Development of reporter genes for multimodality molecular imaging is highly important. In contrast to the conventional strategies which have focused on fusing several reporter genes together to serve as multimodal reporters, human tyrosinase (TYR) – the key enzyme in melanin production – was evaluated in this study as a stand-alone reporter gene for in vitro and in vivo photoacoustic imaging (PAI), magnetic resonance imaging (MRI) and positron emission tomography (PET). Human breast cancer cells MCF-7 transfected with a plasmid that encodes TYR (named as MCF-7-TYR) and non-transfected MCF-7 cells were used as positive and negative controls, respectively. Melanin targeted N-(2-(diethylamino)ethyl)-18F-5-fluoropicolinamide was used as a PET reporter probe. In vivo PAI/MRI/PET imaging studies showed that MCF-7-TYR tumors achieved significant higher signals and tumor-to-background contrasts than those of MCF-7 tumor. Our study demonstrates that TYR gene can be utilized as a multifunctional reporter gene for PAI/MRI/PET both in vitro and in vivo.
Poised RNA polymerase II is predominantly found at developmental control genes and is thought to allow their rapid and synchronous induction in response to extracellular signals. How the recruitment of poised RNA Pol II is regulated during development is not known. By isolating muscle tissue from Drosophila embryos at five stages of differentiation, we show that the recruitment of poised Pol II occurs at many genes de novo and this makes them permissive for future gene expression. When compared to other tissues, these changes are stage-specific and not tissue-specific. In contrast, Polycomb group repression is tissue-specific and in combination with Pol II (the balanced state) marks genes with highly dynamic expression. This suggests that poised Pol II is temporally regulated and is held in check in a tissue-specific fashion. We compare our data to mammalian embryonic stem cells and discuss a framework for predicting developmental programs based on chromatin state.
Axillary web syndrome is a cause of significant morbidity in the early postoperative period after axillary surgery.
A patient developed axillary web syndrome after secondary breast surgery and recovered in 3 weeks through physical therapy and using Aescuven Forte.
The pathogenesis of axillary web syndrome is not clear. It is reported that axillary surgery is the main cause. The presented case indicates that tissue injury might be an important cause of axillary web syndrome. Though axillary web syndrome is self-limiting, special physical therapy and Aescuven Forte can shorten the natural duration.
Secondary breast surgery could cause axillary web syndrome. Physical therapy and Aescuven Forte could shorten the duration of the self-limited morbidity.
Axillary web syndrome; Breast cancer; Sentinel lymph node biopsy; Thrombophlebitis
The use of quantum dots (QDs) in biomedical research has grown tremendously, yet successful examples of clinical applications are absent due to many clinical concerns. Here, we report on a new type of stable and biocompatible dendron-coated InP/ZnS core/shell QDs as a clinically translatable nanoprobe for molecular imaging applications. The QDs (QD710-Dendron) were demonstrated to hold several significant features: near-infrared (NIR) emission, high stability in biological media, suitable size with possible renal clearance and ability of extravasation. More importantly, a pilot mouse toxicity study confirmed that QD710-Dendron lacks significant toxicity at the doses tested. The acute tumor uptake of QD710-Dendron resulted in good contrast from the surrounding non-tumorous tissues, indicating the possibility of passive targeting of the QDs. The highly specific targeting of QD710-Dendron-RGD2 to integrin αvβ3–positive tumor cells resulted in high tumor uptake and long retention of the nanoprobe at tumor sites. In summary, QD710-Dendron and RGD modified nanoparticles demonstrate small size, high stability, biocompatibility, favorable in vivo pharmacokinetics, and successful tumor imaging properties. These features satisfy the requirements for clinical translation and should promote efforts to further investigate the possibility of using QD710-Dendron based nanoprobes in the clinical setting in the near future.
