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Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using single-particle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm ( xy / z ) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups—immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

Objective Whether pathologic stage at radical nephroureterectomy (RNU) can serve as an appropriate surrogate for oncologic outcomes in patients with high-grade (HG) upper tract urothelial carcinoma (UTUC) treated with neoadjuvant chemotherapy (NAC) is not defined. We sought to determine whether patients who achieve pathologically non-muscle-invasive (ypT0, ypTa, ypT1, ypTis) HG UTUC after receipt of NAC exhibit oncologic outcomes comparable to those who are inherently low stage without chemotherapy. Methods We identified 647 UTUC patients who underwent RNU among 3 institutions from 1993to2016. Patients with low or unknown grade, pathologic muscle invasion, or receipt of adjuvant chemotherapy were excluded. We compared clinicopathologic data and oncologic outcomes between pT0-1 and ypT0-1 patients. Kaplan-Meier analysis was used to assess overall (OS), cancer-specific (CSS), and systemic recurrence-free (RFS) survival. Predictors of these endpoints were identified using Cox regression. Results 234 (43 ypT0-1, 191 pT0-1) patients with HG UTUC were included. Two patients exhibited pathologic complete response after NAC. OS (P = 0.055), CSS (P = 0.152), and RFS (P = 0.098) were similar between ypT0-1 and pT0-1 patients. Predictors of worse outcomes included African-American race (RFS, CSS, and OS), Charlson score (OS), and systemic recurrence (OS and CSS). Conclusions Patients with HG UTUC who achieve ypT0-1 stage after NAC exhibit favorable oncologic outcomes comparable to those inherently non-muscle-invasive who do not receive chemotherapy. Improvements in clinical staging will play an important role in better defining candidacy for NAC in treating HG UTUC while minimizing overtreatment. Furthermore, pathologic stage may serve as an appropriate early surrogate for oncologic endpoints in designing clinical trials.

anemia had a greater risk of having a high-grade tumor, an advanced tumor stage, and lymph node metastasis. They also showed that preoperative anemia was significantly associated with extraurothelial recurrence and worse cancer-specific survival (CSS). However, their well-organized study could still benefit from additional discussion, in particular, regarding the role of anemia in UTUC at different stages of chronic kidney disease (CKD). Anemia is important from a clinical perspective, because this biomolecular marker can assist in treatment and follow-up planning. Nevertheless, anemia can be attributed to a variety of causes, including CKD. In addition, both preoperative anemia and CKD are not uncommon in patients with UTUC. 1-4 From 2000 to 2013, we enrolled 352 patients who had undergone RNU with bladder cuff excision for nonmetastatic UTUC at our institution. Of the 352 patients, 234 (66.5%) met the World Health Organization criteria for anemia. A total of 81 patients (23.0%) experienced disease progression during the follow-up period. We found that the metastasis-free survival (MFS) rates did not significantly correlate with the presence of preoperative anemia in the Kaplan-Meier analysis (P ¼ .079; Figure 1A). In addition, 66 patients (18.8%) died of cancer-specific causes in our cohort. The Kaplan-Meier analysis also indicated that the CSS rates were not significantly influenced by the presence of preoperative anemia (P ¼ .074; Figure 1B). The results seem to indicate that anemia is not associated with disease progression and prognosis; however, we observed interesting findings after additional subgroup analysis in which we considered the patients’ renal function. A total of 72 patients (20.5%) had end-stage renal disease (ESRD) before RNU. Of these 72 patients, 68 (94.4%) were anemic preoperatively. In contrast, 166 of 280 patients (59.3%) without ESRD had anemia, and 70 (25.0%) experienced metastatic progression. The Kaplan-Meier analysis demonstrated significantly different MFS rates according to the presence of preoperative anemia in patients without ESRD (P ¼ .010; Figure 2A). Moreover, 58 of the patients (20.7%) without ESRD had died of UTUC. Also, the CSS rates were significantly associated with the presence of preoperative anemia (P ¼ .009; Figure 2B).

