Paradoxical Mitophagy Regulation by PINK1 and TUFm.

Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.

A study of skin marker alignment using different diamond-shaped light fields for prone breast external-beam radiation therapy.

For breast cancer patients treated in the prone position with tangential fields, a diamond-shaped light field (DSLF) can be used to align with corresponding skin markers for image-guided radiation therapy (IGRT). This study evaluates and compares the benefits of different DSLF setups. Seventy-one patients who underwent daily tangential kilovoltage (kV) IGRT were categorized retrospectively into four groups: (1) DSLF field size (FS) = 10 × 10 cm2 , gantry angle = 90° (right breast)/270° (left breast), with the same isocenter as treatment tangential beams; (2) same as group 1, except DSLF FS = 4 × 4 cm2 ; (3) DSLF FS = 4 × 4-6 × 8 cm2 , gantry angle = tangential treatment beam, off-isocenter so that the DSLF was at the approximate breast center; and (4) No-DSLF. We compared their total setup time (including any DSLF/marker-based alignment and IGRT) and relative kV-based couch shift corrections. For groups 1-3, DSLF-only dose distributions (excluding kV-based correction) were simulated by reversely shifting the couch positions from the computed tomography plans, which were assumed equivalent to the delivered dose when both DSLF and IGRT were used. For patient groups 1-4, the average daily setup time was 2.6, 2.5, 5.0, and 8.3 min, respectively. Their mean and standard deviations of daily kV-based couch shifts were 0.64 ± 0.4, 0.68 ± 0.3, 0.8 ± 0.6, and 1.0 ± 0.6 cm. The average target dose changes after excluding kV-IGRT for groups 1-3 were-0.2%, -0.1%, and +0.4%, respectively, whereas DSLF-1 was most efficient in sparing heart and chest wall, DSLF-2 had lowest lung Dmax ; and DSLF-3 maintained the highest target coverage at the cost of highest OAR dose. In general, the use of DSLF greatly reduces patient setup time and may result in smaller IGRT corrections. If IGRT is limited, different DSLF setups yield different target coverage and OAR dose sparing. Our findings will help DSLF setup optimization in the prone breast treatment setting.

Knowledge-based planning in robotic intracranial stereotactic radiosurgery treatments.

PURPOSE: To develop a knowledge-based planning (KBP) model that predicts dosimetric indices and facilitates planning in CyberKnife intracranial stereotactic radiosurgery/radiotherapy (SRS/SRT). METHODS: Forty CyberKnife SRS/SRT plans were retrospectively used to build a linear KBP model which correlated the equivalent radius of the PTV (req_PTV ) and the equivalent radius of volume that receives a set of prescription dose (req_Vi , where Vi  = V10% , V20% … V120% ). To evaluate the model's predictability, a fourfold cross-validation was performed for dosimetric indices such as gradient measure (GM) and brain V50% . The accuracy of the prediction was quantified by the mean and the standard deviation of the difference between planned and predicted values, (i.e., ΔGM = GMpred - GMclin and fractional ΔV50%  = (V50%pred - V50%clin )/V50%clin ) and a coefficient of determination, R2 . Then, the KBP model was incorporated into the planning for another 22 clinical cases. The training plans and the KBP test plans were compared in terms of the new conformity index (nCI) as well as the planning efficiency. RESULTS: Our KBP model showed desirable predictability. For the 40 training plans, the average prediction error from cross-validation was only 0.36 ± 0.06 mm for ΔGM, and 0.12 ± 0.08 for ΔV50% . The R2 for the linear fit between req_PTV and req_vi was 0.985 ± 0.019 for isodose volumes ranging from V10% to V120% ; particularly, R2  = 0.995 for V50% and R2  = 0.997 for V100% . Compared to the training plans, our KBP test plan nCI was improved from 1.31 ± 0.15 to 1.15 ± 0.08 (P < 0.0001). The efficient automatic generation of the optimization constraints by using our model requested no or little planner's intervention. CONCLUSION: We demonstrated a linear KBP based on PTV volumes that accurately predicts CyberKnife SRS/SRT planning dosimetric indices and greatly helps achieve superior plan quality and planning efficiency.

S-Nitrosocaptopril prevents cancer metastasis in vivo by creating the hostile bloodstream microenvironment against circulating tumor cells.