Nanoprobes; fluorescence; renal clearance; clinical translation; molecular imaging
Cancer metastasis is a highly coordinated and dynamic multistep process in which cancer cells interact with a variety of host cells. Morphological studies have documented the association of circulating tumor cells with host platelets. Tumor cell-induced platelet aggregation (TCIPA) contributes significantly to hematogenous metastasis; however, the molecular mechanisms involved in breast cancer TCIPA are poorly characterized. In this study, MCF-7 metastatic human breast cancer cells induced dose-dependent aggregation of washed platelets. Four major platelet activation pathways, glycoprotein (GP)-Ib-IX, GPIIb/IIIa, thromboxane (TX)-A2 and adenosine diphosphate (ADP) were activated during TCIPA and were inhibited by their respective inhibitors, 7E3, SZ-1, aspirin and apyrase. Pretreatment of platelets with 7E3, SZ-1 or apyrase significantly inhibited TCIPA, while pretreatment with aspirin had no effect. Moreover, combined pretreatment of platelets with 7E3, SZ-1 and apyrase significantly inhibited TCIPA, compared to single inhibitors. Combinations of antiplatelet drugs may represent a promising strategy to prevent cancer metastasis.
breast cancer; tumor cell-induced platelet aggregation; glycoprotein-Ib-IX; glycoprotein-IIb/IIIa; thromboxane A2; adenosine diphosphate
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Current therapies are insufficient, making HCC an intractable disease. Our previous studies confirmed that inhibition of protein phosphatase 2A (PP2A) may provide a promising therapeutic strategy for cancer. Unfortunately, constitutive expression of PP2A in normal tissues limits the application of PP2A inhibition. Thus, a HCC-specific gene delivery system should be developed. The α-fetoprotein (AFP) promoter is commonly used in HCC-specific gene therapy strategies; however, the utility of this approach is limited due to the weak activity of the AFP promoter. It has been shown that linking the AFP enhancer with the promoter of the non-tissue-specific, human housekeeping phosphoglycerate kinase (pgk) gene can generate a strong and HCC-selective promoter.
We constructed a HCC-specific gene therapy system to target PP2A using the AFP enhancer/pgk promoter, and evaluated the efficiency and specificity of this system both in vitro and in vivo.
AFP enhancer/pgk promoter-driven expression of the dominant negative form of the PP2A catalytic subunit α (DN-PP2Acα) exerted cytotoxic effects against an AFP-positive human hepatoma cell lines (HepG2 and Hep3B), but did not affect AFP-negative human hepatoma cells (SK-HEP-1) or normal human liver cells (L-02). Moreover, AFP enhancer/pgk promoter driven expression of DN-PP2Acα inhibited the growth of AFP-positive HepG2 tumors in nude mice bearing solid tumor xenografts, but did not affect AFP-negative SK-HEP-1 tumors.
The novel approach of AFP enhancer/pgk promoter-driven expression of DN-PP2Acα may provide a useful cancer gene therapy strategy to selectively target HCC.
Hepatocellular carcinoma; AFP; Pgk; PP2A
Early evaluation of cancer response to a therapeutic regimen can help increase the effectiveness of treatment schemes and, by enabling early termination of ineffective treatments, minimize toxicity, and reduce expenses. Biomarkers that provide early indication of tumor therapy response are urgently needed. Solid tumors require blood vessels for growth, and new anti-angiogenic agents can act by preventing the development of a suitable blood supply to sustain tumor growth. The purpose of this study is to develop a class of novel molecular imaging probes that will predict tumor early response to an anti-angiogenic regimen with the humanized VEGF antibody bevacizumab.
Using a bevacizumab sensitive LS174T colorectal cancer model and a 12-mer bacteriophage (phage)-display peptide library, a bevacizumab responsive peptide (BRP) was identified after six rounds of biopanning and tested in vitro and in vivo.
This 12-mer peptide was metabolically stable and had low toxicity to both endothelial cells and tumor cells. Near-infrared dye IRDye800-labeled BRP phage showed strong binding to bevacizumab treated tumors, but not to untreated control LS174T tumors. In addition, both IRDye800 and 18F-labeled BRP peptide had significantly higher uptake in tumors treated with bevacizumab than in controls treated with phosphate buffered saline (PBS). Ex vivo histopathology confirmed the specificity of the BRP peptide to bevacizumab-treated tumor vasculature.
