Dye Labeling Research Articles

Quantification of the asymmetric migration of the lipophilic dyes, DiO and DiD, in homotypic co-cultures of chondrosarcoma SW-1353 cells.

DiO and DiD are lipophilic cell labelling dyes used in the staining of cells in vivo and in vitro. The aim of the present study was to quantify the asymmetrical distribution of dyes in co-cultured cells and to measure the intercellular transfer of DiO and DiD. DiO and DiD were applied separately to stain two identical populations of SW-1353 human chondrosarcoma cells that were subsequently co-cultured (homotypic co-culture). The intercellular migration of dyes in the co-cultured cells was measured by flow cytometry and recorded under a fluorescent microscope. DiD and DiO caused no effect on the proliferation of cells, the degradation rate of the two dyes was comparable and crossover effects between dyes were negligible. The results of the present study suggested that asymmetrical intercellular migration of DiD and DiO was responsible for the asymmetrical distribution of these dyes in co-cultured cells. To take advantage of the lipophilic dyes migration in the double-stained co-cultured cells we suggest to apply mixed-dyes controls prior to the flow cytometric analysis. These controls are performed by staining cells with a 1:1 mix of the two dyes and would enable the estimation of the intensity of intercellular contact in co-culture systems. A 1:1 premix of DiO and DiD was applied to estimate cellular effect on intercellular exchange of lipid dyes in co-cultures incubated with cycloheximide and cytochalasin B. The cellular effect contributed 6–7% of intercellular migration of the lipophilic dyes, DiO and DiD. The majority of the observed intercellular transfer of these dyes was due to non-cellular, passive transfer.

Bioorthogonal Modification of the Major Sheath Protein of Bacteriophage M13: Extending the Versatility of Bionanomaterial Scaffolds.

With a mass of ∼1.6 × 107 Daltons and composed of approximately 2700 proteins, bacteriophage M13 has been employed as a molecular scaffold in bionanomaterials fabrication. In order to extend the versatility of M13 in this area, residue-specific unnatural amino acid incorporation was employed to successfully display azide functionalities on specific solvent-exposed positions of the pVIII major sheath protein of this bacteriophage. Employing a combination of engineered mutants of the gene coding for the pVIII protein, the methionine (Met) analog, l-azidohomoalanine (Aha), and a suitable Escherichia coli Met auxotroph for phage production, conditions were developed to produce M13 bacteriophage labeled with over 350 active azides (estimated by fluorescent dye labeling utilizing a strain-promoted azide-alkyne cycloaddition) and capable of azide-selective attachment to 5 nm gold nanoparticles as visualized by transmission electron microscopy. The capability of this system to undergo dual labeling utilizing both chemical acylation and bioorthogonal cycloaddition reactions was also verified. The above stratagem should prove particularly advantageous in the preparation of assemblies of larger and more complex molecular architectures based on the M13 building block.

B6 Super-resolution fluorescence imaging of the seeding and polymerizatoin of the huntingtin exon 1 protein

Background Huntington’s disease (HD) is caused by the expansion of the CAG repeat in exon 1 of the gene coding for the huntingtin (HTT) protein. The mutant version of the polyglutamine tract can result in misfolding and aggregation of N-terminal fragments of HTT under physiological conditions. Therefore, it’s crucial to understand the mechanisms underlying the seeding and polymerization of HTT exon 1. However, the formation of HTT aggregates has been difficult to investigate by conventional fluorescence microscopy due to their small size. Recent advances in super-resolution microscope have made this possible by permitting researchers to investigate protein aggregation in much greater detail. Aims To use highly novel super-resolution microscope techniques to understand the mechanisms underlying the seeding and polymerization of HTT exon 1 Methods/techniques Purify native HTT exon 1 protein from newly designed constructs and use two-colour TIRF (total internal reflection fluorescence microscope)-SIM (Structured illumination microscopy) microscope to directly observe the growth of HTT exon 1 at the single fibril level. Results/outcome We have designed and cloned new constructs ideal for making native HTT exon 1 protein by precise protease cleavage and fluorescent dye labelling for microscope imaging. The characterisation and application of these constructs will be presented. Conclusions Our methods will enable us to directly visualise the dynamics of the HTT exon 1 aggregation and study the aggregation kinetics which is important to HD therapeutics.