Objectives To investigate the association of tumor location on oncological outcomes in patients treated with radical nephroureterectomy (RNU) for primary ureteral urothelial carcinoma (UC). Materials and methods From January 1990 to December 2007, 127 patients with primary solitary ureteral UC who underwent RNU at our institution were included. The patients were divided into 3 groups based on tumor location—proximal, middle, or distal ureter. Patients' medical records were reviewed retrospectively. The clinicopathologic data and oncologic outcomes were compared among the groups. Results Of the 127 patients, 40 (31.5%) had tumors in the proximal ureter, 40 (31.5%) in the middle ureter, and 47 (37.0%) in the distal ureter. Patients with distal ureteral UC were more likely to undergo open procedures to manage the bladder cuff (P = 0.005). Other clinical and histopathologic variables were not different among the 3 groups. Comparing the proximal, middle, or distal ureteral UC, bladder recurrence developed in, respectively, 25.0%, 25.0%, and 21.3% cases (P = 0.892); local retroperitoneal recurrence in 2.5%, 12.5%, and 4.3% (P = 0.141); contralateral recurrence in 0%, 0%, and 4.3%(P = 0.177); and distant metastasis in 17.5%, 10.0%, and 4.3% (P = 0.147). Recurrence-free and cancer-specific survival among the 3 groups were not different (P = 0.781 and 0.192, respectively). Conclusions Tumor location cannot be used to predict oncologic outcomes in patients treated with RNU for primary ureteral UC. Therefore, clinical decisions or follow-up protocol should not differ among patients with primary proximal, middle, or distal ureteral UC.

Androgen-deprivation therapy (ADT) is important in the treatment of prostate cancer. However, the relationship between ADT and the risk of diabetes remains unclear, and the association between duration and types of ADT has not been fully investigated.To examine the risk of developing type 2 diabetes mellitus (T2DM) in men who underwent ADT for prostate cancer.Data were collected retrospectively from the Longitudinal Health Insurance Database of Taiwan. In total, 4604 prostate cancer patients ≥40 years old who underwent ADT were included in the study cohort, and 4604 prostate cancer patients without ADT were included as controls, after adjusting for age and other comorbidities.During the four-year follow-up period, the incidence of new-onset T2DM was 27.49 and 11.13 per 1000 person-years in the ADT and ADT-never cohorts, respectively. The ADT cohort was 2.19 times more likely to develop T2DM than the control group (95% CI 1.90-2.53, P 0.001). Furthermore, the association was particularly striking in the subgroup of patients receiving complete androgen blockade (adjusted HR 2.33, 95% CI 1.96-2.78, P 0.001).Men with prostate cancer who received ADT are at risk for developing diabetes.

Measuring the co-localization of fluorescent constructs is a powerful technique in life science microscopy that can provide critical functional information about coincident events in the cell. Fluorescence coincidence can be used to determine the separation distance of binding targets, binding kinetics, conformational change, or even the orientation of a target construct. Techniques such as FRET and plasmonic resonance energy transfer can act as nanometric “rulers” to provide a separation of two targets, but their instrumentation relies on a slow camera based approach that is not amenable to three-dimensional freely diffusing targets, and they provide no rotational information. Here we present a dual-particle tracking microscope that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution down to 1 ms. The targets can be tracked with a separation distance up to 400 nm with 20 nm accuracy. And since each target is individually localized, a wealth of data may be extracted; such as, 3D position, 2D rotation, separation distance, and fluorescent lifetime of each target. The approach is a multicolor extension to an existing technique known as, TSUNAMI, Tracking Single-particles Using Nonlinear And Multiplexed Illumination. Just like TSUNAMI, this dual-color extension is capable of tracking within thick and scattering samples up to 200 μm deep. The technique is applied to the measurement of DNA flexing dynamics in free solution. DNA constructs with both red and green fluorescent microspheres labelled at the ends are observed in free diffusion. The trajectories from different lengths of DNA linker constructs are analyzed for binding distance and positional information to elucidate the differences in DNA dynamics during free diffusion.

