Search

The antibody Trastuzumab (Tz) targeting ERBB2 has improved the prognosis of patients with breast cancer (BCa) that overexpress this receptor. Resistance to Tz negatively impacts prognosis. Several mechanisms have been reported to cause Tz resistance. The objective of this study was to identify common mechanisms in in vitro models of acquired BCa Tz resistance. In particular, we used three widely available ERBB2+ BCa cell lines adapted to grow in Tz. We performed several analyses to address possible changes in phenotype, proliferation, and ERBB2 membrane expres-sion common to the three Tz-R cell lines compared to wt, but none was found. High-resolution mass spectrometry analysis identified, instead, a common set of differentially expressed proteins (DEPs) in Tz-R vs. wt cells. Furthermore, bioinformatic tools revealed that all three Tz-R cell models shared a modulation of proteins involved in the metabolism of lipids, organophosphate biosynthetic process, and macromolecule methylation. Ultrastructural analysis confirmed altera-tion of lipid droplets in resistant cells. Our data strongly support the hypothesis that complex metabolic adaptation, including lipid metabolism, protein phosphorylation, and possibly chro-matin remodeling, may fuel Tz resistance. The identification of a common set of 10 DEPs in all three TZ-resistant cell lines may provide novel targets for therapeutic intervention.

Background and Purpose The pathogenesis of amyotrophic lateral sclerosis (ALS) is not fully clarified, although excessive glutamate (Glu) transmission and the downstream cytotoxic cascades are major mechanisms for motor neuron death. Two metabotropic glutamate receptors (mGlu1 and mGlu5) are overexpressed in ALS and regulate cellular disease processes. Expression and function of mGlu5 receptors are altered at early symptomatic stages in the SOD1G93A mouse model of ALS and knockdown of mGlu5 receptors in SOD1G93A mice improved disease progression. Experimental Approach We treated male and female SOD1G93A mice with 2‐chloro‐4‐((2,5‐dimethyl‐1‐(4‐(trifluoromethoxy)phenyl)‐1H‐imidazol‐4‐yl)ethynyl)pyridine (CTEP), an orally available mGlu5 receptor negative allosteric modulator (NAM), using doses of 2 mg·kg−1 per 48 h or 4 mg·kg−1 per 24 h from Day 90, an early symptomatic disease stage. Disease progression was studied by behavioural and histological approaches. Key Results CTEP dose‐dependently ameliorated clinical features in SOD1G93A mice. The lower dose increased survival and improved motor skills in female mice, with barely positive effects in male mice. Higher doses significantly ameliorated disease symptoms and survival in both males and females, females being more responsive. CTEP also reduced motor neuron death, astrocyte and microglia activation, and abnormal glutamate release in the spinal cord, with equal effects in male and female mice. No differences were also observed in CTEP access to the brain. Conclusion and Implications Our results suggest that mGlu5 receptors are promising targets for the treatment of ALS and highlight mGlu5 receptor NAMs as effective pharmacological tools with translational potential.

To date, the 5-year overall survival rate of 60% for early-stage non-small cell lung cancer (NSCLC) is still unsatisfactory. Therefore, reliable prognostic factors are needed. Growing evidence shows that cancer progression may depend on an interconnection between cancer cells and the surrounding tumor microenvironment; hence, circulating molecules may represent promising markers of cancer recurrence. In order to identify a prognostic score, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs (Exo-miR) and peptides, in 67 radically resected NSCLCs. The miRnome profile selected the Exo-miR-130a-3p as the most overexpressed in relapsed patients. Peptidome analysis identified four progressively more degraded forms of fibrinopeptide A (FpA), which were depleted in progressing patients. Notably, stepwise Cox regression analysis selected Exo-miR-130a-3p and the greatest FpA (2-16) to build a score predictive of recurrence, where high-risk patients had 18 months of median disease-free survival. Moreover, in vitro transfections showed that higher levels of miR-130a-3p lead to a deregulation of pathways involved in metastasis and angiogenesis, including the coagulation process and metalloprotease increase which might be linked to FpA reduction. In conclusion, by integrating circulating markers, the identified risk score may help clinicians predict early-stage NSCLC patients who are more likely to relapse after primary surgery.

Background Fibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α- and β-chains N-termini (GPR, GHR; A-, B-knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' γ- and (B)β-chains C-terminal regions. A role for mostly disordered (A)α-chains C-terminal regions "bridging" between fibrin molecules/fibrils has been proposed. Objectives Fibrinogen Detroit is a clinically observed mutation (AαR19 → S) with nonengaging GPS A-knobs. By analogy, a similar Bβ-chain mutation, BβR17 → S, should produce nonengaging GHS B-knobs. A homozygous "Double-Detroit" mutant (AαR19 → S, BβR17 → S; DD-FG) was developed: with A-a and B-b engagements endogenously blocked, other interactions would become apparent. Methods DD-FG, wild-type recombinant (WT-FG), and human plasma (hp-FG) fibrinogen self-association was studied by turbidimetry coupled with fibrinopeptides release high-performance liquid chromatography (HPLC)/mass spectrometry analyses, and by light-scattering following size-exclusion chromatography (SE-HPLC). Results In contrast to WT-FG and hp-FG, DD-FG produced no turbidity increase, irrespective of thrombin concentration. The SE-HPLC profile of concentrated DD-FG was unaffected by thrombin treatment, and light-scattering, at lower concentration, showed no intensity and hydrodynamic radius changes. Compared with hp-FG, both WT-FG and DD-FG showed no FpA cleavage difference, while ~50% FpB was not recovered. Correspondingly, SDS-PAGE/Western-blots revealed partial Bβ-chain N-terminal and Aα-chain C-terminal degradation. Nevertheless, ~70% DD-FG molecules bearing (A)αC-regions potentially able to associate were available. Higher-concentration, nearly intact hp-FG with 500-fold molar excess GPRP-NH2 /GHRP-NH2 knobs-mimics experiments confirmed these no-association findings. Conclusions (A)αC-regions interactions appear too weak to assist native fibrin polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered.

