An evaluation of different Cripto-1 antibodies and their variable results.

Cripto-1 is a protein expressed during embryonal development and has been linked to several malignant processes in cancer. Since the discovery of cripto-1 in the late 1980s, it has become a subject of biomarker investigation in several types of cancer which in many cases relies on immunolocalization of cripto-1 using antibodies. Investigating cripto-1 expression and localization in primary glioblastoma cells, we discovered nonspecific binding of cripto-1 antibody to the extracellular matrix Geltrex. A panel of four cripto-1 antibodies was investigated with respect to their binding to the Geltrex matrix and to the cripto-1 positive control cells NTERA2. The cripto-1 expression was varied for the different antibodies with respect to cellular localization and fixation methods. To further elaborate on these findings, we present a systematic review of cripto-1 antibodies found in the literature and highlight some possible cross reactants with data on sequence alignments and structural comparison of EGF domains.

A tumorsphere model of glioblastoma multiforme with intratumoral heterogeneity for quantitative analysis of cellular migration and drug response.

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor and is characterized by its sudden onset and invasive growth into the brain parenchyma. The invasive tumor cells evade conventional treatments and are thought to be responsible for the ubiquitous tumor regrowth. Understanding the behavior of these invasive tumor cells and their response to therapeutic agents could help improve patient outcome. In this study, we present a GBM tumorsphere migration model with high biological complexity to study migrating GBM cells in a quantitative and qualitative manner. We demonstrated that the in vitro migration model could be used to investigate both inhibition and stimulation of cell migration with oxaliplatin and GBM-derived extracellular vesicles, respectively. The intercellular heterogeneity within the GBM tumorspheres was examined by immunofluorescent staining of nestin/vimentin and GFAP, which showed nestin and vimentin being highly expressed in the periphery of tumorspheres and GFAP mostly in cells in the tumorsphere core. We further showed that this phenotypic gradient was present in vivo after implanting dissociated GBM tumorspheres, with the cells migrating away from the tumor being nestin-positive and GFAP-negative. These results indicate that GBM tumorsphere migration models, such as the one presented here, could provide a more detailed insight into GBM cell biology and prove highly relevant as a pre-clinical platform for drug screening and assessing drug response in the treatment of GBM.

Oxaliplatin enhances gap junction-mediated coupling in cell cultures of mouse trigeminal ganglia.

Communications between satellite glial cells and neighboring neurons within sensory ganglia may contribute to neuropathic and inflammatory pain. To elucidate the role of satellite glial cells in chemotherapy-induced pain, we examined the effects of oxaliplatin on the gap junction-mediated coupling between these cells. We also examined whether the gap junction blocker, carbenoxolone, can reverse the coupling. Primary cultures of mice trigeminal ganglia, 24-48h after cell isolation, were used. Satellite glial cells were injected with Lucifer yellow in the presence or absence of oxaliplatin (60 µM). In addition, the effect of carbenoxolone (100 µM) on coupling, and the expression of connexin 43 proteins were evaluated. Dye coupling between adjacent satellite glial cells was significantly increased (2.3-fold, P<0.05) following a 2h incubation with oxaliplatin. Adding carbenoxolone to the oxaliplatin-treated cultures reversed oxaliplatin-evoked coupling to baseline (P<0.05). Immunostaining showed no difference between expression of connexin 43 in control and oxaliplatin-treated cultures. Our findings indicated that oxaliplatin-increased gap junction-mediated coupling between satellite glial cells in primary cultures of mouse trigeminal ganglia, and carbenoxolone reversed this effect. Hence, it is proposed that increased gap junction-mediated coupling was seen between satellite glial cells in TG. This observation together with our previous data obtained from a behavioral study suggests that this phenomenon might contribute to chemotherapy-induced nociception following oxaliplatin treatment.

A comprehensive overview of exosomes as drug delivery vehicles - endogenous nanocarriers for targeted cancer therapy.

Exosomes denote a class of secreted nanoparticles defined by size, surface protein and lipid composition, and the ability to carry RNA and proteins. They are important mediators of intercellular communication and regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest them to be important both for diagnostic and therapeutic purposes, prompting the idea of using exosomes as drug delivery vehicles, especially for gene therapy. This review covers the current status of evidence presented in the field of exosome-based drug delivery systems. Components for successful exosome-based drug delivery, such as choice of donor cell, therapeutic cargo, use of targeting peptide, loading method and administration route are highlighted and discussed with a general focus pertaining to the results obtained in models of different cancer types. In addition, completed and on-going clinical trials are described, evaluating exosome-based therapies for the treatment of different cancer types. Due to their endogenous origin, exosome-based drug delivery systems may have advantages in the treatment of cancer, but their design needs further refinement to justify their usage on the clinical scale.