In summary, a novel 12-mer peptide BRP selected usmg phage display techniques allowed noninvasive visualization of early responses to anti-angiogenic treatment. Suitably labeled BRP peptide may be potentially useful pre-clinically and clinically for monitoring treatment response.
Phage display; Angiogenesis; Therapy response; Bevacizumab; Molecular imaging
Radioiodine therapy has proven to be a safe and effective approach in the treatment of differentiated thyroid cancer. Similar treatment strategies have been exploited in nonthyroidal malignancies by transfecting hNIS gene into tumor cells or xenografts. However, rapid radioiodine efflux is often observed after radioiodine uptake, limiting the overall antitumor effects. In this study, we aimed at constructing multicistronic co-expression of hNIS and hTPO genes in tumor cells to enhance the radioiodine uptake and prolong the radioiodine retention. Driven by the cytomegalovirus promoter, hNIS and hTPO were simultaneously inserted into the expression cassette of adenoviral vector. An Ad5 viral vector (Ad-CMV-hTPO-T2A-hNIS) was assembled as a gene therapy vehicle by Gateway technology and 2A method. The co-expression of hNIS and hTPO genes was confirmed by a double-label immunofluorescence assay. The radioiodine (125I) uptake and efflux effects induced by co-expression of hNIS and hTPO genes were determined in transfected and non-transfected PC-3 cells. Significantly higher uptake (6.58 ± 0.56 fold, at 1 h post-incubation) and prolonged retention (5.47 ± 0.36 fold, at 1 h of cell efflux) of radioiodine (125I) were observed in hNIS and hTPO co-expressed PC-3 cells as compared to non-transfected PC-3 cells. We concluded that the new virus vector displayed favorable radioiodine uptake and retention properties in hNIS-hTPO transfected PC-3 cells. Our study will provide valuable information on improving the efficacy of hNIS-hTPO co-mediated radioiodine gene therapy.
Gene therapy; prostate cance; hNIS; hTPO; gateway cloning system
Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [18F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers.
The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3′ end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence.
Minimally invasive video-assisted thyroidectomy (MIVAT), the modified Miccoli’s thyroid surgery, is the most widespread minimally invasive technique and has been widely used for treatment of thyroid disease. This study aimed to verify the potential benefits of the modified Miccoli’s thyroid surgery, determine the feasibility of the MIVAT for early-stage differential thyroid carcinoma and evaluate the likelihood of the surgical method as a standard operation for early malignant thyroid carcinoma.
A total of 135 patients were retrospectively compared which included two groups of patients: the first group underwent the conventional thyroidectomy; the other group underwent MIVAT. Patients with thyroid nodule smaller than 20 mm and without previous neck surgery were included while those with wide-ranging and distant metastases of cervical tissues, or any suspected thyroid nodal metastases were excluded for analysis. MIVAT and the central compartment (level VI) lymph nodes dissection (LND) were considered as a new treatment method for this retrospective study. In addition to the comparison of surgical outcomes between the new treatment and the conventional thyroid surgery, other surgical parameters including operative time, operative volume of hemorrhage, incisional length, postoperative volume of drainage, length of hospitalization, accidence of hoarse voice, accidence of bucking, accidence of hypocalcemia and peak angle of cervical axial rotation were also compared.
Out of 135 patients, 111 patients underwent conventional thyroid surgery and 24 patients underwent MIVAT plus level VI LND for treatment of early-stage differential malignant carcinoma. Patients who received the new surgical treatment had significantly shorter incisional length (3.1 cm vs. 6.9 cm, p < 0.0001), shorter operative time (109 min vs. 139 min, p = 0.014) and fewer operative hemorrhage (29.5 ml vs. 69.7 ml, p < 0.0001) when compared to the conventional treatment. Postoperative peak angle of cervical axial rotation of patients treated with MIVAT was less than those treated with conventional surgery (L: 31.5° vs. 39.0°, p < 0.0001; R: 31.5° vs. 38.0°, p < 0.0001). Incisional wound infection, postoperative hoarse voice, bucking and hypocalcemia were not observed in all patients. Postoperative analgetica was not required as well.
