Prediction of outcome in patients with low-grade squamous intraepithelial lesions by fluorescence in situ hybridization analysis of human papillomavirus, TERC, and MYC

BACKGROUND Cytology is an excellent method with which to diagnose preinvasive lesions of the uterine cervix, but it suffers from limited specificity for clinically significant lesions. Supplementary methods might predict the natural course of the detected lesions. The objective of the current study was to test whether a multicolor fluorescence in situ hybridization (FISH) assay might help to stratify abnormal results of Papanicolaou tests. METHODS A total of 219 liquid-based cytology specimens of low-grade squamous intraepithelial lesions (LSIL), 49 atypical squamous cells of undetermined significance (ASCUS) specimens, 52 high-grade squamous intraepithelial lesion (HSIL) specimens, and 50 normal samples were assessed by FISH with probes for the human papillomavirus (HPV), MYC, and telomerase RNA component (TERC). Subtyping of HPV by polymerase chain reaction (PCR) was performed in a subset of cases (n=206). RESULTS There was a significant correlation found between HPV detection by FISH and PCR (P<.0001). In patients with LSILs, the presence of HPV detected by FISH was significantly associated with disease progression (P<.0001). An increased MYC and/or TERC gene copy number (>2 signals in>10% of cells) prevailed in 43% of ASCUS specimens and was more frequent in HSIL (85%) than in LSIL (33%) (HSIL vs LSIL: P<.0001). Increased TERC gene copy number was significantly correlated with progression of LSIL (P<.01; odds ratio, 7.44; area under the receiver operating characteristic curve, 0.73; positive predictive value, 0.30; negative predictive value, 0.94) CONCLUSIONS: The detection of HPV by FISH analysis is feasible in liquid-based cytology and is significantly correlated with HPV analysis by PCR. The analysis of TERC gene copy number may be useful for risk stratification in patients with LSIL.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Quantitative analysis of fluorescence lifetime measurements of the macula using the fluorescence lifetime imaging ophthalmoscope in healthy subjects.

PURPOSE Fundus autofluorescence (FAF) cannot only be characterized by the intensity or the emission spectrum, but also by its lifetime. As the lifetime of a fluorescent molecule is sensitive to its local microenvironment, this technique may provide more information than fundus autofluorescence imaging. We report here the characteristics and repeatability of FAF lifetime measurements of the human macula using a new fluorescence lifetime imaging ophthalmoscope (FLIO). METHODS A total of 31 healthy phakic subjects were included in this study with an age range from 22 to 61 years. For image acquisition, a fluorescence lifetime ophthalmoscope based on a Heidelberg Engineering Spectralis system was used. Fluorescence lifetime maps of the retina were recorded in a short- (498-560 nm) and a long- (560-720 nm) spectral channel. For quantification of fluorescence lifetimes a standard ETDRS grid was used. RESULTS Mean fluorescence lifetimes were shortest in the fovea, with 208 picoseconds for the short-spectral channel and 239 picoseconds for the long-spectral channel, respectively. Fluorescence lifetimes increased from the central area to the outer ring of the ETDRS grid. The test-retest reliability of FLIO was very high for all ETDRS areas (Spearman's ρ = 0.80 for the short- and 0.97 for the long-spectral channel, P < 0.0001). Fluorescence lifetimes increased with age. CONCLUSIONS The FLIO allows reproducible measurements of fluorescence lifetimes of the macula in healthy subjects. By using a custom-built software, we were able to quantify fluorescence lifetimes within the ETDRS grid. Establishing a clinically accessible standard against which to measure FAF lifetimes within the retina is a prerequisite for future studies in retinal disease.

Effectiveness of fluorescence-based methods to detect in situ demineralization and remineralization on smooth surfaces.

