A novel low-molecular-weight chitosan/gamma-polyglutamic acid polyplexes for nucleic acid delivery into zebrafish larvae.

Many challenges, such as virus infection, extreme weather and long cultivation periods, during the development of fish larvae have been observed, especially in aquaculture. Gene delivery is a useful method to express functional genes to defend against these challengers. However, the methods for fish larvae are insufficient. In our earlier report, low-molecular-weight chitosan (LMWCS) showed a strong positive charge and may be useful for polyplex formulation. Herein, we present a simple self-assembly of LMWCS polyplexes (LMWCSrNPs) for gene delivery into zebrafish larvae. Different weight ratios of LMWCS/gamma-polyglutamic acid (γ-PGA)/plasmid DNA were analyzed by gel mobility assay. Delivery efficiency determined by green fluorescent protein (GFP) expression in zebrafish liver (ZFL) cells showed that delivery efficiency at a weight ratio of 20:8:1 was higher than others. Zeta potential and transmission electron microscopy (TEM) analysis showed that the round shape of the particle size varied. In our earlier reports, IRF9S2C could induce interferon-stimulated gene (ISG) expression to induce innate immunity in zebrafish and pufferfish. Further delivery of pcDNA3-IRF9S2C-HA plasmid DNA into ZFL cells and zebrafish larvae by LMWCSrNP successfully induced ISG expression. Collectively, LMWCSrNP could be a novel gene delivery system for zebrafish larvae and might be used to improve applications in aquaculture.

X-ray-Activated Simultaneous Near-Infrared and Short-Wave Infrared Persistent Luminescence Imaging for Long-Term Tracking of Drug Delivery.

Both short-wave infrared (SWIR: 900-1700 nm) and near-infrared (NIR: 650-900 nm) luminescence possess lower optical scattering and higher signal-to-noise in deep tissues than conventional luminescence, gaining increasing attention in biomedicine. Herein, we designed mesoporous silica-coated Yb-doped magnesium germanate nanoparticles (mMGOs) with excellent two-in-one NIR and SWIR persistent luminescence after X-ray irradiation by simply regulating the valence of rare-earth ions, which also possess high cargo loading and a controlled release profile in the tumor region. The investigations in vitro and in vivo showed that mMGOs were repeatedly activated to realize rechargeable persistent luminescence imaging for tracking cargo delivery in mice. Moreover, the stimulative drug-release profile inhibited tumor growth effectively. Both of the X-ray excited two-in-one NIR and SWIR persistent luminescence imaging not only allowed for rechargeable imaging of deep tumors but also achieved long-term tracking with a remarkable tumor inhibition effect.

Neuroprotective Effects of Emodin against Ischemia/Reperfusion Injury through Activating ERK-1/2 Signaling Pathway.

BACKGROUND: Stroke is one of the leading causes of death and disability worldwide and places a heavy burden on the economy in our society. Current treatments, such as the use of thrombolytic agents, are often limited by a narrow therapeutic time window. However, the regeneration of the brain after damage is still active days, even weeks, after stroke occurs, which might provide a second window for treatment. Emodin, a traditional Chinese medicinal herb widely used to treat acute hepatitis, has been reported to possess antioxidative capabilities and protective effects against myocardial ischemia/reperfusion injury. However, the underlying mechanisms and neuroprotective functions of Emodin in a rat middle cerebral artery occlusion (MCAO) model of ischemic stroke remain unknown. This study investigates neuroprotective effects of Emodin in ischemia both in vitro and in vivo. METHODS: PC12 cells were exposed to oxygen-glucose deprivation to simulate hypoxic injury, and the involved signaling pathways and results of Emodin treatment were evaluated. The therapeutic effects of Emodin in ischemia animals were further investigated. RESULTS: Emodin reduced infarct volume and cell death following focal cerebral ischemia injury. Emodin treatment restored PC12 cell viability and reduced reactive oxygen species (ROS) production and glutamate release under conditions of ischemia/hypoxia. Emodin increased Bcl-2 and glutamate transporter-1 (GLT-l) expression but suppressed activated-caspase 3 levels through activating the extracellular signal-regulated kinase (ERK)-1/2 signaling pathway. CONCLUSION: Emodin induced Bcl-2 and GLT-1 expression to inhibit neuronal apoptosis and ROS generation while reducing glutamate toxicity via the ERK-1/2 signaling pathway. Furthermore, Emodin alleviated nerve cell injury following ischemia/reperfusion in a rat MCAO model. Emodin has neuroprotective effects against ischemia/reperfusion injury both in vitro and in vivo, which may be through activating the ERK-1/2 signaling pathway.

