Proteomics: A new dimension to decode small cell lung cancer.

Small cell lung cancer (SCLC) is a recalcitrant malignancy. Conquering it will require deep insight into its biology. In this issue of Cell, Liu and colleagues describe proteomic and phosphoproteomic landscapes of resected SCLC tumors and illustrate the potential of this knowledge to identify new SCLC vulnerabilities.

Cigarette Smoke Enhances the Malignant Phenotype of Esophageal Adenocarcinoma Cells by Disrupting a Repressive Regulatory Interaction Between miR-145 and LOXL2.

Although linked to esophageal carcinogenesis, the mechanisms by which cigarette smoke mediates initiation and progression of esophageal adenocarcinomas (EAC) have not been fully elucidated. In this study, immortalized esophageal epithelial cells and EAC cells (EACCs) were cultured with or without cigarette smoke condensate (CSC) under relevant exposure conditions. Endogenous levels of microRNA (miR)-145 and lysyl-likeoxidase 2 (LOXL2) were inversely correlated in EAC lines/tumors compared with that in immortalized cells/normal mucosa. The CSC repressed miR-145 and upregulated LOXL2 in immortalized esophageal epithelial cells and EACCs. Knockdown or constitutive overexpression of miR-145 activated or depleted LOXL2, respectively, which enhanced or reduced proliferation, invasion, and tumorigenicity of EACC, respectively. LOXL2 was identified as a novel target of miR-145 as well as a negative regulator of this miR in EAC lines/Barrett's epithelia. Mechanistically, CSC induced recruitment of SP1 to the LOXL2 promoter; LOXL2 upregulation coincided with LOXL2 enrichment and concomitant reduction of H3K4me3 levels within the promoter of miR143HG (host gene for miR-145). Mithramycin downregulated LOXL2 and restored miR-145 expression in EACC and abrogated LOXL2-mediated repression of miR-145 by CSC. These findings implicate cigarette smoke in the pathogenesis of EAC and demonstrate that oncogenic miR-145-LOXL2 axis dysregulation is potentially druggable for the treatment and possible prevention of these malignancies.

High-Precision Mapping of Membrane Proteins on Synaptic Vesicles using Spectrally Encoded Super-Resolution Imaging.

The spatial resolution of single-molecule localization microscopy is limited by the photon number of a single switching event because of the difficulty of correlating switching events dispersed in time. Here we overcome this limitation by developing a new class of photoswitching semiconducting polymer dots (Pdots) with structured and highly dispersed single-particle spectra. We imaged the Pdots at the first and the second vibronic emission peaks and used the ratio of peak intensities as a spectral coding. By correlating switching events using the spectral coding and performing 4-9 frame binning, we achieved a 2-3 fold experimental resolution improvement versus conventional superresolution imaging. We applied this method to count and map SV2 and proton ATPase proteins on synaptic vesicles (SVs). The results reveal that these proteins are trafficked and organized with high precision, showing unprecedented level of detail about the composition and structure of SVs.

Joint-Predominant Rheumatic Complications of Immune Checkpoint Inhibitor Therapy in Patients with Thymic Epithelial Tumors.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced cancers. However, activation of the immune system can occasionally cause life-threatening toxicity involving critical organs. Induction of immune-mediated toxicity is a significant concern for patients with thymic epithelial tumors (TETs) due to defects in immune tolerance. An increased risk of skeletal and cardiac muscle inflammation following treatment with ICIs is well recognized in patients with advanced TETs. However, uncommon musculoskeletal and rheumatic complications can also occur. The cases presented in this report highlight the spectrum of presentation of immune-mediated, joint-predominant musculoskeletal adverse events in patients with advanced TETs treated with ICIs, including polymyalgia rheumatica-like illness and inflammatory arthritis.

Multimode Time-Resolved Superresolution Microscopy Revealing Chain Packing and Anisotropic Single Carrier Transport in Conjugated Polymer Nanowires.

