Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15N-labeling approach, the fates of supplemented 15N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the 15N atoms percent of NH4+, NO2-, NO3-, NH2OH, and N2H4, where liquid samples (<0.5 mL) were mixed with colorimetric reagents (naphthylethylenediamine for NO2-, indophenol for NH4+, and p-aminobenzaldehyde for N2H4), and the mass spectra of the formed N complex dyes were obtained by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. NH2OH and NO3- were chemically converted to NO2- by iodine oxidation and copper/hydrazine reduction reaction, respectively, prior to the above colorimetric reaction. The intensity of the isotope peak (M + 1 or M + 2) increased when the N complex dye was formed by coupling with a 15N-labeled compound, and a linear relationship was found between the determined 15N/14N peak ratio and 15N atom% for the tested N species. The developed method was applied to bacterial cultures to examine their N-transformation reactions, enabling us to observe the occurrence of NO2- oxidation and NO3- reduction in a hypoxic Nitrobacter winogradskyi culture. IMPORTANCE15N/14N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15N atom% of NO2- can be determined from >1,000 liquid samples daily at <$1 (U.S.) per 384 samples for routine analysis. This convenient MS method is a powerful tool that will advance our ability to explore the N-transformation reactions that occur in various environments and biological samples.

Effectiveness of tenofovir alafenamide for chronic hepatitis B patients with a poor response to the previously used nucleos(t)ide analogs.

BACKGROUND: Few studies have demonstrated the potency of tenofovir alafenamide (TAF) in patients with poor response to other nucleos(t)ide analogs (NAs). METHODS: We conducted a retrospective study comprising consecutive 40 patients exhibiting a poor response to other NAs, who subsequently received TAF-containing regimens. The primary outcome was the prevalence of virological response (VR) at each time and maintained virological response (MVR) under TAF-containing regimens until week 96. RESULTS: In the entire cohort, the prevalence of MVR was 71.1% (27/38). Further, poor tenofovir disoproxil fumarate (TDF) response was significantly associated with a lower prevalence of MVR (p = 0.014). In TDF-naïve patients, the prevalence of MVR was 92.3% (12/13) and 62.5% (5/8) in patients with lamivudine resistance (LAM-r) and entecavir resistance (ETV-r), respectively. Further, viral load and HBeAg status at baseline were associated with a lower prevalence of MVR (p = 0.013). Among the seven patients with prior TDF exposure, 2 patients achieved MVR. Among them, one patient with development of viral breakthrough during TDF/LAM achieved MVR after switching to TAF/ETV. In contrast, one of the five patients with non-MVR had three substitutions (rtS106C, rtD134N/S, and rtL269I) of quadruple mutations in addition to ETV-r. Other patients with rtA181T + rtN236T also could not achieve MVR. CONCLUSION: TAF exhibited high antiviral potency in patients with LAM-r and ETV-r. However, TAF potency was associated with previous TDF response, viral load, and HBeAg status at baseline. Additionally, a quadruple mutation may impact tenofovir resistance; however, further studies are needed to verify this.

Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase.

Antimicrobial resistance is a global public threat and raises the need for development of new antibiotics with a novel mode of action. The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to a new class of serine peptidases, family S46. Because S46 peptidases are not found in mammals, these enzymes are attractive targets for novel antibiotics. However, potent and selective inhibitors of these peptidases have not been developed to date. In this study, a high-resolution crystal structure analysis of PgDPP11 using a space-grown crystal enabled us to identify the binding of citrate ion, which could be regarded as a lead fragment mimicking the binding of a substrate peptide with acidic amino acids, in the S1 subsite. The citrate-based pharmacophore was utilized for in silico inhibitor screening. The screening resulted in an active compound SH-5, the first nonpeptidyl inhibitor of S46 peptidases. SH-5 and a lipophilic analog of SH-5 showed a dose-dependent inhibitory effect against the growth of P. gingivalis. The binding mode of SH-5 was confirmed by crystal structure analysis. Thus, these compounds could be lead structures for the development of selective inhibitors of PgDPP11.

Tumor-specific CD8-positive T cell-mediated antitumor immunity is implicated in the antitumor effect of local hyperthermia.

