. Furthermore, the relative abundance of highly-branched sialyted N-glycans is significantly up-regulated on Panc-1 and MIA PaCa-2 pancreatic cancer cells compared to that of normal HPDE pancreas cells. Taken together, these results indicate that specific N-glycosylation profile changes in pancreatic cancer cells can be used to not only distinguish between normal and cancerous cells but also provide more information on their location and metastatic potential.

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics.

MALDI-MS-based quantitative analysis for ketone containing homoserine lactones in Pseudomonas aeruginosa.

N-Acyl homoserine lactones (AHLs), quorum sensing molecules produced by Gram-negative bacteria, are used as important secondary metabolites for antibacterial drug development and cell-to-cell communication. Although various analytical techniques have been developed for detection and quantitation of AHLs from more complex bacterial culture media, only a few methods have been applied to AHL identification in physiological samples. Here, we developed a highly sensitive and reliable MALDI-based 3-oxo AHL quantitation method by employing Girard's reagent T (GT) to produce a permanent cationic charge state [M](+) at the ketone group of AHLs. After extracting AHLs from the supernatant of bacterial cultures using ethyl acetate, the extracts were subsequently derivatized with GT without any additional purification or desalting steps. The chemical derivatization of 3-oxo AHLs dramatically enhanced sensitivity (up to 60 000 times) by lowering the limit of detection (LOD, ∼0.5 fmol)/limit of quantitation (LOQ, ∼2.5 fmol). Additionally, the GT-derivatized 3-oxo AHLs allowed more accurate quantitative analysis from the Pseudomonas aeruginosa PAO1 culture supernatants. This method may be applied for developing high-throughput and sensitive detection methods of quorum sensing signal molecules in biofilm-related clinical applications such as virulence factor characterization and antibacterial drug development.

Comparative N-linked glycan analysis of wild-type and α1,3-galactosyltransferase gene knock-out pig fibroblasts using mass spectrometry approaches.

Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after α1,3-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (α-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.

Platelet-activating factor enhances tumour metastasis via the reactive oxygen species-dependent protein kinase casein kinase 2-mediated nuclear factor-κB activation.

Platelet-activating factor (PAF) promotes tumour metastasis via activation of the transcription factor nuclear factor-κB (NF-κB). We here investigated the role of the protein kinase CK2 (formerly Casein Kinase 2 or II) in PAF-induced NF-κB activation and tumour metastasis, given that PAF has been reported to increase CK2 activity, and that CK2 plays a key role in NF-κB activation. PAF increased CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. CK2 inhibitors inhibited the PAF-mediated NF-κB activation and expression of NF-κB-dependent pro-inflammatory cytokines and anti-apoptotic factors. Pre-treatment with the antioxidant N-Acetyl-L-Cysteine (NAC) resulted in a significant inhibition in PAF-induced enhancement of CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. H2 O2 and known reactive oxygen species inducers, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) enhanced CK2 activity, phosphorylation and protein expression, which was again inhibited by antioxidant. PAF, LPS and TNF-α induced increased CK2 activity, phosphorylationand protein expression, which were inhibited by p38 inhibitor. PAF, LPS or TNF-α increased pulmonary metastasis of B16F10, which was inhibited by antioxidants, CK2 inhibitor and p38 inhibitor. Our data suggest that (i) reactive oxygen species activate CK2 via p38, which, in turn, induces NF-κB activation, and (ii) PAF, LPS and TNF-α increase pulmonary tumour metastasis via the induction of the reactive oxygen species (ROS)/p38/CK2/NF-κB pathway.