A facile, sensitive, and highly specific trinitrophenol assay based on target-induced synergetic effects of acid induction and electron transfer towards DNA-templated copper nanoclusters

Reliable, selective and sensitive approaches for trinitrophenol (TNP) detection are highly desirable with respect to national security and environmental protection. Herein, a simple and novel fluorescent strategy for highly sensitive and specific TNP assay has been successfully developed, which is based on the quenching of the fluorescent poly(thymine)-templated copper nanoclusters (DNA-CuNCs), through the synergetic effects of acid induction and electron transfer. Upon the addition of TNP, donor-acceptor complexes between the electron-deficient nitro-groups in TNP and the electron-donating DNA templates are formed, resulting in the close proximity between TNP and CuNCs. Moreover, the acidity of TNP contributes to the pH decrease of the system. These factors combine to dramatically quench the fluorescence of DNA-CuNCs, providing a “signal-off” strategy for TNP sensing. The as-proposed strategy demonstrates high sensitivity for TNP assay, and a detection limit of 0.03μM is obtained, which is lower than those reported by using organic fluorescent materials. More significantly, this approach shows outstanding selectivity over a number of TNP analogues, such as 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4-dinitrophenol (DNP), 3-nitrophenol (NP), nitrobenzene (NB), phenol (BP), and toluene (BT). Compared with previous studies, this method does not need complex DNA sequence design, fluorescent dye labeling, or sophisticated organic reactions, rendering the strategy with additional advantages of simplicity and cost-effectiveness. In addition, the as-proposed strategy has been adopted for the detection of TNP in natural water samples, indicating its great potential to be applied in the fields of public safety and environmental monitoring.

Developmental Validation of a novel 5 dye Y-STR System comprising the 27 YfilerPlus loci.

In this study, a new STRtyper-27 system, including the same Yfiler Plus loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA H4, DYS449, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627 and DYF387S1a/b), was established using a set of 5 fluorescent dye labels. Primers, internal size standard, allelic ladders and matrix standard set were designed and created in-house for this multiplex system. This paper describes the validation studies conducted with the STRtyper-27Y system using a 3130XL genetic analyzer for fragment length detection that included the analysis of the following parameters and aspects: sensitivity, species specificity, inhibition, haplotype concordance, precision, stutter, DNA mixtures, and stability studies with crime scene samples. The studies demonstrated, that the STRtyper-27Y system provided equivalent overall performance comparable to the latest Yfiler Plus kit, but with enhanced compatibility in terms of instrument platforms and software allowing forensic laboratories to conduct its forensic application and evaluate its performance, all in their own 5 dye Y-STR chemistry system /environment without software or instrument upgrades.

Application of Optical Clearing Methods on the Songbird Brain

Abstract Songbirds provide an opportunity to explore the neural mechanism underlying the learning and production of complex sound sequences. Imaging the neural structure at single-cell resolution in large volume tissue helps to disclose the functions of neural circuits involved in the song system. Recent advancements in optical clearing techniques may contribute to successful imaging of large-volume samples of songbird brains. These new clearing methods were originally optimized for rodent nervous tissues and previously have not been tested on avian brains. Here we examined whether SeeDB and ClearT, two of the latest optical clearing methods, are suitable for songbird brains, in addition to rodent brains. These two methods were chosen because of the shortness of time required to complete them and because of their compatibility with common lipophilic dye labeling. We applied these methods to brain sections of Bengalese Finches Lonchura striata var. domestica and laboratory rats Rattus norvegicus, then quan...

Incorporation of Hydrophobic Dyes within Cellulose Acetate and Acetate Phthalate Based Nanoparticles

Polysaccharide based nanoparticles (NP) have demonstrated a unique potential for biomedical and biotechnological applications. Various functionalities can be covalently linked to these NP, however, physical entrapment of functional compounds is still a highly desired approach, e.g., for drug delivery. Using different dyes, it is demonstrated how hydrophobic compounds can be incorporated into composite particles derived from cellulosic esters and how the particle properties are affected by the composition. The dye loaded NP are studied by UV–vis spectroscopy to gain insight in the interaction of the hydrophobic compounds with the cellulosic matrix. By using functional cellulose derivatives, in particular carboxylate group bearing acetate phthalates, it is possible to introduce reactive moieties on the NP surface that can be exploited for coupling additional functionalities such as antibodies. By this approach, NP can be obtained that are well suited as dye labels in immunoassay applications.image

Photostability Enhancement of Fluorenone-Based Two-Photon Fluorescent Probes for Cellular Nucleus Monitoring and Imaging