Cripto-1: an extracellular protein - connecting the sequestered biological dots.

Cripto-1 (CR-1) is a multifunctional embryonic protein that is re-expressed during inflammation, wound repair, and malignant transformation. CR-1 can function either as a tethered co-receptor or shed as a free ligand underpinning its flexible role in cell physiology. CR-1 has been shown to mediate cell growth, migration, invasion, and induce epithelial to mesenchymal transition (EMT). The main signaling pathways mediating CR-1 effects include Nodal-dependent (Smad2/3) and Nodal-independent (Src/p44/42/Akt) signaling transduction pathways. In addition, there are several naturally occurring binding partner proteins (BPPs) for CR-1 that can either agonize or antagonize its bioactivity. We will review the collective role of CR-1 as an extracellular protein, discuss caveats to consider in developing a quantitation assay, define possible mechanistic avenues applicable for drug discovery, and report on our experimental approaches to overcome these problematic issues.

MicroRNAs as modulators and biomarkers of inflammatory and neuropathic pain conditions.

The post-transcriptional regulator molecules, microRNAs, have emerged as important biomarkers and modulators of numerous pathophysiological processes including oncogenesis and cardiovascular diseases. Recently, a significant number of dysregulations in microRNAs have been reported in patients suffering from painful disorders such as complex regional pain syndrome, cystitis-induced chronic pain and irritable bowel disorder, in both affected tissues and the circulation. Moreover, microRNAs are known to be involved in pain processing based on several recent findings in animal models of inflammatory and neuropathic pain. The basis of this review was to cover and summarize available articles in English encompassing "microRNA and pain". In animal pain models widespread microRNA modulation is present and manifests on multiple levels i.e.: the dorsal root ganglia, the spinal dorsal horn and the brain. Numerous functional in vivo studies have found that dysregulated microRNAs are involved in the post-transcriptional modulation of genes implicated in pain generation and maintenance. Lastly, a few animal studies have delivered promising results as to the possibility of applying microRNAs as therapeutics to alleviate established pain and several clinical studies have highlighted the potential in applying microRNAs as biomarkers in painful conditions such as complex regional pain syndrome and fibromyalgia. This review briefly introduces the basics of microRNAs, their biogenesis and function, and mainly focuses on the recent advances made in understanding the role of microRNAs in relation to pain processing and painful conditions. It also provides an overview of widely diverse methodological approaches and results with a potential for future implications of microRNAs in the diagnosis and treatment of pain.

Participant comprehension and perspectives regarding the convenience, security, and satisfaction with teleconsent compared to in-person consent: A parallel-group pilot study among Danish citizens.

Background: Teleconsent via video conferencing enables decentralized trials with remote consent and has the additional benefit of allowing a real-time reaction to potential misunderstandings. However, participant acceptance of and satisfaction with teleconsent versus in-person consent processes are unknown. Methods: We conducted a parallel-group pilot study to evaluate participant comprehension and perspectives regarding the convenience, security, and satisfaction with teleconsent compared to in-person consent among Danish citizens for a hypothetical research study. Results: There were no statistically significant differences in perceptions of security or satisfaction between teleconsent and in-person consent arms. However, participants viewed teleconsent as more convenient than in-person consent, as no transportation was needed and the process was less time-consuming. Recruitment was also faster in the teleconsent arm, and more people dropped out of the in-person arm, citing difficulties with transportation and time. Conclusion: Decentralized clinical trials have been demonstrated to increase recruitment and enrollment rates, improve trial efficiency, and decrease dropout rates and trial delays. We add to this literature by suggesting that patients perceive teleconsent as similar to in-person consent, suggesting this is a feasible and acceptable substitution for in-person consent in multisite, decentralized trials. Future work should include patient perspectives from a larger, more diverse group of participants.

Direct site-directed photocoupling of proteins onto surfaces coated with beta-cyclodextrins.