The aim of this research was to evaluate the effects of different lens types on the availability and efficacy of anti-inflammatory and antibiotic drugs. Three lens types were examined: (1) nonionic hydrogel lenses, (2) ionic hydrogel lenses, and (3) silicone hydrogel lenses. The lenses were incubated with (a) dexamethasone, (b) betamethasone, (c) bromophenacyl bromide, and (d) chloramphenicol. Drug availability was quantified by gradient HPLC, and chloramphenicol antibacterial activity was quantified by testing the inhibition of Salmonella typhimurium growth on agar. The lens allowing the most abundant passage of betamethasone was the ionic hydrogel lens, followed by the silicone hydrogel lens and nonionic hydrogel lens. The lens allowing the most abundant passage of dexamethasone was the ionic hydrogel lens, but only at 0.5 h and 1 h. Regarding chloramphenicol, the ionic hydrogel lens and silicone hydrogel lens allowed more abundant passage than the nonionic hydrogel lens. These results highlight the relevance of adapting lenses to anti-inflammatory therapy, thus allowing a personalized medical approach.

Colorectal cancer (CRC) is the second cause of death in men and the third in women. This work deals with the study of the low molecular weight protein fraction of sera from patients who underwent surgery for CRC and who were followed for several years thereafter. MALDI-TOF MS was used to identify serum peptidome profiles of healthy controls, non-metastatic CRC patients and metastatic CRC patients. A multiple regression model was applied to signals preliminarily selected by SAM analysis to take into account the age and gender differences between the groups. We found that, while a signal m/z 2021.08, corresponding to the C3f fragment of the complement system, appears significantly increased only in serum from metastatic CRC patients, a m/z 1561.72 signal, identified as a prothrombin fragment, has a significantly increased abundance in serum from non-metastatic patients as well. The findings were also validated by a bootstrap resampling procedure. The present results provide the basis for further studies on large cohorts of patients in order to confirm C3f and prothrombin as potential serum biomarkers. Thus, new and non-invasive tests might be developed to improve the classification of colorectal cancer. This work was supported by grants from Italian Ministry of Health (5×1000 Funds 2013 and 2015) and IT8-11/13 from the Basque Government.

Integrity of serum samples is a major concern for biomarker discovery in many fields, including oncology. A gradual degradation of the fibrinopeptide A (fpA) as a result of the preservation process was already observed. In this work, we investigated this process to define a method for the assessment of sera integrity based on the total amount of fpA and its degradation pattern. SeraDeg software was developed. It identifies fpA fragments in spectra and extracts their abundances. The SeraDeg Quality Score (SDQS) is finally determined for each spectrum depending on its total fpA abundance and on the ratio of its higher mass versus lower mass fragments abundances. Spectra were evaluated for the total amount of fpA and the percent contribution of its fragments. The total fpA abundance is significantly lower in cryo-preserved samples than in fresh sera. Moreover, fresh sera present higher percentages of the most intact forms of fpA. Four relevant fpA fragments were identified. We developed and made available on-line SeraDeg, a software able to screen serum spectra, compute fpA related parameters and assign quality scores. SeraDeg was validated using spectra from a set of 250 samples. Biological significance The susceptibility of fpA to degradation suggests its use as quality indicator of cryo-preserved serum samples. SeraDeg can support the adoption of this method which may especially be useful in differential sero-proteomics for wide-range biomarker discovery. Moreover, it discloses the doors to a critical re-evaluation of previous experiments in light of the quality of serum samples as assessed by fpA degradation analysis.

Aloe vera extracts have been used for drug therapy due to their antimicrobial, anti-inflammatory, and epithelia-regenerative actions. Considering the possible adverse side effects on the corneal surface of anti-microbial agent administration after ocular surgery, it is of interest to identify the ability of Aloe vera extract release from a therapeutic lens to prevent ocular irritation. The aim of this research was to evaluate the effects of different lens types on the availability and efficacy of releasing Aloe vera extracts in which they are soaked. Two lens types were examined: (1) non-ionic hydrogel lenses and (2) ionic hydrogel lenses. Aloe vera availability was quantified by LC&ndash, MS, and activity was quantified by a spectrophotometer (Nanodrop) at different times: (a) 30 min, (b) 60 min, (c) 2 h, (d) 6 h, and (e) 24 h. The lens allowing for the most abundant passage of Aloe vera extracts was the ionic hydrogel lens, followed by the non-ionic hydrogel lens. The obtained results demonstrate that the best lens allowing for the high and stable release of Aloe vera extract to the corneal surface are those composed of ionic hydrogels.