PURPOSE: This study aimed to elucidate the contribution of T cell-mediated antitumor immunity in the antitumor effect of local hyperthermia (LH). MATERIALS AND METHODS: C57BL/6J mice were injected with the mouse lymphoma cell line, E.G7-OVA, in the right femur on day 0. LH was induced by immersing the right femur in a water bath at 42 °C for 60 min on day 7, followed by administration of anti-CD8 monoclonal antibodies (mAb) or anti-CTLA-4 mAb (days 8, 11, and 14). The effect of LH on tumor growth (TG) was assessed by measuring the duration until tumor volume reached 1000 mm3 and survival time. Tumor-specific T cell responses were measured using enzyme-linked immunospot (ELISpot) assay. RESULTS: TG with and without LH treatment was 9.0 ± 9.6 and 7.0 ± 1.6 days, respectively. TG was significantly slower with LH treatment (p = .01). The therapeutic effect of LH was mitigated by addition of anti-CD8 mAb (p < .05 for both TG and survival) compared with the untreated (control) group. Furthermore, addition of anti-CTLA-4 mAb did not significantly affect the therapeutic effect of LH. The ELISpot assay showed that the number of spots in the LH group (276.3 ± 14.5) was significantly greater than in the control group (59.0 ± 4.5, p < .001). CONCLUSION: CD8-positive T cell-mediated antitumor immunity significantly contributes to the antitumor effect of LH.

Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant GM1 ß-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations.

HLA class I expression and its alteration by preoperative hyperthermo-chemoradiotherapy in patients with rectal cancer.

OBJECTIVE: Enhancing immunologic responses, including human leukocyte antigen (HLA) class I expression on tumor cells and recognition and elimination of tumor cells by tumor-specific cytotoxic T lymphocyte (CTL), is considered a novel concept of radiotherapy. The present study examined patients who underwent preoperative hyperthermo-chemoradiotherapy (HCRT) for locally advanced rectal cancer to assess the correlation between HLA class I expression and clinical outcome. MATERIALS AND METHODS: Seventy-eight patients with locally advanced rectal adenocarcinoma who received preoperative HCRT were enrolled. The median age of the patients was 64 years (range, 33-85 years) and 4, 18, and 56 patients had clinical stage I, II and III disease, respectively. Formalin-fixed and paraffin-embedded tissues excised before and after HCRT were subjected to immunohistochemical analysis with an anti-HLA class I-A, B, C antibody. HLA class I expression was graded according to tumor cell positivity. RESULTS: In pre-HCRT, the number of specimens categorized as Grade 0 and 1 were 19 (24%) and 58 (74%), respectively. Only 1 patient (1%) showed Grade 2 expression. However, 6 (8%), 27 (35%), 7 (9%), and 12 (15%) post-HCRT specimens were graded as Grade 0, 1, 2, and 3, respectively. There was a significant increase in HLA class I expression in post-HCRT specimens (p<0.01). However, neither pre- nor post-HCRT HLA class I expression affected overall survival and distant metastasis-free survival in clinical stage III patients. Univariate analysis revealed that Post-HCRT HLA class I expression showed a significant negative relationship with LC (p<0.05). Nevertheless, multivariate analysis showed that there was no correlation between HLA class I expression and clinical outcome. CONCLUSION: HCRT increased HLA class I expression in rectal cancer patients. However, multivariate analysis failed to show any correlation between the level of HLA class I expression and prognosis.

Candidate molecules for chemical chaperone therapy of GM1-gangliosidosis.

A growing body of evidence suggests that misfolding of a mutant protein followed by its aggregation or premature degradation in the endoplasmic reticulum is one of the main mechanisms that underlie inherited neurodegenerative diseases, including lysosomal storage diseases. Chemical or pharmacological chaperones are small molecules that bind to and stabilize mutant lysosomal enzyme proteins in the endoplasmic reticulum. A number of chaperone compounds for lysosomal hydrolases have been identified in the last decade. They have gained attention because they can be orally administrated, and also because they can penetrate the blood-brain barrier. In this article, we describe two chaperone candidates for the treatment of GM1-gangliosidosis. We also discuss the future direction of this strategy targeting other lysosomal storage diseases as well as protein misfolding diseases in general.

Transcatheter arterial chemotherapy using miriplatin-lipiodol suspension with or without embolization for unresectable hepatocellular carcinoma.