This study aimed to evaluate the effectiveness of fluorescence-based methods (DIAGNOdent, LF; DIAGNOdent pen, LFpen, and VistaProof fluorescence camera, FC) in detecting demineralization and remineralization on smooth surfaces in situ. Ten volunteers wore acrylic palatal appliances, each containing 6 enamel blocks that were demineralized for 14 days by exposure to a 20% sucrose solution and 3 of them were remineralized for 7 days with fluoride dentifrice. Sixty enamel blocks were evaluated at baseline, after demineralization and 30 blocks after remineralization by two examiners using LF, LFpen and FC. They were submitted to surface microhardness (SMH) and cross-sectional microhardness analysis. The integrated loss of surface hardness (ΔKHN) was calculated. The intraclass correlation coefficient for interexaminer reproducibility ranged from 0.21 (FC) to 0.86 (LFpen). SMH, LF and LFpen values presented significant differences among the three phases. However, FC fluorescence values showed no significant differences between the demineralization and remineralization phases. Fluorescence values for baseline, demineralized and remineralized enamel were, respectively, 5.4 ± 1.0, 9.2 ± 2.2 and 7.0 ± 1.5 for LF; 10.5 ± 2.0, 15.0 ± 3.2 and 12.5 ± 2.9 for LFpen, and 1.0 ± 0.0, 1.0 ± 0.1 and 1.0 ± 0.1 for FC. SMH and ΔKHN showed significant differences between demineralization and remineralization phases. There was a negative and significant correlation between SMH and LF and LFpen in the remineralization phase. In conclusion, LF and LFpen devices were effective in detecting demineralization and remineralization on smooth surfaces provoked in situ.

Monte Carlo modeling of polarized light propagation: Stokes vs Jones - Part I

This bipartite comparative study aims at inspecting the similarities and differences between the Jones and Stokes–Mueller formalisms when modeling polarized light propagation with numerical simulations of the Monte Carlo type. In this first part, we review the theoretical concepts that concern light propagation and detection with both pure and partially/totally unpolarized states. The latter case involving fluctuations, or “depolarizing effects,” is of special interest here: Jones and Stokes–Mueller are equally apt to model such effects and are expected to yield identical results. In a second, ensuing paper, empirical evidence is provided by means of numerical experiments, using both formalisms.

Monte Carlo modeling of polarized light propagation: Stokes vs Jones - Part II

In this second part of our comparative study inspecting the (dis)similarities between “Stokes” and “Jones,” we present simulation results yielded by two independent Monte Carlo programs: (i) one developed in Bern with the Jones formalism and (ii) the other implemented in Ulm with the Stokes notation. The simulated polarimetric experiments involve suspensions of polystyrene spheres with varying size. Reflection and refraction at the sample/air interfaces are also considered. Both programs yield identical results when propagating pure polarization states, yet, with unpolarized illumination, second order statistical differences appear, thereby highlighting the pre-averaged nature of the Stokes parameters. This study serves as a validation for both programs and clarifies the misleading belief according to which “Jones cannot treat depolarizing effects.”

Fluorescence-encoded gold nanoparticles: library design and modulation of cellular uptake into dendritic cells.

In order to harness the unique properties of nanoparticles for novel clinical applications and to modulate their uptake into specific immune cells we designed a new library of homo- and hetero-functional fluorescence-encoded gold nanoparticles (Au-NPs) using different poly(vinyl alcohol) and poly(ethylene glycol)-based polymers for particle coating and stabilization. The encoded particles were fully characterized by UV-Vis and fluorescence spectroscopy, zeta potential and dynamic light scattering. The uptake by human monocyte derived dendritic cells in vitro was studied by confocal laser scanning microscopy and quantified by fluorescence-activated cell sorting and inductively coupled plasma atomic emission spectroscopy. We show how the chemical modification of particle surfaces, for instance by attaching fluorescent dyes, can conceal fundamental particle properties and modulate cellular uptake. In order to mask the influence of fluorescent dyes on cellular uptake while still exploiting its fluorescence for detection, we have created hetero-functionalized Au-NPs, which again show typical particle dependent cellular interactions. Our study clearly prove that the thorough characterization of nanoparticles at each modification step in the engineering process is absolutely essential and that it can be necessary to make substantial adjustments of the particles in order to obtain reliable cellular uptake data, which truly reflects particle properties.

Predicting the "usefulness" of 5-ALA-derived tumor fluorescence for fluorescence-guided resections in pediatric brain tumors: a European survey.