Fibrin gels (FBGs) are potential delivery vehicles for many drugs, and can be easily prepared from purified components. We previously demonstrated their applicability for the release of different doxorubicin (Dox) nanoparticles used clinically or in an experimental stage, such as its inclusion complex with the amino β-cyclodextrin polymer (oCD-NH2/Dox). Here we extend these studies by in vitro and in vivo evaluations. An in vitro cytotoxicity model consisting of an overlay of a neuroblastoma (NB) cell-containing agar layer above a drug-loaded FBG layer was used. Local toxicity in vivo (histology and blood analysis) was studied in a mouse orthotopic NB model (SHSY5YLuc+ cells implanted into the left adrenal gland). In vitro data show that FBGs loaded with oCD-NH2/Dox have a slightly lower cytotoxicity against NB cell lines than those loaded with Dox. Fibrinogen (FG), and Ca2+ concentrations may modify this activity. In vivo data support a lower general and local toxicity for FBGs loaded with oCD-NH2/Dox than those loaded with Dox. Our results suggest a possible increase of the therapeutic index of Dox when locally administered through FBGs loaded with oCD-NH2/Dox, opening the possibility of using these releasing systems for the treatment of neuroblastoma.

It has been reported that the ATM kinase inhibitor KU60019 preferentially radiosensitizes orthotopic high grade gliomas (HGG) driven by established U87 and U1242 cell lines bearing specific TP53 mutations. We wished to determine whether those results could be extended to tumors driven by primary glioma initiating cells (GIC) that closely mimic clinical tumors. Orthotopic HGG were developed in immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by intracranial injection of primary GIC isolated from the adult glioblastoma COMI (acronym of patient’s name) and the pediatric anaplastic astrocytoma 239/12. Similar to the clinical tumors of origin, the orthotopic tumors COMI and 239/12 displayed different growth properties with a voluminous expansive lesion that exerted considerable mass effect on the adjacent structures and an infiltrating, gliomatosis-like growth pattern with limited compressive attitude, respectively. Significant elongations of median animal survival bearing the adult COMI tumor was observed after one KU60019 convection enhanced delivery followed by total 7.5 Gy of ionizing radiation delivered in fifteen 0.5 Gy fractions, as compared to animals treated with vehicle + ionizing radiation (105 vs 89 days; ratio: 0.847; 95% CI of ratio 0.4969 to 1.198; P:0.0417). Similarly, a trend to increased median survival was observed with the radiosensitized pediatric tumor 239/12 (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891). Our results indicate that radiosensitization by KU60019 is effective towards different orthotopic gliomas that faithfully mimic the clinical tumors and that multiple GIC-based animal models may be essential to develop novel therapeutic protocols for HGG transferable to the clinics.

meso-(p-acetamidophenyl)-calix[4]pyrrole 3 was found to exhibit remarkable cytotoxicity towards A549 cancer cells. A comparative study including the isomer of 3meso-(m-acetamidophenyl)-calix[4]pyrrole 5, as well as molecules containing ‘fragments’ of these structures, demonstrated that both the calix[4]pyrrole and the acetamidophenyl units are essential for high cytotoxicity. Although calix[4]pyrroles and other anion-complexing ionophores have recently been reported to induce apoptosis by perturbing cellular chloride concentrations, in our study an alternative mechanism has emerged, as proven by the isolation of covalent DNA adducts revealed by the 32P postlabelling technique. Preliminary pharmacokinetic studies indicate that 3 is able to cross the Blood-Brain-Barrier, therefore being a potential drug that could kill primary and brain metastatic cancer cells simultaneously.

It has been reported that the ATM kinase inhibitor KU60019 preferentially radiosensitizes orthotopic high grade gliomas (HGG) driven by established U87 and U1242 cell lines bearing specific TP53 mutations. We wished to determine whether those results could be extended to tumors driven by primary glioma initiating cells (GIC) that closely mimic clinical tumors. Orthotopic HGG were developed in immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice by intracranial injection of primary GIC isolated from the adult glioblastoma COMI (acronym of patient's name) and the pediatric anaplastic astrocytoma 239/12. Similar to the clinical tumors of origin, the orthotopic tumors COMI and 239/12 displayed different growth properties with a voluminous expansive lesion that exerted considerable mass effect on the adjacent structures and an infiltrating, gliomatosis-like growth pattern with limited compressive attitude, respectively. Significant elongations of median animal survival bearing the adult COMI tumor was observed after one KU60019 convection enhanced delivery followed by total 7.5 Gy of ionizing radiation delivered in fifteen 0.5 Gy fractions, as compared to animals treated with vehicle + ionizing radiation (105 vs 89 days; ratio: 0.847; 95% CI of ratio 0.4969 to 1.198; P:0.0417) [ARRIVE 16]. Similarly, a trend to increased median survival was observed with the radiosensitized pediatric tumor 239/12 (186 vs 167 days; ratio: 0.8978; 95% CI of ratio: 0.5352 to 1.260; P: 0.0891) [ARRIVE 16]. Our results indicate that radiosensitization by KU60019 is effective towards different orthotopic gliomas that faithfully mimic the clinical tumors and that multiple GIC-based animal models may be essential to develop novel therapeutic protocols for HGG transferable to the clinics.