OBJECTIVE: The purpose of this retrospective study was to compare the anti-tumor and adverse effects of transcatheter arterial chemoembolization and transcatheter arterial infusion chemotherapy using miriplatin-lipiodol suspension in patients with unresectable hepatocellular carcinoma. METHODS: From 2007 to 2010, 162 consecutive patients with unresectable hepatocellular carcinoma were treated using miriplatin. Of these, 122 patients were treated by transcatheter arterial chemoembolization and 40 were treated by transcatheter arterial infusion chemotherapy. There were no significant differences in baseline characteristics between the two groups, except for prothrombin activity. Assessments were performed 1-3 months after treatment. RESULTS: Objective responses were achieved in 13 patients undergoing transcatheter arterial infusion chemotherapy and 70 patients undergoing transcatheter arterial chemoembolization (33 versus 57%, P = 0.003). By multivariate logistic regression analysis, objective response was significantly associated with (i) a Lens culinaris agglutinin-reactive fraction of α-fetoprotein ≤10% (P = 0.004; risk ratio = 3.09; 95% confidence interval = 1.42-6.70), (ii) no previous transcatheter arterial chemoembolization (P = 0.007; risk ratio = 4.41; 95% confidence interval = 1.49-13.07) and (iii) transcatheter arterial chemoembolization using gelatin sponge 1 mm particles (P = 0.021; risk ratio = 2.97; 95% confidence interval = 1.17-7.49). Fever, anorexia and elevated serum transaminase levels were observed in most patients after miriplatin administration; there were no significant differences in the number of adverse effects between the two groups. CONCLUSIONS: These results suggest that the addition of embolizing agents to a treatment regimen using miriplatin-lipiodol suspension can be safely used for patients with unresectable hepatocellular carcinoma. Objective response was achieved in a significantly higher number of transcatheter arterial chemoembolization patients than transcatheter arterial infusion chemotherapy patients.

Expression of hypoxia-inducible factor 1α predicts clinical outcome after preoperative hyperthermo-chemoradiotherapy for locally advanced rectal cancer.

Hypoxia-inducible factor 1α (HIF-1α) is an intrinsic marker of tumor hypoxia. It has been considered that hypoxic conditions reduce radiosensitivity, but the role of HIF-1α in patients treated with preoperative therapy for rectal cancer is still unclear. The aim of this study was to evaluate the predictive value of tumor response to preoperative hyperthermo-chemoradiotherapy (HCRT) and the prognostic significance of HIF-1α expression in patients with locally advanced rectal cancer. Between 2003 and 2006, 50 patients with histologically proven rectal adenocarcinoma who underwent HCRT followed by surgery were investigated. HIF-1α expression was immunohistochemically evaluated using pre-treatment biopsies. The total radiation dose was 40-50 Gy and chemotherapy consisted of 5-FU and LV administered by continuous infusion on Day 1-5, Day 15-19, and Day 29-33 during radiotherapy. Hyperthermia treatment was performed for once a week for 2-5 sessions. The surgical operation was performed 8 weeks after HCRT and each resected specimen was graded by histological criteria of the Japanese Classification of Colorectal Carcinoma. The effects of HIF-1α on clinical outcomes were analyzed by univariate and multivariate analysis. Positive HIF-1α expression was recognized in 42.0% of samples (21/50). Resected specimens that showed pathological grades 1, 2, and 3 numbered 17, 24, and 9 cases, respectively. There were no significant differences between the HIF-1α-positive group and HIF-1α-negative group for pathological grading and pCR. Overall survival (OS) rate at 3 years in the HIF-1α-negative group was 85.2%, which was significantly better than the 60.6% in the HIF-1α-positive group. Recurrence-free survival (RFS) rate at 3 years in the HIF-1α-negative group was 82.8%, being significantly better than 47.6% in the HIF-1α-positive group. In addition, elevated HIF-1α expression was significantly correlated with recurrence-free survival and metastasis-free survival rate in multivariate analysis. HIF-1α expression might be predictive of recurrence-free survival and metastasis-free survival rate for rectal cancer patients treated with HCRT.

Previous chemoembolization response after transcatheter arterial chemoembolization (TACE) can predict the anti-tumor effect of subsequent TACE with miriplatin in patients with recurrent hepatocellular carcinoma.

AIM: The purpose of this retrospective study was to evaluate the efficacy and safety of transcatheter arterial chemoembolization (TACE) with miriplatin in patients with unresectable hepatocellular carcinoma (HCC). METHODS: From 2007 to 2010, 122 consecutive patients with unresectable HCC were treated by TACE with miriplatin-lipiodol suspension in our institute. Twenty-two patients (18%) had a solitary nodule and 100 patients (82%) had multiple nodules. Ninety-eight patients (80%) had a history of TACE. RESULTS: Thirty-five of the 122 treated patients (29%) showed complete response (CR). And no serious complications were observed. Patients who had shown CR after previous TACE (pre-CR) were significantly more likely to show CR in the current study compared with patients who had shown less successful responses after previous TACE (56 vs. 20%, p = 0.003). Multivariate analysis revealed that response after previous TACE (pre-CR, risk ratio: 4.76; p = 0.035), tumor multiplicity (solitary, risk ratio: 9.69; p = 0.003), and injection artery (peripheral to segmental hepatic artery, risk ratio: 5.28;p = 0.040) were significant independent predictors associated with CR after TACE using miriplatin. CONCLUSION: In repetition of TACE treatment, switching the TACE agent from epirubicin or cisplatin to miriplatin offered a favorable treatment effect, especially in patients who had shown a CR after previous TACE.