BACKGROUND Five-aminolevulinic acid (Gliolan, medac, Wedel, Germany, 5-ALA) is approved for fluorescence-guided resections of adult malignant gliomas. Case reports indicate that 5-ALA can be used for children, yet no prospective study has been conducted as of yet. As a basis for a study, we conducted a survey among certified European Gliolan users to collect data on their experiences with children. METHODS Information on patient characteristics, MRI characteristics of tumors, histology, fluorescence qualities, and outcomes were requested. Surgeons were further asked to indicate whether fluorescence was "useful", i.e., leading to changes in surgical strategy or identification of residual tumor. Recursive partitioning analysis (RPA) was used for defining cohorts with high or low likelihoods for useful fluorescence. RESULTS Data on 78 patients <18 years of age were submitted by 20 centers. Fluorescence was found useful in 12 of 14 glioblastomas (85 %), four of five anaplastic astrocytomas (60 %), and eight of ten ependymomas grades II and III (80 %). Fluorescence was found inconsistently useful in PNETs (three of seven; 43 %), gangliogliomas (two of five; 40 %), medulloblastomas (two of eight, 25 %) and pilocytic astrocytomas (two of 13; 15 %). RPA of pre-operative factors showed tumors with supratentorial location, strong contrast enhancement and first operation to have a likelihood of useful fluorescence of 64.3 %, as opposed to infratentorial tumors with first surgery (23.1 %). CONCLUSIONS Our survey demonstrates 5-ALA as being used in pediatric brain tumors. 5-ALA may be especially useful for contrast-enhancing supratentorial tumors. These data indicate controlled studies to be necessary and also provide a basis for planning such a study.

Fluorescence lifetime imaging of the ocular fundus in mice

PURPOSE Fundus autofluorescence (AF) is characterized not only by its intensity or excitation and emission spectra but also by the lifetimes of the fluorophores. Fluorescence lifetime is influenced by the fluorophore's microenvironment and may provide information about the metabolic tissue state. We report quantitative and qualitative autofluorescence lifetime imaging of the ocular fundus in mice. METHODS A fluorescence lifetime imaging ophthalmoscope (FLIO) was used to measure fluorescence lifetimes of endogenous fluorophores in the murine retina. FLIO imaging was performed in 1-month-old C57BL/6, BALB/c, and C3A.Cg-Pde6b(+)Prph2(Rd2)/J mice. Measurements were repeated at monthly intervals over the course of 6 months. For correlation with structural changes, an optical coherence tomogram was acquired. RESULTS Fundus autofluorescence lifetime images were readily obtained in all mice. In the short spectral channel (498-560 nm), mean ± SEM AF lifetimes were 956 ± 15 picoseconds (ps) in C57BL/6; 801 ± 35 ps in BALB/c mice; and 882 ± 37 ps in C3A.Cg-Pde6b(+)Prph2(Rd2)/J mice. In the long spectral channel (560-720 nm), mean ± SEM AF lifetimes were 298 ± 14 ps in C57BL/6 mice, 241 ± 10 ps in BALB/c mice, and 288 ± 8 ps in C3A.Cg-Pde6b(+)Prph2(Rd2)/J mice. There was a general decrease in mean AF lifetimes with age. CONCLUSIONS Although fluorescence lifetime values differ among mouse strains, we found little variance within the groups. Fundus autofluorescence lifetime imaging in mice may provide additional information for understanding retinal disease processes and may facilitate monitoring of therapeutic effects in preclinical studies.

Development of the First Fluorescence Screening Assay for the SLC39A2 Zinc Transporter.