A perfect microenvironment facilitates the activated circulating tumor cells (CTCs) to spark the adhesion-invasion-extravasation metastatic cascade in their premetastatic niche. Platelet-CTC interaction contributes to the progression of tumor malignancy by protecting CTCs from shear stress and immunological assault, aiding CTCs entrapment in the capillary bed, enabling CTCs to successfully exit the bloodstream and enter the tissue, inducing epithelial-mesenchymal-like transition (EMT), and assisting in the establishment of metastatic foci. To prevent the cascade from sparking, we show that, the multifunctional S-nitrosocaptopril (CapNO) acts on both CTCs and platelets to interrupt platelet/CTCs interplay and adhesion to endothelium, thus inhibiting CTC-based pulmonary metastasis in vivo. The activated platelets cloak cancer HT29 cells, resulting in HT29-exhibiting platelet biomarkers CD61 and P-selectin positive. CapNO inhibits both sialyl Lewisx (Slex) expression on HT29 and ADP-induced activation of platelets through P-selectin- and GPIIb/IIIa-dependent mechanisms, confirmed by the corresponding antibody assay. CapNO inhibits platelet- or interleukin (IL)-1ß-mediated adhesion between HT29 and endothelial cells, and micrometastatic formation in the lungs of immunocompetent syngeneic mouse models. CapNO have also shown the effects of vasodilation, anticoagulation, inhibition of matrix metalloproteinase-2 (MMP2) expression on cancer cells, and inhibition of cell adhesion molecules (CAMs) expression on vascular endothelium. Due to a series of the beneficial effects of CapNO, CTCs remain exposed to the hostile bloodstream environment and are vulnerable to death induced by shear stress and immune elimination. This new discovery provides a basis for CapNO used for cancer metastatic chemoprevention, and might suggest regulation of the CTCs bloodstream microenvironment as a new avenue for cancer metastatic prevention.

Thirty-day rat toxicity study reveals reversible liver toxicity of mifepristone (RU486) and metapristone.

OBJECTIVE: Mifepristone (RU486) is an oral first-line contraceptive used by hundreds of millions of women, and recently it was tested for anticancer activity in both genders worldwide. We are developing metapristone (the N-monodemethyl RU486) as a potential metastasis chemopreventive. The present acute and 30-d subacute toxicity study aimed at examining and compared in parallel the potential toxicity of the two drugs. METHODS: The single-dose acute toxicity and 30-d subacute toxicity studies were conducted in mice and rats, respectively, by gavaging metapristone or mifepristone at various doses. Blood samples and organs were collected for blood chemistry, hematology and histology analyses. RESULTS: Oral mifepristone (3000 mg/kg) caused 30% and 40% death in female and male mice, respectively, within 15 h post-dosing. In comparison, the same dose of metapristone produced 30% acute death in males only. Thirty-day oral administration of the two drugs to rats (12.5, 50 and 200 mg/kg/day) caused reversible hepatotoxicity that only occurred at 200 mg/kg/day group, evidenced by the elevated liver enzyme activity and liver organ weight. CONCLUSION: The present study, for the first time, reveals reversible hepatotoxicity in rats caused by the 30-d consecutive administration at the high dose, and warns the potential hepatotoxicity caused by long-term administrations of high doses of mifepristone or metapristone in clinical trials but not by the acute single abortion doses.

Isolation and characterization of living circulating tumor cells in patients by immunomagnetic negative enrichment coupled with flow cytometry.

BACKGROUND: This study was aimed at establishing a sensitive and specific isolation, characterization, and enumeration method for living circulating tumor cells (CTCs) in patients with colorectal carcinoma. METHODS: Quantitative isolation and characterization of CTCs were performed through a combination of immunomagnetic negative enrichment and fluorescence-activated cell sorting. Isolated CTCs were identified by immunofluorescence staining. The viability and purity of the sorted cells were determined by flow cytometry. Blood samples spiked with HCT116 cells (range, 3-250 cells) were used to determine specificity, recovery, and sensitivity. The method was used to enumerate, characterize, and isolate living CTCs in 10 mL of blood from patients with colorectal carcinoma. RESULTS: The average recovery of HCT116 cells was 61% or more at each spiking level, and the correlation coefficient was 0.992. An analysis of samples from all 18 patients with colorectal carcinoma revealed that 94.4% were positive for CTCs with an average of 33 ± 24 CTCs per 10 mL of blood and with a diameter of 14 to 20 µm (vs 8-12 µm for lymphoma). All patients were CD47(+) , with only 4.3% to 61.2% being CD44(+) . The number of CTCs was well correlated with the patient TNM stage and could be detected in patients at an early cancer stage. The sorted cells could be recultured, and their viability was preserved. CONCLUSIONS: This method provides a novel technique for highly sensitive and specific detection and isolation of CTCs in patients with colorectal carcinoma. This method complements the existing approaches for the de novo functional identification of a wide variety of CTC types. It is likely to help in predicting a patient's disease progression and potentially in selecting the appropriate treatment.

