Depletion of the histone chaperone tNASP inhibits proliferation and induces apoptosis in prostate cancer PC-3 cells

<p>Abstract</p> <p>Background</p> <p>NASP (Nuclear Autoantigenic Sperm Protein) is a histone chaperone that is present in all dividing cells. NASP has two splice variants: tNASP and sNASP. Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP splice variant. We examined the consequences of tNASP depletion for prostate cancer PC-3 cells.</p> <p>Methods</p> <p>tNASP was depleted from prostate cancer PC-3 cells, cervical cancer HeLa cells, and prostate epithelial PWR-1E cells using lentivirus expression of tNASP shRNA. Cell cycle changes were studied by proliferation assay with CFSE labeling and double thymidine synchronization. Gene expression profiles were detected using RT²Profiler PCR Array, Western and Northern blotting.</p> <p>Results</p> <p>PC-3 and HeLa cells showed inhibited proliferation, increased levels of cyclin-dependant kinase inhibitor p21 protein and apoptosis, whereas non-tumorigenic PWR-1E cells did not. All three cell types showed decreased levels of HSPA2. Supporting in vitro experiments demonstrated that tNASP, but not sNASP is required for activation of HSPA2.</p> <p>Conclusions</p> <p>Our results demonstrate that PC-3 and HeLa cancer cells require tNASP to maintain high levels of HSPA2 activity and therefore viability, while PWR-1E cells are unaffected by tNASP depletion. These different cellular responses most likely arise from changes in the interaction between tNASP and HSPA2 and disturbed tNASP chaperoning of linker histones. This study has demonstrated that tNASP is critical for the survival of prostate cancer cells and suggests that targeting tNASP expression can lead to a new approach for prostate cancer treatment.</p

Molecular Acoustic Angiography: A New Technique for High-resolution Superharmonic Ultrasound Molecular Imaging

Ultrasound molecular imaging utilizes targeted microbubbles to bind to vascular targets such as integrins, selectins, and other extracellular binding domains. After binding, these microbubbles are typically imaged using low pressures and multi-pulse imaging sequences. In this article, we present an alternative approach for molecular imaging using ultrasound which relies on superharmonic signals produced by microbubble contrast agents. Bound bubbles were insonified near resonance using a low frequency (4 MHz) and superharmonic echoes were received at high frequencies (25–30 MHz). While this approach was observed to produce declining image intensity during repeated imaging in both in vitro and in vivo experiments due to bubble destruction, the feasibility of superharmonic molecular imaging was demonstrated for transmit pressures which are sufficiently high to induce shell disruption in bound microbubbles. This approach was validated using microbubbles targeted to the αvβ3 integrin in a rat fibrosarcoma model (n=5), and combined with superharmonic images of free microbubbles to produce high contrast, high resolution 3D volumes of both microvascular anatomy and molecular targeting. Image intensity over repeated scans and the effect of microbubble diameter were also assessed in vivo, indicating that larger microbubbles yield increased persistence in image intensity. Using ultrasound-based acoustic angiography images rather than conventional B-mode ultrasound to provide the underlying anatomical information facilitates anatomical localization of molecular markers. Quantitative analysis of relationships between microvasculature and targeting information indicated that most targeting occurred within 50 µm of a resolvable vessel (>100 µm diameter). The combined information provided by these scans may present new opportunities for analyzing relationships between microvascular anatomy and vascular targets, subject only to limitations of the current mechanically-scanned system and microbubble persistence to repeated imaging at moderate mechanical indices

Classification of Mouse Sperm Motility Patterns Using an Automated Multiclass Support Vector Machines Model1