Compared with conventional thyroid surgery for early-stage differential thyroid carcinoma, the new surgical treatment could be considered as an alternative surgical method for treatment of early-stage thyroid carcinoma since it was feasible, safe and clinically effective with better surgical and cosmetic outcomes.
Plasma cell mastitis is distinct from the common form of mastitis and clinically resembles breast carcinoma. The lesion occurs in non-lactating young women, and the incidence rate is rising. Surgical resection is the main treatment, but cannot prevent recurrence of the disease. Disfigurement or removal of breast after the operations can cause marked physical and psychological distress. The etiology of plasma cell mastitis is unclear up till now. It is therefore necessary to investigate further the underlying immunological changes of the disease.
The lesions of plasma cell mastitis removed from patients through aseptic operation were mixed with normal saline into homogenate tube machine (homogenate tubes were disinfected and sterilized prior to treatment). The mixture was homogenized at medium speed and grinded in ultrasonic cell disruptor. The homogenate obtained was made into oil emulsion with Freund's adjuvant. Thirty female BALB/c mice (6 weeks after sexual maturity) were divided into five groups A-E: group A was blank control; group B was normal saline control; group C was inoculated with 0.02 ml water-in-oil emulsion; group D was inoculated with 0.04 ml water-in-oil emulsion; group E was complete Freund's adjuvant control.
Pathology results showed that mouse mammary gland acinar cells remained integral without any abnormal changes observed in control groups A and B. Experimental groups C and D showed dilation of mouse mammary ductal tissue with a large number of epithelial cells and debris in the lumen, and fibrosis around ducts accompanied by large duct cells, neutrophils, lymphocytes, and especially plasma cell infiltration. Pathological changes were observed in 3 (50%) mice and 5 (83.3%) mice in group C and D respectively. In group E, neutrophil infiltration in mammary gland was observed in 5 mice, but neither infiltration of plasma cells nor other abnormal pathological changes were observed.
The lesions of patient with plasma cell mastitis could make the female BALB/c mice experience the similar clinical and pathological manifestation. High-dose group can successfully establish a mouse model of plasma cell mastitis.
We hypothesized that imaging-based assessment of cellular proliferation in prostate cancer may improve tumor characterization. We therefore evaluated the biodistribution and effect of androgen on tumor uptake of the cellular proliferation imaging marker [18F]-2′-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) in xenograft mouse models of human prostate cancer. Castrated and noncastrated athymic male mice were implanted with androgen-independent PC3 and androgen-sensitive CWR22 human prostate cancer cells. Dynamic microPET/CT imaging was performed for 1h followed by 10 minute static scans at 2h and 3h. Animals were sacrificed after imaging for biodistribution studies and immunohistochemical staining of tumors for androgen receptor and Ki-67/MIB expression. 18F-FMAU uptake was significantly higher in all major organs of the castrated animals in comparison to noncastrated mice with the highest uptake in liver and the lowest uptake in muscle and bone. When compared to PC3 tumors, CWR22 xenografts showed significantly higher tumor-to-muscle (2.56±0.30 vs. 1.99±0.30, p=0.008) and tumor-to-liver uptake ratios (1.72±0.12 vs. 1.26±0.17, p=0.0003) in the noncastrated animal at 3h time point. Androgen receptor and Ki-67/MIB expressions were higher in CWR22 than PC3 xenografts. Our initial preclinical observations suggest that there may be an association between androgen signaling and thymidine metabolism and that 18F-FMAU PET may be useful in prostate tumor characterization.
18F-FMAU; Prostate; Cancer; Proliferation; microPET
Endothelial dysfunction contributes to the development of atherosclerosis in patients with diabetes mellitus, but the mechanisms of endothelial dysfunction in this setting are incompletely understood. Recent studies have shown altered mitochondrial dynamics in diabetes mellitus with increased mitochondrial fission and production of reactive oxygen species (ROS). We investigated the contribution of altered dynamics to endothelial dysfunction in diabetes.