MK615, a prospective anti-proliferative agent, enhances CD4/CD8 ratio after exposure to irradiation.

PURPOSE: Recently, it was found that MK615 possessed an anti-proliferative ability on treated cancer cells as a consequence of triterpenoid compounds. It is well known that radiation affects cellular-mediated immunity in cancer patients who are treated with radiotherapy. Similarly, the ability of triterpenoid compounds to enhance the cellular-mediated immunity has been observed. Therefore, in the present study, we attempted to investigate the effect of MK615 on both cancer cells and cellular-mediated immunity after irradiation. MATERIALS AND METHODS: After mice were inoculated with mouse mammary carcinoma (FM3A) cells, they were categorised as follows: Non-treated, irradiated with 5 Gy, treated with 660 µg/day MK615 (MK615, an extract from the Japanese apricot) and lastly exposed to both irradiation and MK615. Afterward, mice were sacrificed and spleens were utilised to measure the cluster of differentiation 4 and 8 (CD4 and CD8) using flowcytometry. Simultaneously, in vitro study, human alveolar basal epithelial carcinomic (A549), mouse lymphoma (EL4) and FM3A cell lines were examined. Growth inhibition was assessed via colony, cell viability and apoptotic assays. RESULTS: The median survival was in favour of the MK615-treated group (26.1 ±â€Š1.9 days) compared with non-treated group (22.3 ±â€Š2.3 days) (p < 0.05). Approximately 50% reduction of the CD4/CD8 ratio was observed following the exposure to irradiation alone. However, this ratio was comparable between the non-treated and both MK615-treated groups. Additionally, only the dual treatment was associated with tumour volume reduction. In contrast, in vitro study showed that MK615 had no significant (p ≥ 0.1) effect on the selected cell lines with or without irradiation. CONCLUSION: MK615 has a potential to reduce tumour volume and may normalise cellular-mediated immunity level following the exposure to irradiation.

Results of concomitant chemoradiation for cervical cancer using high dose rate intracavitary brachytherapy: study of JROSG (Japan Radiation Oncology Study Group).

The purpose of this study was to clarify outcome for concurrent chemoradiation (CT-RT) in locally advanced cervix cancer in Japan. This is a non-randomized retrospective analysis of 226 patients treated with definitive CT-RT or radiotherapy alone (RT alone) in nine institutions between 2001 and 2003. External irradiation consisted of whole pelvic irradiation and pelvic side wall boost irradiation, using a central shield during the latter half of the treatment with the anteroposterior parallel opposing technique. The external beam irradiation was performed with 1.8 or 2 Gy per fraction. High-dose-rate intracavitary brachytherapy (HDR) was performed in all cases. In chemotherapy, platinum based drugs were used alone or in combination with other drugs such as 5FU. Grade of late complications was scaled retrospectively with CTCv2.0. Overall survival rate at 50 months of stage Ib, II and III, IV was 82% and 66% in CR-RT and 81% and 43% in R alone, respectively. Disease-free survival rate at 50 months of stage Ib, II and III, IV was 74% and 59% in CR-RT and 76% and 52% in R alone, respectively. There was no significant difference between CT-RT and RT for overall survival and disease free survival. Univariate analysis suggested that loco-regional control was better with CT-RT, but multivariate analysis could not confirm this finding. Compared to RT alone, CT-RT caused significantly more acute and late complications. Thus, late complication (grade 3-4) free survival rate at 50 month was 69% for CT-RT and 86% for RT alone (p<0.01). The therapeutic window with concomitant radiochemotherapy and HDR brachytherapy may be narrow, necessitating a close control of dose volume parameters and adherence to systems for dose prescription.

Lipiodol and dye at the site of ablation decreases during RFA.