Vigorous sperm motility, including the transition from progressive to hyperactivated motility that occurs in the female reproductive tract, is required for normal fertilization in mammals. We developed an automated, quantitative method that objectively classifies five distinct motility patterns of mouse sperm using Support Vector Machines (SVM), a common method in supervised machine learning. This multiclass SVM model is based on more than 2000 sperm tracks that were captured by computer-assisted sperm analysis (CASA) during in vitro capacitation and visually classified as progressive, intermediate, hyperactivated, slow, or weakly motile. Parameters associated with the classified tracks were incorporated into established SVM algorithms to generate a series of equations. These equations were integrated into a binary decision tree that sequentially sorts uncharacterized tracks into distinct categories. The first equation sorts CASA tracks into vigorous and nonvigorous categories. Additional equations classify vigorous tracks as progressive, intermediate, or hyperactivated and nonvigorous tracks as slow or weakly motile. Our CASAnova software uses these SVM equations to classify individual sperm motility patterns automatically. Comparisons of motility profiles from sperm incubated with and without bicarbonate confirmed the ability of the model to distinguish hyperactivated patterns of motility that develop during in vitro capacitation. The model accurately classifies motility profiles of sperm from a mutant mouse model with severe motility defects. Application of the model to sperm from multiple inbred strains reveals strain-dependent differences in sperm motility profiles. CASAnova provides a rapid and reproducible platform for quantitative comparisons of motility in large, heterogeneous populations of mouse sperm

Therapeutic ultrasound as a potential male contraceptive: power, frequency and temperature required to deplete rat testes of meiotic cells and epididymides of sperm determined using a commercially available system

<p>Abstract</p> <p>Background</p> <p>Studies published in the 1970s by Mostafa S. Fahim and colleagues showed that a short treatment with ultrasound caused the depletion of germ cells and infertility. The goal of the current study was to determine if a commercially available therapeutic ultrasound generator and transducer could be used as the basis for a male contraceptive.</p> <p>Methods</p> <p>Sprague-Dawley rats were anesthetized and their testes were treated with 1 MHz or 3 MHz ultrasound while varying power, duration and temperature of treatment.</p> <p>Results</p> <p>We found that 3 MHz ultrasound delivered with 2.2 Watt per square cm power for fifteen minutes was necessary to deplete spermatocytes and spermatids from the testis and that this treatment significantly reduced epididymal sperm reserves. 3 MHz ultrasound treatment reduced total epididymal sperm count 10-fold lower than the wet-heat control and decreased motile sperm counts 1,000-fold lower than wet-heat alone. The current treatment regimen provided nominally more energy to the treatment chamber than Fahim's originally reported conditions of 1 MHz ultrasound delivered at 1 Watt per square cm for ten minutes. However, the true spatial average intensity, effective radiating area and power output of the transducers used by Fahim were not reported, making a direct comparison impossible. We found that germ cell depletion was most uniform and effective when we rotated the therapeutic transducer to mitigate non-uniformity of the beam field. The lowest sperm count was achieved when the coupling medium (3% saline) was held at 37 degrees C and two consecutive 15-minute treatments of 3 MHz ultrasound at 2.2 Watt per square cm were separated by 2 days.</p> <p>Conclusions</p> <p>The non-invasive nature of ultrasound and its efficacy in reducing sperm count make therapeutic ultrasound a promising candidate for a male contraceptive. However, further studies must be conducted to confirm its efficacy in providing a contraceptive effect, to test the result of repeated use, to verify that the contraceptive effect is reversible and to demonstrate that there are no detrimental, long-term effects from using ultrasound as a method of male contraception.</p

Prospects of Detecting Baryon and Quark Superfluidity from Cooling Neutron Stars

Baryon and quark superfluidity in the cooling of neutron stars are investigated. Observations could constrain combinations of the neutron or Lambda-hyperon pairing gaps and the star's mass. However, in a hybrid star with a mixed phase of hadrons and quarks, quark gaps larger than a few tenths of an MeV render quark matter virtually invisible for cooling. If the quark gap is smaller, quark superfluidity could be important, but its effects will be nearly impossible to distinguish from those of other baryonic constituents.Comment: 4 pages, 3 ps figures, uses RevTex(aps,prl). Submitted to Phys. Rev. Let

Ultrasound Molecular Imaging of Secreted Frizzled Related Protein-2 Expression in Murine Angiosarcoma