The unique pharmacological characteristics of mifepristone (RU486): from terminating pregnancy to preventing cancer metastasis.

Mifepristone (RU486) is a born-for-woman molecule discovered three decades ago. Unlike those antihypertensive and antipsychotic pharmaceutical blockbusters, this abortifacient offers relatively low profit potential. Current understanding of mechanism of action of mifepristone and its on-going clinical trials are changing our views on the drug beyond its abortifacient scope. Here we briefly review its metabolism and pharmacokinetic properties including its unique enterohepatic circulation, its mechanisms of actions involving antiprogesterone and antiglucocorticoid, growth inhibition of various cancer cell lines, suppression of invasive and metastatic cancer potential, downregulation of Cdk2, Bcl-2, and NF-kappa B, interference of heterotypic cell adhesion to basement membrane, and cell migration. We comprehensively analyze recent results from preclinical and clinical studies using mifepristone as an anticancer drug for breast, meningioma, and gliomas tumors in the central nervous system, prostate cancer, ovarian and endometrial cancer, and gastric adenocarcinoma. Although mifepristone has more benefits for global public health than we originally thought, its effect as a postmetastatic chemotherapeutic agent is limited. Nonetheless, owing to its unique safe, metabolism and other pharmacological properties, metapristone (the primary metabolite of mifepristone) may have potential for cancer metastatic chemoprevention.

In utero diagnosis of long QT syndrome by magnetocardiography.

BACKGROUND: The electrophysiology of long QT syndrome (LQTS) in utero is virtually unstudied. Our goal here was to evaluate the efficacy of fetal magnetocardiography (fMCG) for diagnosis and prognosis of fetuses at risk of LQTS. METHODS AND RESULTS: We reviewed the pre/postnatal medical records of 30 fetuses referred for fMCG because of a family history of LQTS (n=17); neonatal/childhood sudden cardiac death (n=3), or presentation of prenatal LQTS rhythms (n=12): 2° atrioventricular block, ventricular tachycardia, heart rate < 3(rd) percentile. We evaluated heart rate and reactivity, cardiac time intervals, T-wave characteristics, and initiation/termination of Torsade de Pointes, and compared these with neonatal ECG findings. After birth, subjects were tested for LQTS mutations. Based on accepted clinical criteria, 21 subjects (70%; 9 KCNQ1, 5 KCNH2, 2 SCN5A, 2 other, 3 untested) had LQTS. Using a threshold of corrected QT= 490 ms, fMCG accurately identified LQTS fetuses with 89% (24/27) sensitivity and 89% (8/9) specificity in 36 sessions. Four fetuses (2 KCNH2 and 2 SCN5A), all with corrected QT ≥ 620 ms, had frequent episodes of Torsade de Pointes, which were present 22-79% of the time. Although some episodes initiated with a long-short sequence, most initiations showed QRS aberrancy and a notable lack of pause dependency. T-wave alternans was strongly associated with severe LQTS phenotype. CONCLUSIONS: Corrected QT prolongation (≥490 ms) assessed by fMCG accurately identified LQTS in utero; extreme corrected QT prolongation (≥620 ms) predicted Torsade de Pointes. FMCG can play a critical role in the diagnosis and management of fetuses at risk of LQTS.

Exosome proteomics study of the effects of traditional cigarettes and electronic cigarettes on human bronchial epithelial cells.