A method called Dock'n'Flash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli. Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A molecular dynamic simulation, with water as solvent, showed high solvent accessibility of the pBpa benzophenone group in N27pBpa-cutinase mutant. The formation of an inclusion complex between the benzophenone group of N27pBpa-cutinase and beta-CD was shown, and an apparent K(d) of 1.65 mM was determined using (1)H NMR. Photocoupling of beta-CD to N27pBpa-cutinase in a 1:1 ratio, upon UVA irradiation at 360 +/- 20 nm, was shown by MALDI-TOF mass spectroscopy. UVA photoimmobilization of N27pBpa-cutinase on quartz slides coated with beta-CD was achieved from liquid or dry films by total internal reflection fluorescence (TIRF). The Dock'n'Flash method offers a solution for direct photocoupling and patterning of recombinant proteins onto surfaces with site-specific attachment.

Conventional Treatment of Glioblastoma Reveals Persistent CD44+ Subpopulations.

Glioblastoma (GBM) is the most frequent and devastating primary tumor of the central nervous system with a median survival of 12 to 15 months after diagnosis. GBM is highly difficult to treat due to its delicate location, inter- and intra-tumoral heterogeneity, and high plasticity in response to treatment. In this study, we intracranially implanted primary GBM cells into mice which underwent conventional GBM treatments, including irradiation, temozolomide, and a combination. We obtained single cell suspensions through a combination of mechanical and enzymatic dissociation of brain tissue and investigated in detail the changes in GBM cells in response to conventional treatments in vivo using multi-color flow cytometry and cluster analysis. CD44 expression was elevated in all treatment groups, which was confirmed by subsequent immunohistochemistry. High CD44 expression was furthermore shown to correlate with poor prognosis of GBM and low-grade glioma (LGG) patients. Together, these results indicate a key role for CD44 in glioma pathogenesis.

Effect of growth media and serum replacements on the proliferation and differentiation of adipose-derived stem cells.

BACKGROUND AIMS: Studies of mesenchymal stromal cells (MSC) from BM and adipose tissue have demonstrated similar differentiation potentials along the adipo-, osteo- and chondrogenic lineages. While most clinical trials have been performed using BM-derived MSC, the focus is shifting toward the use of stem cells derived from fat tissue. The aim of the current investigation was to define optimal culture conditions that would facilitate clinical use of adipose-derived stem cells (ASC). METHODS: Different types and concentrations of serum replacers and basal media were tested with respect to the optimal expansion and subsequent differentiation of primary human ASC. The effect of initial seeding density on the growth of ASC was also determined. RESULTS: While several of the serum replacements proved to be clearly inferior to fetal calf serum (FCS) in promoting ASC growth, the knockout serum replacement (KOSR) had expansion properties similar to those of FCS. However, with respect to the capacity to support adipo-, osteo- and chondrogenic differentiation, KOSR proved to be less consistent than FCS. Among the media formulations, modified Eagle medium alpha supported a significantly faster cell expansion than the other basal media while still maintaining the full differentiation potential of ASC. Regarding the plating density most favorable for rapid expansion, we found that initial plating densities ranging from 100 to 200 cell/cm(2) resulted in significantly shorter doubling times than plating densities both below and above that range. CONCLUSIONS: Identification of the optimal basal medium and serum replacer, together with the most favorable plating density, will facilitate cell-based and tissue-engineering applications employing ASC in pre-clinical and clinical settings.

Printing novel molecular architectures with micrometer resolution using light.

In this paper we present a new photonic technology and demonstrate that it allows for precise immobilisation of proteins to sensor surfaces. The new technology secures spatially controlled molecular immobilisation since the immobilisation of each molecule to a support surface can be limited to the focal point of the UV laser beam, with dimensions as small as a few micrometers. We have demonstrated that we are not limited to immobilising molecules according to conventional patterns like microarrays. We can immobilise molecules on a surface with any arbitrary pattern. The different illumination/immobilisation setups presented expand the capabilities and usefulness of the new technology, since immobilisation can both be achieved with a laser system and with an affordable Xenon lamp setup. Of extreme relevance to the success of this technology is the precise knowledge of photon flux, energy flux, total number of photons per area, fluency and peak intensity. The expected resolution, taken into account the size of the focused laser beam, the precision of translation stage, and the scanner resolution of our laser scanner is in good agreement with the experimental resolution obtained. The flexibility of this new technology allows creating any patterns/ structures of molecules, with micrometer resolution, thus being of relevance for present and future nanotechnological applications.

Cripto-1 localizes to dynamic and shed filopodia associated with cellular migration in glioblastoma cells.