Fibrin gels are attractive biomaterials for local delivery of a variety of agents, from drugs to proteins. Similarly, polymer-anticancer-drug conjugates and nanoparticles are emerging as potential candidates for cancer treatment. Combining these different approaches, we have studied the efficacy of fibrin gels loaded with cisplatin (DDP) and a complex of DDP with hyaluronate (DDP-HA) for tumor growth inhibition in a melanoma model. Loaded gels prepared at relatively high fibrinogen concentration (22 mg/ml) showed good in vitro antiproliferative activities, prolonged release of the anticancer drug, and a long persistence (10-15 days) in vivo when implanted subcutaneously (sc) in immunodeficient mice. Gels loaded with DDP or DDP-HA containing 1/3 or even 1/6 of their systemic dose (6 mg/kg) and positioned under the tumor mass in mice bearing a sc human SK-Mel-28 tumor showed an antitumor activity better than that of the original parent compound given intraperitoneally (ip). Moreover, in an additional experiment in vivo, fibrin gels loaded with N-trimethyl chitosan-based nanoparticles containing a DDP-HA complex were assayed, resulting in a further 8 % improvement of anticancer activity, with lesser adverse systemic toxic effects. Taken together, these results suggest that the combination of fibrin gels and drugs complexed with suitable macromolecules holds great promise for loco-regional anticancer therapy of melanoma and other surgically removable cancer types.

MALDI-TOF MS was used to recognise serum peptidome profiles predictive of mortality in women affected by early BCa. Mortality was analysed based on signal profiling, and appropriate statistics were used. The results indicate that four signals were increased in deceased patients compared with living patients. Three of the four signals were individually associated with all-cause mortality, but only one having mass/charge ratio (m/z) 1,046.49 was associated with BCa-specific mortality and was the only peak to maintain an independent prognostic role after multivariate analysis. Two groups exhibiting different mortality probabilities were identified after clustering patients based on the expression of the four peptides, but m/z 1,046.49 was exclusively expressed in the cluster exhibiting the worst mortality outcome, thus confirming the crucial value of this peptide. The specific role of this peak was confirmed by competing risk analysis. MS findings were validated by ELISA analysis after demonstrating that m/z 1,046.49 structurally corresponded to Angiotensin II (ATII). In fact, mortality results obtained after arbitrarily dividing patients according to an ATII serum value of 255 pg/ml (which corresponds to the 66(th) percentile value) were approximately comparable to those previously demonstrated when the same patients were analysed according to the expression of signal m/z 1,046.49. Similarly, ATII levels were specifically correlated with BCa-related deaths after competing risk analysis. In conclusion, ATII levels were increased in women who exhibited worse mortality outcomes, reinforcing the evidence that this peptide potentially significantly affects the natural history of early BCa. Our findings also confirm that MALDI-TOF MS is an efficient screening tool to identify novel tumour markers and that MS findings can be rapidly validated through less complex techniques, such as ELISA.

Geena 2 is a tool for filtering, averaging, and aligning MALDI/TOF mass spectra, designed to assist scientists in the analysis of high volumes of data and support them for comparative studies. Three web interfaces are available with different levels of complexity. In this manuscript, we explain how to use Geena 2 with these three interfaces to perform analyses of one's own data. Two support protocols showing how to check the example input file and how to create an input file with own data are also presented. © 2018 by John Wiley & Sons, Inc.

Background Local delivery of anticancer drugs represents a desirable type of treatment. Nevertheless, characteristics such as availability, biocompatibility, ease of operation, and efficacy sometimes represent difficult to overcome hurdles. Fibrin gels (FBGs) may be attractive biomaterials for local treatment when loaded with different chemotherapeutics or with polymer-anticancer-drug conjugates and nanoparticles. These components, linked together, might represent candidates to counteract local recurrences or reduce the volume of inoperable tumors. In the present study we analyzed the features of in vitro release of different formulations of doxorubicin (DOXO) from FBGs, and in vivo FBGs degradation. Methods In vitro DOXO release from FBGs was studied as a function of thrombin and Ca2+ ion concentrations. DOXO was loaded in FBGs either alone or pre-incorporated in nanoparticles characterized by different physical features. The FBGs in vivo degradation was analyzed after sc or ip positioning. Results Our results suggest that each of the factors involved in the FBGs preparation may have different effects on drug release. In particular, the fibrinogen (FG) concentration and, above all, the DOXO formulation were found to have the greatest impact. Not surprisingly, we have also found a longer permanence in vivo of FBGs prepared at the highest thrombin, Ca2+ ion, and FG concentrations. Conclusions The aim of this work was to study the effect of several conditions for preparing drug delivery systems based on FBGs loaded with different clinical or experimental formulations of DOXO. Our data identify some of these modalities that will be tested in vivo to evaluate their antitumor activity.

