Mouse Strain- and Charge-Dependent Vessel Permeability of Nanoparticles at the Lower Size Limit.

Remarkable advancement has been made in the application of nanoparticles (NPs) for cancer therapy. Although NPs have been favorably delivered into tumors by taking advantage of the enhanced permeation and retention (EPR) effect, several physiological barriers present within tumors tend to restrict the diffusion of NPs. To overcome this, one of the strategies is to design NPs that can reach lower size limits to improve tumor penetration without being rapidly cleared out by the body. Several attempts have been made to achieve this, such as selecting appropriate nanocarriers and modifying surface properties. While many studies focus on the optimal design of NPs, the influence of mouse strains on the effectiveness of NPs remains unknown. Therefore, this study aimed to assess whether the vascular permeability of NPs near the lower size limit differs among mouse strains. We found that the vessel permeability of dextran NPs was size-dependent and dextran NPs with a size below 15 nm exhibited leakage from postcapillary venules in all strains. Most importantly, the leakage rate of 8-nm fluorescein isothiocyanate dextran was significantly higher in the BALB/c mouse strain than in other strains. This strain dependence was not observed in slightly positive TRITC-dextran with comparable sizes. Our results indicate that the influence on mouse strains needs to be taken into account for the evaluation of NPs near the lower size limit.

Noninvasive assessment of liver function reserve with fluorescent dosimetry of indocyanine green.

Using in vivo multiphoton fluorescent dosimetry, we demonstrate that the clearance dynamics of Indocyanine Green (ICG) in the blood can quickly reveal liver function reserve. In normal rats, the ICG retention rate was below 10% at the 15-minute post-administration; While in the rat with severe hepatocellular carcinoma (HCC), the 15-minute retention rate is over 40% due to poor liver metabolism. With a 785 nm CW laser, the fluorescence dosimeter can evaluate the liver function reserve at a 1/10 clinical dosage of ICG without any blood sampling. In the future, this low-dosage ICG 15-minute retention dosimetry can be applied for the preoperative assessment of hepatectomy or timely perioperative examination.

Accelerating precision anti-cancer therapy by time-lapse and label-free 3D tumor slice culture platform.

The feasibility of personalized medicine for cancer treatment is largely hampered by costly, labor-intensive and time-consuming models for drug discovery. Herein, establishing new pre-clinical models to tackle these issues for personalized medicine is urgently demanded. Methods: We established a three-dimensional tumor slice culture (3D-TSC) platform incorporating label-free techniques for time-course experiments to predict anti-cancer drug efficacy and validated the 3D-TSC model by multiphoton fluorescence microscopy, RNA sequence analysis, histochemical and histological analysis. Results: Using time-lapse imaging of the apoptotic reporter sensor C3 (C3), we performed cell-based high-throughput drug screening and shortlisted high-efficacy drugs to screen murine and human 3D-TSCs, which validate effective candidates within 7 days of surgery. Histological and RNA sequence analyses demonstrated that 3D-TSCs accurately preserved immune components of the original tumor, which enables the successful achievement of immune checkpoint blockade assays with antibodies against PD-1 and/or PD-L1. Label-free multiphoton fluorescence imaging revealed that 3D-TSCs exhibit lipofuscin autofluorescence features in the time-course monitoring of drug response and efficacy. Conclusion: This technology accelerates precision anti-cancer therapy by providing a cheap, fast, and easy platform for anti-cancer drug discovery.

Functionalizing Collagen with Vessel-Penetrating Two-Photon Phosphorescence Probes: A New In Vivo Strategy to Map Oxygen Concentration in Tumor Microenvironment and Tissue Ischemia.

The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio-probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (ReI ) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, ReI -diimine complex already exhibits excellent emission yield (34%, λem   = 583 nm) and large two-photon absorption cross-sections (σ2   = 300 GM @ 800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen-bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 µs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post-extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial-empowered phosphorescence sensing and imaging.

Low-toxicity FePt nanoparticles for the targeted and enhanced diagnosis of breast tumors using few centimeters deep whole-body photoacoustic imaging.

A considerable amount of early breast tumors grown at a depth over 2 cm in breast tissues. With high near-infrared absorption of iron-platinum (FePt) nanoparticles, we achieved few centimeters deep photoacoustic (PA) imaging for the diagnosis of breast tumors. The imaging depth can extend over 5 cm in chicken breast tissues at the low laser energy density of 20 mJ/cm2 (≤ ANSI safety limit). After anti-VEGFR conjugation and the tail-vein injection, we validated their targeting on tumor sites by the confocal microscopy and PA imaging. Using a home-made whole-body in vivo PA imaging, we found that the nanoparticles were rapidly cleared away from the site of the tumor and majorly metabolized through the liver. These results validated the clinical potential of the FePt nanoparticles in the low-toxicity PA theragnosis of early breast cancer and showed the capacity of our whole-body PA imaging technique on monitoring the dynamic biodistribution of nanoparticles in the living body.