Here, we developed a novel, multimode superresolution method to perform full-scale structural mapping and measure the energy landscape for single carrier transport along conjugated polymer nanowires. Through quenching of the local emission, the motion of a single photogenerated hole was tracked using blinking-assisted localization microscopy. Then, utilizing binding and unbinding dynamics of quenchers onto the nanowires, local emission spectra were collected sequentially and assembled to create a superresolution map of emission sites throughout the structure. The hole polaron trajectories were overlaid with the superresolution maps to correlate structures with charge transport properties. Using this method, we compared the efficiency of inter- and intrachain hole transport inside the nanowires and for the first time directly measured the depth of carrier traps originated from torsional disorder and chemical defects.

Improving the Accuracy of Pdot-Based Continuous Glucose Monitoring by Using External Ratiometric Calibration.

Continuous glucose monitoring (CGM) allows type I and II diabetes patients to track changes in their glucose levels, allowing detection of impending hypoglycemia or hyperglycemia. Polymer dots (Pdots) are candidates for use in implanted CGM systems due to their exceptional brightness, photostability, sensitivity, and biocompatibility. However, Pdot glucose transducers are oxygen-dependent, and changes in tissue oxygen levels affect their measurement accuracy. Here, we describe an external ratiometric calibration method that corrects for changes in tissue oxygen levels to improve measurement accuracy. This method uses the ratio of oxygen concentrations inside and outside the Pdot glucose transducer as an indicator of glucose concentration to correct for signal deviations caused by tissue oxygen fluctuations. A second oxygen-sensitive Pdot that is not conjugated with glucose oxidase is used to measure the oxygen concentration outside the Pdot glucose transducer. We describe the theoretical basis for this approach and demonstrate its effectiveness experimentally in a subcutaneous mouse implant model. This external ratiometric system achieves higher accuracy glucose measurements than previous Pdot-based CGM systems and comparable accuracy to current commercial CGM systems, demonstrating the utility of the external ratiometric calibration strategy.

Reversible Ratiometric NADH Sensing Using Semiconducting Polymer Dots.

Reduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in living cells due to its role as an electron carrier in redox reactions, and its concentration is an important indicator of cell metabolic state. Abnormal NADH levels are associated with age-related metabolic diseases and neurodegenerative disorders, creating a demand for a simple, rapid analytical method for point-of-care NADH sensing. Here we develop a series of NADH-sensitive semiconducting polymer dots (Pdots) as nanoprobes for NADH measurement, and test their performance in vitro and in vivo. NADH sensing is based on electron transfer from semiconducting polymer chains in the Pdot to NADH upon UV excitation, quenching Pdot fluorescence emission. In polyfluorene-based Pdots, this mechanism resulted in an on-off NADH sensor; in DPA-CNPPV Pdots, UV excitation resulted in NADH-sensitive emission at two wavelengths, enabling ratiometric detection. Ratiometric NADH detection using DPA-CNPPV Pdots exhibits high sensitivity (3.1 µM limit of detection), excellent selectivity versus other analytes, reversibility, and a fast response (less than 5 s). We demonstrate applications of the ratiometric NADH-sensing Pdots including smartphone-based NADH imaging for point-of-care use.

High-Throughput Counting and Superresolution Mapping of Tetraspanins on Exosomes Using a Single-Molecule Sensitive Flow Technique and Transistor-like Semiconducting Polymer Dots.

A method for high-throughput counting and superresolution mapping of surface proteins on exosomes is described. The method combines a single-molecule sensitive flow technique and an adaptive superresolution imaging method. Exosomes stained with membrane dye and dye-conjugated antibodies were analyzed using a microfluidic platform at a flow rate of 100 exosome s-1 to determine size and protein copy number. Superresolution mapping was performed with exosomes labeled with novel transistor-like, semiconducting polymer dots (Pdots), which exhibit spontaneous blinking with <5 nm localization error and a broad range of optical-adjustable duty cycles. Based on the copy numbers extracted from the flow analysis, the switch-on frequency of the Pdots were finely adjusted so that structures of hundreds of exosomes were obtained within five minutes. The high throughput and high sensitivity of this method offer clear advantages for characterization of exosomes and similar biological vesicles.

Monitoring Metabolites Using an NAD(P)H-sensitive Polymer Dot and a Metabolite-Specific Enzyme.