Fibrinogen is a large heterogeneous aggregation/degradation-prone protein playing a central role in blood coagulation and associated pathologies, whose structure is not completely resolved. When a high-molecular-weight fraction was analyzed by size-exclusion high-performance liquid chromatography/small-angle X-ray scattering (HPLC-SAXS), several composite peaks were apparent and because of the stickiness of fibrinogen the analysis was complicated by severe capillary fouling. Novel SAS analysis tools developed as a part of theUltraScan Solution Modeler(US-SOMO; http://somo.uthscsa.edu/), an open-source suite of utilities with advanced graphical user interfaces whose initial goal was the hydrodynamic modeling of biomacromolecules, were implemented and applied to this problem. They include the correction of baseline drift due to the accumulation of material on the SAXS capillary walls, and the Gaussian decomposition of non-baseline-resolved HPLC-SAXS elution peaks. It was thus possible to resolve at least two species co-eluting under the fibrinogen main monomer peak, probably resulting from in-column degradation, and two others under an oligomers peak. The overall and cross-sectional radii of gyration, molecular mass and mass/length ratio of all species were determined using the manual or semi-automated procedures available within theUS-SOMOSAS module. Differences between monomeric species and linear and sideways oligomers were thus identified and rationalized. This newUS-SOMOversion additionally contains several computational and graphical tools, implementing functionalities such as the mapping of residues contributing to particular regions ofP(r), and an advanced module for the comparison of primaryI(q)versus qdata with model curves computed from atomic level structures or bead models. It should be of great help in multi-resolution studies involving hydrodynamics, solution scattering and crystallographic/NMR data.

// Barbara Banelli 1, 2, * , Antonio Daga 3, * , Alessandra Forlani 1, * , Giorgio Allemanni 1 , Daniela Marubbi 3, 4 , Maria Pia Pistillo 1 , Aldo Profumo 5 , Massimo Romani 1 1 Laboratory of Tumor Epigenetics, IRCCS AOU San Martino–IST, Genova, Italy 2 Department of Health Sciences, University of Genova, Genova, Italy 3 Laboratory of Regenerative Medicine, IRCCS AOU San Martino–IST, Genova, Italy 4 Department of Experimental Medicine (DIMES), University of Genova, Genova, Italy 5 Biopolymers and Proteomic Unit, IRCCS AOU San Martino–IST, Genova, Italy * These authors contributed equally to this work Correspondence to: Massimo Romani, email: tumor.epigenetics@gmail.com , massimo.romani@hsanmartino.it Keywords: glioblastoma, drug resistance, epigenetics, temozolomide, histone demethylase Received: September 24, 2016 Accepted: March 22, 2017 Published: April 04, 2017 ABSTRACT In glioblastoma several histone demethylase genes (KDM) are overexpressed compared to normal brain tissue and the development of Temozolomide (TMZ) resistance is accompanied by the transient further increased expression of KDM5A and other KDMs following a mechanism that we defined as “epigenetic resilience”. We hypothesized that targeting KDMs may kill the cells that survive the cytotoxic therapy. We determined the effect of JIB 04 and CPI-455, two KDM inhibitors, on glioblastoma cells and found that both molecules are more effective against TMZ-resistant rather than native cells. Because of its lower IC50, we focused on JIB 04 that targets KDM5A and other KDMs as well. We have shown that this molecule activates autophagic and apoptotic pathways, interferes with cell cycle progression, inhibits cell clonogenicity and dephosphorylates Akt thus inactivating a potent pro-survival pathway. We performed combination temozolomide/JIB 04 in vitro treatments showing that these two molecules, under certain conditions, have a strong synergic effect and we hypothesize that JIB 04 intercepts the cells that escape the G2 block exerted by TMZ. Finally we studied the permeability of JIB 04 across the blood-brain barrier and found that this molecule reaches bioactive concentration in the brain; furthermore a pilot in vivo experiment in an orthotopic GB xenograft model showed a trend toward longer survival in treated mice with an Hazard Ratio of 0.5. In conclusion we propose that the combination between cytotoxic drugs and molecules acting on the epigenetic landscape may offer the opportunity to develop new therapies for this invariably lethal disease.

The levels of urinary catecholamine metabolites, such as homovanillic acid (HVA) and vanillylmandelic acid, are routinely used as a clinical tool in the diagnosis and follow-up of neuroblastoma (NB) patients. Recently, in the Clinical Pathology Laboratory Unit of G. Gaslini Children Hospital, a commercial method that employs liquid chromatography coupled to electrochemical detection (LC-EC) has been introduced for the measurement of these metabolites in the routine laboratory practice. Using this LC-EC method, an unknown peak could be observed only in samples derived from NB patients. To investigate the nature of this peak, we used a combination of liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) and liquid chromatography-ion trap tandem mass spectrometry (LC-IT-MS). The first approach was used to obtain the elemental composition of the ions present in this new signal. To get additional structural information useful for the elucidation of unknown compounds, the ion trap analyzer was exploited. We were able to identify not just one, but three unknown signals in urine samples from NB patients which corresponded to three conjugated products of HVA: HVA sulfate and two glucuronoconjugate isomers. The enzymatic hydrolysis with β-glucuronidase confirmed the proposed structures, while the selective alkaline hydrolysis allowed us to distinguish the difference between phenol- and acyl-glucuronide of HVA. The latter was the unknown peak observed in LC-EC separations of urine samples from NB patients.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

We describe a method for the simultaneous determination of the five fibrinopeptide forms derived from the thrombin-promoted activation of human fibrinogen by capillary zone electrophoresis (CZE). The fibrinopeptide mixture was first desalted by a solid-phase extraction (SPE) step. The analysis was performed in reversed polarity in a highly cross-linked polyethylene glycol (PEG)-coated capillary with UV-light absorption detection at 200 nm. Several parameters including buffer concentration and pH, presence of an organic modifier, temperature, and applied voltage, have been tested. The best separations were obtained within 20 min, utilizing a 20 mM sodium phosphate buffer without organic modifier, in the narrow 6.1-6.2 pH range, at 25 degrees C, with an applied voltage of 20 kV. Quantitative analysis is made possible by the use of sheep fibrinopeptide A as an internal standard to correct for both extraction and injection errors.