Rab18 Collaborates with Rab7 to Modulate Lysosomal and Autophagy Activities in the Nervous System: an Overlapping Mechanism for Warburg Micro Syndrome and Charcot-Marie-Tooth Neuropathy Type 2B.

Mutations in RAB18, a member of small G protein, cause Warburg micro syndrome (WARBM), whose clinical features include vision impairment, postnatal microcephaly, and lower limb spasticity. Previously, our Rab18-/- mice exhibited hind limb weakness and spasticity as well as signs of axonal degeneration in the spinal cord and lumbar spinal nerves. However, the cellular and molecular function of RAB18 and its roles in the pathogenesis of WARBM are still not fully understood. Using immunofluorescence staining and expression of Rab18 and organelle markers, we find that Rab18 associates with lysosomes and actively traffics along neurites in cultured neurons. Interestingly, Rab18-/- neurons exhibit impaired lysosomal transport. Using autophagosome marker LC3-II, we show that Rab18 dysfunction leads to aberrant autophagy activities in neurons. Electron microscopy further reveals accumulation of lipofuscin-like granules in the dorsal root ganglion of Rab18-/- mice. Surprisingly, Rab18 colocalizes, cofractionates, and coprecipitates with the lysosomal regulator Rab7, mutations of which cause Charcot-Marie-Tooth (CMT) neuropathy type 2B. Moreover, Rab7 is upregulated in Rab18-deficient neurons, suggesting a compensatory effect. Together, our results suggest that the functions of RAB18 and RAB7 in lysosomal and autophagic activities may constitute an overlapping mechanism underlying WARBM and CMT pathogenesis in the nervous system.

Comparative health risk of inhaled exposure to organic solvents, toxic metals, and hexavalent chromium from the use of spray paints in Taiwan.

The study investigated the exposure of spray painters to organic solvents, toxic metals, and hexavalent chromium over 21 working days in 2017. The results found these concentrations of 12 VOCs to be below the short-term exposure limit (STEL) established by the US Occupational Safety and Health Administration (OSHA). The mass concentration of total particulate matter (PM) exposure to workers was 20.01 ± 10.78 mg/m3, which exceeds OSHA's permissible exposure level of 15 mg/m3. The mean concentration of the total metals for all particle sizes was 109.1 ± 12.0 µg/m3, and those for lead (496,017.0 ng/m3) and iron (252,123.8 ng/m3) were the highest of metal elements. Significantly, the mean concentrations of Pb and As exceeded OSHA's permissible exposure limits (PELs) of 0.05 and 0.01 mg/m3, respectively. The total hexavalent chromium concentration was 1163.01 ng/m3, and the individual particle sizes (PM1-2.5, PM1, and PM0.25) were strongly and positively correlated with the Cr(VI) concentrations for PM2.5. The study determined that approximately 56.14% of the hexavalent chromium inhaled during the spray-painting process was deposited in the upper respiratory system of the head airway region, followed by the alveolar and tracheobronchial regions, with fractions of 11.93 and 0.05%, respectively. Although the mean ratio of hexavalent chromium to total chromium was only 3.6% for all particle sizes, the cancer risk of the total particles in Cr(VI) (1.6 × 10-3) exceeded the acceptable risk value (10-6). The cancer risks of As and Cr(VI) associated with quasi-ultrafine particles, PM0.5-1, PM1-2.5, and PM> 2.5, also exceeded 10-6. Comparison of the carcinogenicity risk of VOCs and metals suggests that the adverse health effect of inhaled particles on spray-painting workers is more serious than that from VOC exposure.

CISD2 Haploinsufficiency Disrupts Calcium Homeostasis, Causes Nonalcoholic Fatty Liver Disease, and Promotes Hepatocellular Carcinoma.

CISD2 is located within the chromosome 4q region frequently deleted in hepatocellular carcinoma (HCC). Mice with Cisd2 heterozygous deficiency develop a phenotype similar to the clinical manifestation of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Cisd2 haploinsufficiency causes a low incidence (20%) of spontaneous HCC and promotes HBV-associated and DEN-induced HCC; conversely, 2-fold overexpression of Cisd2 suppresses HCC in these models. Mechanistically, Cisd2 interacts with Serca2b and mediates its Ca2+ pump activity via modulation of Serca2b oxidative modification, which regulates ER Ca2+ uptake and maintains intracellular Ca2+ homeostasis in the hepatocyte. CISD2 haploinsufficiency disrupts calcium homeostasis, causing ER stress and subsequent NAFLD and NASH. Hemizygous deletion and decreased expression of CISD2 are detectable in a substantial fraction of human HCC specimens. These findings substantiate CISD2 as a haploinsufficient tumor suppressor and highlights Cisd2 as a drug target when developing therapies to treat NAFLD/NASH and prevent HCC.