We introduce an NAD(P)H-sensitive polymer dot (Pdot) biosensor for point-of-care monitoring of metabolites. The Pdot is combined with a metabolite-specific NAD(P)H-dependent enzyme that catalyzes the oxidation of the metabolite, generating NAD(P)H. Upon UV illumination, the NAD(P)H quenches the fluorescence emission of Pdot at 627 nm via electron transfer, and also fluoresces at 458 nm, resulting in a shift from red to blue emission at higher NAD(P)H concentrations. Metabolite concentration is quantified ratiometrically-based on the ratio of blue-to-red channel emission intensities, with a digital camera-with high sensitivity and specificity. We demonstrate phenylalanine biosensing in human plasma for a phenylketonuria screening test, quantifying several other disease-related metabolites (lactate, glucose, glutamate, and ß-hydroxybutyrate), and a paper-based assay with smartphore imaging for point-of-care use.

Dual-Mode Superresolution Imaging Using Charge Transfer Dynamics in Semiconducting Polymer Dots.

In a conjugated polymer-based single-particle heterojunction, stochastic fluctuations of the photogenerated hole population lead to spontaneous fluorescence switching. We found that 405 nm irradiation can induce charge recombination and activate the single-particle emission. Based on these phenomena, we developed a novel class of semiconducting polymer dots that can operate in two superresolution imaging modes. The spontaneous switching mode offers efficient imaging of large areas, with <10 nm localization precision, while the photoactivation/deactivation mode offers slower imaging, with further improved localization precision (ca. 1 nm), showing advantages in resolving small structures that require high spatial resolution. Superresolution imaging of microtubules and clathrin-coated pits was demonstrated, under both modes. The excellent localization precision and versatile imaging options provided by these nanoparticles offer clear advantages for imaging of various biological systems.

Compact Conjugated Polymer Dots with Covalently Incorporated Metalloporphyrins for Hypoxia Bioimaging.

Hypoxia is closely related to multiple diseases, especially in tumors, which increases the aggressiveness and drug resistance of cancer cells. Precise hypoxia imaging is of great significance for cancer diagnosis and the evaluation of therapeutic effects. A kind of hydrophobic polymer (i.e., PFPtTFPP) as an imaging probe for hypoxia with fluorene as an energy donor and an oxygen-sensitive PtII porphyrin as an energy acceptor was developed. Compact polymer dots (Pdots) with a small size were prepared by nanoprecipitation. The PFPtTFPP Pdots showed excellent hypoxia sensing in solution with high sensitivity and full reversibility. The emission intensity, quantum yields, lifetime, and single-particle brightness significantly increased under hypoxia conditions. Remarkably, hypoxia imaging in vitro and in vivo was realized, and a clear increase in brightness was observed under hypoxia conditions and in the tumor area. Excellent hypoxia imaging ability is beneficial to potential applications in cancer diagnosis.

Semiconducting Polymer Nanocavities: Porogenic Synthesis, Tunable Host-Guest Interactions, and Enhanced Drug/siRNA Delivery.

Nanocavities composed of lipids and block polymers have demonstrated great potential in biomedical applications such as sensors, nanoreactors, and delivery vectors. However, it remains a great challenge to produce nanocavities from fluorescent semiconducting polymers owing to their hydrophobic rigid polymer backbones. Here, we describe a facile, yet general strategy that combines photocrosslinking with nanophase separation to fabricate multicolor, water-dispersible semiconducting polymer nanocavities (PNCs). A photocrosslinkable semiconducting polymer is blended with a porogen such as degradable macromolecule to form compact polymer dots (Pdots). After crosslinking the polymer and removing the porogen, this approach yields semiconducting polymer nanospheres with open cavities that are tunable in diameter. Both small molecules and macromolecules can be loaded in the nanocavities, where molecular size can be differentiated by the efficiency of the energy transfer from host polymer to guest molecules. An anticancer drug doxorubicin (Dox) is loaded into the nanocavities and the intracellular release is monitored in real time by the fluorescence signal. Finally, the efficient delivery of small interfering RNA (siRNA) to silence gene expression without affecting cell viability is demonstrated. The combined features of bright fluorescence, tunable cavity, and efficient drug/siRNA delivery makes these nanostructures promising for biomedical imaging and drug delivery.