Dysregulated trafficking of receptor tyrosine kinases has been linked to oncogenesis. Here we study the trafficking patterns and dynamics of epidermal growth factor receptor (EGFR) trafficking of benign (BPH1), non-invasive malignant (LNCaP), and highly invasive malignant (PC-3) prostate cells using an advanced 3D single-particle tracking technique termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination). As a feedback-control tracking system, TSUNAMI is capable of tracking fluorescent nanoparticle-tagged EGFR for up to 10 minutes and in the z-direction for up to ±50 microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, a trajectory analysis algorithm is developed to classify trajectories and extract the dynamic parameters, such as diffusivity, inward movement, and internalization duration. These parameters can be used to quantify the metastatic potentials of prostate cancer cell lines. For instance, the diffusivities of EGFRs on the highly invasive malignant PC-3 cells (0.010 ± 0.014 µm2/s) are around one-quarter of those estimated from the benign BPH1 cells (0.036 ± 0.058 µm2/s), possibly due to the abnormally high expression of EGFRs on the PC-3 cells. The highly invasive PC-3 cells also exhibit longer (2.83 ± 0.23 µm vs. 1.45 ± 0.16 µm) and faster (0.021 ± 0.016 µm/s vs. 0.005 ± 0.002 µm/s) inward movement as compared with the non-invasive LNCaP prostate cancer cells, which could be due to the high endocytotic activity associated with the invasive PC-3 cells. In addition, the dynamics parameters extracted from the EGFR trajectories are correlated with the expression levels of a number of epithelial-mesenchymal-transition (EMT)-related genes. The high EGFR expression is related to the decrease of EGFR diffusivity, and the increase of dynamins coheres with more active inward movement. After EMT induction, the non-invasive LNCaP prostate cancer cells also exhibit gene expression profiles and EGFR trafficking patterns similar to those shown in invasive PC-3 cells. Our work demonstrates that EGFR trajectory-derived dynamics parameters are linked to metastatic potentials. A new class of biophysical markers can be established based on the analysis of membrane protein trajectories.

We aimed to identify the effect of preoperative anemia on oncologic outcomes in patients with upper tract urothelial carcinoma (UTUC) who had different levels of renal function.Between 2000 and 2013, we enrolled 370 patients who underwent radical nephroureterectomy for nonmetastatic UTUC. Preoperative anemia was defined as hemoglobin130g/l in men and120g/l in women based on the World Health Organization classification. Kaplan-Meier method was applied to estimate the effect anemia on survival, and hazard ratios (HR) of anemia and other clinicopathological parameters were evaluated by Cox regression model. The analyses were also performed in patients with different chronic kidney disease (CKD) stages.In all, 242 (65.4%) patients were anemic before surgery. Those with preoperative anemia had worse CKD stage (P0.001) and higher pathological tumor stage (P = 0.023). In univariate analysis, metastasis-free and cancer-specific survival rates were not significantly associated with preoperative anemia (HR = 1.51, 95% CI: 0.93-2.44, P = 0.093 and HR = 1.59, 95% CI: 0.93-2.72, PP = 0.094, respectively). However, in patients without stage 5 CKD, those with preoperative anemia had apparently inferior metastasis-free and cancer-specific survival than those without (HR = 1.88, 95% CI: 1.14-3.01, P = 0.014 and HR = 2.03, 95% CI: 1.16-3.56, P = 0.010, respectively). A multivariate Cox proportional hazards model indicated that preoperative anemia was an independent predictor for both metastasis-free (HR = 2.17, 95% CI: 1.21-3.90, P = 0.010) and cancer-specific survival (HR = 2.21, 95% CI: 1.15-4.21, P = 0.017).Among patients without stage 5 CKD, preoperative anemia was a significant prognostic factor to predict metastatic progression and cancer-specific death in UTUC following radical nephroureterectomy. It was important to be aware of patients׳ renal function while evaluating the effect of anemia on outcome of UTUC.