A series of fluorenone-based two-photon fluorescent probes with high photostability for nucleus imaging are prepared and developed. The one- and two-photon photophysical properties exhibit these new probes possess 0.448–0.634 of fluorescence quantum yields and 469–495 GM of two-photon absorption cross sections at 800 nm femtosecond laser excitation. The luminescence “turn-on” experiment in buffer solutions indicates that 35-fold of fluorescence intensity and 68-fold fluorescence quantum yield enhancement appear between new probes and calf thymus DNA. In the nuclear double-staining experiment, the high mean colocalization coefficients of 0.92–0.96 between new probes and nuclear labeling dye Hoechst 33342 are acquired, demonstrating excellent nuclear localization in 3T3 cells. The counterstain studies by introducing commercial mitochondrial staining reagent MTR and nuclear staining dye DAPI further show good membrane permeability and counterstain compatibility in multicolor cell labeling application. The ph...

Single-Molecule FRET States, Conformational Interchange, and Conformational Selection by Dye Labels in Calmodulin.

We investigate the roles of measurement time scale and the nature of the fluorophores in the FRET states measured for calmodulin, a calcium signaling protein known to undergo pronounced conformational changes. The measured FRET distributions depend markedly on the measurement time scale (nanosecond or microsecond). Comparison of FRET distributions measured by donor fluorescence decay with FRET distributions recovered from single-molecule burst measurements binned over time scales of 90 μs to 1 ms reveals conformational averaging over the intervening time regimes. We find further that, particularly in the presence of saturating Ca(2+), the nature of the measured single-molecule FRET distribution depends markedly on the identity of the FRET pair. The results suggest interchange between conformational states on time scales of hundreds of microseconds or less. Interaction with a fluorophore such as the dye Texas Red alters both the nature of the measured FRET distributions and the dynamics of conformational interchange. The results further suggest that the fluorophore may not be merely a benign reporter of protein conformations in FRET studies, but may in fact alter the conformational landscape.

Mapping Calcium-Sensitive Regions in the Neuronal Calcium Sensor GCAP2 by Site-Specific Fluorescence Labeling.

Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 μM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

Improved Tracking and Resolution of Bacteria in Holographic Microscopy Using Dye and Fluorescent Protein Labeling.

Digital holographic microscopy (DHM) is an emerging imaging technique that permits instantaneous capture of a relatively large sample volume. However, large volumes usually come at the expense of lower spatial resolution, and the technique has rarely been used with prokaryotic cells due to their small size and low contrast. In this paper we demonstrate the use of a Mach-Zehnder dual-beam instrument for imaging of labeled and unlabeled bacteria and microalgae. Spatial resolution of 0.3 μm is achieved, providing a sampling of several pixels across a typical prokaryotic cell. Both cellular motility and morphology are readily recorded. The use of dyes provides both amplitude and phase contrast improvement and is of use to identify cells in dense samples.

Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials.

Novel asymmetric monomethine cyanine dyes derived from sulfobetaine benzothiazolium moiety as potential fluorescent dyes for non-covalent labeling of DNA

Four novel monomethine cyanine dyes derived from a condensation reaction between N-quaternary 2-thiomethylbenzothiazolic, and 4-methylquinolinuim choromophores have been synthesized and characterized by 1H NMR, APT carbon NMR, ESI mass spectrometry, IR, absorption and fluorescence spectroscopy. The main synthetic approach for the target cyanines involves variations on both the substituent on the aromatic ring of the benzothiazolium derivative, as well as the size of the component yielding the additional positive charge linked to the quinolinium moiety. The interaction between novel fluorophores and dsDNA has been studied. The dyes were found to have negligible fluorescence in the buffer solution, but exhibited a significant emission increase upon binding to dsDNA. The binding parameters of cyanine dyes have been determined by fluorescence titration using the McGhee & von Hippel site-excluded model. The results obtained are consistent with an intercalative binding mode between cyanines and DNA.

Engraftment and regenerative effects of bone marrow stromal cell transplantation on damaged rat olfactory mucosa.

