Functionalized Carbon Nanoparticles as Theranostic Agents and Their Future Clinical Utility in Oncology.

Over the years, research of nanoparticle applications in pre-clinical and clinical applications has greatly advanced our therapeutic and imaging approaches to many diseases, most notably neoplastic disorders. In particular, the innate properties of inorganic nanomaterials, such as gold and iron oxide, as well as carbon-based nanoparticles, have provided the greatest opportunities in cancer theranostics. Carbon nanoparticles can be used as carriers of biological agents to enhance the therapeutic index at a tumor site. Alternatively, they can also be combined with external stimuli, such as light, to induce irreversible physical damaging effects on cells. In this review, the recent advances in carbon nanoparticles and their use in cancer theranostics will be discussed. In addition, the set of evaluations that will be required during their transition from laboratory investigations toward clinical trials will be addressed.

Plasmonic and label-free real-time quantitative PCR for point-of-care diagnostics.

In response to the world's medical community's need for accurate and immediate infectious pathogen detection, many researchers have focused on adapting the standard molecular diagnostic method of polymerase chain reaction (PCR) for point-of-care (POC) applications. PCR technology is not without its shortcomings; current platforms can be bulky, slow, and power-intensive. Although there have been some advances in microfluidic PCR devices, a simple-to-operate and fabricate PCR device is still lacking. In the first part of this paper, we introduce a compact plasmonic PCR thermocycler in which fast DNA amplification is derived from efficient photothermal heating of a colloidal reaction mixture containing gold nanorods (AuNRs) using a small-scale vertical-cavity surface-emitting laser (VCSEL). Using this method, we demonstrate 30 cycle-assay time of sub-ten minutes for successful Chlamydia trachomatis DNA amplification in 20 µL total PCR sample volume. In the second part, we report an ultrasensitive real-time amplicon detection strategy which is based on cycle-by-cycle monitoring of 260 nm absorption of the PCR sample. This was accomplished by irradiating the PCR sample using a UV LED and collecting the transmitted optical power with a photodetector. The UV absorption dependency on the nucleotides' structural degree of freedom gives rise to distinctive features in the shape of UV amplification curves for the determination of PCR results, thus circumventing the need for the complicated design of target-specific probes or intercalating fluorophores. This amplicon quantification method has a high detection sensitivity of one DNA copy. This is the first demonstration of a compact plasmonic thermocycler combined with a real-time fluorophore-free quantitative amplicon detection system. The small footprint of our PCR device stems from hardware miniaturization, while abundant sample volume facilitates highly sensitive detection and fluid handling required for in-field sample analysis, thereby making it an excellent candidate for POC molecular diagnostics.

Real time plasmonic qPCR: how fast is ultra-fast? 30 cycles in 54 seconds.

Polymerase Chain Reaction (PCR) is a critical tool for biological research investigators but recently it also has been making a significant impact in clinical, veterinary and agricultural applications. Plasmonic PCR, which employs the very efficient heat transfer of optically irradiated metallic nanoparticles, is a simple and powerful methodology to drive PCR reactions. The scalability of next generation plasmonic PCR technology will introduce various forms of PCR applications ranging from small footprint portable point of care diagnostic devices to large footprint central laboratory multiplexing devices. In a significant advance, we have introduced a real time plasmonic PCR and explored the ability of ultra-fast cycling compatible with both label-free and fluorescence-based monitoring of amplicon production. Furthermore, plasmonic PCR has been substantially optimized to now deliver a 30 cycle PCR in 54 seconds, with a detectable product. The advances described here will have an immediate impact on the further development of the use of plasmonic PCR playing a critical role in rapid point of care diagnostics.

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

Correction: Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells.

[This corrects the article DOI: 10.1371/journal.pone.0149723.].

Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells.

