Purpose.— To update key recommendations of the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2) testing in breast ...

Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update

Metallic and metal oxide nanoparticles (NPs), including titanium dioxide NPs, among polymeric NPs, liposomes, micelles, quantum dots, dendrimers, or fullerenes, are becoming more and more important due to their potential use in novel medical therapies. Titanium dioxide (titanium(IV) oxide, titania, TiO2) is an inorganic compound that owes its recent rise in scientific interest to photoactivity. After the illumination in aqueous media with UV light, TiO2 produces an array of reactive oxygen species (ROS). The capability to produce ROS and thus induce cell death has found application in the photodynamic therapy (PDT) for the treatment of a wide range of maladies, from psoriasis to cancer. Titanium dioxide NPs were studied as photosensitizing agents in the treatment of malignant tumors as well as in photodynamic inactivation of antibiotic-resistant bacteria. Both TiO2 NPs themselves, as well as their composites and combinations with other molecules or biomolecules, can be successfully used as photosensitizers in PDT. Moreover, various organic compounds can be grafted on TiO2 nanoparticles, leading to hybrid materials. These nanostructures can reveal increased light absorption, allowing their further use in targeted therapy in medicine. In order to improve efficient anticancer and antimicrobial therapies, many approaches utilizing titanium dioxide were tested. Results of selected studies presenting the scope of potential uses are discussed in this review.

https://www.mdpi.com/2079-4991/10/2/387/pdf

Titanium Dioxide Nanoparticles: Prospects and Applications in Medicine.

Nuclear pore complexes (NPCs) perforate the nuclear envelope and represent the exclusive passageway into and out of the nucleus of the eukaryotic cell. Apart from their essential transport function, components of the NPC have important, direct roles in nuclear organization and in gene regulation. Because of its central role in cell biology, it is of considerable interest to determine the NPC structure at atomic resolution. The complexity of these large, 40–60 MDa protein assemblies has for decades limited such structural studies. More recently, exploiting the intrinsic modularity of the NPC, structural biologists are making progress toward understanding this nanomachine in molecular detail. Structures of building blocks of the stable, architectural scaffold of the NPC have been solved, and distinct models for their assembly proposed. Here we review the status of the field and lay out the challenges and the next steps toward a full understanding of the NPC at atomic resolution.

The nuclear pore complex has entered the atomic age

Significance: Optical imaging using the endogenous fluorescence of metabolic cofactors has enabled nondestructive examination of dynamic changes in cell and tissue function both in vitro and in vivo. Quantifying NAD(P)H and FAD fluorescence through an optical redox ratio and fluorescence lifetime imaging (FLIM) provides sensitivity to the relative balance between oxidative phosphorylation and glucose catabolism. Since its introduction decades ago, the use of NAD(P)H imaging has expanded to include applications involving almost every major tissue type and a variety of pathologies. Recent Advances: This review focuses on the use of two-photon excited fluorescence and NAD(P)H fluorescence lifetime techniques in cancer, neuroscience, tissue engineering, and other biomedical applications over the last 5 years. In a variety of cancer models, NAD(P)H fluorescence intensity and lifetime measurements demonstrate a sensitivity to the Warburg effect, suggesting potential for early detection or high-throughp...

Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD.

Fluorescence lifetime imaging (FLI) provides unique quantitative information in biomedical and molecular biology studies but relies on complex data-fitting techniques to derive the quantities of interest. Herein, we propose a fit-free approach in FLI image formation that is based on deep learning (DL) to quantify fluorescence decays simultaneously over a whole image and at fast speeds. We report on a deep neural network (DNN) architecture, named fluorescence lifetime imaging network (FLI-Net) that is designed and trained for different classes of experiments, including visible FLI and near-infrared (NIR) FLI microscopy (FLIM) and NIR gated macroscopy FLI (MFLI). FLI-Net outputs quantitatively the spatially resolved lifetime-based parameters that are typically employed in the field. We validate the utility of the FLI-Net framework by performing quantitative microscopic and preclinical lifetime-based studies across the visible and NIR spectra, as well as across the 2 main data acquisition technologies. These results demonstrate that FLI-Net is well suited to accurately quantify complex fluorescence lifetimes in cells and, in real time, in intact animals without any parameter settings. Hence, FLI-Net paves the way to reproducible and quantitative lifetime studies at unprecedented speeds, for improved dissemination and impact of FLI in many important biomedical applications ranging from fundamental discoveries in molecular and cellular biology to clinical translation.

https://www.pnas.org/content/pnas/116/48/24019.full.pdf

Fast fit-free analysis of fluorescence lifetime imaging via deep learning.

A method of rapidly differentiating lung tumor from healthy tissue is extraordinarily needed for both the diagnosis and the intraoperative margin assessment. We assessed the ability of fluorescence lifetime imaging microscopy (FLIM) for differentiating human lung cancer and normal tissues with the autofluorescence, and also elucidated the mechanism in tissue studies and cell studies. A 15-patient testing group was used to compare FLIM results with traditional histopathology diagnosis. Based on the endogenous fluorescence lifetimes of the testing group, a criterion line was proposed to distinguish normal and cancerous tissues. Then by blinded examined 41 sections from the validation group of other 16 patients, the sensitivity and specificity of FLIM were determined. The cellular metabolism was studied with specific perturbations of oxidative phosphorylation and glycolysis in cell studies. The fluorescence lifetime of cancerous lung tissues is consistently lower than normal tissues, and this is due to the both decrease of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) lifetimes. A criterion line of lifetime at 1920 ps can be given for differentiating human lung cancer and normal tissues.The sensitivity and specificity of FLIM for lung cancer diagnosis were determined as 92.9% and 92.3%. These findings suggest that NADH and FAD can be used to rapidly diagnose lung cancer. FLIM is a rapid, accurate and highly sensitive technique in the judgment during lung cancer surgery and it can be potential in earlier cancer detection.

