Functional Graphene for Peritumoral Brain Microenvironment Modulation Therapy in Glioblastoma.

Peritumoral brain invasion is the main target to cure glioblastoma. Chemoradiotherapy and targeted therapies fail to combat peritumoral relapse. Brain inaccessibility and tumor heterogeneity explain this failure, combined with overlooking the peritumor microenvironment. Reduce graphene oxide (rGO) provides a unique opportunity to modulate the local brain microenvironment. Multimodal graphene impacts are reported on glioblastoma cells in vitro but fail when translated in vivo because of low diffusion. This issue is solved by developing a new rGO formulation involving ultramixing during the functionalization with polyethyleneimine (PEI) leading to the formation of highly water-stable rGO-PEI. Wide mice brain diffusion and biocompatibility are demonstrated. Using an invasive GL261 model, an anti-invasive effect is observed. A major unexpected modification of the peritumoral area is also observed with the neutralization of gliosis. In vitro, mechanistic investigations are performed using primary astrocytes and cytokine array. The result suggests that direct contact of rGO-PEIUT neutralizes astrogliosis, decreasing several proinflammatory cytokines that would explain a bystander tumor anti-invasive effect. rGO also significantly downregulates several proinvasive/protumoral cytokines at the tumor cell level. The results open the way to a new microenvironment anti-invasive nanotherapy using a new graphene nanomaterial that is optimized for in vivo brain delivery.

Fast-field-cycling NMR at very low magnetic fields: water molecular dynamic biomarkers of glioma cell invasion and migration.

Our objective was to study NMR relaxometry of glioma invasion/migration at very low field (<2 mT) by fast-field-cycling NMR (FFC-NMR) and to decipher the pathophysiological processes of glioma that are responsible for relaxation changes in order to open a new diagnostic method that can be extended to imaging. The phenotypes of two new glioma mouse models, Glio6 and Glio96, were characterized by T2w -MRI, HE histology, Ki-67 immunohistochemistry (IHC) and CXCR4 RT-qPCR, and were compared with the U87 model. R1 dispersions of glioma tissues were acquired at low field (0.1 mT-0.8 T) ex vivo and were fitted with Lorentzian and power-law models to extract FFC biomarkers related to the molecular dynamics of water. In order to decipher relaxation changes, three main invasion/migration pathophysiological processes were studied: hypoxia, H2 O2 function and the water-channel aquaporin-4 (AQP4). Glio6 and Glio96 were characterized with invasion/migration phenotype and U87 with high cell proliferation as a solid glioma. At very low field, invasion/migration versus proliferation was characterized by a decrease in the relaxation-rate constant (ΔR1 ≈ -32% at 0.1 mT) and correlation time (≈-40%). These decreases corroborated the AQP4-IHC overexpression (Glio6/Glio96: +92%/+46%), suggesting rapid transcytolemmal water exchange, which was confirmed by the intracellular water-lifetime τIN decrease (ΔτIN ≈ -30%). In functional experiments, AQP4 expression, τIN and the relaxation-rate constant at very low field were all found to be sensitive to hypoxia and to H2 O2 stimuli. At very low field the role of water exchanges in relaxation modulation was confirmed, and for the first time it was linked to the glioma invasion/migration and to its main pathophysiological processes: hypoxia, H2 O2 redox signaling and AQP4 expression. The method appears appropriate to evaluate the effect of drugs that can target these pathophysiological mechanisms. Finally, FFC-NMR operating at low field is demonstrated to be sensitive to invasion glioma phenotype and can be straightforwardly extended to FFC-MRI as a new cancer invasion imaging method in the clinic.

Genomic duplication in the 19q13.42 imprinted region identified as a new genetic cause of intrauterine growth restriction.