BACKGROUND: The incidence of papillary thyroid carcinoma (PTC) has risen steadily over the past few decades as well as the recurrence rates. It has been proposed that targeted ablative physical therapy could be a therapeutic modality in thyroid cancer. Targeted bio-affinity functionalized multi-walled carbon nanotubes (BioNanofluid) act locally, to efficiently convert external light energy to heat thereby specifically killing cancer cells. This may represent a promising new cancer therapeutic modality, advancing beyond conventional laser ablation and other nanoparticle approaches. METHODS: Thyroid Stimulating Hormone Receptor (TSHR) was selected as a target for PTC cells, due to its wide expression. Either TSHR antibodies or Thyrogen or purified TSH (Thyrotropin) were chemically conjugated to our functionalized Bionanofluid. A diode laser system (532 nm) was used to illuminate a PTC cell line for set exposure times. Cell death was assessed using Trypan Blue staining. RESULTS: TSHR-targeted BioNanofluids were capable of selectively ablating BCPAP, a TSHR-positive PTC cell line, while not TSHR-null NSC-34 cells. We determined that a 2:1 BCPAP cell:α-TSHR-BioNanofluid conjugate ratio and a 30 second laser exposure killed approximately 60% of the BCPAP cells, while 65% and >70% of cells were ablated using Thyrotropin- and Thyrogen-BioNanofluid conjugates, respectively. Furthermore, minimal non-targeted killing was observed using selective controls. CONCLUSION: A BioNanofluid platform offering a potential therapeutic path for papillary thyroid cancer has been investigated, with our in vitro results suggesting the development of a potent and rapid method of selective cancer cell killing. Therefore, BioNanofluid treatment emphasizes the need for new technology to treat patients with local recurrence and metastatic disease who are currently undergoing either re-operative neck explorations, repeated administration of radioactive iodine and as a last resort external beam radiation or chemotherapy, with fewer side effects and improved quality of life.

Selection and mutation in the "new" genetics: an emerging hypothesis.

It has been anticipated that new, much more sensitive, next generation sequencing (NGS) techniques, using massively parallel sequencing, will likely provide radical insights into the genetics of multifactorial diseases. While NGS has been used initially to analyze individual human genomes, and has revealed considerable differences between healthy individuals, we have used NGS to examine genetic variation within individuals, by sequencing tissues "in depth", i.e., oversequencing many thousands of times. Initial studies have revealed intra-tissue genetic heterogeneity, in the form of multiple variants of a single gene that exist as distinct "majority and "minority" variants. This highly specialized form of somatic mosaicism has been found within both cancer and normal tissues. If such genetic variation within individual tissues is widespread, it will need to be considered as a significant factor in the ontogeny of many multifactorial diseases, including cancer. The discovery of majority and minority gene variants and the resulting somatic cell heterogeneity in both normal and diseased tissues suggests that selection, as opposed to mutation, might be the critical event in disease ontogeny. We, therefore, are proposing a hypothesis to explain multifactorial disease ontogeny in which pre-existing multiple somatic gene variants, which may arise at a very early stage of tissue development, are eventually selected due to changes in tissue microenvironments.

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method.

The proteasome is the primary subcellular organelle responsible for protein degradation. It is a dynamic assemblage of 34 core subunits and many differentially expressed, transiently interacting, modulatory proteins. This paper describes a novel affinity chromatography method for the purification of functional human holoproteasome complexes using mild conditions. Human proteasomes purified by this simple procedure maintained the ability to proteolytically process synthetic peptide substrates and degrade ubiquitinated parkin. Furthermore, the entire purification fraction was analyzed by mass spectrometry in order to identify proteasomal proteins and putative proteasome-interacting proteins. The mild purification conditions maintained transient physical interactions between holoproteasomes and a number of known modulatory proteins. In addition, several classes of putative interacting proteins co-purified with the proteasomes, including proteins with a role in the ubiquitin proteasome system for protein degradation or DNA repair. These results demonstrate the efficacy of using this affinity purification strategy for isolating functional human proteasomes and identifying proteins that may physically interact with human proteasomes.

Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity.