We have recently reported that glioblastoma (GB)-initiating cells (GIC) with low expression and/or mutation of TP53 and high expression of PI3K ("responder" genetic profile) can be effectively and safely radiosensitized by the ATM inhibitor KU60019. We report here on drug's diffusion and elimination from the animal body and brain, its effects on orthotopic GB and efficacy toward pediatric GIC. Healthy mice were infused by convection enhanced delivery (CED) with KU60019 and the drug kinetics followed by high performance liquid chromatography-mass spectrometry. Already at the end of CED, KU60019 had diffused from the injection site to the ipsilateral and, to a lower extent, controlateral hemisphere. After 24 hr, no drug could be detected all over the brain or in other organs, indicating rapid draining and excretion. After intraperitoneal injection, traces only of KU60019 could be detected in the brain, indicating inability to cross the brain-blood barrier. Consistent with the induction of cell cycle progression previously observed in vitro, KU60019 stimulated proliferation of orthotopic GB cells with the highest effect observed 96 hr after drug delivery. Adult GIC with high expression of TP53 and low expression of PI3K could be radiosensitized by KU60019, although less promptly than GIC bearing the "responder" profile. Consistent with the kinetics of proliferation induction, the highest radiosensitizing effect was observed 96 hr after delivery of KU60019 to GIC. Pediatric GIC could be similarly radiosensitized after exposure to KU60019. The results indicate that ATM inhibition may allow to radiosensitize a wide range of adult and pediatric high-grade gliomas.

The azobenzene unit used as a photochemically and thermally switchable linker in the assembly of a bis-calix[4]pyrrole receptor provides a means to modulate the binding of bis-carboxylates of significant biological importance in cancer research. Conversely, the complexation of different bis-anionic guests has significant kinetic effects on both the photochemical and thermal trans/cis isomerization of the azobenzene unit.

Many biological supramolecular structures are formed by polymerization of macromolecular monomers. Light scattering techniques can provide structural information from such systems, if suitable procedures are used to collect the data and then to extract the relevant parameters. We present an experimental set-up in which a commercial multiangle laser light scattering photometer is linked to a stopped-flow mixer, allowing, in principle, the time-resolved extrapolation of the weight-average molecular weight M(w) and of the z-average square radius of gyration (z) of the polymers from Zimm-like plots. However, if elongated structures are formed as the polymerization proceeds, curved plots rapidly arise, from which M(w) and (z) cannot be recovered by linear fitting. To verify the correctness of a polynomial fitting procedure, polydisperse collections of rod-like or worm-like particles of different lengths, generated at various stages during bifunctional polycondensations of rod-like macromolecular monomers, were considered. Then, the angular dependence of their time-averaged scattered intensity was calculated in the Rayleigh-Gans-Debye approximation, with random and systematic noise also added to the data. For relatively narrow size distributions, a third-degree polynomial fitting gave satisfactory results across a broad range of conversion degrees, yielding M(w) and (z) values within 2% and no greater than 10-20%, respectively, of the calculated values. When more broad size distributions were analyzed, the procedure still performed well for semiflexible polymers, but started to seriously underestimate both M(w) and (z) when rigid rod-like particles were analyzed, even at relatively low conversion degrees. The data were also analyzed in the framework of the Casassa approximation, from which the mass per unit length of the polymers can be derived. These procedures were applied to a set of data taken on the early stages of the thrombin-catalyzed polymerization of fibrinogen, a rod-like macromolecule approximately 50 nm long. The polymers, grown in the absence of Ca(2+) by rate-limiting amounts of thrombin, appeared to be characterized by a much broader size distribution than the one expected for a classical Flory bifunctional polycondensation, and they seem to behave as relatively flexible worm-like double-stranded chains. Evidence for the formation of fibrinogen-fibrin monomer complexes is also inferred from the time dependence of the mass/length ratio. However, our data are also compatible with the presence of limited amounts of single-stranded structures in the very early stages, either as a secondary, less populated pathway, or as transient intermediates to the classical double-stranded fibrils.

Trastuzumab, a humanized monoclonal antibody directed against the epidermal growth factor receptor 2 (HER2), is a milestone in the treatment of HER2-overexpressing breast cancer patients. An enzyme-linked immunosorbent assay (ELISA) for trastuzumab has been developed for routine use in the laboratory to support clinical and pharmacokinetic studies to optimize therapy. The method relies on an antigen peptide linked to a 96-well plate via the streptavidin/biotin system. The peptide sequence mimics the extracellular portion of the HER2 receptor that is recognized by trastuzumab. The calibration range of the assay is 10 to 360 ng/mL per well, corresponding to a trastuzumab serum concentration from 5 to 180 μg/mL with a lower limit of quantification of 10 μg/mL. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum using this assay. The procedure was also tested in sera obtained from breast cancer patients to evaluate trastuzumab serum levels, confirming the applicability of method that could be a valid assay to use in daily laboratory practice.