To develop a new therapeutic method to treat olfactory deficits, we investigated the engraftment and regenerative effects of transplanted bone marrow stromal cells (BMSCs) on damaged rat olfactory mucosa. To induce olfactory nerve degeneration, one side of the olfactory mucosa of Sprague-Dawley rats was damaged via Triton X-100 irrigation. Phosphate-buffered saline containing syngeneic BMSCs was injected into the olfactory mucosa for transplantation. PKH fluorescent cell dye labeling of BMSCs was used to monitor the transplanted cells. After transplantation of BMSCs, the thickness and regeneration of olfactory mucosa were analyzed using hematoxylin-eosin (H&E) staining. S100 immunohistochemical staining was used to measure nerve sheath regeneration. The increase in NGF (nerve growth factor) level in the olfactory mucosa was measured by Western blot analysis. Transplanted bone marrow stromal cells were engrafted to the lamia propria of damaged mucosa. The mean time for normalization of thickness and morphological recovery of the olfactory mucosa was 4weeks in the therapeutic group and 9weeks in the control group. S100 immunoreactivity was higher on the BMSC-treated side than on the control side. During regeneration, the expression of NGF increased in the olfactory mucosa of the experimental group. Based on these results, BMSC transplantation accelerated regeneration of olfactory mucosa damaged by Triton X-100, and NGF may be essential to this regenerative process.

Hybrid theatre and alternative localization techniques in conventional and single-port video-assisted thoracoscopic surgery.

Management of pulmonary nodules in terms of diagnosis and intraoperative localization can be challenging, especially in the minimal invasive video-assisted thoracoscopic surgery (VATS) approach, and may be even more difficult with single port VATS with limited access. The ability to localize small lesions intraoperatively is particularly important for excisional biopsy for diagnostic frozen section, as well as to guide sublobar resection. Some of the common techniques to aid localization include preoperative percutaneous hookwire localization, colour dye or radio-dye labelling injection of the nodule or adjacent site to allowing visualization or detection by radioactive counter intraoperatively. The use of hybrid operating room (OR) for intraoperative localization of lung nodules was first reported in 2013, and was called image guided VATS (iVATS). Subsequently, we have expanded the iVATS application for single port VATS major lung resection of small or ground-glass opacity lesions. By performing an on-table cone-beam CT scan, real-time and accurate assessment of the pulmonary lesion can be made, which can aid the localization process. Other types of physical or colour marker that can be deployed percutaneously in the hybrid OR immediate before surgery can enhance haptic feedback and sensitivity of digital palpation, as well as provide a radiopaque nidus for radiological confirmation. In the past decade, the electromagnetic navigation bronchoscopy (ENB) technology had developed into a useful adjunct technology for the localization of peripheral lung nodules by injection of marking agent or deployment of fiducial to the lesion through the endobronchial route causing much lower marking agent diffusion and artefacts. Recently, the combination of hybrid OR and ENB for lung nodule localization and marking has further increased the accuracy and applicability of the technology. The article will be exploring the latest development of the above approaches to lung nodule localization, and discuss some of the techniques' advantages and flaws.

Abstract P2-01-06: Differential proteomics identifies complement factor H proteolytic species as early breast cancer biomarkers

Abstract Background Breast cancer is the most common cancer among female population worldwide. We have been particularly interested in whether proteolytic species in human blood plasma can be biomarkers for detection of early breast cancer. Thus, a new differential proteomics approach has been implemented to evaluate this hypothesis. Methods We first collected plasma samples from 6 patients before and after neoadjuvant chemotherapy. These pairs of samples were then subjected to a modified two-dimensional differential gel electrophoresis (modified 2-D DIGE), comprising fluorescent dye labeling, macroporous reverse phase (mRP) HPLC and reducing/non-reducing SDS-PAGE. The difference protein species were analyzed with LC-MS/MS. Cleavage site-specific antibodies have been produced to perform a large-scale examination of total 379 plasma samples. These samples include 75, 74, 46, 48, and 48 patients at stage 0, 1, 2, 3 and 4, respectively. Also, there are 29 samples from normal individuals and 59 from other cancers/diseases to serve as control. Results A group of proteolytic species in some breast cancer patients were found by modified 2-D DIGE. Notably, these species disappeared from the plasma after the diseased tissue was surgically removed. A pair of complement factor H (CFH) derivatives were identified using LC-MS/MS analyses. Through a series of examination, we concluded that proteolytic removal of Arg-341 is likely the molecular mechanism that leads to these findings. According to these data, we have generated antibodies that can specifically recognize these proteolytic products. We used antibodies to tested over 350 clinical samples and found these biomarkers can be specifically detected in plasma of breast cancer patients. It is quite encouraging that positive detection is shown in 15% of stage 0 pts(11/75) and 20% of stage 1 pts (15/74). Other 20 patients (21∼22%) with stage 2 and 3 are found positive. 13 out of 48 patients (27%) with metastatic tumor have also been detected. Surprisingly, the signal did not observed in normal individuals or patients with other diseases, strongly suggesting that these biomarkers are highly specific to breast cancer. Conclusion Our initial results show the utility of this novel strategy in detection of cancer-specific proteolysis. About 20% of early breast cancer patients including stage 0 disease can be detected through these biomarkers. The promise of these proteolytic species as early cancer biomarkers and use of the cleavage site-specific antibodies are particularly remarkable, since early breast cancer detection can be applied to establish higher cure rates and thus lead to better prognosis for patients. Keyword: Breast cancer, Early cancer biomarker, Proteolytic processing. Citation Format: Kuo W-H, Huang Y-C, Tsay Y-G. Differential proteomics identifies complement factor H proteolytic species as early breast cancer biomarkers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-01-06.