Structural studies of the ligand-binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand binding, H12 is stabilized in a precise position to seal the ligand-binding pocket and finalize the assembly of the activation function (AF-2) domain. In this study, we investigated the role of the conserved proline 892 of the androgen receptor (AR) in directing the dynamic location and orientation of the AR-H12. We used a combined approach including kinetic and biochemical assays with molecular dynamic simulations to analyze two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome. Our analyses revealed distinct mechanisms by which these substitutions impair H12 function resulting in severely defective receptors. The AR-P892A receptor exhibited reduced ligand binding and transactivational potential because of an increased flexibility in H12. The AR-P892L substitution renders the receptor inactive due to a distorted, unstructured and misplaced H12. To confirm the mutants' inability to stabilize H12 in an active position, we have developed a novel in vivo assay to evaluate the accessibility of the H12-docking site on the AR-LBD surface. An extrinsic AR-H12 peptide was able to interact with wild-type and mutant LBDs in the absence of ligand. Ligand-induced proper positioning of the intrinsic H12 of wild-type AR prevented these interactions, whereas the misplacement of the mutants' H12 did not. Proline at this position may be critical for H12 dynamics not only in the AR, but also in other nuclear receptors where this proline is conserved.

Molecular pathology of the androgen receptor in male (in)fertility.

Idiopathic male infertility, accounting for 40% of all male infertility cases, is postulated to have a genetic basis. The androgen receptor (AR) plays a crucial post-meiotic role during male germ cell differentiation, which includes terminal differentiation of spermatids and their release from the seminiferous epithelium. Mutations in the AR gene result in a condition known as androgen insensitivity syndrome (AIS) affecting normal male morphogenesis. Depending on the severity of the syndrome, the external phenotype can range from normal female to normal male. In almost all cases affected individuals are infertile. In seven reported cases individuals appeared to suffer primarily or solely from male infertility, suggesting these AR mutations specifically cause male infertility. Three of these mutations are possibly population specific. Longer CAG repeats present in exon 1 of the AR have been studied as a possible risk factor for male infertility. Results are contradictory, with a trend to significance (Asian populations) and non-significance (European populations). Recent advances in protein modelling techniques may result in a much better understanding of the mechanism of action of the known infertility mutations. The determination of the significance of longer CAG repeats is likely to require studies that examine CAG repeat lengths in spermatozoa as well as patients' blood.

An examination of how different mutations at arginine 855 of the androgen receptor result in different androgen insensitivity phenotypes.

Two substitutions at an identical location in the ligand-binding domain (LBD) of the human androgen receptor (AR), R855C and R855H, are associated with complete androgen insensitivity syndrome (AIS) and partial AIS, respectively. Kinetic analysis of the mutant receptors in genital skin fibroblasts and in transfected cells revealed very low total binding (Bmax) and increased rate constants of dissociation (k) for the R855C mutant; and normal Bmax and k, with slightly elevated equilibrium affinity constants (Kd), but decreased transactivational capacity for the R855H mutant. Further analysis of the R855H mutant revealed both thermolability and decreased N/C-terminal inter-actions in the presence and absence of the co-activator transcriptional intermediary factor 2. To establish the nature of these functional differences we have used molecular dynamic modeling to create four-dimensional models of each of the mutant receptors. Molecular dynamic modeling produced profoundly different models for each of the mutants: in modeling of R855C a surprisingly significant distant alteration in the position of helix 12 of the helix 12 positioning of the AR ligand binding domain (AR-LBD) occurs, which would predict severe ligand binding abnormalities and complete AIS; in modeling of R855H, no dramatic effect on the position of helix 12 was seen; thus, binding properties of the receptor are not compromised. Molecular dynamics four-dimensional modeling clearly supports the biochemical and kinetic studies of both mutants. Such novel computational modeling may lead to a better understanding of the structure-function relationships and the molecular mechanics of ligand binding not only of the AR-LBD but also of other nuclear receptors.

Transglutaminase potentiates ligand-dependent proteasome dysfunction induced by polyglutamine-expanded androgen receptor.