Electronic cigarette (e-Cig) has been promoted as a safer alternative to traditional cigarette (t-Cig) recently. However, there are limited scientific data on the potential health effects of e-Cig use. In this study, we evaluated the cytotoxicities of e-Cig and t-Cig condensate solutions (e-CigCS and t-CigCS) on human bronchial epithelial cells (16HBE cells) in vitro, and employed the exosome proteomic technique to systematically assess the effects of e-CigCS and t-CigCS on 16HBE cells. Cytotoxicity assay showed 16HBE cells were more sensitive to t-CigCS than e-CigCS. Proteomic analysis demonstrated that there are 431 differential expressed exosomal proteins (DEEPs) in test groups compared to the control air group (P-value<0.05) and t-CigCS has a greater influence than e-CigCS on exosomal protein expression. Bioinformatic analysis showed the DEEPs from the t-Cig group were significantly enriched in pathways in cancer while tobacco-flavored e-Cig (e-Cigt) and menthol-flavored e-Cig (e-Cigm) groups were not. Further validations of some DEEPs, such as NF-κB p65, Sulfiredoxin-1(SRXN1) and Thioredoxin-interacting protein (TXNIP), were carried out using immunoblot and Real-time PCR analysis, showing that t-Cig may have a greater influence than e-Cig on tumor development and metastasis. Taken together, the finding reported here strongly support our hypothesis that electronic cigarettes are significantly less toxic compared with traditional cigarette.

Hairpin-inserted cross-shaped DNA nanoprobe for ultrasensitive microRNA detection based on built-in target analogue cycle amplification.

It remains technically challenging to develop a sensitive assay system to isothermally amplify the signal for miRNA detection because of its low abundance in tested sample, sequence similarities and existence in complex biological environments. In this study, using miRNA-21 as target model, a hairpin-inserted cross-shaped DNA nanoprobe (CP) with four functional arms is constructed for the ultrasensitive detection of miRNA via one-step built-in target analogue (BTA) cycle-mediated signal amplification. BTA is pre-locked in one arm of CP probe and inactive. In the presence of target miRNA, BTA can be unlocked and initiate an isothermal amplification process. Utilizing as-designed CP probe, miRNA-21 can be detected to down to 500 fM, and the linear response range spans over five orders of magnitude. The nonspecific signal is less than 1% upon nontarget miRNAs. CP probe exhibits âˆ¼six times enhancement in resistance to nuclease degradation and no obvious degradation-induced fluorescence change is detected during the assay period. The recovery yield ranges from 98.2～105.5% in FBS solution. Because of the high sensitivity, desirable specificity, strong anti-interference ability and substantial increase in nuclease resistance, CP probe is a promising tool for the detection of miRNAs in a complex biological milieu.

Radiation Oncology: Future Vision for Quality Assurance and Data Management in Clinical Trials and Translational Science.

The future of radiation oncology is exceptionally strong as we are increasingly involved in nearly all oncology disease sites due to extraordinary advances in radiation oncology treatment management platforms and improvements in treatment execution. Due to our technology and consistent accuracy, compressed radiation oncology treatment strategies are becoming more commonplace secondary to our ability to successfully treat tumor targets with increased normal tissue avoidance. In many disease sites including the central nervous system, pulmonary parenchyma, liver, and other areas, our service is redefining the standards of care. Targeting of disease has improved due to advances in tumor imaging and application of integrated imaging datasets into sophisticated planning systems which can optimize volume driven plans created by talented personnel. Treatment times have significantly decreased due to volume driven arc therapy and positioning is secured by real time imaging and optical tracking. Normal tissue exclusion has permitted compressed treatment schedules making treatment more convenient for the patient. These changes require additional study to further optimize care. Because data exchange worldwide have evolved through digital platforms and prisms, images and radiation datasets worldwide can be shared/reviewed on a same day basis using established de-identification and anonymization methods. Data storage post-trial completion can co-exist with digital pathomic and radiomic information in a single database coupled with patient specific outcome information and serve to move our translational science forward with nimble query elements and artificial intelligence to ask better questions of the data we collect and collate. This will be important moving forward to validate our process improvements at an enterprise level and support our science. We have to be thorough and complete in our data acquisition processes, however if we remain disciplined in our data management plan, our field can grow further and become more successful generating new standards of care from validated datasets.

Peroxiredoxin IV regulates pro-inflammatory responses in large yellow croaker (Pseudosciaena crocea) and protects against bacterial challenge.