An Integrated Bioinformatics Analysis Repurposes an Antihelminthic Drug Niclosamide for Treating HMGA2-Overexpressing Human Colorectal Cancer.

Aberrant overexpression of high mobility group AT-hook 2 (HMGA2) is frequently found in cancers and HMGA2 has been considered an anticancer therapeutic target. In this study, a pan-cancer genomics survey based on Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) data indicated that HMGA2 was mainly overexpressed in gastrointestinal cancers including colorectal cancer. Intriguingly, HMGA2 overexpression had no prognostic impacts on cancer patients' overall and disease-free survivals. In addition, HMGA2-overexpressing colorectal cancer cell lines did not display higher susceptibility to a previously identified HMGA2 inhibitor (netroposin). By microarray profiling of HMGA2-driven gene signature and subsequent Connectivity Map (CMap) database mining, we identified that S100 calcium-binding protein A4 (S100A4) may be a druggable vulnerability for HMGA2-overexpressing colorectal cancer. A repurposing S100A4 inhibitor, niclosamide, was found to reverse the HMGA2-driven gene signature both in colorectal cancer cell lines and patients' tissues. In vitro and in vivo experiments validated that HMGA2-overexpressing colorectal cancer cells were more sensitive to niclosamide. However, inhibition of S100A4 by siRNAs and other inhibitors was not sufficient to exert effects like niclosamide. Further RNA sequencing analysis identified that niclosamide inhibited more cell-cycle-related gene expression in HMGA2-overexpressing colorectal cancer cells, which may explain its selective anticancer effect. Together, our study repurposes an anthelminthic drug niclosamide for treating HMGA2-overexpression colorectal cancer.

Using multivariate regression model with least absolute shrinkage and selection operator (LASSO) to predict the incidence of Xerostomia after intensity-modulated radiotherapy for head and neck cancer.

PURPOSE: The aim of this study was to develop a multivariate logistic regression model with least absolute shrinkage and selection operator (LASSO) to make valid predictions about the incidence of moderate-to-severe patient-rated xerostomia among head and neck cancer (HNC) patients treated with IMRT. METHODS AND MATERIALS: Quality of life questionnaire datasets from 206 patients with HNC were analyzed. The European Organization for Research and Treatment of Cancer QLQ-H&N35 and QLQ-C30 questionnaires were used as the endpoint evaluation. The primary endpoint (grade 3(+) xerostomia) was defined as moderate-to-severe xerostomia at 3 (XER3m) and 12 months (XER12m) after the completion of IMRT. Normal tissue complication probability (NTCP) models were developed. The optimal and suboptimal numbers of prognostic factors for a multivariate logistic regression model were determined using the LASSO with bootstrapping technique. Statistical analysis was performed using the scaled Brier score, Nagelkerke R(2), chi-squared test, Omnibus, Hosmer-Lemeshow test, and the AUC. RESULTS: Eight prognostic factors were selected by LASSO for the 3-month time point: Dmean-c, Dmean-i, age, financial status, T stage, AJCC stage, smoking, and education. Nine prognostic factors were selected for the 12-month time point: Dmean-i, education, Dmean-c, smoking, T stage, baseline xerostomia, alcohol abuse, family history, and node classification. In the selection of the suboptimal number of prognostic factors by LASSO, three suboptimal prognostic factors were fine-tuned by Hosmer-Lemeshow test and AUC, i.e., Dmean-c, Dmean-i, and age for the 3-month time point. Five suboptimal prognostic factors were also selected for the 12-month time point, i.e., Dmean-i, education, Dmean-c, smoking, and T stage. The overall performance for both time points of the NTCP model in terms of scaled Brier score, Omnibus, and Nagelkerke R(2) was satisfactory and corresponded well with the expected values. CONCLUSIONS: Multivariate NTCP models with LASSO can be used to predict patient-rated xerostomia after IMRT.