OBJECTIVES: Intrahepatic metastasis is one of the serious complications of radiofrequency ablation (RFA) therapy for hepatocellular carcinoma. The aim of this study was to investigate how liver tissue could be disseminated during the RFA procedure with different devices and protocols in an in vivo porcine model. METHODS: Three pigs underwent RFA procedures using 2 different devices: needles that could be expanded (LeVeen needle) and those that could not (cool-tip needle). A LeVeen needle was used with a single-step full extension method or a stepwise extension method. Before RFA, a mixture of lipiodol and blue dye was injected intrahepatically into a precoagulated area. After the ablation procedure, the specimen was cut to evaluate the amount of dye remaining in the ablated region and the distribution of the dye outside the ablated area. RESULTS: The stepwise extension method resulted in the disappearance of the smallest amount of the dye and lipiodol at the ablation site, compared with the full extension method and cool-tip needle. Dye was found at sites distant from the ablated area in all cases using the cool-tip needle, but in none with the stepwise extension method. CONCLUSIONS: The stepwise procedure using the expandable needle can reduce tumor cell scattering, which can cause intrahepatic metastasis, compared with other methods.

Impact of a clinical pathway in cases of transurethral resection of the prostate.

Due to the recent dramatic increase in health care costs, costs containment for medical care has been recognized as an important issue. We evaluated the effects of a clinical pathway on hospital charges and the clinical outcome. The subjects consisted of 48 patients who underwent transurethral resection of the prostate (TUR-P) at the Department of Urology, Nerima General Hospital via its own clinical pathway during the two-year period from April 1998 to March 2000. The mean length of hospital stay, clinical outcome and the medical insurance charges for these patients were compared with those of 68 patients who had undergone TUR-P before the implementation of the clinical pathway. As a result of the clinical pathway implementation for TUR-P, the length of hospital stay decreased from 17.2 days to 3.8 days and the total medical insurance charges (insurance points) also decreased from 37,484.6 to 31,278.9 yen. The postoperative complications did not substantially differ before and after the implementation of the clinical pathway. These results demonstrate that the establishment of clinical pathway can improve the treatment efficiency for almost all patients. It is, however, important to take into account the individuality of patients.

Fibroblast screening for chaperone therapy in beta-galactosidosis.

We performed screening of beta-galactosidase-deficient fibroblasts for possible chemical chaperone therapy using N-octyl-4-epi-beta-valienamine (NOEV) in patients with GM1-gangliosidosis and Morquio B disease (beta-galactosidosis). Fibroblasts were cultured with NOEV for 4 days and beta-galactosidase activity was measured. Mutation analysis was performed simultaneously. Two separate criteria were set for evaluation of the chaperone effect: a relative increase of enzyme activity (more than 3-fold), and an increase up to more than 10% normal enzyme activity. Among the 50 fibroblast strains tested, more than 3-fold increase was achieved in 17 cell strains (34%), and more than 10% normal activity in 10 (20%). Both criteria were satisfied in 6 (12%), and either of them in 21 (42%). Juvenile GM1-gangliosidosis was most responsive, and then infantile GM1-gangliosidosis. This enhancement was mutation-specific. We estimate that the NOEV chaperone therapy will be effective in 20-40% of the patients, mainly in juvenile and infantile GM1-gangliosidosis patients. A molecular design may produce mutation-specific chaperone compounds for the other disease phenotypes. This cellular screening will be useful for identification of human patients with beta-galactosidase deficiency for chaperone therapy to be started in the near future.

G-CSF treatment increases side population cell infiltration after myocardial infarction in mice.

Granulocyte-colony stimulating factor (G-CSF) has been reported to mobilize bone marrow multi-potent stem cells, which differentiate into cardiac myocytes after myocardial infarction (MI). However, there have not been any reports regarding the effect of G-CSF on stem cell infiltration in the MI site. Hearts of mice that had undergone coronary occlusion were isolated and digested with collagenase. Infiltrating cells in the heart were collected using Percoll density gradients. The infiltrating cells were sorted for side population (SP) cells using Hoechst 33342 dye. Hundreds of infiltrating SP cells were found in the heart from 1 to 14 d after MI. There were only a few SP cells in hearts without infarction. Infiltrating SP cells were increased in the 4-d G-CSF treated group compared with the vehicle group (1106 +/- 106 vs. 323 +/- 26/heart, P < 0.05). The infiltration of inflammatory cells was not influenced by the G-CSF treatment. In a separate series of experiments, we confirmed that the infiltrating SP cells were derived from bone marrow. That is, SP cells in the infarcted hearts of mice, which had been transplanted with bone marrow from ROSA 26 (beta-galactosidase transgenic) mice, were positive for beta-galactosidase. In the immunohistochemical examination, Sca-1(+)/CD45(-) cells were existed in the infarcted site after MI. Therefore, SP cells may infiltrate into infarcted heart. G-CSF augmented this kind of stem cell infiltration without increasing inflammatory cells. These results suggest that G-CSF may enhance myocardial regeneration without aggravated inflammation in the infarcted heart.