A long-term health risk assessment based on a multi-media, multi-pathway approach for biodegradable volatile organic compounds is presented in this pap

Oligonucleotide-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that have seen applications in cellular imaging and chemical/biological detection. Recently we discovered the red fluorescence emission of DNA/Ag NCs could be enhanced more than 500 fold when brought into close proximity to a guanine-rich DNA sequence (Yeh et al., Nano Letters, 10 (8): 3106-3110, 2010). Based on this finding, we developed a new type of molecular probe (termed NanoCluster Beacon, NCB) that fluoresces upon target DNA binding. Compared to molecular beacons, NCBs require only a single labeling step and do not rely on F [[Unable to Display Character: ő]]rster energy transfer as fluorescence switching mechanism. Moreover, there is no need to remove the silver nanocluster precursors (Ag+ and BH4-) used during nanocluster formation, as these are essentially non-fluorescent. Our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers. It is also the first demonstration that a turn-on probe can be made based on fluorescent noble metal nanoclusters.Our initial investigation demonstrated NCB detection of an influenza target with a signal-to-background (S/B) ratio five times better than that of a conventional molecular beacon. Here, we expand upon this work to demonstrate a method of using NCBs to differentiate single-nucleotide variations. Our method discriminates single-nucleotide variants by colorimetric change of the NCB probes rather than fluorescence intensity change. This added dimensionality enables the increase in fluorescence intensity (on/off switching) to quantify the amount of target, whereas the fluorescence color identifies single-nucleotide variants. Samples with single-nucleotide variations can be unambiguously identified on a common gel imager with naked eyes, making this method a reliable and low-cost assay with simple readout format.Ref: H.-C. Yeh et al., “A DNA-silver nanocluster probe that fluoresces upon hybridization,” Nano Letters 10 (8): 3106-3110, 2010.

DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences, termed enhancer sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that “lights up” upon target binding. Since NCBs do not rely on Forster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. In addition, we show the fluorescence emission color of a NCB can change substantially (a shift of 60-70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. We have exploited this color shift to directly detect single nucleotide polymorphisms (SNPs). This SNP detection method has been validated on all single-nucleotide substitution scenarios in three synthetic DNA targets, in six disease-related SNP targets, and in two clinical samples taken from patients with ovarian serous borderline tumors. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers.

Oligonucleotide-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that have seen applications in cellular imaging and chemical/biological detection. We discovered the red fluorescence emission of DNA/Ag NCs could be enhanced more than 500 fold when these silver nanoclusters were brought close to a guanine-rich DNA sequence (called an enhancer). Based on this finding, we developed a new type of molecular probe termed NanoCluster Beacon (NCB) that fluoresces upon target DNA binding (1). Expanding upon this work, recently we found non-emissive DNA/Ag NCs can light up into three distinct colors (green, yellow, and red) through interactions with three different DNA enhancers respectively (2), enabling the use of NCBs in multiplexed assays.In order to investigate the enhancer-mediated, color switching behavior of DNA/Ag NCs, a toehold DNA displacement technique was used to replace old enhancers with new enhancers in the proximity of nanoclusters at room temperature. Our results showed that the light-up color can change from green to red if the green enhancer is replaced with the red enhancer, but not vice versa. Similar behavior was also seen from yellow to red color switching. This information may be useful in future silver nanocluster probe design.Reference:[1] H.-C. Yeh, J. Sharma, J.J. Han, J.S. Martinez and J.H. Werner, “A DNA-silver nanocluster probe that fluoresces upon hybridization,” Nano Letters 10(8): 3106-3110, 2010.[2] H.-C. Yeh, J. Sharma, J.J. Han, J.S. Martinez and J.H. Werner, “A beacon of light: a new molecular probe for homogeneous detection of nucleic acid targets,” IEEE Nanotechnology Magazine 5(2): 28-33, 2011.