Double-Layer Magnetic Nanoparticle-Embedded Silica Particles for Efficient Bio-Separation

Superparamagnetic Fe₃O₄ nanoparticles (NPs) based nanomaterials have been exploited in various biotechnology fields including biomolecule separation. However, slow accumulation of Fe₃O₄ NPs by magnets may limit broad applications of Fe₃O₄ NP-based nanomaterials. In this study, we report fabrication of Fe₃O₄ NPs double-layered silica nanoparticles (DL MNPs) with a silica core and highly packed Fe₃O₄ NPs layers. The DL MNPs had a superparamagnetic property and efficient accumulation kinetics under an external magnetic field. Moreover, the magnetic field-exposed DL MNPs show quantitative accumulation, whereas Fe₃O₄ NPs single-layered silica nanoparticles (SL MNPs) and silica-coated Fe₃O₄ NPs produced a saturated plateau under full recovery of the NPs. DL MNPs are promising nanomaterials with great potential to separate and analyze biomolecules.     

Effect of different concentrations of oxygen on expression of sigma 1 receptor and superoxide dismutases in human colon adenocarcinoma cell lines

Context: Tumor cells due to distance from capillary vessels exist in different oxygenation conditions (anoxia, hypoxia, normoxia). Changes in cell oxygenation lead to reactive oxygen species production and oxidative stress. Sigma 1 receptor (Sig1R) is postulated to be stress responding agent and superoxide dismutases (SOD1 and SOD2) are key antioxidant enzymes. It is possible that they participate in tumor cells adaptation to different concentrations of oxygen.

Objective: Evaluation of Sig1R, SOD1, and SOD2 expression in different concentrations of oxygen (1%, 10%, 21%) in colon adenocarcinoma cell lines.

Materials and methods: SW480 (primary adenocarcinoma) and SW620 (metastatic) cell lines were cultured in standard conditions in Dulbecco’s modified Eagle’s medium for 5 days, and next cultured in Hypoxic Chamber in 1% O₂, 10% O₂, 21% O₂. Number of living cells was determined by trypan blue assay. Level of mRNA for Sig1R, SOD1, and SOD2 was determined by standard PCR method. Statistical analysis was conducted using Statistica 10.1 software.

Results: We observed significant changes in expression of Sig1R, SOD1, SOD2 due to different oxygen concentrations. ANOVA analysis revealed significant interactions between studied parameters mainly in hypoxia conditions in SW480 cells and between Sig1R and SOD2 in SW620 cells. It also showed that changes in expression of studied proteins depend significantly on type of the cell line.

Conclusion: Changes of Sig1R and SOD2 expression point to mitochondria as main organelle responsible for survival of tumor cells exposed to hypoxia or oxidative stress. Studied proteins are involved in intracellular response to stress related with different concentrations of oxygen.     

Inhibitory effect of Tanshinone IIA on inverted formin-2 protects HaCaT cells against oxidative injury via regulating mitochondrial stress

Context: Epidermal cells play an important role in regulating the regeneration of skin after burns and wounds.

Objective: The aim of our study is to explore the role of Tanshinone IIA (Tan IIA) in the apoptosis of epidermal HaCaT cells induced by H₂O₂, with a focus on mitochondrial homeostasis and inverted formin-2 (INF2).

Materials and methods: Cellular viability was determined using the MTT assay, TUNEL staining, western blot analysis and LDH release assay. Adenovirus-loaded INF2 was transfected into HaCaT cells to overexpress INF2 in the presence of Tan IIA treatment. Mitochondrial function was determined using JC-1 staining, mitochondrial ROS staining, immunofluorescence and western blotting.

Results: Oxidative stress promoted the death of HaCaT cells and this effect could be reversed by Tan IIA. At the molecular levels, Tan IIA treatment sustained mitochondrial energy metabolism, repressed mitochondrial ROS generation, stabilized mitochondrial potential, and blocked the mitochondrial apoptotic pathway. Furthermore, we demonstrated that Tan IIA modulated mitochondrial homeostasis via affecting INF2-related mitochondrial stress. Overexpression of INF2 could abolish the protective effects of Tan IIA on HaCaT cells viability and mitochondrial function. Besides, we also reported that Tan IIA regulated INF2 expression via the ERK pathway; inhibition of this pathway abrogated the beneficial effects of Tan IIA on HaCaT cells survival and mitochondrial homeostasis.