We report findings from a male fetus of 26 weeks' gestational age with severe isolated intrauterine growth restriction (IUGR). Chromosomal microarray analysis (CMA) on amniotic fluid cells revealed a 1.06-Mb duplication in 19q13.42 inherited from the healthy father. This duplication contains 34 genes including ZNF331, a gene encoding a zinc-finger protein specifically imprinted (paternally expressed) in the placenta. Study of the ZNF331 promoter by methylation-specific-multiplex ligation-dependent probe amplification showed that the duplicated allele was not methylated in the fetus unlike in the father's genome, suggesting both copies of the ZNF331 gene are expressed in the fetus. The anti-ZNF331 immunohistochemical analysis confirmed that ZNF331 was expressed at higher levels in renal and placental tissues from this fetus compared to controls. Interestingly, ZNF331 expression levels in the placenta have previously been reported to inversely correlate with fetal growth parameters. The original observation presented in this report showed that duplication of ZNF331 could be a novel genetic cause of isolated IUGR and underlines the usefulness of CMA to investigate the genetic causes of isolated severe IUGR.

CEST-MRI for glioma pH quantification in mouse model: Validation by immunohistochemistry.

In glioma, the acidification of the extracellular tumor microenvironment drives proliferation, angiogenesis, immunosuppression, invasion and chemoresistance. Therefore, quantification of glioma extracellular pH (pHe) is of crucial importance. This study is focused on the application of the YbHPDO3A (ytterbium 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane) probe for in vivo glioma pHe quantification using chemical exchange saturation transfer (CEST)-MRI and its correlation with tumor metabolism assessed by immunohistochemistry. The U87 glioma mouse model was used (n = 18) and MRI performed at 4.7 T. CEST-MRI of YbHPDO3A solutions at different pH values showed two resolved CEST spectra at 71 ppm and 99 ppm, both sensitive to pH variations, allowing therefore calculation of the ratiometric curve for in vivo pH quantification. In vivo MRI sequences consisted of T2w for tumor localization, T2w * to assess YbHPDO3A biodistribution by exploiting its magnetic susceptibility effect and CEST for glioma pHe mapping. T2w * images show that YbHPDO3A extravasates in tumor in regions with damaged blood-brain barrier. The pHe is calculated only in these regions. Hematoxylin/eosin histology and Ki-67, CA-IX (carbonic anhydrase 9) and NHE-1 immunohistochemical staining were performed; their expression rates were compared with the in vivo pHe values. On the basis of the cell proliferation marker Ki-67, two groups were defined: one group with a lower mitotic index (MI% < 20% = mean value) and a mean pHe value of 7.00 (low-proliferation/high-pH group) and the other with MI% > 20% and an acidic pHe of 6.6 (high-proliferation/low-pH group). CA-IX and NHE-1 were over-expressed in the high-proliferation/low-pH group (CA-IX, 92 ± 7% versus 30 ± 13%; NHE-1, 84 ± 8% versus 35 ± 11%), indicating an acidic/hypoxic microenvironment. These immunohistochemical results are consistent with our pHe mapping (Pearson correlation coefficient > 0.70) and provide evidence for the feasibility of the CEST-MRI method with the YbHPDO3A probe for glioma pHe quantification at 4.7 T. Importantly, the YbHPDO3A probe has similar chemical and biological properties to the clinically approved MRI contrast agent GdHPDO3A. This makes the method promising for a clinical translation.

Microduplication of the ARID1A gene causes intellectual disability with recognizable syndromic features.

PURPOSE: To determine whether duplication of the ARID1A gene is responsible for a new recognizable syndrome. METHODS: We describe four patients with a 1p36.11 microduplication involving ARID1A as identified by array-comparative genomic hybridization . We performed comparative transcriptomic analysis of patient-derived fibroblasts using RNA sequencing and evaluated the impact of ARID1A duplication on the cell cycle using fluorescence-activated cell sorting. Functional relationships between differentially expressed genes were investigated with ingenuity pathway analysis (IPA). RESULTS: Combining the genomic data, we defined a small (122 kb), minimally critical region that overlaps the full ARID1A gene. The four patients shared a strikingly similar phenotype that included intellectual disability and microcephaly. Transcriptomic analysis revealed the deregulated expression of several genes previously linked to microcephaly and developmental disorders as well as the involvement of signaling pathways relevant to microcephaly, among which the polo-like kinase (PLK) pathway was especially notable. Cell-cycle analysis of patient-derived fibroblasts showed a significant increase in the proportion of cells in G1 phase at the expense of G2-M cells. CONCLUSION: Our study reports a new microduplication syndrome involving the ARID1A gene. This work is the first step in clarifying the pathophysiological mechanism that links changes in the gene dosage of ARID1A with intellectual disability and microcephaly.Genet Med advance online publication 01 December 2016.