Treatment of nasopharyngeal carcinoma by tomotherapy: five-year experience.

PURPOSE: To analyze of survival curve and toxicity outcomes for patients treated for nasopharyngeal carcinoma (NPC) by intensity-modulated radiotherapy (IMRT) delivered by helical TomoTherapy (HT). MATERIALS AND METHODS: Since May 2006, 72 patients with primary NPC were treated. In 67 cases PET-CT was used to help delineate the gross tumor volume (GTV); in 4 of these cases distant metastases in bone, mediastinal lymph nodes and unexpected small neck nodes were detected by high SUV uptake. 3, 22, 19, and 27 patients, respectively, had AJCC stage I to IV disease. Patients received a median total dose of 72 Gy to the GTV, 64.8 Gy to the elective PTV, and 54 Gy to the clinically negative neck region. RESULTS: At a median follow-up of 41 months (range 0.2 to 67 months), no patient has recurred locally. Two patients with stage IIb disease, both of whom received chemotherapy, recurred regionally. Ten patients developed distant metastases. One died from progressive disease with initial proved bony metastasis. Two patients with stage IIb disease, both of whom received chemotherapy, experienced neck node recurrence. 5-year locoregional control rate was 97%; freedom from distant metastases was 84.6% at 5 years. No evidence of disease was detected in 13 early stage (I/IIa/IIb) patients who did not receive chemotherapy. Acute grade 3 toxicity occurred in four patients and grade 4 in two patients. Late toxicities were low, with no grade 3+ xerostomia, grade 2 xerostomia in two patients (3%), and grade 3 hearing loss in two patients (3%). CONCLUSIONS: HT resulted in excellent long-term disease control and survival in heterogeneous NPC patients. Generally mild acute and late toxicity, with low rates of xerostomia, were obtained. Image-guided HT offers the ability to deliver conformal, OAR-sparing dose distributions to a wide variety of NPC patients with good long-term clinical outcomes.

Six-year clinical follow-up after treatment of diffuse in-stent restenosis with cutting balloon angioplasty followed by intracoronary brachytherapy with liquid rhenium-188-filled balloon via transradial approach.

BACKGROUND: Long-term follow-up studies revealed a significant decline in the benefits of intracoronary radiation for in-stent restenosis. METHODS AND RESULTS: A total of 25 study and 25 contemporaneous control patients with diffuse in-stent restenosis who underwent cutting balloon angioplasty (CBA) transradially, followed by subsequent intracoronary irradiation with a liquid ß-emitter Rhenium-188 (¹88Re)-filled balloon were enrolled in the study. The mean clinical follow-up durations were 64.9 ± 13.0 and 66.3 ± 13.8 months for the irradiated and control patients, respectively. Six-month angiographic restenosis was observed in 16% (4 of 25) of the patients in the irradiated group and 48% (12 of 25) of the patients in the control groups (P = 0.03). The 6-month major adverse cardiac events (MACE) rate was 12% and 44%, respectively (P = 0.025). The 3-year follow-up angiography was performed in 16 of 21 (76%) irradiated patients and in 4 of 13 (31%) control patients who had no significant restenosis at the 6-month angiographic follow-up. Restenosis occurred in 1 of 16 (7%) irradiated patients and 2 of 4 (50%) control patients. Late target lesion revascularization was performed in 1 irradiated and 2 control patients. The MACE rate within 6 years was significantly reduced in the irradiated group (20% vs. 56%, P = 0.019). CONCLUSIONS: Brachytherapy using ¹88Re-filled balloon following CBA for diffuse in-stent restenotic native coronary arteries is effective in reducing target lesion restenosis and improving long-term outcomes.