Cell death caused by the synergistic effects of zinc and dopamine is mediated by a stress sensor gene Gadd45b - implication in the pathogenesis of Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) is not completely understood, Zinc (Zn(2+) ) and dopamine (DA) have been shown to involve in the degeneration of dopaminergic cells. By microarray analysis, we identified Gadd45b as a candidate molecule that mediates Zn(2+) and DA-induced cell death; the mRNA and protein levels of Gadd45b are increased by Zn(2+) treatment and raised to an even higher level by Zn(2+) plus DA treatment. Zn(2+) plus DA treatment-induced PC12 cell death was enhanced when there was over-expression of Gadd45b and was decreased by knock down of Gadd45b. MAPK p38 and JNK signaling was able to cross-talk with Gadd45b during Zn(2+) and DA treatment. The synergistic effects of Zn(2+) and DA on PC12 cell death can be accounted for by an activation of the Gadd45b-induced cell death pathway and an inhibition of p38/JNK survival pathway. Furthermore, the in vivo results show that the levels of Gadd45b protein expression and phosphorylation of p38 were increased in the substantia nigra by the infusion of Zn(2+) /DA in the mouse brain and the level of Gadd45b mRNA is significantly higher in the substantia nigra of male PD patients than normal controls. The novel role of Gadd45b and its interactions with JNK and p38 will help our understanding of the pathogenesis of PD and help the development of future treatments for PD. Zinc and dopamine are implicated in the degeneration of dopaminergic neurons. We previously demonstrated that zinc and dopamine induced synergistic effects on PC12 cell death. Results from this study show that these synergistic effects can be accounted for by activation of the Gadd45b-induced cell death pathway and inhibition of the p38/JNK survival pathway. We provide in vitro and in vivo evidence to support a novel role for Gadd45b in the pathogenesis of Parkinson's disease.

Infrared-active quadruple contrast FePt nanoparticles for multiple scale molecular imaging.

A single nanomaterial with multiple imaging contrasts and functions is highly desired for multiscale theragnosis. Herein, we demonstrate single 1-1.9 µm infrared-active FePt alloy nanoparticles (FePt NPs) offering unprecedented four-contrast-in-one molecular imaging - computed tomography (CT), magnetic resonance imaging (MRI), photoacoustic (PA) imaging, and high-order multiphoton luminescence (HOMPL) microscopy. The PA response of FePt NPs outperforms that of infrared-active gold nanorods by 3- to 5.6-fold under identical excitation fluence and particle concentrations. HOMPL (680 nm) of an isolated FePt NP renders spatial full-width-at-half-maximum values of 432 nm and 300 nm beyond the optical diffraction limit for 1230-nm and 920-nm excitation, respectively. The in vivo targeting function was successfully visualized using HOMPL, PA imaging, CT, and MRI, thereby validating FePt as a single nanomaterial system covering up to four types (Optical/PA/CT/MRI) of molecular imaging contrast, ranging from the microscopic level to whole-body scale investigation.

In vivo quantification of the structural changes of collagens in a melanoma microenvironment with second and third harmonic generation microscopy.

Using in vivo second harmonic generation (SHG) and third harmonic generation (THG) microscopies, we tracked the course of collagen remodeling over time in the same melanoma microenvironment within an individual mouse. The corresponding structural and morphological changes were quantitatively analyzed without labeling using an orientation index (OI), the gray level co-occurrence matrix (GLCM) method, and the intensity ratio of THG to SHG (RTHG/SHG). In the early stage of melanoma development, we found that collagen fibers adjacent to a melanoma have increased OI values and SHG intensities. In the late stages, these collagen networks have more directionality and less homogeneity. The corresponding GLCM traces showed oscillation features and the sum of squared fluctuation VarGLCM increased with the tumor sizes. In addition, the THG intensities of the extracellular matrices increased, indicating an enhanced optical inhomogeneity. Multiplying OI, VarGLCM, and RTHG/SHG together, the combinational collagen remodeling (CR) index at 4 weeks post melanoma implantation showed a 400-times higher value than normal ones. These results validate that our quantitative indices of SHG and THG microscopies are sensitive enough to diagnose the collagen remodeling in vivo. We believe these indices have the potential to help the diagnosis of skin cancers in clinical practice.