Microscopic DTI accurately identifies early glioma cell migration: correlation with multimodal imaging in a new glioma stem cell model.

Monitoring glioma cell infiltration in the brain is critical for diagnosis and therapy. Using a new glioma Glio6 mouse model derived from human stem cells we show how diffusion tensor imaging (DTI) may predict glioma cell migration/invasion. In vivo multiparametric MRI was performed at one, two and three months of Glio6 glioma growth (Glio6 (n = 6), sham (n = 3)). This longitudinal study reveals the existence of a time window to study glioma cell/migration/invasion selectively. Indeed, at two months only Glio6 cell invasion was detected, while tumor mass formation, edema, blood-brain barrier leakage and tumor angiogenesis were detected later, at three months. To robustly confirm the potential of DTI for detecting glioma cell migration/invasion, a microscopic 3D-DTI (80 µm isotropic spatial resolution) technique was developed and applied to fixed mouse brains (Glio6 (n = 6), sham (n = 3)). DTI changes were predominant in the corpus callosum (CC), a known path of cell migration. Fractional anisotropy (FA) and perpendicular diffusivity (D⊥ ) changes derived from ex vivo microscopic 3D-DTI were significant at two months of tumor growth. In the caudate putamen an FA increase of +38% (p < 0.001) was observed, while in the CC a - 28% decrease in FA (p < 0.005) and a + 95% increase in D⊥ (p < 0.005) were observed. In the CC, DTI changes and fluorescent Glio6 cell density obtained by two-photon microscopy in the same brains were correlated (p < 0.001, r = 0.69), validating FA and D⊥ as early quantitative biomarkers to detect glioma cell migration/invasion. The origin of DTI changes was assessed by electron microscopy of the same tract, showing axon bundle disorganization. During the first two months, Glio6 cells display a migratory phenotype without being associated with the constitution of a brain tumor mass. This offers a unique opportunity to apply microscopic 3D-DTI and to validate DTI parameters FA and D⊥ as biomarkers for glioma cell invasion.

The brain tissue response to surgical injury and its possible contribution to glioma recurrence.

Surgery is the first line therapy for glioma. However, glioma recurs in 90 % of the patients in the resection margin. The impact of surgical brain injury (SBI) on glioma recurrence is largely overlooked. Herein, we review some of the mechanisms involved in tissue repair that may impact glioma recurrence at the resection margin. Many processes or molecules involved in tissue repair after brain injury are also critical for glioma growth. They include a wide array of secreted growth factors, cytokines and transcription factors including NFКB and STAT3 which in turn activate proliferative and anti-apoptotic genes and processes such as angiogenesis and inflammation. Because some residual glioma cells always remain in the tumor resection margin, there are now compelling arguments to suggest that some aspects of the brain tissue response to SBI can also participate to glioma recurrence at the resection margin. Brain tissue response to SBI recruits angiogenesis and inflammation that precede and then follow tumor recurrence at the resection margin. The healing response to SBI is double edged, as inflammation is involved in regeneration and healing, and has both pro- and anti-tumorigenic functions. A promising therapeutic approach is to normalize and re-educate the molecular and cellular responses at the resection margin to promote anti-tumorigenic processes involved in healing while inhibiting pro-tumorigenic activities. Manipulation of the inflammatory response to SBI to prevent local recurrence could also enhance the efficacy of other therapies such as immunotherapy. However, our current knowledge is far from sufficient to achieve this goal. Acknowledging, understanding and manipulating the double-edged role played by SBI in glioma recurrence is surely challenging, but it cannot be longer delayed.

An in-house assay is superior to Sepsityper for direct matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification of yeast species in blood cultures.

We developed an in-house assay for the direct identification, by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, of yeasts in blood culture. Sixty-one representative strains from 12 species were analyzed in spiked blood cultures. Our assay accurately identified 95 of 107 (88.8%) positive blood cultures and outperformed the commercial Sepsityper kit (81.7% identification).