In this study, we applied a comparative proteomic approach to the analysis of differentially expressed proteins in the spleens of large yellow croaker following treatment with an inactivated trivalent bacterial vaccine. Twenty-four altered proteins were identified by MALDI-TOF or MALDI-TOF-TOF, including immune-related proteins, antioxidant proteins, signal transducers, protein biosynthesis and catabolism modulators, and carbonic anhydrases. Three Prx family members, namely, Prx I, Prx II, and Prx IV, were upregulated after treatment with the vaccine, indicating potentially important roles for these antioxidant proteins in the antibacterial immune response. Large yellow croaker Prx IV (LycPrxIV), which has thiol-dependent peroxidase activity, was constitutively expressed in all tissues examined. Immunoelectron microscopy showed that LycPrxIV was primarily localized to the rER or peroxisome in spleen cells of healthy fish, and its synthesis on the rER increased following treatment with bacterial vaccine. Suppression of LycPrxIV by siRNA resulted in an increase in NF-kappaB activity in spleen tissues, while in vivo administration of recombinant LycPrxIV (rLycPrxIV) caused a decrease in NF-kappaB activity, indicating that LycPrxIV negatively regulates NF-kappaB activation. Likewise, siRNA-mediated knockdown of LycPrxIV increased the expression of TNF-alpha and CC chemokine, and downregulated the expression of IL-10. However, injection of fish with rLycPrxIV induced the opposite expression pattern of these cytokines, suggesting a role for LycPrxIV in regulating pro-inflammatory responses. Bacterial challenge experiments showed that suppression of LycPrxIV expression by siRNA significantly increased fish mortality as compared to controls, whereas rLycPrxIV provided a protective effect. Together, our data suggest that LycPrxIV may regulate pro-inflammatory responses to protect large yellow croaker from bacterial challenge, revealing a novel antibacterial mechanism in fish.

Beta2-microglobulin is involved in the immune response of large yellow croaker to Aeromonas hydrophila: a proteomic based study.

Outbreaks of infectious diseases in cultured large yellow croaker have resulted in great economic losses. However, information regarding its immune defense is limited. In the present study, an approach by the combination of differential proteomics with EST resource was applied for investigation of a profile of serum immune response by large yellow croaker to Aeromonas hydrophila challenge after immunization and for characterizing of one of the targeted immune molecules. Of the twelve altered proteins involved in the response, eight were identified by MS, in which three were randomly selected for antiserum preparation and were further confirmed by Western blotting. Furthermore, three beta(2)m clones, one of the altered molecules, were obtained from a previously constructed Kidney Smart cDNA library of this fish, and were compared for their identity, which contributed to the identification of beta(2)m cDNA diversity. Meanwhile, the up-regulated beta(2)m in response to the bacterial immunization and challenge was further confirmed by Western blotting. Our results indicate that beta(2)m is involved in the immune response of large yellow croaker to the challenge by A. hydrophila after immunization, which suggests an efficient approach for characterizing of targeted molecules at both the gene and protein levels.

Molecular characterization and expression analysis of MHC class II alpha and beta genes in large yellow croaker (Pseudosciaena crocea).

MHC class II molecules play an important role in the activation of CD4(+) T cells, which are the central orchestrating cells of an immune response. Here, we report the cloning of MHC class II alpha and beta cDNAs from large yellow croaker (Pscr-DAAs and Pscr-DAB) by expressed sequence tags analysis and RACE-PCR techniques. Three different class II alpha and two class II beta sequences were obtained from spleens of two individual fish. Each of the three class II alpha sequences encodes a polypeptide of 239 amino acids while the two class II beta cDNA sequences encode for a protein of 249 aa. All the characteristic features of MHC class II chain structure could be identified in the deduced proteins of three class II alpha and two class II beta sequences, including the leader peptide, alpha1/beta1 and alpha2/beta2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. RT-PCR analysis showed that MHC class II alpha and beta mRNAs were broadly expressed in various tissues examined, although at different levels. Upon stimulation with inactivated trivalent bacterial vaccine or polyinosinic polycytidylic acid (poly(I:C)), the expression levels of both alpha and beta genes were obviously up-regulated in intestine, kidney and spleen. Real-time PCR analysis demonstrated that the expression levels of class II alpha and beta were quickly up-regulated in spleen, kidney, and intestine at 12 h after induction with poly(I:C), while their expression levels significantly increased at 48 h upon immunization with bacterial vaccine, indicating that the up-regulation of both class II alpha and beta expression was induced by bacterial vaccine or poly(I:C) at the early phase of induction, and that class II alpha and beta transcripts were quicker up-regulated by poly I:C than by bacterial vaccine.

Dosimetric and planning efficiency comparison for lung SBRT: CyberKnife vs VMAT vs knowledge-based VMAT.