Increased expression of nuclear NF-kappaB after coronary artery balloon injury can be inhibited by intracoronary beta-irradiation.

PURPOSE: Molecular mechanisms by which balloon angioplasty injury-induced neointimal hyperplasia can be reduced by intravascular brachytherapy are unclear. We investigated the role of nuclear factor-kappaB (NF-kappaB) in neointimal hyperplasia following intracoronary irradiation. MATERIALS AND METHODS: Fifty-four coronary arteries from 30 pigs were divided into 6 groups: sham control, balloon angioplasty injury alone, beta-irradiation at doses of 14 or 20 Gy, and 14 or 20 Gy beta-irradiation immediately followed by balloon injury. Coronary arteries were injured by overstretch balloon angioplasty and then the arteries were irradiated using a Rhenium-188 ((188)Re) beta-emitting solution-filled balloon. Pigs were scarified one day or one week after coronary interventions for molecular detection and six weeks after the procedures for histological examination. RESULTS: Six weeks after coronary interventions, the histological results show that balloon angioplasty injury had induced intimal hyperplasia in coronary artery but the response was significantly reduced by 28% and 60% when the injury was immediately treated by 14 and 20 Gy (188)Re beta-irradiation, respectively. The expression of arterial NF-kappaB p65, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) were detected at one day and one week after the procedures. The treatment of balloon injury could significantly induce the NF-kappaB p65 expression in both gene and protein levels, and such induction could be significantly reduced by (188)Re beta-irradiation at dose of 20 Gy. However, the similar result on the regulation of gene expression affected by the beta-irradiation could not be observed in ICAM-1 and VCAM-1. CONCLUSION: The inhibitory effect of intracoronary brachytherapy on neointimal formation following overstretch balloon angioplasty could involve inhibition of NF-kappaB p65.

Incidence of needle tract seeding and responses of soft tissue metastasis by hepatocellular carcinoma postradiotherapy.

AIM: To determine the incidence of needle tract seeding after fine needle aspiration (FNA) or percutaneous ethanol injection (PEI) and compare iatrogenic or spontaneous soft tissue metastasis (STM) by hepatocellular carcinoma (HCC) postradiotherapy (RT) in responses. METHODS: From November 1997 to January 2006, those who presented with STM by HCC after our invasive procedures or developed spontaneously were enrolled into this retrospective study. Metastatic lesions could be divided into procedure related (PR), which were located at the liver span and were related to invasive procedures, and non-procedure related (NPR), which were in extrahepatic areas. STM was treated with an electron or photon beam. RESULTS: A total of 39 HCC cases with developed STM were referred for RT, including 17 in the PR group and 22 in the NPR group. During the same period, a total of 18,227 person-times of FNA or PEI were performed on these HCC patients. The overall incidence of HCC with STM that was caused by invasive procedures was estimated at 0.13%. According to the Cox' regression model, the initial treatment modality influences the time duration after the initial diagnosis of HCC when STM has not occurred. None of these patients' soft tissue tumor increased in size during RT. The PR group had lower rates of bone metastasis (P=0.003) and coexisting extrahepatic metastasis (P=0.011) and a longer survival rate (P=0.003) than the NPR group. The estimated rates of 18-gauge and 22-gauge needle-induced HCC-related STM were 0.60% and 0.11%, respectively (P=0.064). CONCLUSION: The PR group bears a better prognosis than the NPR group post-RT.