Angiosarcoma is a biologically aggressive vascular malignancy with a high metastatic potential. In the era of targeted medicine, knowledge of specific molecular tumor characteristics has become more important. Molecular imaging using targeted ultrasound contrast agents can monitor tumor progression non-invasively. Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. We hypothesize that SFRP2-directed imaging could be a novel approach to imaging the tumor vasculature. To develop an SFRP2 contrast agent, SFRP2 polyclonal antibody was biotinylated and incubated with streptavidin-coated microbubbles. SVR angiosarcoma cells were injected into nude mice, and when tumors were established the mice were injected intravenously with the SFRP2 -targeted contrast agent, or a control streptavidin-coated contrast agent. SFRP2 -targeted contrast agent detected tumor vasculature with significantly more signal intensity than control contrast agent: the normalized fold-change was 1.6±0.27 (n = 13, p = 0.0032). The kidney was largely devoid of echogenicity with no significant difference between the control contrast agent and the SFRP2-targeted contrast agent demonstrating that the SFRP2-targeted contrast agent was specific to tumor vessels. Plotting average pixel intensity obtained from SFRP2-targeted contrast agent against tumor volume showed that the average pixel intensity increased as tumor volume increased. In conclusion, molecularly-targeted imaging of SFRP2 visualizes angiosarcoma vessels, but not normal vessels, and intensity increases with tumor size. Molecular imaging of SFRP2 expression may provide a rapid, non-invasive method to monitor tumor regression during therapy for angiosarcoma and other SFRP2 expressing cancers, and contribute to our understanding of the biology of SFRP2 during tumor development and progression

Conditionally Replicating Adenovirus Expressing TIMP2 Increases Survival in a Mouse Model of Disseminated Ovarian Cancer

Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer

Optimizing ultrasound molecular imaging of secreted frizzled related protein 2 expression in angiosarcoma.

Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. Previously, we showed ultrasound molecular imaging with SFRP2-targeted contrast increased average video pixel intensity (VI) of angiosarcoma vessels by 2.2 ± 0.6 VI versus streptavidin contrast. We hypothesized that redesigning our contrast agents would increase imaging performance. Improved molecular imaging reagents were created by combining NeutrAvidin™-functionalized microbubbles with biotinylated SFRP2 or IgY control antibodies. When angiosarcoma tumors in nude mice reached 8 mm, time-intensity, antibody loading, and microbubble dose experiments optimized molecular imaging. 10 minutes after injection, the control-subtracted time-intensity curve (TIC) for SFRP2-targeted contrast reached a maximum, after subtracting the contribution of free-flowing contrast. SFRP2 antibody-targeted VI was greater when contrast was formulated with 10-fold molar excess of maleimide-activated NeutrAvidin™ versus 3-fold (4.5 ± 0.18 vs. 0.32 ± 0.15, VI ± SEM, 5 x 106 dose, p < 0.001). Tumor vasculature returned greater average video pixel intensity using 5 x 107 versus 5 x 106 microbubbles (21.2 ± 2.5 vs. 4.5 ± 0.18, p = 0.0011). Specificity for tumor vasculature was confirmed by low VI for SFRP2-targeted, and control contrast in peri-tumoral vasculature (3.2 ± 0.52 vs. 1.6 ± 0.71, p = 0.92). After optimization, average video pixel intensity of tumor vasculature was 14.2 ± 3.0 VI units higher with SFRP2-targeted contrast versus IgY-targeted control (22.1 ± 2.5 vs. 7.9 ± 1.6, p < 0.001). After log decompression, 14.2 ΔVI was equal to ~70% higher signal, in arbitray acoustic units (AU), for SFRP2 versus IgY. This provided ~18- fold higher acoustic signal enhancement than provided previously by 2.2 ΔVI. Basing our targeted contrast on NeutrAvidin™-functionalized microbubbles, using IgY antibodies for our control contrast, and optimizing our imaging protocol significantly increased the SFRP2-specific signal returned from angiosarcoma vasculature, and may provide new opportunities for targeted molecular imaging