Conclusions: Overall, our results indicated that oxidative stress-mediated HaCaT cells apoptosis could be reversed by Tan IIA treatment via reducing INF2-related mitochondrial stress in a manner dependent on the ERK signaling pathway.     

Effect of lipid composition on incorporation of trastuzumab-PEG-lipid into nanoliposomes by post-insertion method: physicochemical and cellular characterization

Context: Anti-HER2 immunoliposomes are promising nanotechnology based systems for active targeting of breast tumors, which depends on the amount of incorporated antibody.

Objective/Aim: In this work, we investigated the possible effect of lipid composition on the incorporation of trastuzumab-PEG-PE micelles into nanoliposomes and on their subsequent specific cellular targeting.

Materials and methods: Trastuzumab (anti-HER2 monoclonal antibody) was monothiolated and conjugated to maleimide-PEG-PE micelles. Liposomes of different lipid compositions were prepared by the thin layer hydration. Trastuzumab-PEG-PE micelles were incorporated into the liposomes by the post-insertion method. The percentage of lipid mixing was determined based on fluorescence resonance energy transfer. Cellular binding and uptake of rhodamine-labeled immunoliposomes were studied in SKBR-3 (HER2⁺⁺⁺) and MCF-7 (HER2⁺) cells. Also, antitumor cell activity of the immunoliposomes was compared to free trastuzumab and the liposomes.

Results: The lipid mixing of trastuzumab-PEG-PE micelles depended on the liposome composition. The immunoliposomes containing DPPC, cholesterol and PEG-PE showed prominent lipid mixing. The lipid mixing was consistent with the cell binding results which showed an efficient and specific binding of the immunoliposomes to SKBR-3 cells. Antitumor cell activity of the immunoliposomes in SKBR-3, unlike MCF-7 cells, depended on the content of trastuzumab.

Discussion: Cholesterol and PEG-PE in the liposome composition are prerequisites for a successful lipid mixing due to their ability to facilitate fusion. The higher lipid mixing results in higher antibody incorporation and consequently higher targeted cell binding.

Conclusions: The lipid mixing depends on the liposome composition, which reflects targeted cell binding of the immunoliposomes.     

Downregulation of IDH2 exacerbates H2O2-mediated cell death and hypertrophy

Objectives: Reactive oxygen species-mediated cell death contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. We recently showed that mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems.

Methods: In the present study, we demonstrated that H₂O₂-induced apoptosis and hypertrophy of H9c2 cardiomyoblasts was markedly exacerbated by small interfering RNA (siRNA) specific for IDH2.

Results: Attenuated IDH2 expression resulted in the modulation of cellular and mitochondrial redox status, mitochondrial function, and cellular oxidative damage. MitoTEMPO, a mitochondria-targeted antioxidant, efficiently suppressed increased caspase-3 activity, increased cell size, and depletion of cellular GSH levels in IDH2 siRNA-transfected cells that were treated with H₂O₂.

Discussion: These results indicated that the disruption of cellular redox balance might be responsible for the enhanced H₂O₂-induced apoptosis and hypertrophy of cultured cardiomyocytes by the attenuated IDH2 expression.     

Roles of M3 receptor in the effect of penehyclidine hydrochloride upregulated beta-arrestin-1 expression in LPS-stimulated HPMVEC

Background: This study is to investigate the roles of muscarinic receptor 3 (M₃ receptor) in the effect of penehyclidine hydrochloride (PHC) upregulated beta-arrestin-1 expression in lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMVEC).

Methods: HPMVECs were transfected with a shRNA-containing plasmid that specifically targets M₃ receptor mRNA. Cells were collected to measure F-actin contents, levels of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as changes of F-actin cytoskeleton arrangement by Laser scanning confocal. Beta-arrestin-1 protein expressions were determined by Western blot and beta-arrestin-1 mRNA expressions were measured by Real-time PCR.