Rapid-Steady-State-T1 signal modeling during contrast agent extravasation: toward tumor blood volume quantification without requiring the arterial input function.

PURPOSE: This study demonstrates how to quantify the tumor blood volume fraction (BVf) using the dynamic Rapid-Steady-State-T1 (RSST1 )-MRI method despite contrast agent (CA) leakage and without arterial input function (AIF) determination. METHODS: For vasculature impermeable to CAs, the BVf is directly quantified from the RSST1 signal amplitude. In case of CA extravasation, we propose a two-compartment model to describe the dynamic RSST1 signal increase. We applied the mathematical model in a pilot-study on a RG2-glioma model to compare extravasation of two Gd-based CAs. The BVf quantification using the mathematical model in a C6-glioma model (n = 8) with the clinical CA Gd-DOTA was validated using a ΔR2 *-steady-state MRI method with an USPIO and by immunohistochemical staining of perfused vessels labeled with Hoechst-33342 dye in the same rats. RESULTS: BVf in tumor and in healthy brain tissues (0.034 ± 0.005 and 0.026 ± 0.004, respectively) derived from the dynamic RSST1 signal were confirmed by ΔR2 *-steady-state MRI (0.036 ± 0.003 and 0.027 ± 0.002, respectively, correlation coefficient rS = 0.74) and by histology (0.036 ± 0.003 and 0.025 ± 0.004 respectively, rS = 0.87). CONCLUSION: Straightforward tumor BVf quantification without AIF determination is demonstrated in presence of CA leakage. The method will facilitate angiogenesis assessment in longitudinal neuro-oncologic studies in particular when monitoring the response to antiangiogenic therapies.

Implantable SDF-1α-loaded silk fibroin hyaluronic acid aerogel sponges as an instructive component of the glioblastoma ecosystem: Between chemoattraction and tumor shaping into resection cavities.

In view of inevitable recurrences despite resection, glioblastoma (GB) is still an unmet clinical need. Dealing with the stromal-cell derived factor 1-alpha (SDF-1α)/CXCR4 axis as a hallmark of infiltrative GB tumors and with the resection cavity situation, the present study described the effects and relevance of a new engineered micro-nanostructured SF-HA-Hep aerogel sponges, made of silk fibroin (SF), hyaluronic acid (HA) and heparin (Hep) and loaded with SDF-1α, to interfere with the GB ecosystem and residual GB cells, attracting and confining them in a controlled area before elimination. 70 µm-pore sponges were designed as an implantable scaffold to trap GB cells. They presented shape memory and fit brain cavities. Histological results after implantation in brain immunocompetent Fischer rats revealed that SF-HA-Hep sponges are well tolerated for more than 3 months while moderately and reversibly colonized by immuno-inflammatory cells. The use of human U87MG GB cells overexpressing the CXCR4 receptor (U87MG-CXCR4+) and responding to SDF-1α allowed demonstrating directional GB cell attraction and colonization of the device in vitro and in vivo in orthotopic resection cavities in Nude rats. Not modifying global survival, aerogel sponge implantation strongly shaped U87MG-CXCR4+ tumors in cavities in contrast to random infiltrative growth in controls. Overall, those results support the interest of SF-HA-Hep sponges as modifiers of the GB ecosystem dynamics acting as "cell meeting rooms" and biocompatible niches whose properties deserve to be considered toward the development of new clinical procedures. STATEMENT OF SIGNIFICANCE: Brain tumor glioblastoma (GB) is one of the worst unmet clinical needs. To prevent the relapse in the resection cavity situation, new implantable biopolymer aerogel sponges loaded with a chemoattractant molecule were designed and preclinically tested as a prototype targeting the interaction between the initial tumor location and its attraction by the peritumoral environment. While not modifying global survival, biocompatible SDF1-loaded hyaluronic acid and silk fibroin sponges induce directional GB cell attraction and colonization in vitro and in rats in vivo. Interestingly, they strongly shaped GB tumors in contrast to random infiltrative growth in controls. These results provide original findings on application of exogenous engineered niches that shape tumors and serve as cell meeting rooms for further clinical developments.

Quantitative rapid steady state T1 magnetic resonance imaging for cerebral blood volume mapping in mice: Lengthened measurement time window with intraperitoneal Gd-DOTA injection.