Rapid diagnosis and intraoperative margin assessment of human lung cancer with fluorescence lifetime imaging microscopy

Titanium dioxide (TiO2) has attracted wide attention as a potential photosensitizer (PS) in photodynamic therapy (PDT). However, bare TiO2 can only be excited by ultraviolet illumination, and it lacks specific targeting ligands, which largely impede its application. In our study, we produced nitrogen-doped TiO2 and linked it with an effective cancer cell targeting agent, folic acid (FA), to obtain N-TiO2-FA nanoconjugates. Characterization of N-TiO2-FA included Zeta potential, absorption spectra and thermogravimetric analysis. The results showed that N-TiO2-FA was successfully produced and it possessed better dispersibility in aqueous solution than unmodified TiO2. The N-TiO2-FA was incubated with human nasopharyngeal carcinoma (KB) and human pulmonary adenocarcinoma (A549) cells. The KB cells that overexpress folate receptors (FR) on cell membranes were used as FR-positive cancer cells, while A549 cells were used as FR-negative cells. Laser scanning confocal microscopy results showed that KB cells had a higher uptake efficiency of N-TiO2-FA, which was about twice that of A549 cells. Finally, N-TiO2-FA is of no cytotoxicity, and has a better photokilling effect on KB cells under visible light irradiation. In conclusion, N-TiO2-FA can be as high-value as a PS in cancer targeting PDT.

/pdf/targeting-and-photodynamic-killing-of-cancer-cell-by-4z7g90r0g4.pdf

Targeting and Photodynamic Killing of Cancer Cell by Nitrogen-Doped Titanium Dioxide Coupled with Folic Acid.

TiO2 nanoparticles modified with phthalocyanines (Pc) have been proven to be a potential photosensitizer in the application of photodynamic therapy (PDT). However, the generation of reactive oxygen species (ROS) by TiO2 nanoparticles modified with Pc has not been demonstrated clearly. In this study, nitrogen-doped TiO2 conjugated with Pc (N-TiO2-Pc) were studied by means of monitoring the generation of ROS. The absorbance and photokilling effect on HeLa cells upon visible light of different regions were also studied and compared with non-doped TiO2-Pc and Pc. Both N-TiO2-Pc and TiO2-Pc can be activated by visible light and exhibited much higher photokilling effect on HeLa cells than Pc. In addition, nitrogen-doping can greatly enhance the formation of 1O2 and •O2−, while it suppresses the generation of OH•. This resulted in significant photodynamic activity. Therefore, N-TiO2-Pc can be an excellent candidate for a photosensitizer in PDT with wide-spectrum visible irradiation.

https://www.mdpi.com/2079-4991/7/10/338/pdf

Study of the Photodynamic Activity of N-Doped TiO2 Nanoparticles Conjugated with Aluminum Phthalocyanine

Either modulated illumination or temporal fluctuation analysis can assist super-resolution techniques in overcoming the diffraction limit of conventional optical microscopy. As they are not contradictory to each other, an effective combination of spatial and temporal super-resolution mechanisms would further improve the resolution of fluorescent images. Here, a super-resolution imaging method called fluctuation-enhanced Airyscan technology (FEAST) is proposed, which achieves ~40 nm lateral imaging resolution and is useful for a range of fluorescent proteins and organic dyes. It was demonstrated not only to obtain different subcellular super-resolution images, but also to improve the accuracy of counting the average human epidermal growth factor receptor 2 (HER2) copy number for diagnosis in breast cancer. Furthermore, the combination of FEAST and sample expansion microscopy (Ex-FEAST) improves the lateral resolution to ~26 nm.

Multicomposite super-resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions.

Fluorescence lifetime measurements are often performed on live as well as fixed cells and tissues. Fixation and mounting processes are routinely used in cellular research or clinical diagnosis. In this paper, the effects of fixation and mounting on the fluorescence lifetime of cellular autofluorescence were studied by fluorescence lifetime imaging microscopy over time. Two endogenous fluorescent fluorophores, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), showed different results between live cells and fixed cells. The average lifetime of NADH in live HeLa cells was about 1.02 ns, while maintained about 1.57 ns during the fixation periods of 14 days. The average lifetimes of FAD in live and fixed HeLa cells within 11 days were similar around 1.75 ns but increased to 2.10 ns after 12 days. The free and bound states of the two kinds of fluorophores were further analyzed. It was found that the bound-FAD had two different groups, which was related to the cell division cycle. The effect of mounting medium on fluorescence lifetimes was also studied, indicating glycerol has a negative impact on the fluorescence lifetime compared with neutral balsam.

/pdf/effect-of-fixation-and-mounting-on-fluorescence-lifetime-of-1x50gasykp.pdf

Longfang Yao

Papers

Rapid diagnosis and intraoperative margin assessment of human lung cancer with fluorescence lifetime imaging microscopy

Targeting and Photodynamic Killing of Cancer Cell by Nitrogen-Doped Titanium Dioxide Coupled with Folic Acid.

Study of the Photodynamic Activity of N-Doped TiO2 Nanoparticles Conjugated with Aluminum Phthalocyanine

Multicomposite super-resolution microscopy: Enhanced Airyscan resolution with radial fluctuation and sample expansions.

Effect of Fixation and Mounting on Fluorescence Lifetime of Cellular Autofluorescence