H3K9 histone methyltransferase, KMT1E/SETDB1, cooperates with the SMAD2/3 pathway to suppress lung cancer metastasis.

Aberrant histone methylation is a frequent event during tumor development and progression. KMT1E (also known as SETDB1) is a histone H3K9 methyltransferase that contributes to epigenetic silencing of both oncogenes and tumor suppressor genes in cancer cells. In this report, we demonstrate that KMT1E acts as a metastasis suppressor that is strongly downregulated in highly metastatic lung cancer cells. Restoring KMT1E expression in this setting suppressed filopodia formation, migration, and invasive behavior. Conversely, loss of KMT1E in lung cancer cells with limited metastatic potential promoted migration in vitro and restored metastatic prowess in vivo. Mechanistic investigations indicated that KMT1E cooperates with the TGFß-regulated complex SMAD2/3 to repress metastasis through ANXA2. Together, our findings defined an essential role for the KMT1E/SMAD2/3 repressor complex in TGFß-mediated lung cancer metastasis.

Synergistic delivery of gold nanorods using multifunctional microbubbles for enhanced plasmonic photothermal therapy.

Plasmonic photothermal therapy (PPTT) using plasmonic nanoparticles as efficient photoabsorbing agents has been proposed previously. One critical step in PPTT is to effectively deliver gold nanoparticles into the cells. This study demonstrates that the delivery of gold nanorods (AuNRs) can be greatly enhanced by combining the following three mechanisms: AuNRs encapsulated in protein-shell microbubbles (AuMBs), molecular targeting, and sonoporation employing acoustic cavitation of microbubbles (MBs). Both in vitro and in vivo tests were performed. For molecular targeting, the AuMBs were modified with anti-VEGFR2. Once bound to the angiogenesis markers, the MBs were destroyed by ultrasound to release the AuNRs and the release was confirmed by photoacoustic measurements. Additionally, acoustic cavitation was induced during MB destruction for sonoporation (i.e., increase in transient cellular permeability). The measured inertial cavitation dose was positively correlated with the temperature increase at the tumor site. The quantity of AuNRs delivered into the cells was also determined by measuring the mass spectrometry and observed using third-harmonic-generation microscopy and two-photon fluorescence microscopy. A temperature increase of 20 °C was achieved in vitro. The PPTT results in vivo also demonstrated that the temperature increase (>45 °C) provided a sufficiently high degree of hyperthermia. Therefore, synergistic delivery of AuNRs was demonstrated.

The EP300, KDM5A, KDM6A and KDM6B chromatin regulators cooperate with KLF4 in the transcriptional activation of POU5F1.

POU5F1 is essential for maintaining pluripotency in embryonic stem cells (ESCs). It has been reported that the constitutive activation of POU5F1 is sustained by the core transcriptional regulatory circuitry in ESCs; however, the means by which POU5F1 is epigenetically regulated remains enigmatic. In this study a fluorescence-based reporter system was used to monitor the interplay of 5 reprogramming-associated TFs and 17 chromatin regulators in the transcription of POU5F1. We show the existence of a stoichiometric effect for SOX2, POU5F1, NANOG, MYC and KLF4, in regulating POU5F1 transcription. Chromatin regulators EP300, KDM5A, KDM6A and KDM6B cooperate with KLF4 in promoting the transcription of POU5F1. Moreover, inhibiting HDAC activities induced the expression of Pou5f1 in mouse neural stem cells (NSCs) in a spatial- and temporal- dependent manner. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) shows that treatment with valproic acid (VPA) increases the recruitment of Kdm5a and Kdm6a to proximal promoter (PP) and proximal enhancer (PE) of Pou5f1 whereas enrichment of Ep300 and Kdm6b was seen in PP but not PE of Pou5f1 promoter. These findings reveal the interplay between the chromatin regulators and histone modifications in the expression of POU5F1.

Distinct mechanisms account for beta-amyloid toxicity in PC12 and differentiated PC12 neuronal cells.

Whether reactive oxygen species (ROS) mediate beta-amyloid (A beta) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by A beta(25-35). The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 microM A beta(25-35). Microscopic examination showed that A beta(25-35) induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 microM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of A beta(25-35). Since H(2)O(2) has been proposed to be involved in A beta toxicity, the effects of H(2)O(2) on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H(2)O(2) than naive PC12 cells. These data suggest that ROS may mediate A beta(25-35) toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for A beta(25-35) toxicity. Therefore, the protective effects of antioxidants against A beta may depend upon the redox state of the cells.