Cripto-1 is a protein participating in tissue orientation during embryogenesis but has also been implicated in a wide variety of cancers, such as colon, lung and breast cancer. Cripto-1 plays a role in the regulation of different pathways, including TGF-ß/Smad and Wnt/ß-catenin, which are highly associated with cell migration both during embryonal development and cancer progression. Little is known about the detailed subcellular localization of cripto-1 and how it participates in the directional movement of cells. In this study, the subcellular localization of cripto-1 in glioblastoma cells was investigated in vitro with high-resolution microscopy techniques. Cripto-1 was found to be localized to dynamic and shed filopodia and transported between cells through tunneling nanotubes. Our results connect the refined subcellular localization of cripto-1 to its functions in cellular orientation and migration.

Characterization of rat primary trigeminal satellite glial cells and associated extracellular vesicles under normal and inflammatory conditions.

Satellite glial cells (SGCs) in sensory ganglia contribute to the pathogenesis of chronic pain, potentially through mediating extracellular or paracrine signaling. Recently, extracellular vesicles (EVs) in the form of exosomes have been found to play an important role in cell-cell communication. However, their release from SGCs and extent in modulating pain remain unknown. An in vitro cell platform using fresh primary SGCs was used to characterize the shed vesicles by size and proteomic profiling following activation of SGCs by lipopolysaccharide (LPS), simulating neurogenic inflammation in vivo. Results demonstrated that SGCs shed vesicles in the size range of exosomes (>150 nm) but with altered protein expression upon LPS-activation. Proteomic profiling of SGCs-shed EVs showed that a number of proteins were differentially regulated upon LPS stimulation such as junction plakoglobin and myosin 9 that are proposed as novel biomarkers of SGCs activation under inflammatory conditions. Findings from this study highlight the utility of using fresh primary SGC cultures as a model to further investigate EVs under normal and inflammatory conditions. SIGNIFICANCE: This study demonstrated that.

Instability of standard PCR reference genes in adipose-derived stem cells during propagation, differentiation and hypoxic exposure.

BACKGROUND: For the accurate determination of gene expression changes during growth and differentiation studies on adipose-derived stem cells (ASCs), quantitative real-time RT-PCR has become a method of choice. The technology is very sensitive, however, without a proper selection of reference genes, to which the genes of interest are normalized, erroneous results may be obtained. RESULTS: In this study, we have compared the gene expression levels of a panel of twelve widely used reference genes during hypoxic culture, osteogenic and chondrogenic differentiation, and passaging of primary human ASCs. We found that several of the commonly used reference genes including 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were unsuitable for normalization in the conditions we tested, whereas tyrosine 3/tryptophan 5-monooxygenase activation protein (YMHAZ), TATAA-box binding protein (TBP), beta-glucuronidase (GUSB) were the most stable across all conditions. CONCLUSION: When determining gene expression levels in adipose-derived stem cells, we recommend normalizing transcription levels to the geometric mean of YMHAZ, TBP and GUSB.

On the use of liposome controls in studies investigating the clinical potential of extracellular vesicle-based drug delivery systems - A commentary.

The field of extracellular vesicle (EV)-based drug delivery systems has evolved significantly through the recent years, and numerous studies suggest that these endogenous nanoparticles can function as efficient drug delivery vehicles in a variety of diseases. Many characteristics of these EV-based drug delivery vehicles suggest them to be superior at residing in the systemic circulation and possibly at mediating therapeutic effects compared to synthetic drug delivery vehicles, e.g. liposomes. In this Commentary, we discuss how some currently published head-to-head comparisons of EVs versus liposomes are weakened by the inadequate choice of liposomal formulation, and encourage researchers to implement better controls to show any potential superiority of EVs over other synthetic nanoparticles.

Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces.

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 microm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.

Serum MicroRNA Signatures in Migraineurs During Attacks and in Pain-Free Periods.