Methods and Results
We observed mitochondrial fragmentation (P=0.002) and increased expression of fission-1 protein (Fis1, P<0.0001) in venous endothelial cells freshly isolated from patients with diabetes mellitus (n=10) compared to healthy controls (n=9). In cultured human aortic endothelial cells exposed to 30 mM glucose, we observed a similar loss of mitochondrial networks and increased expression of Fis1 and dynamin-related protein-1 (Drp1), proteins required for mitochondrial fission. Altered mitochondrial dynamics was associated with increased mitochondrial ROS production and a marked impairment of agonist-stimulated activation of endothelial nitric oxide synthase (eNOS) and cGMP production. Silencing Fis1 or DRP1 expression with siRNA blunted high glucose-induced alterations in mitochondrial networks, ROS production, eNOS activation, and cGMP production. An intracellular ROS scavenger provided no additional benefit, suggesting that increased mitochondrial fission may impair endothelial function via increased ROS.
These findings implicate increased mitochondrial fission as a contributing mechanism for endothelial dysfunction in diabetic states.
endothelium; mitochondria; mitochondrial dynamics; reactive oxygen species
The aggregation kinetics of silver nanoparticles (AgNPs) that were coated with two commonly used capping agents—citrate and polyvinylpyrrolidone (PVP)—were investigated. Time-resolved dynamic light scattering (DLS) was employed to measure the aggregation kinetics of the AgNPs over a range of monovalent and divalent electrolyte concentrations. The aggregation behavior of citrate-coated AgNPs in NaCl was in excellent agreement with the predictions based on Derjaguin–Landau–Verwey–Overbeek (DLVO) theory, and the Hamaker constant of citrate-coated AgNPs in aqueous solutions was derived to be 3.7 × 10-20 J. Divalent electrolytes were more efficient in destabilizing the citrate-coated AgNPs, as indicated by the considerably lower critical coagulation concentrations (2.1 mM CaCl2 and 2.7 mM MgCl2 vs. 47.6 mM NaCl). The PVP-coated AgNPs were significantly more stable than citrate-coated AgNPs in both NaCl and CaCl2, which is likely due to steric repulsion imparted by the large, non-charged polymers. The addition of humic acid resulted in the adsorption of the macromolecules on both citrate- and PVP-coated AgNPs. The adsorption of humic acid induced additional electrosteric repulsion that elevated the stability of both nanoparticles in suspensions containing NaCl or low concentrations of CaCl2. Conversely, enhanced aggregation occurred for both nanoparticles at high CaCl2 concentrations due to interparticle bridging by humic acid clusters.
We evaluated noninvasive positron emission tomography (PET) imaging for monitoring tumor response to the VEGFR-2 tyrosine kinase (TK) inhibitor ZD4190 during cancer therapy.
Orthotopic MDA-MB-435 tumor-bearing mice were treated with ZD4190 (100 mg/kg orally per day for three consecutive days). Tumor growth was monitored by caliper measurement. During the therapeutic period, longitudinal PET scans were acquired using 18F-FDG, 18F-FLT and 18F-FPPRGD2 as imaging tracers to evaluate tumor glucose metabolism, tumor cell proliferation, and angiogenesis, respectively. Imaging metrics were validated by immunohistochemical analysis of Ki67, GLUT-1, F4/80, CD31, murine integrin β3, and human integrin αvβ3.
Three consecutive daily oral administrations of 100 mg/kg of ZD4190 were effective in delaying MDA-MB-435 tumor growth. A significant difference in tumor volume was observed on day 7 between the treatment group and the control group (p<0.01). After the final treatment, tumor growth resumed after a short delay. In the control tumors, 18F-FPPRGD2 uptake was stable between days 0 and 7. In ZD4190-treated tumors, 18F-FPPRGD2 uptake had decreased significantly relative to baseline by 26.74±8.12% (p<0.05) on day 1 and by 41.19±6.63% (p<0.01) on day 3, then had returned to baseline on day 7. Tumor uptake of 18F-FLT had also decreased on both day 1 and day 3 after initiation of ZD4190 treatment. No significant change in 18F-FDG uptake in ZD4190-treated tumors was observed, however, compared with the control group. All of the imaging findings were supported by ex vivo analysis of related biomarkers.