This work demonstrates how the rapid steady state T1 MRI technique for cerebral blood volume fraction (BVf) quantification can be used with intraperitoneal Gd-DOTA injections in mice at 4.7 T. The peak signal amplitude after intravenous administration (0.7 mmol/kg) and the steady state signal amplitude reached 15 min after intraperitoneal administration (6 mmol/kg) in the same mice lead to equivalent BVf measures in the order of 0.02 in the brain. The resulting time window for BVf quantification is ≈30 min and allows for cerebral BVf mapping with increased spatial resolution or signal-to-noise ratio, or for monitoring functional BVf changes. A cerebral BVf increase of up to 25% induced by the vasodilator acetazolamide was observed, validating the vascular origin of the signal. The noninvasive and quantitative rapid steady state T1 technique can be used in serial studies to evaluate new drugs or disease models, such as antiangiogenic therapies in tumors.

Shark cartilage extract induces cytokines expression and release in endothelial cells and induces E-selectin, plasminogen and t-PA genes expression through an antioxidant-sensitive mechanism.

Neovastat® is a standardized extract of marine cartilage, an avascular tissue, which contains many biologically active molecules and has multiple antiangiogenic properties. In addition to VEGFR2 and MMPs inhibition, shark cartilage extract (SCE) has recently been shown to induce tissue plasminogen activator gene (PLAT) expression in bovine endothelial cells in a TNF like manner, by inducing the typical mediators NF-κB and JNK. There is now compelling evidences that the NF-κB and JNK pathways are activated by cytokines induced generation of reactive oxygen species (ROS). We used macroarray genes expression analysis on human umbilical vein endothelial cells, to investigate if that mechanism could mediate the effect of SCE. Transcriptomic results showed that SCE induced expression of several cytokines. Their impact must be important, given that treatment of endothelial cells with the cytokine TNF-α was able to reproduce most of the effects of cartilage extract on genes expression. In addition, most of the genes, known to be inducible by NF-κB or JNK following cytokines stimulation, were less induced by SCE when endothelial cells were pretreated with the antioxidant N-Acetylcysteine (NAC), suggesting a role of ROS in endothelial cell activation by SCE. Finally, the possible effects of PLAT, PLG, SELE, IL8 and PRDX2 (those validated by q-PCR) on angiogenesis, will also be discussed.

Hypoxia-induced expression of VE-cadherin and filamin B in glioma cell cultures and pseudopalisade structures.

Most of our knowledge regarding glioma cell biology comes from cell culture experiments. For many years the standards for glioma cell culture were the use of cell lines cultured in the presence of serum and 20 % O2. However, in vivo, normoxia in many brain areas is in close to 3 % O2. Hence, in cell culture, the experimental value referred as the norm is hyperoxic compared to any brain physiological value. Likewise, cells in vivo are not usually exposed to serum, and low-passaged glioma neurosphere cultures maintained in serum-free medium is emerging as a new standard. A consequence of changing the experimental normoxic standard from 20 % O2 to the more brain physiological value of 3 % O2, is that a 3 % O2 normoxic reference point enabled a more rigorous characterization of the level of regulation of genes by hypoxia. Among the glioma hypoxia-regulated genes characterized using this approach we found VE-cadherin that is required for blood vessel formation, and filamin B a gene involved in endothelial cell motility. Both VE-cadherin and filamin B were found expressed in pseudopalisades, a glioblastoma pathognomonic structure made of hypoxic migrating cancer cells. These results provide additional clues on the role played by hypoxia in the acquisition of endothelial traits by glioma cells and on the functional links existing between pseudopalisades, hypoxia, and tumor progression.

Translation of the ecological trap concept to glioma therapy: the cancer cell trap concept.

Viewing tumors as ecosystems offers the opportunity to consider how ecological concepts can be translated to novel therapeutic perspectives. The ecological trap concept emerged approximately half a century ago when it was observed that animals can prefer an environment of low quality for survival over other available environments of higher quality. The presence of such a trap can drive a local population to extinction. The cancer cell trap concept is the translation of the ecological trap into glioma therapy. It exploits and diverts the invasive potential of glioma cells by guiding their migration towards specific locations where a local therapy can be delivered efficiently. This illustrates how an ecological concept can change therapeutic obstacles into therapeutic tools.