MicroRNAs have emerged as important biomarkers and modulators of pathophysiological processes including oncogenesis and neurodegeneration. MicroRNAs are found to be involved in the generation and maintenance of pain in animal models of inflammation and neuropathic pain. Recently, microRNA dysregulation has been reported in patients with painful conditions such as complex regional pain syndrome and fibromyalgia. The aim of this study was to assess whether serum microRNA alterations occur during migraine attacks and whether migraine manifests in chronic serum microRNA aberrations. Two cohorts of 24 migraineurs, and age- and sex-matched healthy controls were included. High-content serum microRNA (miRNA) arrays were used to assess the serum microRNA profiles of migraineurs during attacks and pain-free periods in comparison with healthy controls. Of the 372 assessed microRNAs, 32 or ≈ 8% were found to be differentially expressed and 4 of these--miR-34a-5p, 29c-5p, -382-5p, and -26b-3p--were selected for further investigation. Migraine attacks were associated with an acute upregulation in miR-34a-5p and miR-382-5p expression. Interestingly, miR-382-5p not only exhibited an upregulation during attack but also proved to be a biomarker for migraine when comparing migraineurs in pain-free periods to the healthy control group (p = <0.01). In conclusion, migraine manifestation is reflected in serum miRNA aberrations, both during attacks and pain-free periods. This finding sheds light on the potential role of microRNAs in the pathophysiology of migraine and adds a new approach towards potential identification of much sought-after serum biomarkers of migraine.

Systematic review of factors influencing extracellular vesicle yield from cell cultures.

The potential therapeutic utility of extracellular vesicles (EVs) has spawned an interest into a scalable production, where the quantity and purity of EV samples is sufficient for clinical applications. EVs can be isolated using several different protocols; however, these isolation protocols and the subsequent methods of quantifying the resulting EV yield have not been sufficiently standardized. Therefore, the possibility of comparing different studies with respect to these parameters is limited. In this review, we have presented factors that might influence the yield and function of EVs from cell culture supernatants. The methods of isolation, downstream quantification, and culture conditions of the EV producing cells have been discussed. In order to examine the inter-study coherency of EV yields, 259 studies were initially screened, and 46 studies were included for extensive downstream analysis of EV yields where information pertaining to the isolation protocols and quantification methods was obtained from each study. Several other factors influencing yield were compared, such as cell type producing EVs, cell confluence level, and cell stimulation. In conclusion, various factors may impact the resulting EV yield, including technical aspects such as EV isolation and quantification procedures, and biological aspects such as cell type and culture conditions. The reflections presented in this review might aid in future standardization of the workflow in EV research.

Evaluation of electroporation-induced adverse effects on adipose-derived stem cell exosomes.

In the recent years, the possibility of utilizing extracellular vesicles for drug delivery purposes has been investigated in various models, suggesting that these vesicles may have such potential. In addition to the choice of donor cell type for vesicle production, a major obstacle still exists with respect of loading the extracellular vesicles efficiently with the drug of choice. One of the proposed solutions to this problem has been drug loading by electroporation, where small pores are created in the membrane of the extracellular vesicles, hereby allowing for free diffusion of the drug compound into the interior of the vesicle. We investigated the utility of adipose-derived stem cells (ASCs) as an efficient exosome donor cell type with a particular focus on the treatment of glioblastoma multiforme (GBM). In addition, we evaluated electroporation-induced effects on the ASC exosomes with respect to their endogenous potential of stimulating GBM proliferation, and morphological changes to single and multiple ASC exosomes. We found that electroporation does not change the endogenous stimulatory capacity of ASC exosomes on GBM cell proliferation, but mediates adverse morphological changes including aggregation of the exosomes. In order to address this issue, we have successfully optimized the use of a trehalose-containing buffer system as a way of maintaining the structural integrity of the exosomes.

MicroRNA expression signatures and their correlation with clinicopathological features in glioblastoma multiforme.

The increasing interest in identifying molecular biomarkers to determine patient prognosis in glioblastoma multiforme (GBM) has resulted in several microRNA (miRNA)-based signatures able to predict progression-free and overall survival. However, the coherency between these signatures is small, and correlations to clinicopathological features other than survival are seldom seen. The aim of this study was to identify any significant relationship between miRNA signatures and clinicopathological data by combining pathological features with miRNA and mRNA analysis in fourteen GBM patients. In total, 161 miRNAs were shown to cluster the GBM tumor samples into long- and short-term-surviving patients. Many of these miRNAs were associated with differential expression in GBM, including a number of miRNAs shown to confer risk or protection with respect to clinical outcome and to modulate the mesenchymal mode of migration and invasion. An inverse relationship between miR-125b and nestin expression was identified and correlated with overall survival in GBM patients, eloquently illustrating how clinicopathological findings and molecular profiling may be a relevant combination to predict patient outcome. The intriguing finding that many of the differentially expressed miRNAs contained exosome-packaging motifs in their mature sequences suggests that we must expand our view to encompass the complex intercellular communication in order to identify molecular prognostic biomarkers and to increase our knowledge in the field of GBM pathogenesis.