Zinc is an essential micronutrient that is crucial for many vital cellular functions such as DNA and protein synthesis, metabolism, and intracellular signaling. Therefore, the intracellular zinc concentration is tightly regulated by zinc transporters and zinc-binding proteins. The members of the SCL39 transporter family transport zinc into the cytosol. The SLC39A2 (hZIP2) protein is highly expressed in prostate epithelial cells and was found to be involved in prostate cancer development. Thus far, there is no specific modulator available for the SLC39 transporters. The aim of this study was to develop a screening assay for compound screening targeting hZIP2. Employing the pIRES2-DsRed Express 2 bicistronic vector, we detected human ZIP2 expression at the plasma membrane in transiently transfected HEK293 cells. Using the FLIPR Tetra fluorescence plate reader, we demonstrated that ZIP2 transports Cd(2+) with an apparent Km value of 53.96 nM at an extracellular pH of 6.5. The cadmium influx via hZIP2 was inhibited by zinc in a competitive manner. We found that hZIP2 activity can be measured using cadmium in the range of 0.1 to 10 µM with our assay. In summary, for the first time we developed an assay for human ZIP2 that can be adapted to other zinc transporters.

Switching on the fluorescence of 2-aminopurine by site-selective microhydration

​2-Aminopurine (​2AP) is a fluorescent isomer of ​adenine and has a fluorescence lifetime of ~11 ns in water. It is widely used in biochemical settings as a site-specific fluorescent probe of DNA and RNA structure and base-flipping and -folding. These assays assume that ​2AP is intrinsically strongly fluorescent. Here, we show this not to be the case, observing that gas-phase, jet-cooled ​2-aminopurine and ​9-methyl-2-aminopurine have very short fluorescence lifetimes (156 ps and 210 ps, respectively); they are, to all intents and purposes, non-fluorescent. We find that the lifetime of ​2-aminopurine increases dramatically when it is part of a hydrate cluster, 2AP·(H2O)n, where n = 1–3. Not only does it depend on the presence of water molecules, it also depends on the specific hydrogen-bonding site to which they attach and on the number of H2O molecules at that site. We selectively microhydrate ​2-aminopurine at its sugar-edge, cis-amino or trans-amino sites and see that its fluorescence lifetime increases by 4, 50 and 95 times (to 14.5 ns), respectively.

In vitro performance of a pen-type laser fluorescence device and bitewing radiographs for approximal caries detection in permanent and primary teeth.

AIM To evaluate the performance of a pen‑type laser fluorescence device (DIAGNOdent 2190; LFpen, KaVo, Germany) and bitewing radiographs (BW) for approximal caries detection in permanent and primary teeth. MATERIALS AND METHODS A total of 246 anterior approximal surfaces (102 permanent and 144 primary) were selected. Contact points were simulated using sound teeth. Two examiners assessed all approximal surfaces using LFpen and BW. The teeth were histologically assessed for the reference standard. Optimal cut‑off limits were calculated for LFpen for primary and permanent teeth. Sensitivity, specificity, accuracy and area under the receiver operating characteristic curve (Az) were calculated for D1 (enamel and dentin lesions) and D3 (dentin lesions) thresholds. The reproducibility was assessed by intraclass correlation coefficient (ICC) and Cohen's weighted kappa values. RESULTS For permanent teeth, the LFpen cut‑off were 0- 27 (sound), 28- 33 (enamel caries) and >33 (dentin caries). For primary teeth, the LFpen cut‑off were 0- 7 (sound), 8- 32 (enamelcaries) and >32 (dentin caries). The LFpen presented higher sensitivity values than BW for primary teeth (0.58 vs. 0.32 at D1 and 0.80 vs. 0.47 at D3) and permanent teeth (0.80 vs. 0.57 at D1 and 0.94 vs. 0.51 at D3). Specificity did not show a significant difference between the methods. Rank correlations with histology were 0.59 and 0.83 (LFpen) and 0.36 and 0.70 (BW) for primary and permanent teeth, respectively, considering all lesions. ICC values for LFpen were 0.71 (inter) and 0.86 (intra) for permanent teeth and 0.94 (inter) and 0.90/0.99 for primary teeth. Kappa values for BW were 0.69 (inter) and 0.68/0.90 (intra) for permanent teeth and 0.64 (inter) and 0.89/0.89 for primary teeth. CONCLUSION LFpen presented better reproducibility for primary and permanent teeth and higher accuracy in detecting caries lesions at D1 threshold than BW for permanent teeth. LFpen should be used as an adjunct method for approximal caries detection.