Insights into Healthcare Professionals' Perceptions and Attitudes toward Nanotechnological Device Application: What Is the Current Situation in Glioblastoma Research?

Nanotechnology application in cancer treatment is promising and is likely to quickly spread worldwide in the near future. To date, most scientific studies on nanomaterial development have focused on deepening the attitudes of end users and experts, leaving clinical practice implications unexplored. Neuro-oncology might be a promising field for the application of nanotechnologies, especially for malignant brain tumors with a low-survival rate such as glioblastoma (GBM). As to improving patients' quality of life and life expectancy, innovative treatments are worth being explored. Indeed, it is important to explore clinicians' intention to use experimental technologies in clinical practice. In the present study, we conducted an exploratory review of the literature about healthcare workers' knowledge and personal opinions toward nanomedicine. Our search (i) gives evidence for disagreement between self-reported and factual knowledge about nanomedicine and (ii) suggests the internet and television as main sources of information about current trends in nanomedicine applications, over scientific journals and formal education. Current models of risk assessment suggest time-saving cognitive and affective shortcuts, i.e., heuristics support both laypeople and experts in the decision-making process under uncertainty, whereas they might be a source of error. Whether the knowledge is poor, heuristics are more likely to occur and thus clinicians' opinions and perspectives toward new technologies might be biased.

In-depth exploration of cerebrospinal fluid by combining peptide ligand library treatment and label-free protein quantification.

Cerebrospinal fluid (CSF) is the biological fluid in closest contact with the brain and thus contains proteins of neural cell origin. Hence, CSF is a biochemical window into the brain and is particularly attractive for the search for biomarkers of neurological diseases. However, as in the case of other biological fluids, one of the main analytical challenges in proteomic characterization of the CSF is the very wide concentration range of proteins, largely exceeding the dynamic range of current analytical approaches. Here, we used the combinatorial peptide ligand library technology (ProteoMiner) to reduce the dynamic range of protein concentration in CSF and unmask previously undetected proteins by nano-LC-MS/MS analysis on an LTQ-Orbitrap mass spectrometer. This method was first applied on a large pool of CSF from different sources with the aim to better characterize the protein content of this fluid, especially for the low abundance components. We were able to identify 1212 proteins in CSF, and among these, 745 were only detected after peptide library treatment. However, additional difficulties for clinical studies of CSF are the low protein concentration of this fluid and the low volumes typically obtained after lumbar puncture, precluding the conventional use of ProteoMiner with large volume columns for treatment of patient samples. The method has thus been optimized to be compatible with low volume samples. We could show that the treatment is still efficient with this miniaturized protocol and that the dynamic range of protein concentration is actually reduced even with small amounts of beads, leading to an increase of more than 100% of the number of identified proteins in one LC-MS/MS run. Moreover, using a dedicated bioinformatics analytical work flow, we found that the method is reproducible and applicable for label-free quantification of series of samples processed in parallel.

Perilymph metabolomic and proteomic MALDI-ToF profiling with porous silicon chips: A proof-of-concept study.

INTRODUCTION: Sensorineural hearing losses (SNHLs) are a significant public health issue, and the hearing loss field is desperately in need of effective therapy. Pathophysiological mechanisms are not yet clearly understood in the absence of validated methods to assess the inner ear content. Proteomic and metabolomic analysis of perilymph is opening new research perspectives for SNHLs. We aimed to demonstrate the feasibility of an innovative mass spectrometry (MS) strategy using porous silicon chips (PSCs) to investigate the low molecular weight (LMW) protein and metabolite content of human perilymph. Our second objective was to stratify perilymph samples according to their MS profiles and compare these results with clinical data. MATERIAL AND METHODS: Perilymph samples obtained during cochlear implant surgery from patients with SNHLs were retrieved from a validated biobank. To focus on LMW entities, we used a PSC enrichment protocol before MALDI-ToF MS analysis. PSCs were used as a LMW molecular preanalytical stabilizer and amplifier. Patients' clinical data and SNHL characteristics were retrieved retrospectively from medical charts. RESULTS: We successfully acquired and compared 59 exploitable MS profiles out of 71 perilymph samples. There was a good correlation between duplicates. Comparing both ears from the same patient, we found good reproducibility even when there was a one-year interval between samplings. We identified three distinct groups when comparing the samples' metabolomic profiles and four homogeneous groups comparing their LMW proteome profiles. Clinical data analysis suggested that some groups shared clinical or preanalytical characteristics. CONCLUSION: This proof-of-concept study confirms that LMW proteome and metabolome content of perilymph can be analyzed with PSCs. Based on protein profiles, we managed to stratify perilymp samples according to their molecular composition. These results must be confirmed with a larger population, and sampling methods require improvement, but this approach seems promising. In the future, this approach may pave the way for companion test strategies to precisely diagnose and define potential molecular targets for audioprotective therapies.