The formation of a fibrin network following fibrinogen enzymatic activation is the central event in blood coagulation and has important biomedical and biotechnological implications. A non-covalent polymerization reaction between macromolecular monomers, it consists basically of two complementary processes: elongation/branching generates an interconnected 3D scaffold of relatively thin fibrils, and cooperative lateral aggregation thickens them more than 10-fold. We have studied the early stages up to the gel point by fast fibrinogen:enzyme mixing experiments using simultaneous small-angle X-ray scattering and wide-angle, multi-angle light scattering detection. The coupled evolutions of the average molecular weight, size, and cross section of the solutes during the fibrils growth phase were thus recovered. They reveal that extended structures, thinner than those predicted by the classic half-staggered, double-stranded mechanism, must quickly form. Following extensive modeling, an initial phase is proposed in which single-bonded "Y-ladder" polymers rapidly elongate before undergoing a delayed transition to the double-stranded fibrils. Consistent with the data, this alternative mechanism can intrinsically generate frequent, random branching points in each growing fibril. The model predicts that, as a consequence, some branches in these expanding "lumps" eventually interconnect, forming the pervasive 3D network. While still growing, other branches will then undergo a Ca(2+)/length-dependent cooperative collapse on the resulting network scaffolding filaments, explaining their sudden thickening, low final density, and basic mechanical properties.

A small library of polytopic receptors has been synthesized from meso-p- and meso-m-aminophenylcalix[4]pyrroles and p- or m-phthaloyl or trimesic chloride. Selected bis-carboxylates and the citrate anion, which either exhibit altered distribution profiles in cancerous tissues in comparison with healthy tissues or are metabolites of carcinogenic substances (for example, trans,trans-muconic acid from benzene exposure in humans) were tested as ligands. Varied affinities and binding modes were observed as a function of the number of calix[4]pyrroles and the topology of amide units present in each of the polytopic receptors. The structures of the 1:1 complexes derived by molecular modeling are in excellent agreement with the results of (1)H NMR complexation studies.

The usefulness of a new high-performance liquid chromatography/small-angle X-ray scattering (HPLC-SAXS) data analysis module within the multi-resolution modeling suite US-SOMO is illustrated with size-exclusion small-angle X-ray scattering (SE-SAXS) data of a crude bovine serum albumin sample. The module is then applied to the SE-SAXS study of a human plasma fibrinogen high-molecular-weight fraction presenting severe aggregation problems and a split non-symmetrical main elution peak probably resulting from in-column degradation., Fibrinogen is a large heterogeneous aggregation/degradation-prone protein playing a central role in blood coagulation and associated pathologies, whose structure is not completely resolved. When a high-molecular-weight fraction was analyzed by size-exclusion high-performance liquid chromatography/small-angle X-ray scattering (HPLC-SAXS), several composite peaks were apparent and because of the stickiness of fibrinogen the analysis was complicated by severe capillary fouling. Novel SAS analysis tools developed as a part of the UltraScan Solution Modeler (US-SOMO; http://somo.uthscsa.edu/), an open-source suite of utilities with advanced graphical user interfaces whose initial goal was the hydrodynamic modeling of biomacromolecules, were implemented and applied to this problem. They include the correction of baseline drift due to the accumulation of material on the SAXS capillary walls, and the Gaussian decomposition of non-baseline-resolved HPLC-SAXS elution peaks. It was thus possible to resolve at least two species co-eluting under the fibrinogen main monomer peak, probably resulting from in-column degradation, and two others under an oligomers peak. The overall and cross-sectional radii of gyration, molecular mass and mass/length ratio of all species were determined using the manual or semi-automated procedures available within the US-SOMO SAS module. Differences between monomeric species and linear and sideways oligomers were thus identified and rationalized. This new US-SOMO version additionally contains several computational and graphical tools, implementing functionalities such as the mapping of residues contributing to particular regions of P(r), and an advanced module for the comparison of primary I(q) versus q data with model curves computed from atomic level structures or bead models. It should be of great help in multi-resolution studies involving hydrodynamics, solution scattering and crystallographic/NMR data.

Micellar electrokinetic chromatography (MEKC) using bile salts has been employed to separate retinoids differing in structure and charge; bile salts in MEKC allows the separation of liposoluble molecules but, to the best of our knowledge, there are only few data on the above-mentioned technique for the separation of highly hydrophobic compounds. The three natural vitamin A derivatives, retinal, retinol and retinoic acid, were successfully separated by MEKC using sodium cholate within a relatively short time (ca. 25 min), whereas the separation of these compounds was not successful using sodium dodecyl sulfate or sodium deoxycholate. Several parameters (pH and organic modifiers, in addition to bile salts concentration) have been tested to provide a system that can be extended to synthetic retinoids, which are often used in treating several diseases, including cancer prevention and therapy.

Gross cystic disease (GCDB) is a breast benign condition predisposing to breast cancer. Cryopreserved sera from GCDB patients, some of whom later developed a cancer (cases), were studied to identify potential risk markers. A MALDI-TOF mass spectrometry analysis found several complement C3f fragments having a significant increased abundance in cases compared to controls. After multivariate analysis, the full-length form of C3f maintained a predictive value of breast cancer risk. Higher levels of C3f in the serum of women affected by a benign condition like GCDB thus appears to be correlated to the development of breast cancer even 20 years later. Biological significance Increased complement system activation has been found in the sera of women affected by GCDB who developed a breast cancer, even twenty or more years later. C3f may predict an increased breast cancer risk in the healthy population and in women affected by predisposing conditions.