Computer tomography-assisted three-dimensional technique to assess rectal and bladder wall dose in intracavitary brachytherapy for uterine cervical cancer.

We used a computer tomography (CT)-assisted three-dimensional (3D) technique to assess dose to the rectum and bladder in intracavitary brachytherapy (ICBT) for patients with cervical cancer, and compared this technique with the conventional method. The results revealed that the difference in dose to the rectal and bladder wall between these two methods were significant. The CT-assisted technique is a feasible method, and it gives different results than the conventional method.

Clinical comparison of two linear-quadratic model-based isoeffect fractionation schemes of high-dose-rate intracavitary brachytherapy for cervical cancer.

PURPOSE: Two linear-quadratic model-based isoeffect fractionation schemes of high-dose-rate intracavitary brachytherapy (HDR-IC) were used to treat cervical cancer in two consecutive periods. Patient outcomes and complications were analyzed and compared. METHODS AND MATERIALS: Between November 1987 and December 1996, a total of 541 women diagnosed with cervical cancer were treated with curative-intent radiotherapy. Patients were categorized into two groups according to the two isoeffect schemes used. Group 1 consisted of 254 patients treated with external beam radiotherapy (EBRT) plus 7.2 Gy HDR-IC to Point A for three fractions in the first period. Group 2 consisted of 284 patients treated with EBRT plus 4.8 Gy HDR-IC for five fractions in the second period. The goal of the new scheme for the latter group was to deliver an isoeffect dose that maintained similar tumor control but reduced normal tissue complications. The calculated biologically effective dose (BED(10), assuming an alpha/beta ratio = 10) of EBRT plus HDR-IC for tumor and acute responding tissue in Groups 1 and 2 was 90 Gy(10) (52.8 + 37.2 Gy) and 88.6 Gy(10) (53.1 + 35.5 Gy), respectively. The corresponding BED(3) for late responding tissue (assuming an alpha/beta ratio = 3) in Groups 1 and 2 was 146.7 Gy(3) (73.3 + 73.4 Gy) and 134.4 Gy(3) (72 + 62.4 Gy), respectively. Patients were followed for 6.1-15.2 years (median, 9.8 years). RESULTS: Overall, 66 patients (12.2%) developed pelvic recurrence. Of these, 53 patients had central recurrence. Of the 53 patients with central recurrence, 24 (9.4%) were in Group 1 and 29 (10.1%) in Group 2 (p = 0.722). The actuarial pelvic control rate for Groups 1 and 2 was 88.2% and 86.3% at 5 years and 87.3% and 85.5% at 10 years, respectively (p = 0.504). The actuarial overall survival rate for Groups 1 and 2 was 63.5% and 56.1% at 5 years and 47.8% and 49.3% at 10 years, respectively (p = 0.734). The actuarial proctitis rate for Groups 1 and 2 was 49.7% and 32.7% at 5 years and 50.5% and 32.7% at 10 years, respectively (p <0.001). Most of the decrease in the rate of proctitis was a result of a decrease in the incidence of low-grade proctitis (38% vs. 22%). The incidence of high-grade complications remained unchanged, 8% vs. 7%. The actuarial cystitis rate for Groups 1 and 2 was 14.3% vs. 11.4% at 5 years and 24.1% vs. 15% at 10 years, respectively (p = 0.134). Multivariate analysis revealed that the fractionation scheme (three fractions vs. five fractions) was a significant factor influencing the proctitis rate (p = 0.004, hazard ratio = 0.807; 95% confidence interval, 0.697-0.934), but not the local pelvic control rate, overall survival rate, or cystitis rate. CONCLUSION: The treatment results of the two groups maintained similar outcomes, while the complications decreased. The linear-quadratic model correctly predicted this outcome. Biologically, the manipulation of the fraction size in our study suggested that the sensitivity of the late responding tissue to the fractional change from 7.2 Gy to 4.8 Gy in HDR-IC is high and detectable clinically. The success, however, had its limitations, and the improvement was confined to low-grade complications.