Results: Similar to normal cells, PHC could also increase F-actin contents and beta-arrestin-1 expressions, reduce ICAM-1 and VCAM-1 expressions, and inhibit LPS-stimulated reorganization of F-actin and formation of stress fiber in M₃ receptor shRNA group. Compared with normal cells, F-actin cytoskeleton was neat, ICAM-1 and VCAM-1 expressions were decreased, as well as F-actin contents were increased in M₃ receptor shRNA group. However, there were no differences in beta-arrestin-1 expressions between normal cell groups and M₃ receptor shRNA groups.

Conclusion: These results indicate that M₃ receptor plays an important role in pulmonary microvascular endothelial barrier function, and knock-out of M₃ receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. However, upregulative effect of PHC on beta-arrestin-1 expression is independent with presence of M₃ receptor.     

Proteomics and phosphoproteomics study of LCMT1 overexpression and oxidative stress: overexpression of LCMT1 arrests H2O2-induced lose of cells viability

Objectives: Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, is also known to be a target of ROS. The methylation of PP2A can be catalyzed by leucine carboxyl methyltransferase-1 (LCMT1), which regulates PP2A activity and substrate specificity.

Methods: In the previous study, we have showed that LCMT1-dependent PP2Ac methylation arrests H₂O₂-induced cell oxidative stress damage. To explore the possible protective mechanism, we performed iTRAQ-based comparative quantitative proteomics and phosphoproteomics studies of H₂O₂-treated vector control and LCMT1-overexpressing cells.

Results: A total of 4480 non-redundant proteins and 3801 unique phosphopeptides were identified by this means. By comparing the H₂O₂-regulated proteins in LCMT1-overexpressing and vector control cells, we found that these differences were mainly related to protein phosphorylation, gene expression, protein maturation, the cytoskeleton and cell division. Further investigation of LCMT1 overexpression-specific regulated proteins under H₂O₂ treatment supported the idea that LCMT1 overexpression induced ageneral dephosphorylation of proteins and indicated increased expression of non-erythrocytic hemoglobin, inactivation of MAPK3 and regulation of proteins related to Rho signal transduction, which were known to be linked to the regulation of the cytoskeleton.

Discussion: These data provide proteomics and phosphoproteomics insights into the association of LCMT1-dependent PP2Ac methylation and oxidative stress and indirectly indicate that the methylation of PP2A plays an important role against oxidative stress.     

Modification of cysteine 457 in plakoglobin modulates the proliferation and migration of colorectal cancer cells by altering binding to E-cadherin/catenins

Objectives: In tissue samples from patients with colorectal cancer (CRC), oxidation of C420 and C457 of plakoglobin (Pg) within tumor tissue was identified by proteomic analysis. The aim of this study was to identify the roles of Pg C420 and C457.

Methods: Human CRC tissues, CRC and breast cancer cells, and normal mouse colon were prepared to validate Pg oxidation. MC38 cells were co-transfected with E-cadherin plus wild type (WT) or mutant (C420S or C457S) Pg to evaluate protein interactions and cellular localization, proliferation, and migration.

Results: Pg was more oxidized in stage III CRC tumor tissue than in non-tumor tissue. Similar oxidation of Pg was elicited by H₂O₂ treatment in normal colon and cancer cells. C457S Pg exhibited diminished binding to E-cadherin and α-catenin, and reduced the assembly of E-cadherin–α-/β-catenin complexes. Correspondingly, immunofluorescent analysis of Pg cellular localization suggested impaired binding of C457S Pg to membranes. Cell migration and proliferation were also suppressed in C457S-expressing cells.

Discussion: Pg appears to be redox-sensitive in cancer, and the C457 modification may impair cell migration and proliferation by affecting its interaction with the E-cadherin/catenin axis. Our findings suggest that redox-sensitive cysteines of Pg may be the targets for CRC therapy.     