Role of Transmembrane Water Exchange in Glioma Invasion/Migration: In Vivo Preclinical Study by Relaxometry at Very Low Magnetic Field.

This work shows that the longitudinal relaxation differences observed at very low magnetic fields between invasion/migration and proliferation processes on glioma mouse models in vivo are related to differences in the transmembrane water exchange basically linked to the aquaporin expression changes. Three glioma mouse models were used: Glio6 and Glio96 as invasion/migration models and U87 as cell proliferation model. In vivo proton longitudinal relaxation-rate constants (R1) at very low fields were measured by fast field cycling NMR (FFC-NMR). The tumor contribution to the observed proton relaxation rate, R1tum (U87: 12.26 ± 0.64 s−1; Glio6: 3.76 ± 0.88 s−1; Glio96: 6.90 ± 0.64 s−1 at 0.01 MHz), and the intracellular water lifetime, τin (U87: 826 ± 19 ms; Glio6: 516 ± 8 ms; Glio96: 596 ± 15 ms), were found to be good diagnostic hallmarks to distinguish invasion/migration from proliferation (p < 0.01 and 0.001). Overexpression of AQP4 and AQP1 were assessed in invasion/migration models, highlighting the pathophysiological role of these two aquaporins in water exchange that, in turn, determine the lower values in the observed R1 relaxation rate constant in glioma invasion/migration. Overall, our findings demonstrate that τin and R1 (measured at very low fields) are relevant biomarkers, discriminating invasion/migration from proliferation in vivo. These results highlight the use of FFC-NMR and FFC-imaging to assess the efficiency of drugs that could modulate aquaporin functions.

Comparative proteomic profiles of Aspergillus fumigatus and Aspergillus lentulus strains by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS).

BACKGROUND: Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was applied to analyze the protein profiles in both somatic and metabolic extracts of Aspergillus species. The study was carried out on some Aspergillus species within the Fumigati section (Aspergillus fumigatus wild-types and natural abnormally pigmented mutants, and Aspergillus lentulus). The aim was to validate whether mass spectrometry protein profiles can be used as specific signatures to discriminate different Aspergillus species or even mutants within the same species. RESULTS: The growth conditions and the SELDI-TOF parameters were determined to generate characteristic protein profiles of somatic and metabolic extracts of Aspergillus fumigatus strains using five different ProteinChips®, eight growth conditions combining two temperatures, two media and two oxygenation conditions. Nine strains were investigated: three wild-types and four natural abnormally pigmented mutant strains of A. fumigatus and two strains of A. lentulus. A total of 242 fungal extracts were prepared. The spectra obtained are protein signatures linked to the physiological states of fungal strains depending on culture conditions. The best resolutions were obtained using the chromatographic surfaces CM10, NP20 and H50 with fractions of fungi grown on modified Sabouraud medium at 37 °C in static condition. Under these conditions, the SELDI-TOF analysis allowed A. fumigatus and A. lentulus strains to be grouped into distinct clusters. CONCLUSIONS: SELDI-TOF analysis distinguishes A. fumigatus from A. lentulus strains and moreover, permits separate clusters of natural abnormally pigmented A. fumigatus strains to be obtained. In addition, this methodology allowed us to point out fungal components specifically produced by a wild-type strain or natural mutants. It offers attractive potential for further studies of the Aspergillus biology or pathogenesis.