International audience; Introduction: Fibrinogen is a key component of the blood coagulation system and plays important, diverse roles in several relevant pathologies such as thrombosis, hemorrhage, and cancer. It is a large glycoprotein whose three-dimensional molecular structure is not fully known. Furthermore, circulating fibrinogen is highly heterogeneous, mainly due to proteolytic degradation and alternative mRNA processing. Recombinant production of human fibrinogen allows investigating the impact on the three-dimensional structure of specific changes in the primary structure. Methods: We performed analytical ultracentrifugation analyses of a full-length recombinant human fibrinogen, its counterpart purified from human plasma, and a recombinant human fibrinogen with both Aα chains truncated at amino acid 251, thus missing their last 359 amino acid residues. Results: We have accurately determined the translational diffusion and sedimentation coefficients (D t(20,w) 0 , s (20,w) 0) of all three species. This was confirmed by derived molecular weights within 1% for the full length species, and 5% for the truncated species, as assessed by comparison with SDS-PAGE/Western blot analyses and primary structure data. No significant differences in the values of D t(20,w) 0 and s (20,w) 0 were found between the recombinant and purified full length human fibrinogens, while slightly lower and higher values, respectively, resulted for the recombinant truncated human fibrinogen compared to a previously characterized purified human fibrinogen fragment X obtained by plasmin digestion. Conclusions: Full-length recombinant fibrinogen is less polydisperse but hydrodynamically indistinguishable from its counterpart purified from human plasma. Recombinant Aα251-truncated human fibrinogen instead behaves differently from fragment X, suggesting a role for the Bβ residues 1-52 in inter-molecular interactions. Overall, these new hydrodynamic data will constitute a reliable benchmark against which models of fibrinogen species could be compared.

The levels of urinary catecholamine metabolites, such as homovanillic acid (HVA) and vanillylmandelic acid, are routinely used as a clinical tool in the diagnosis and follow-up of neuroblastoma (NB) patients. Recently, in the Clinical Pathology Laboratory Unit of G. Gaslini Children Hospital, a commercial method that employs liquid chromatography coupled to electrochemical detection (LC-EC) has been introduced for the measurement of these metabolites in the routine laboratory practice. Using this LC-EC method, an unknown peak could be observed only in samples derived from NB patients. To investigate the nature of this peak, we used a combination of liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) and liquid chromatography-ion trap tandem mass spectrometry (LC-IT-MS). The first approach was used to obtain the elemental composition of the ions present in this new signal. To get additional structural information useful for the elucidation of unknown compounds, the ion trap analyzer was exploited. We were able to identify not just one, but three unknown signals in urine samples from NB patients which corresponded to three conjugated products of HVA: HVA sulfate and two glucuronoconjugate isomers. The enzymatic hydrolysis with β-glucuronidase confirmed the proposed structures, while the selective alkaline hydrolysis allowed us to distinguish the difference between phenol- and acyl-glucuronide of HVA. The latter was the unknown peak observed in LC-EC separations of urine samples from NB patients.

Fibrin polymerization occurs in two steps: the assembly of fibrin monomers into protofibrils and the lateral aggregation of protofibrils into fibers. Here we describe a novel fibrinogen that apparently impairs only lateral aggregation. This variant is a hybrid, where the human αC region has been replaced with the homologous chicken region. Several experiments indicate this hybrid human-chicken (HC) fibrinogen has an overall structure similar to normal. Thrombin-catalyzed fibrinopeptide release from HC fibrinogen was normal. Plasmin digests of HC fibrinogen produced fragments that were similar to normal D and E; further, as with normal fibrinogen, the knob 'A' peptide, GPRP, reversed the plasmin cleavage associated with addition of EDTA. Dynamic light scattering and turbidity studies with HC fibrinogen showed polymerization was not normal. Whereas early small increases in hydrodynamic radius and absorbance paralleled the increases seen during the assembly of normal protofibrils, HC fibrinogen showed no dramatic increase in scattering as observed with normal lateral aggregation. To determine whether HC and normal fibrinogen could form a copolymer, we examined mixtures of these. Polymerization of normal fibrinogen was markedly changed by HC fibrinogen, as expected for mixed polymers. When the mixture contained 0.45 μM normal and 0.15 μM HC fibrinogen, the initiation of lateral aggregation was delayed and the final fiber size was reduced relative to normal fibrinogen at 0.45 μM. Considered altogether, our data suggest that HC fibrin monomers can assemble into protofibrils or protofibril-like structures, but these either cannot assemble into fibers or assemble into very thin fibers.

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API-MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API-MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at -80°C for 18 months or at -20°C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API-MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.

The shape and solution properties of fibrinogen are affected by the location of the C-terminal portion of the Aalpha chains, which is presently still controversial. We have measured the hydrodynamic properties of a human fibrinogen fraction with these appendages mostly intact, of chicken fibrinogen, where they lack 11 characteristic 13-amino acids repeats, and of human fragment X, a plasmin early degradation product in which they have been removed. The human fibrinogen/fragment X samples were extensively characterized by SDS-PAGE/Western blotting and mass spectrometry, allowing their composition to be precisely determined. The solution properties of all samples were then investigated by analytical ultracentrifugation and size-exclusion HPLC coupled with multi-angle light scattering and differential pressure viscometry detectors. The measured parameters suggest that the extra repeats have little influence on the overall fibrinogen conformation, while a significant change is brought about by the removal of the C-terminal portion of the Aalpha chains beyond residue Aalpha200.