This is the first study that compared treatment plan quality and planning efficiency for lung stereotactic body radiation therapy (SBRT) using CyberKnife (CK) Multiplan vs Varian Eclipse treatment planning systems, including volumetric modulated arc therapy (VMAT) and knowledge-based VMAT (KBP-VMAT). Thirteen lung SBRT patients treated with 50 to 55 Gy in 3 or 5 fractions were retrospectively included in this study. CK plans created with Multiplan V. 4.6.1 using 2 fixed circular cones were previously approved used for treatment. For the comparison, the computed tomography (CT) data sets and contours from the CK plans were used to generate VMAT and KBP-VMAT plans (University of California San Diego publicly-shared RapidPlan model) using Eclipse V. 13.7. Metrics used for the comparison of CK, VMAT, and KBP-VMAT plans included monitor units (MUs), conformity indices, dose heterogeneity, high-dose spillage, low-dose spillage, adjacent organs at risk (OAR) doses, and treatment planning time. One-way analysis of variance with post-hoc Tukey tests and paired t-tests were used to analyze the difference of these metrics corresponding to the different planning techniques. All of the 3 planning techniques achieved our clinical goals. With similar planning target volume (PTV) coverage, CK plans yielded the most MU (p< 0.001), the least dose homogeneity (p < 0.002), and the least D2cm dose (p < 0.001), while KBP-VMAT plans resulted in the most OAR sparing. No significant difference was found among other dosimetric metrics such as high-dose spillage, lung V20 and volume receiving 50% of the prescription dose. Compared to VMAT, KBP-VMAT improved OAR sparing (p < 0.05), but required significantly more MU (p < 0.001). KBP-VMAT was associated with the shortest planning time. Eclipse-based VMAT can achieve comparable plan quality for lung SBRT as CK, in a more efficient manner. RapidPlan can facilitate the planning process of KBP-VMAT, with potentially better OAR sparing but higher MU requirements. Further improvement for KBP-VMAT is likely achievable by developing site-specific patient models.

Palindromic probe-mediated strand displacement amplification for highly sensitive and selective microRNA imaging.

MicroRNAs (miRNAs) are involved in a variety of biological processes, and the accurate detection of miRNAs is of great importance for early diagnosis of various cancers. Herein, we have developed a highly sensitive method for the intracellular imaging of miRNAs based on a palindromic probe-induced strand displacement amplification (pSDA). The sensing element is a partly complementary hybrid consisting of two DNA components: one fluorescent dye-labeled signaling probe containing a palindromic sequence and loop-based target recognition site and one quencher moiety-attached locking probe. In the presence of target miRNA, the target species can hybridize with the loop site and release the terminal palindromic fragment, initiating the pSDA reaction. Thus, a considerable amount of fluorescent moieties are spatially separated from the quenchers, generating a dramatically enhanced fluorescence signal. As a result, the target miRNAs can be quantified down to 25 pM with the linear response range over four orders of magnitude. The detection specificity is high enough to eliminate the interference from nontarget miRNAs and other biospecies co-existing in samples, and thus the diseased cells are easily distinguished from healthy cells. Strikingly, the pSDA-based system possesses the desirable capability to discriminate tumor cells from healthy cells, indicating a promising diagnostic tool for the detection of cancers and other diseases in early stage.

Molecular characterization and expression analysis of beta2-microglobulin in large yellow croaker Pseudosciaena crocea.

Beta(2)-microglobulin (beta(2)m), a protein necessary for proper folding, peptide binding, and surface display of class I antigens plays an important role in immune response. The full-length cDNA containing beta(2)m was cloned from the spleen cDNA library of large yellow croaker Pseudosciaena crocea (Pscr-beta ( 2 ) m) by expressed sequence tag (EST) analysis. The Pscr-beta ( 2 ) m is 926 nucleotides (nt) long, including an open reading frame (ORF) of 348 nt encoding a polypeptide of 116 amino acids (aa). The deduced Pscr-beta ( 2 ) m possessed all characteristic domains of beta(2)m in other species, including a 16-aa leader peptide and a typical immunoglobulin (Ig) and major histocompatibility complex protein (MHC) signature YSCRVTH at residues 81-87. Homology modeling showed that the 3D structure of Pscr-beta ( 2 ) m protein is similar to that of human beta(2)m, except for a beta-strand (G) being lost in Pscr-beta ( 2 ) m due to amino acid deletion (positions 94-95). Tissue expression profile analysis revealed that the Pscr-beta ( 2 ) m was constitutively expressed in all tissues examined, such as kidney, spleen, liver, gills, heart, intestine, brain, and muscle, although at different levels. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, the expression of Pscr-beta ( 2 ) m was significantly up-regulated in intestine, kidney and spleen at 24 h post-induction, and increase of Pscr-beta ( 2 ) m transcripts was also observed in liver post-induction with poly(I:C). Real-time PCR further revealed that the expression of Pscr-beta ( 2 ) m in intestine, kidney and spleen tissues was differentially regulated by poly(I:C) and bacterial vaccine during 72 h of induction. These results suggested that Pscr-beta ( 2 ) m might be involved in both antiviral and antibacterial mechanisms in large yellow croaker.