Using multiple biomarkers and determinants to obtain a better measurement of oxidative stress: a latent variable structural equation model approach

Purpose: Since oxidative stress involves a variety of cellular changes, no single biomarker can serve as a complete measure of this complex biological process. The analytic technique of structural equation modeling (SEM) provides a possible solution to this problem by modelling a latent (unobserved) variable constructed from the covariance of multiple biomarkers.

Methods: Using three pooled datasets, we modelled a latent oxidative stress variable from five biomarkers related to oxidative stress: F₂-isoprostanes (FIP), fluorescent oxidation products, mitochondrial DNA copy number, γ-tocopherol (Gtoc) and C-reactive protein (CRP, an inflammation marker closely linked to oxidative stress). We validated the latent variable by assessing its relation to pro- and anti-oxidant exposures.

Results: FIP, Gtoc and CRP characterized the latent oxidative stress variable. Obesity, smoking, aspirin use and β-carotene were statistically significantly associated with oxidative stress in the theorized directions; the same exposures were weakly and inconsistently associated with the individual biomarkers.

Conclusions: Our results suggest that using SEM with latent variables decreases the biomarker-specific variability, and may produce a better measure of oxidative stress than do single variables. This methodology can be applied to similar areas of research in which a single biomarker is not sufficient to fully describe a complex biological phenomenon.     

Cholest-4-en-3-one attenuates TGF-β responsiveness by inducing TGF-β receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells

Purpose: The transforming growth factor-beta (TGF-β) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-β signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro.

Methods: The inhibitory effects of cholest-4-en-3-one on TGF-β-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-β receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation.

Results: Cholest-4-en-3-one attenuated TGF-β signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-β-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling.

Conclusions: Our results suggest that cholest-4-en-3-one inhibits TGF-β signaling may be due, in part to the translocation of TGF-β receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-β deficiency.     

Cheilolejeunea morganii Bever. & Glenny (Lejeuneaceae,Marchantiopsida), a newly described species from lowland indigenous forest sites in New Zealand

Introduction. Cheilolejeunea morganii Bever. & Glenny, a new species of Cheilolejeunea from a lowland forest habitat in eastern Taranaki in the North Island of New Zealand, is described and illustrated.

Methods. DNA sequences were extracted from recently collected material at two locations and compared with those for species in a published phylogeny of the genus to establish the position of C. morganii. Photographic images were obtained of key features of the species for the preparation of illustrations for publication.

Key results. Cheilolejeunea morganii does not match any species described for New Zealand or Australia. The sequencing results indicate its position in the phylogeny is close to Section Paroicae. A key to the New Zealand species of Cheilolejeunea is provided.

Conclusions. In the Australasian flora, C. morganii is distinguished from other species by its combination of monoicy, pycnolejeuneoid gynoecial innovations, lobule length less than 50% lobe length and a multicellular second lobule tooth with 3–4 cells uniseriate. A case is presented for the recognition of the species as a New Zealand endemic.     

Vancomycin-sensitized photooxidation in the presence of the natural pigment vitamin B2: Interaction with excited states and photogenerated ROS

Sensitized photooxidation processes in the presence of natural pigments may provide an alternative to antibiotics degradation since these compounds are transparent to natural light irradiation, therefore, they can be degraded by the action of photosensitizers which absorb light and produce highly reactive species, especially those derived from molecular oxygen (ROS). Most antibiotics used currently belong to a group of pharmaceutical substances that have been considered a new type of contaminants due to their persistence and bioaccumulation in the environment.

Objective: In this context, we decided to investigate the kinetic and mechanistic aspects of Vancomycin (Vanco) photosensitized degradation in the presence of the natural pigment Riboflavin (Vitamin B2, Rf) and the artificial dye Rose Bengal (RB) for comparative purposes.

Methods: The study have been done by using Stationary photolysis, Laser flash photolysis, Time-resolved phosphorence detection of O₂(1Δg) experiments and Bactericidal activity evaluation. The experiments were carried out in aqueous solution at different pH values in order to establish relationships between the structure of the compound and its susceptibility to ROS-mediated photooxidation.