Enhanced bandwidth of white light communication using nanomaterial phosphors.

The bandwidth of white light emitting diodes (WLEDs) is an important factor that affects most of the system performances in visible light communication (VLC). It is mainly limited by the down-conversion phosphors. We propose in this paper to employ nanomaterial phosphors with short fluorescence lifetime and high quantum yield in VLC. The white-emitting device of bandwidth-based lifetime was fabricated by using several kinds of nanophosphors with different fluorescence lifetimes. Moreover, we proposed two theoretical models to analyze the factors that affect bandwidth. Compared with the commercial YAG-based WLEDs, the bandwidth of nanophosphor-based WLEDs can be improved over three times and close to the blue excitation sources. Our study indicates that nanophosphors can become promising fluorescent materials in VLC, and provides a new direction for developing wide-bandwidth VLC systems.

Therapeutic Considerations and Conjugated Polymer-Based Photosensitizers for Photodynamic Therapy.

Conjugated polymers have recently attracted a great deal of attention for applications in photodynamic therapy (PDT) because of their light-harvesting capability, efficient energy transfer, and singlet oxygen generation properties. This review describes recent advances in PDT development, including therapeutic mechanisms of PDT in cancer treatments, light excitation methods, and especially recent advances of conjugated polyelectrolytes and conjugated polymer nanoparticles as photosensitizers. The future direction on PDT and further development of conjugated polymer photosensitizers are discussed. The aim of this review is to stimulate innovative ideas to synthesize a new generation of conjugated polymer photosensitizers and promote their translation to clinical applications of PDT.

Enhanced Phototherapy by Nanoparticle-Enzyme via Generation and Photolysis of Hydrogen Peroxide.

Light has been widely used for cancer therapeutics such as photodynamic therapy (PDT) and photothermal therapy. This paper describes a strategy called enzyme-enhanced phototherapy (EEPT) for cancer treatment. We constructed a nanoparticle platform by covalent conjugation of glucose oxidase (GOx) to small polymer dots, which could be persistently immobilized into a tumor. While the malignant tumors have high glucose uptake, the GOx efficiently catalyzes the glucose oxidation with simultaneous generation of H2O2. Under light irradiation, the in situ generated H2O2 was photolyzed to produce hydroxyl radical, the most reactive oxygen species, for killing cancer cells. In vitro assays indicated that the cancer cells were destroyed by using a nanoparticle concentration at 0.2 µg/mL and a light dose of ∼120 J/cm2, indicating the significantly enhanced efficiency of the EEPT method when compared to typical PDT that requires a photosensitizer of >10 µg/mL for effective cell killing under the same light dose. Furthermore, remarkable inhibition of tumor growth was observed in xenograft-bearing mice, indicating the promise of the EEPT approach for cancer therapeutics.

Effect of Mechanical Axis Correction on Outcomes of Hallux Valgus Surgery.

A new method of mechanical axis planning has recently been suggested to aid in corrective surgery for hallux valgus (HV) deformity, which aims to identify the ideal position for the first metatarsal after correction. We investigated the influence of the mechanical axis angle (MAA) correction on the outcomes of corrective HV surgery. We reviewed 50 radiographs to identify the "normal" MAA range within the population. We also reviewed the medical records of 100 patients who had undergone scarf osteotomy at our institution from January 2011 to December 2013. These patients were segregated into 2 groups according to their postoperative MAA: those within the normal range (normal group) and those outside this range (outlier group). We compared the pre- and postoperative functional scores between the 2 groups using statistical analysis. The normal MAA range within our population was 12.5° ± 0.8° (range 11.0° to 14.3°). We found that the physical component summary score of the short-form 36-item health survey was significantly poorer for the outlier group at 6 and 24 months postoperatively compared with the normal group, although the other postoperative scores were comparable. Surgical correction of the MAA to the normal range of the patient population can be recommended because it provides improved quality of life. However, further studies are required to investigate the influence of MAA planning on other standardized foot and ankle scores.

Multicolor Photo-Crosslinkable AIEgens toward Compact Nanodots for Subcellular Imaging and STED Nanoscopy.