The longitudinal imaging results demonstrated the usefulness of quantitative 18F-FLT and 18F-FPPRGD2 PET imaging in evaluating the early antiproliferative and antiangiogenic effects of ZD4190. The quantification data from the PET imaging were consistent with the pattern of initial growth inhibition with treatment, followed by tumor relapse after treatment cessation.
Positron emission tomography (PET); Antiangiogenic treatment; 18F-FDG; 18F-FLT; 18F-FPPRGD2; Integrin; Therapy response; Tyrosine kinase inhibitor
Much recent research effort in traumatic brain injury (TBI) has been devoted to the discovery of a reliable biomarker correlating with severity of injury. Currently, no consensus has been reached regarding a representative marker for traumatic brain injury. In this study, we explored the potential of epithelial/endothelial tyrosine kinase (Etk) as a novel marker for TBI.
TBI was induced in Sprague Dawley (SD) rats by controlled cortical impact. Brain tissue samples were analyzed by Western blot, Q-PCR, and immunofluorescence staining using various markers including glial fibrillary acidic protein, and epithelial/endothelial tyrosine kinase (Etk). Results show increased Etk expression with increased number and severity of impacts. Expression increased 2.36 to 7-fold relative to trauma severity. Significant upregulation of Etk appeared at 1 hour after injury. The expression level of Etk was inversely correlated with distance from injury site. Etk and trauma/inflammation related markers increased post-TBI, while other tyrosine kinases did not.
The observed correlation between Etk level and the number of impacts, the severity of impact, and the time course after impact, as well as its inverse correlation with distance away from injury site, support the potential of Etk as a possible indicator of trauma severity.
DNA mimic proteins have evolved to control DNA-binding proteins by competing with the target DNA for binding to the protein. The Ocr protein of bacteriophage T7 is the most studied DNA mimic and functions to block the DNA-binding groove of Type I DNA restriction/modification enzymes. This binding prevents the enzyme from cleaving invading phage DNA. Each 116 amino acid monomer of the Ocr dimer has an unusual amino acid composition with 34 negatively charged side chains but only 6 positively charged side chains. Extensive mutagenesis of the charges of Ocr revealed a regression of Ocr activity from wild-type activity to partial activity then to variants inactive in antirestriction but deleterious for cell viability and lastly to totally inactive variants with no deleterious effect on cell viability. Throughout the mutagenesis the Ocr mutant proteins retained their folding. Our results show that the extreme bias in charged amino acids is not necessary for antirestriction activity but that less charged variants can affect cell viability by leading to restriction proficient but modification deficient cell phenotypes.
20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor involved in vascular dysfunction and blood pressure regulation. Studies have revealed strong associations between 20-HETE and endothelial dysfunction however the signalling mechanisms are largely unknown. Therefore we sought to investigate the effect of 20-HETE on endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (Hsp90) association.20-HETE significantly enhanced the constriction and inhibited the relaxation of mouse aortic rings in response to phenylephrine and acetylcholine, respectively (p=0.05 versus control ring). In mice with chronic AMP-activated protein kinase (AMPK) activation this protected against the negative effects of 20-HETE (p<0.05). Immunoprecipitation of eNOS in cells treated with 20-HETE revealed a decrease in basal and vascular endothelial growth factor (VEGF) stimulated Hsp90 association with eNOS (p<0.05). Pre-treatment of the cells with AICAR (a chronic activator of AMPK) prevented the loss of Hsp90 association with eNOS following 20-HETE treatment. Treatment with 20-HETE for 24h induces an increase in eNOS phosphorylation, not observed following acute treatment (30mins). This was accompanied by transient changes in Akt phosphorylation.20-HETE impairs eNOS-Hsp90 association which can be reversed via chronic activation of AMPK. This provides a mechanism for reduced NO bioactivity and endothelial dysfunction in diseases with elevated 20-HETE levels, such as hypertension.