Results: Experimental evidence indicates that in the presence of Rf there is considerable contribution of the radical-mediated mechanism, while in the presence of RB the photooxidation process occurs exclusively through O₂(1Δg) and the reactivity to this excited species increases with increasing pH of the environment.

Discussion: The results obtained, have been shown that Rf can raise the photodegradation of Vanco by both the radical pathway and the O₂(¹Δ_g) mediated. Furthermore, the antibiotic is able to interact with the excited electronic states of Rf as well as O₂(¹Δ_g) generated by energy transfer between the excited triplet state of the photosensitizer and the oxygen ground state. The predominant mechanism for photodegradation of Vanco in the presence of the Rf is the radical via because of the considerable interaction with the excited triplet state of the photosensitizer demonstrated by laser flash photolysis experiments.

Microbiological test on Staphylococcus aureus ATCC25923 showed that the bactericidal activity of the antibiotic on the strain studied was affected by the sensitized photodegradation process, suggesting that photoproducts generated eventually do not retain the bactericidal properties of the original antibiotic.     

Serum tau fragments as predictors of death or poor neurological outcome after out-of-hospital cardiac arrest

Background: Anoxic brain injury is the primary cause of death after resuscitation from out-of-hospital cardiac arrest (OHCA) and prognostication is challenging. The aim of this study was to evaluate the potential of two fragments of tau as serum biomarkers for neurological outcome.

Methods: Single-center sub-study of 171 patients included in the Target Temperature Management (TTM) Trial randomly assigned to TTM at 33?°C or TTM at 36?°C for 24?h after OHCA. Fragments (tau-A and tau-C) of the neuronal protein tau were measured in serum 24, 48 and 72?h after OHCA. The primary endpoint was neurological outcome.

Results: Median (quartile 1 – quartile 3) tau-A (ng/ml) values were 58 (43–71) versus 51 (43–67), 72 (57–84) versus 71 (59–82) and 76 (61–92) versus 75 (64–89) for good versus unfavourable outcome at 24, 48 and 72?h, respectively (p_group = 0.95). Median tau C (ng/ml) values were 38 (29–50) versus 36 (29–49), 49 (38–58) versus 48 (33–59) and 48 (39–59) versus 48 (36–62) (p_group = 0.95). Tau-A and tau-C did not predict neurological outcome (area under the receiver-operating curve at 48?h; tau-A: 0.51 and tau-C: 0.51).

Conclusions: Serum levels of tau fragments were unable to predict neurological outcome after OHCA.     

Circulating levels of apoptotic markers and oxidative stress parameters in women with polycystic ovary syndrome: a case-controlled descriptive study

Context: Apoptotic dysregulation plays a role in the pathogenesis of polycystic ovary syndrome (PCOS).

Objective: To evaluate circulatory apoptotic markers and oxidative stress in patients with PCOS.

Materials and methods: Forty-four women with PCOS, and 44 healthy women as controls were enrolled in the study. Oxidative stress parameters and caspases levels were measured in serum.

Results: The caspase 9 level was significantly lower and related with oxidant status in patients with PCOS, while the circulating levels of caspases 3 and 7 were statistically similar in both groups.

Discussion: This study is the first report demonstrating the circulating levels of apoptotic markers and their relationship with oxidant status in PCOS.

Conclusion: The circulating caspase 9 and oxidant status might contribute to apoptotic dysregulation in PCOS.     

The relationship of single-strand breaks in DNA to breast cancer risk and to tissue concentrations of oestrogens

Context: Clinical study of breast cancer patients in Chicago, IL, USA.

Objective: Ascertain the utility of measurements of single-strand breaks (SSB) in DNA for assessment of breast cancer risk.

Methods: Fine-needle aspirates of the breast, SSB by nick translation, percent breast density (PBD), Gail model risk, cumulative methylation index (CMI), enzymes of DNA repair and tissue antioxidants.

Results: DNA repair enzymes and 4-hydroxyestradiol were negatively associated with SSB; CMI and PBD were positively associated.