Expansion of the CAG trinucleotide repeat encoding glutamine in the androgen receptor gene leads to spinobulbar muscular atrophy (SBMA), a neurodegenerative disorder in a family of polyglutamine diseases with enigmatic pathogenic mechanisms. One established property of glutamine residues is their ability to act as an amine accepter in a transglutaminase-catalyzed reaction, resulting in a proteolytically resistant glutamyl-lysine cross-link. To examine underlying disease mechanisms we investigated the relationship between polyglutamine-expanded androgen receptor and transglutaminase. We found androgen receptor N-terminal fragments are a substrate for transglutaminase. Western blots of the proteins following incubation with transglutaminase show that several different epitopes of the AR appear to be lost. We propose that this is due to the transglutaminase cross-linking of the AR, which interferes with antibody recognition. Furthermore, HEK GFP(u)-1 cells expressing polyglutamine-expanded androgen receptor and transglutaminase exhibit ligand-dependent proteasome dysfunction; this effect was not observed in the presence of cystamine, a transglutaminase inhibitor. In addition, transglutaminase-mediated isopeptide bonds were detected in brains of SBMA transgenic mice, but not in controls, suggesting involvement of transglutaminase-catalyzed reactions in polyglutamine disease pathogenesis. Our hypothesis is that cross-linked AR cannot to be degraded by the proteasome and obstructs the proteasome pore, preventing normal function. Because of the central role the ubiquitin-proteasome degradation system plays in fundamental cellular processes, any alteration in its function could cause cell death, ultimately contributing to SBMA pathogenesis.

The use of androgen receptor amino/carboxyl-terminal interaction assays to investigate androgen receptor gene mutations in subjects with varying degrees of androgen insensitivity.

Five mutations in the ligand-binding domain (LBD) of the human androgen receptor (hAR) found in patients with varying degrees of androgen insensitivity syndrome (AIS) were investigated for their effects on receptor dynamics. These were Arg(871)Gly (mild), Ser(814)Asn (partial), Glu(772)Ala (partial), Val(866)Met (complete), and Arg(774)Cys (complete). Previous analysis showed that the mutant receptors exhibited near-normal kinetics, except Arg(774)Cys, which had severely reduced androgen binding, and Val(866)Met, which showed increased equilibrium dissociation constant (K(d)) and elevated dissociation rate (k) values. Ser(814)Asn exhibited ligand-selective k values, i.e. increased for dihydrotestosterone and mibolerone, but normal for methyltrenolene. Using mammalian two-hybrid assays, hAR amino/carboxyl (N/C)-terminal interactions of the mutant receptors were analyzed in the presence and absence of the hAR coactivator transcription intermediary factor 2 (TIF2). The mutations conferred decreased hAR N/C-terminal interaction, i.e. mild (approximately 1.5-fold), partial (2-fold), and complete (10-fold), that mirrored the degree of AIS. All mutant LBDs showed a 2- to 3-fold increase in N/C-terminal interactions when TIF2 was cotransfected, although of a magnitude still less than that of wild-type LBD with TIF2. The ligand-selective properties of the Ser(814)Asn mutant were also clearly reflected by the N/C-terminal interactions. Thus, measurement of N/C-terminal interactions may assist in the molecular analysis of mutant hARs associated with AIS.

A functional analysis of disease-associated mutations in the androgen receptor gene.

Mutations in the androgen receptor (AR) are associated with a variety of diseases including androgen insensitivity syndrome and prostate cancer, but the way in which these mutations cause disease is poorly understood. We present a method for distinguishing likely disease-causing mutations from mutations that are merely associated with disease but have no causal role. Our method uses a measure of nucleotide conservation, and we find that conservation often correlates with severity of the clinical phenotype. Further, by only including mutations whose pathogenicity has been proven experimentally, this correlation is enhanced in the case of prostate cancer-associated mutations. Our method provides a means for assessing the significance of single nucleotide polymorphisms (SNPs) and cancer-associated mutations.