20-HETE; endothelial dysfunction; eNOS-Hsp90 association; AMPK
In premenopausal women, endocrine adjuvant therapy for breast cancer primarily consists of tamoxifen alone or with ovarian suppressive strategies. Toremifene is a chlorinated derivative of tamoxifen, but with a superior risk-benefit profile. In this retrospective study, we sought to establish the role of toremifene as an endocrine therapy for premenopausal patients with estrogen and/or progesterone receptor positive breast cancer besides tamoxifen.
Patients with early invasive breast cancer were selected from the breast tumor registries at the Sun Yat-Sen Memorial Hospital (China). Premenopausal patients with endocrine responsive breast cancer who underwent standard therapy and adjuvant therapy with toremifene or tamoxifen were considered eligible. Patients with breast sarcoma, carcinosarcoma, concurrent contralateral primary breast cancer, or with distant metastases at diagnosis, or those who had not undergone surgery and endocrine therapy were ineligible. Overall survival and recurrence-free survival were the primary outcomes measured. Toxicity data was also collected and compared between the two groups.
Of the 810 patients reviewed, 452 patients were analyzed in the study: 240 received tamoxifen and 212 received toremifene. The median and mean follow up times were 50.8 and 57.3 months, respectively. Toremifene and tamoxifen yielded similar overall survival values, with 5-year overall survival rates of 100% and 98.4%, respectively (p = 0.087). However, recurrence-free survival was significantly better in the toremifene group than in the tamoxifen group (p = 0.022). Multivariate analysis showed that recurrence-free survival improved independently with toremifene (HR = 0.385, 95% CI = 0.154-0.961; p = 0.041). Toxicity was similar in the two treatment groups with no women experiencing severe complications, other than hot flashes, which was more frequent in the toremifene patients (p = 0.049). No patients developed endometrial cancer.
Toremifene may be a valid and safe alternative to tamoxifen in premenopausal women with endocrine-responsive breast cancer.
Tamoxifen; Toremifene; Breast cancer; Adjuvant endocrine therapy; Premenopausal
This article reports the affibody-based nanoprobes specifically target and image human epidermal growth factor receptor type 2 (HER2)-expressing cells and tumors. The simple, robust, and precise structure of affibody molecules are a promising class of targeting ligands with high affinity. Using near-infrared (NIR) quantum dots (QDs) and iron oxide (IO) nanoparticles as two representative nanomaterials, we designed anti–HER2 affibody molecules with an N-terminus cysteine residue (Cysteine-ZHER2:342) and precisely conjugated with maleimide-functionalized nanoparticles to make nanoparticle-affibody conjugates. The in vitro and in vivo study showed the conjugates are highly specific to target and image HER2-expressing cells and tumors. This work indicated the nanoparticle-affibody conjugates may be excellent candidates as targeting probes for molecular imaging and diagnosis.
Affibody; bioconjugation; nanoprobes; HER2; molecular imaging
Campylobacter jejuni is a common cause of pediatric diarrhea worldwide. Cytolethal distending toxin, produced by Campylobacter jejuni, is a putative virulence factor that induces cell cycle arrest and apoptosis in eukaryotic cells. Cellular cholesterol, a major component of lipid rafts, has a pivotal role in regulating signaling transduction and protein trafficking as well as pathogen internalization. In this study, we demonstrated that cell intoxication by Campylobacter jejuni cytolethal distending toxin is through the association of cytolethal distending toxin subunits and membrane cholesterol-rich microdomains. Cytolethal distending toxin subunits cofractionated with detergent-resistant membranes, while the distribution reduced upon the depletion of cholesterol, suggesting that cytolethal distending toxin subunits are associated with lipid rafts. The disruption of cholesterol using methyl-β-cyclodextrin not only reduced the binding activity of cytolethal distending toxin subunits on the cell membrane but also impaired their delivery and attenuated toxin-induced cell cycle arrest. Accordingly, cell intoxication by cytolethal distending toxin was restored by cholesterol replenishment. These findings suggest that membrane cholesterol plays a critical role in the Campylobacter jejuni cytolethal distending toxin-induced pathogenesis of host cells.