Aggregation induced emission (AIE) has attracted considerable interest for the development of fluorescence probes. However, controlling the bioconjugation and cellular labeling of AIE dots is a challenging problem. Here, this study reports a general approach for preparing small and bioconjugated AIE dots for specific labeling of cellular targets. The strategy is based on the synthesis of oxetane-substituted AIEgens to generate compact and ultrastable AIE dots via photo-crosslinking. A small amount of polymer enriched with oxetane groups is cocondensed with most of the AIEgens to functionalize the nanodot surface for subsequent streptavidin bioconjugation. Due to their small sizes, good stability, and surface functionalization, the cell-surface markers and subcellular structures are specifically labeled by the AIE dot bioconjugates. Remarkably, stimulated emission depletion imaging with AIE dots is achieved for the first time, and the spatial resolution is significantly enhanced to ≈95 nm. This study provides a general approach for small functional molecules for preparing small sized and ultrastable nanodots.

OCT imaging detection of brain blood vessels in mouse, based on semiconducting polymer nanoparticles.

Optical Coherence Tomography (OCT) is a valuable technology that has been used to obtain microstructure images of tissue, and has several advantages, though its applications are limited in high-scattering tissues. Therefore, semiconducting polymer nanoparticles (SPNs) that possess strong absorption characteristics are applied to decrease light scattering in tissues and used as exogenous contrast agents for enhancing the contrast of OCT imaging detection. In this paper, we prepared two kinds of SPNs, termed PIDT-TBZ SPNs and PBDT-TBZ SPNs, as the contrast agents for OCT detection to enhance the signal. Firstly, we proved that they were good contrast agents for OCT imaging in agar-TiO2. After that, the contrast effects of these two SPNs were quantitatively analyzed, and then cerebral blood vessels were monitored by a home-made SD-OCT system. Finally, we created OCT images in vitro and in vivo with these two probes and performed quantitative analysis using the images. The results indicated that these SPNs created a clear contrast enhancement of small vessels in the OCT imaging process, which provides a basis for the application of SPNs as contrast agents for bioimaging studies.

Conjugated polymer dots for ultra-stable full-color fluorescence patterning.

Stable full-color fluorescence patterning are achieved by multicolor polymer-dot inks. The fluorescent patterns show extraordinary stability upon various treatments, offering a superior combination of bright fluorescence, excellent photostability, chemical resistance, and eco-friendship.

A Comparative Genomic Analysis of Parathyroid Adenomas and Carcinomas Harboring Heterozygous Germline CDC73 Mutations.

CONTEXT: Parathyroid cancer has been linked to germline mutations of the CDC73 gene. However, carriers harboring cancer-associated germline CDC73 mutations may develop only parathyroid adenoma or no parathyroid disease. This incomplete penetrance indicates that additional genomic events are required for parathyroid tumorigenesis. OBJECTIVE: (1) Determine the status of the second CDC73 allele in parathyroid tumors harboring germline CDC73 mutations, and (2) compare the genomic landscapes between parathyroid carcinomas and adenomas. DESIGN: Whole-exome and RNA sequencing of 12 parathyroid tumors harboring germline CDC73 mutations (6 adenomas and 6 carcinomas) and their matched normal tissues. RESULTS: All 12 parathyroid tumors had gained one somatic event predicted to cause a complete inactivation of the second CDC73 allele. Several distinctive genomic features were identified in parathyroid carcinomas compared to adenomas, including more single nucleotide variants bearing the C>G transversion and APOBEC deamination signatures, frequent mutations of the genes involved in the PI-3K/mTOR signaling, a greater number of copy number variations, and substantially more genes with altered expression. Parathyroid carcinomas also share some genomic features with adenomas. For instance, both have recurrent somatic mutations and copy number loss that impact the genes involved in T-cell receptor signaling and tumor antigen presentation, suggesting a shared strategy to evade immune surveillance. CONCLUSIONS: Biallelic inactivation of CDC73 is essential for parathyroid tumorigenesis in carriers harboring germline mutations of this gene. Despite sharing some genomic features with adenomas, parathyroid carcinomas have more distinctive alterations in the genome, some of which may be critical for cancer formation.