Abstract A62: TRX-1 targets chemoresistant tumor-initiating cells and prolongs survival in a recurrent ovarian cancer animal model.

Abstract Background: Ovarian cancer is the leading cause of mortality from gynecologic cancers due to the high incidence of chemoresistance and disease recurrence. Disease recurrence is thought to occur due to the presence of residual disease following first-line standard of care (i.e. optimal debulking and chemotherapy). Residual disease post chemotherapy is composed of a unique population of chemoresistant cancer cells with stemness properties and a high capacity for tumor repair. Currently, there are no available options that can target these cells. The objective of this study was to develop a new therapeutic modality that will target chemoresistant ovarian cancer stem cells (OCSC) and consequently prevent recurrence and improve survival. To achieve this objective we developed a library of super-benzopyran (SBP) analogues and identified TRX-1 as the most potent analogue able to induce OCSC cell death in a short period of time. Moreover, in contrast to Cisplatin and Paclitaxel, TRX-1 is able to induce a persistent growth inhibitory effect both in vitro and in vivo thus improving overall survival in a mouse model of recurrent ovarian cancer. Materials and methods: A panel of SBP analogues were generated and activity was determined by testing against pure clones of CD44+/MyD88+ OCSC. In vitro efficacy was assessed using the IncucyteTM kinetic imaging platform complemented by CelltoxTM dye labeling. In vivo efficacy was tested using an intra-peritoneal (i.p.) a mouse model of recurrent ovarian cancer1. Results: TRX-1 was the most potent analogue identified and is able to induce cell death in all OCSC clones tested (IC50 of 136 nM). We observed that 2h in vitro exposure to 2μM TRX-1 was sufficient to induce a sustained growth inhibitory effect in OCSC, hence the cells were not able to recover growth potential even after removal of the drug. In contrast, OCSC exposed for up to 24h with the same dose of Cisplatin or Paclitaxel were able to recover. In vivo, animals bearing residual tumors after Paclitaxel treatment demonstrated tumor progression when further maintained with vehicle or Paclitaxel indicating Paclitaxel resistance. In contrast, maintenance with TRX-1 was able to effectively decrease tumor burden (p = 0.02) and prevent recurrence. In addition, combination treatment with TRX-1 and Cisplatin was able to significantly improve survival (p<0.001) compared to Cisplatin alone. Conclusion: We describe the in vitro and in vivo anti-tumoral effect of a novel compound, TRX-1, which exhibits significant efficacy against chemoresistant OCSC and is able to prevent recurrence in a chemoresistant in vivo model. Recurrence characterized by chemoresistance is the main cause of mortality in ovarian cancer patients. Previous studies from our laboratory have shown that conventional chemotherapy is not effective against OCSC and cannot prevent recurrence. Our finding that TRX-1, by targeting OCSC can prevent recurrence in vivo as maintenance therapy or in combination with chemotherapy provides a new opportunity for developing new therapeutic strategies that can improve survival in ovarian cancer patients. Citation Format: Ayesha Alvero, Eydis Lima, Mary Pitruzzello, Yang Yang-Hartwich, David Brown, Andrew Heaton, Gil Mor. TRX-1 targets chemoresistant tumor-initiating cells and prolongs survival in a recurrent ovarian cancer animal model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A62.

Preferential Attachment of Specific Fluorescent Dyes and Dye Labeled DNA Sequences in a Surface Enhanced Raman Scattering Multiplex.