The effect of nanoparticle size (30–120 nm) on magnetic resonance imaging (MRI) of hepatic lesions in vivo has been systematically examined using polyvinylpyrrolidone (PVP)-coated iron oxide nanoparticles (PVP-IOs). Such biocompatible PVP-IOs with different sizes were synthesized by a simple one-pot pyrolysis method. These PVP-IOs exhibited good crystallinity and high T2 relaxivities, and the relaxivity increased with the size of the magnetic nanoparticles. It was found that cellular uptake changed with both size and surface physiochemical properties, and that PVP-IO-37 with a core size of 37 nm and hydrodynamic particle size of 100 nm exhibited higher cellular uptake rate and greater distribution than other PVP-IOs and Feridex. We systematically investigated the effect of nanoparticle size on MRI of normal liver and hepatic lesions in vivo. The physical and chemical properties of the nanoparticles influenced their pharmacokinetic behavior, which ultimately determined their ability to accumulate in the liver. The contrast enhancement of PVP-IOs within the liver was highly dependent on the overall size of the nanoparticles, and the 100 nm PVP-IO-37 nanoparticles exhibited the greatest enhancement. These results will have implications in designing engineered nanoparticles that are optimized as MR contrast agents or for use in therapeutics.
magnetic nanoparticles; iron oxide; magnetic resonance imaging; size dependent
Horizontal transfer of the chlorothalonil hydrolytic dehalogenase gene (chd) is proposed based on the high conservation of the chd gene and its close association with a novel insertion sequence, ISOcsp1, in 16 isolated chlorothalonil-dechlorinating strains belonging to eight different genera. The ecological role of horizontal gene transfer is assumed to facilitate bacterial adaptation to chlorothalonil-contaminated sites, through detoxification of chlorothalonil to less toxic 2,4,5-trichloro-6-hydroxybenzene-1,3-dicarbonitrile.
Osteosarcoma represents the most common malignant primary bone tumor in childhood; however, the survival rate has remained unchanged for past 20 years. To improve existing diagnosis and treatment methods and broaden the spectrum of imaging agents that can be used for early detection and assessment of tumor response to therapy, we performed a phage display-based screening for peptide sequences that bind specifically to osteosarcoma cells.
From the Ph.D.™ −12 phage display peptide library comprising 2.7 ×109 different displayed peptides, one peptide was enriched after 4 rounds of in vitro selection on 143B osteosarcoma tumor cells with 293T human embryonic kidney cells as a control. Both the peptide and the phage clone displaying the peptide were conjugated with fluorescent dyes for in vitro cell and ex vivo tumor tissue staining. The peptide was further labeled with 18F for positron emission tomography (PET) imaging studies. Cell uptake and efflux and ex vivo biodistribution were also performed with 18F labeled osteosarcoma specific peptide.
ASGALSPSRLDT was the dominant sequence isolated from biopanning and named as OSP-1. OSP-1 shares a significant homology with heparinase II/III family protein, which binds and reacts with heparan sulfate proteoglycans (HSPGs). The fluorescence staining showed that FITC-OSP-1-phage or Cy5.5-OSP-1 had high binding with a panel of osteosarcoma cell lines, much less binding with UM-SCC1 human head and neck squamous cell carcinoma cells, and almost no binding with 293T cells; whereas the scrambled peptide OSP-S had virtually no binding to all the cell lines. 18F-OSP-1 had significantly higher accumulation in 143B tumor cells both in vitro and in vivo than 18F-OSP-S. 18F-OSP-1 also had higher uptake in 143B tumors than UM-SCC-1 tumors.
Our data suggest that OSP-1 peptide is osteosarcoma specific, and the binding site of OSP-1 might be related to heparan sulfate proteoglycans. Appropriately labeled OSP-1 peptide has the potential to serve as a novel probe for osteosarcoma imaging.
Osteosarcoma (OS); Phage display; Positron emission tomography (PET); Molecular imaging; Heparan sulphate proteoglycans (HSPGs)