Conclusions: Quantitative measurement of SSBs by this procedure indicates the relative number of SSBs and is related to promoter methylation, antioxidant availability and percent breast density.     

Synthesis and preliminary biological evaluation for the anticancer activity of organochalcogen (S/se) tethered chrysin-based organometallic RuII(η6-p-cymene) complexes

Organochalcogen (S/Se) functionalized chrysin derivatives were synthesized and coordinated with Ru^II(η⁶-p-cymene) to efficiently form ruthenium-based chemotherapeutic drug entities [C₃₁H₃₅O₄SRuCl]; [C₃₁H₃₅O₄SeRuCl]; [C₃₃H₃₁O₄SRuCl]; and [C₃₃H₃₁O₄SeRuCl]. The complexes were thoroughly characterized by analytical and various spectroscopic techniques which include elemental analysis, UV–vis, IR, NMR (¹H, ¹³C, and ⁷⁷Se NMR), and HR-MS. The interaction studies of these Ru(II) complexes were carried out with CT DNA/HSA by employing UV–vis, fluorescence and circular dichroic techniques in view to examine their chemotherapeutic potential. The complexes demonstrated predominant binding toward CTDNA via electrostatic interaction while, the extent of binding was quantified by calculating intrinsic binding constant (K_b) and binding constant (K) values which revealed higher binding affinity of selenium-based chrysin complexes as compared to their thio-analogs, following the order [C₃₁H₃₅O₄SeRuCl]?>?[C₃₃H₃₁O₄SeRuCl]?>?[C₃₁H₃₅O₄SRuCl]?>?[C₃₃H₃₁O₄SRuCl]. Moreover, interaction of these complexes with human serum albumin (HSA) was also investigated which suggested spontaneous interactions of complexes with the protein by hydrogen bonding and van der Waals forces. To visualize the preferential binding sites and affinity of complexes with DNA and HSA molecular docking studies were performed. Additionally, in vitro anticancer activity of the complexes were evaluated by SRB assay on selected cancer cell lines viz., HeLa (cervical), MIA-PA-CA-2 (pancreatic), MCF-7 (breast), Hep-G2 (Hepatoma), and SK-OV-3 (ovarian) which exhibited the superior cytotoxicity of complex [C₃₁H₃₅O₄SeRuCl] as compared to other analogs on selective cancer phenotypes.

Communicated by Ramaswamy H. Sarma     

Dynamic variation of histone H3 trimethyl Lys4 (H3K4me3) and heterochromatin protein 1 (HP1) with employment length in nickel smelting workers

Objective: To investigate the dynamic variation in H3K4me3 and HP1 with employment length in nickel smelting workers.

Methods: Blood samples were collected from 140 nickel smelting workers and 140 age-matched office workers to test for H3K4me3, and HP1 levels.

Results: H3K4me3 was statistically significantly different (p?<?0.05) between the two groups and positively correlated with employment length (r_s?=_?0.267). HP1 was not correlated with employment length (p?=?0.066) but was significantly different between the two groups.

Conclusions: Chronic exposure to nickel can induce oxidative damage, and increase H3K4me3 expression and inhibit HP1 expression.     

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer.

We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB.

These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.     

Biomarker discovery for drug-induced phospholipidosis: phenylacetylglycine to hippuric acid ratio in urine and plasma as potential markers

Context: Drug-induced phospholipidosis is one of the significant concerns in drug development, especially in safety assessment and noninvasive diagnostic tool is highly desirable.

Objective: The objective of this study is to explored novel biomarkers for phospholipidosis using a metabolomic approach.

Method: NMR spectrometry and LC/MS/MS analyses were applied to urine and plasma of rats administrated cationic amphiphilic drugs.

Results: The phenylacetylglycine to hippuric acid ratio in plasma was increased in time and dose-dependent manners; and it was well correlated with histopathological observation.

Conclusion: The plasma phenylacetylglycine to hippuric acid ratio is a potential marker in monitoring drug-induced phospholipidosis.