A significant advantage of using surface enhanced Raman scattering (SERS) for DNA detection is the capability to detect multiple analytes simultaneously within the one sample. However, as the analytes approach the metallic surface required for SERS, they become more concentrated and previous studies have suggested that different dye labels will have different affinities for the metal surface. Here, the interaction of single stranded DNA labeled with either fluorescein (FAM) or tetramethylrhodamine (TAMRA) with a metal surface, using spermine induced aggregated silver nanoparticles as the SERS substrate, is investigated by analyzing the labels separately and in mixtures. Comparison studies were also undertaken using the dyes in their free isothiocyanate forms, fluorescein isothiocyanate (F-ITC) and tetramethylrhodamine isothiocyanate (TR-ITC). When the two dyes are premixed prior to the addition of nanoparticles, TAMRA exerts a strong masking effect over FAM due to a stronger affinity for the metal surface. When parameters such as order of analyte addition, analysis time, and analyte concentration are investigated, the masking effect of TAMRA is still observed but the extent changes depending on the experimental parameters. By using bootstrap estimation of changes in SERS peak intensity, a greater insight has been achieved into the surface affinity of the two dyes as well as how they interact with each other. It has been shown that the order of addition of the analytes is important and that specific dye related interactions occur, which could greatly affect the observed SERS spectra. SERS has been used successfully for the simultaneous detection of several analytes; however, this work has highlighted the significant factors that must be taken into consideration when planning a multiple analyte assay.

Sculpting the internal architecture of fluorescent silica particles via a template-free approach

Particles with an open, porous structure can be used to deliver payloads. It is often of interest to detect such particles in tissue or materials, which is facilitated by addition of dye. A straightforward approach leading to fluorescent, porous silica particles is described. The particles are etched with 3mM aqueous sodium hydroxide, taking advantage of the etching rate difference between normal silica and an interior band of silica that contains covalently attached dye. No additional steps, such as dye labeling or thermal annealing, are required. Etching modeled the internal structure of the fluorescent silica particles by creating meso/macropores and voids, as reflected by nitrogen absorption measurements. In order to investigate whether a polymer shell influences etching, certain composite particles are top-coated with poly(l-lysine) representing neutral or positive charged surfaces under typical pH conditions in living systems. The polypeptide-coated fluorescent silica cores exhibit the same porous morphology as uncoated homologs. The polypeptide topcoat does little to alter the permeation by the etching agent. Preservation of size during etching, confirmed by dynamic light scattering, transmission electron microscopy and small-angle X-ray scattering, simplifies the use of these template-free porous fluorescent particles as platforms for drug encapsulation, drug carriers and in vivo imaging.

HER Specific TKIs Exert Their Antineoplastic Effects on Breast Cancer Cell Lines through the Involvement of STAT5 and JNK.

BackgroundHER-targeted tyrosine kinase inhibitors (TKIs) have demonstrated pro-apoptotic and antiproliferative effects in vitro and in vivo. The exact pathways through which TKIs exert their antineoplastic effects are, however, still not completely understood.MethodsUsing Milliplex assays, we have investigated the effects of the three panHER-TKIs lapatinib, canertinib and afatinib on signal transduction cascade activation in SKBR3, T47D and Jurkat neoplastic cell lines. The growth-inhibitory effect of blockade of HER and of JNK and STAT5 signaling was measured by proliferation- and apoptosis-assays using formazan dye labeling of viable cells, Western blotting for cleaved PARP-1 and immunolabeling for active caspase 3, respectively.ResultsAll three HER-TKIs clearly inhibited proliferation and increased apoptosis in HER2 overexpressing SKBR3 cells, while their effect was less pronounced on HER2 moderately expressing T47D cells where they exerted only a weak antiproliferative and essentially no pro-apoptotic effect. Remarkably, phosphorylation/activation of JNK and STAT5A/B were inhibited by HER-TKIs only in the sensitive, but not in the resistant cells. In contrast, phosphorylation/activation of ERK/MAPK, STAT3, CREB, p70 S6 kinase, IkBa, and p38 were equally affected by HER-TKIs in both cell lines. Moreover, we demonstrated that direct pharmacological blockade of JNK and STAT5 abrogates cell growth in both HER-TKI-sensitive as well as -resistant breast cancer cells, respectively.ConclusionWe have shown that HER-TKIs exert a HER2 expression-dependent anti-cancer effect in breast cancer cell lines. This involves blockade of JNK and STAT5A/B signaling, which have been found to be required for in vitro growth of these cell lines.