Intracoronary beta-irradiation with liquid rhenium-188: results of the Taiwan radiation in prevention of post-pure balloon angioplasty restenosis study.

STUDY OBJECTIVE: To assess the feasibility and short-term outcome of intracoronary irradiation after pure balloon angioplasty (POBA) of de novo and post-POBA restenotic lesions with a liquid beta-emitter (188)Re-filled balloon. DESIGN AND SETTING: Nonrandomized prospective study with contemporaneous control group in a single medical center. PATIENTS AND METHODS: In the Taiwan Radiation in Prevention of Post-Pure Balloon Angioplasty Restenosis study, 40 patients underwent 14-Gy irradiation and 15 patients underwent 20-Gy irradiation at a tissue depth of 0.5 mm after POBA. Thirty control patients received a 5-min inflation with a perfusion balloon catheter after POBA. RESULTS: No procedural or in-hospital complications, or 30-day major adverse cardiac events were noted. Six-month angiographic restenosis rates were 49% in the 14-Gy group, 20% in the 20-Gy group, and 57% in the control group (p = 0.05, 20-Gy group vs control group). In the lesions with an arc of calcification of < 180 degrees, restenosis occurred in 15 of the 34 lesions (44%) in the 14-Gy group and in none of the 11 lesions (0%) in the 20-Gy group (p = 0.007). In a vessel with a reference diameter < 3.0 mm, restenosis occurred in 1 of the 8 lesions (13%) in the 20-Gy group, and in 8 of the 11 lesions (73%) in the control group (p = 0.02). In the post-POBA restenotic lesions, restenosis occurred in none of the six lesions (0%) in the 20-Gy group, and in five of the six lesions (83%) in the control group (p = 0.008). CONCLUSIONS: Post-POBA, catheter-based brachytherapy in nonstented native coronary artery with a (188)Re-filled balloon can effectively reduce target lesion restenosis with 20-Gy irradiation at a tissue depth of 0.5 mm and seems to be more effective in the treatment of lesions with an arc of calcification < 180 degrees, in a vessel with a reference diameter of < 3.0 mm, and in post-POBA restenotic lesions.

Intracoronary beta-irradiation with liquid rhenium-188 to prevent restenosis following pure balloon angioplasty: results from the TRIPPER-1 study.

BACKGROUND: Patients who receive percutaneous transluminal coronary angioplasty (PTCA) are often haunted by restenosis of the target vessel within 6 months. Intracoronary irradiation has been shown to alter the luminal narrowing response after balloon angioplasty. METHODS: The Taiwan Radiation in Prevention of Post-Pure Balloon Angioplasty Restenosis-I (TRIPPER-I) study evaluated the feasibility, safety, and 6-month angiographic restenosis with intracoronary irradiation after pure balloon angioplasty (POBA) of de novo and post-POBA restenotic lesions in native coronary arteries using a self-centering beta-emitter rhenium-188 (Re-188)-filled balloon. RESULTS: Forty patients received 14 Gy at a 0.5-mm tissue depth with a Re-188 solution-filled perfusion balloon catheter, and 25 control patients received 5-min inflation with a perfusion balloon catheter. There were no procedural complications or in-hospital or 30-day major adverse cardiac events. Six-month angiographic follow-up was performed on 39 Re-188 (97.5%) and 25 control patients (100%). The restenosis rate was 49% in the Re-188 and 56% in the control groups (p=0.62). The composite end-points of death, myocardial infarction, and target-vessel revascularization were 40% in the Re-188 group and 36% in the control group (p=0.80). CONCLUSIONS: Catheter-based radiotherapy after POBA of de novo and post-POBA restenotic lesions with a Re-188-filled balloon is feasible but was ineffective in reducing target lesion restenosis with a dose of 14 Gy delivered at a 0.5-mm tissue depth in this study.