Dosimetric comparison of deep-inspiration breath-hold and free-breathing treatment delivery techniques for left-sided breast cancer using 3D surface tracking.

INTRODUCTION: While radiation therapy has been shown to increase local control and overall survival for breast cancer, late cardiac toxicity remains a concern. Morbidity and mortality have been shown to increase proportionally to the mean heart dose. Deep inspiration breath-hold (DIBH) can reduce heart dose compared to free-breathing (FB) by increasing the heart-to-chest wall distance, especially in left-sided breast cancer. We present our clinical experience with DIBH in left breast and chest-wall irradiation using 3D optical surface tracking. MATERIALS & METHODS: 29 patients were treated with DIBH using a surface tracking system that provides a real time 3D surface image of the patient. Comparisons of maximum and mean heart dose, heart-chest wall separation, and the percentage of lung volume that receives 20 or more Gy (V20) between the DIBH and hypothetical FB treatment plans were conducted with the Student's t-test. Correlation coefficients were also calculated for heart-chest wall separation, heart volume, and lung volume. RESULTS: Comparing DIBH and FB plans showed a decrease in mean and maximum heart doses in all patients. Individual mean heart doses decreased by an average of 1.12 Gy, and the average mean heart dose for DIBH plans was significantly lower than corresponding FB plans (1.02 vs. 2.12 Gy; p < 0.0001). Maximum heart dose decreased by an average of 11.88 Gy and was significantly lower in DIBH versus FB plans (28.33 vs. 43.7 Gy; p = 0.0001). The average difference in heart to chest-wall separation between DIBH and FB images was 2.41 cm. DIBH left lung volume and measured increases in volume on inspiration inversely correlated with maximum heart dose (R = 0.39) and left lung V20 (R = 0.32). CONCLUSIONS: DIBH with 3D surface tracking can significantly benefit patients with left sided disease by limiting the mean and maximum heart dose. DIBH appears to viably reduce heart dose for left-breast cancer patients and thus potentially reduce long-term complications without prolonging treatment delivery.

RU486 Metabolite Inhibits CCN1/Cyr61 Secretion by MDA-MB-231-Endothelial Adhesion.

Successful adhesion of circulating tumor cells (CTCs) to microvascular endothelium of distant metastatic tissue is the key starting step of metastatic cascade that could be effectively chemoprevented as we demonstrated previously. Here, we hypothesize that the hetero-adhesion may produce secretory biomarkers that may be important for both premetastatic diagnosis and chemoprevention. We show that co-incubation of triple-negative breast cancer (TNBC) cell line MDA-MB-231 with human pulmonary microvascular endothelial monolayers (HPMEC) secretes Cyr61 (CCN1), primarily from MDA-MB-231. However, addition of metapristone (RU486 metabolite) to the co-incubation system inhibits Cyr61 secretion probably via the Cyr61/integrin αvß1 signaling pathway without significant cytotoxicity on both MDA-MB-231 and HPMEC. Transfection of MDA-MB-231 with Cyr61-related recombinant plasmid or siRNA enhances or reduces Cyr61 expression, accordingly. The transfection significantly changes hetero-adhesion and migration of MDA-MB-231, and the changed bioactivities by overexpressed CYR61 could be antagonized by metapristone in vitro. Moreover, the circulating MDA-MB-231 develops lung metastasis in mice, which could be effectively prevented by oral metapristone without significant toxicity. The present study, for the first time, demonstrates that co-incubation of MDA-MB-231 with HPMEC secrets CYR61 probably via the CYR61/integrin αvß1 signaling pathway to promote adhesion-invasion of TNBC (early metastatic step). Metapristone, by interfering the adhesion-invasion process, prevents metastasis from happening.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis.