TCAD Analysis of Leakage Current and Breakdown Voltage in Small Pitch 3D Pixel Sensors.

Small-pitch 3D pixel sensors have been developed to equip the innermost layers of the ATLAS and CMS tracker upgrades at the High Luminosity LHC. They feature 50 × 50 and 25 × 100 µm2 geometries and are fabricated on p-type Si-Si Direct Wafer Bonded substrates of 150 µm active thickness with a single-sided process. Due to the short inter-electrode distance, charge trapping effects are strongly mitigated, making these sensors extremely radiation hard. Results from beam test measurements of 3D pixel modules irradiated at large fluences (1016neq/cm2) indeed demonstrated high efficiency at maximum bias voltages of the order of 150 V. However, the downscaled sensor structure also lends itself to high electric fields as the bias voltage is increased, meaning that premature electrical breakdown due to impact ionization is a concern. In this study, TCAD simulations incorporating advanced surface and bulk damage models are used to investigate the leakage current and breakdown behavior of these sensors. Simulations are compared with measured characteristics of 3D diodes irradiated with neutrons at fluences up to 1.5 × 1016neq/cm2. The dependence of the breakdown voltage on geometrical parameters (e.g., the n+ column radius and the gap between the n+ column tip and the highly doped p++ handle wafer) is also discussed for optimization purposes.

Effect of antibiotic monensin on cell proliferation and IGF1R signaling pathway in human colorectal cancer cells.

BACKGROUND/AIMS: Colorectal cancer is the third leading cause of death in patients with cancers in America. Monensin has represented anti-cancer effect on various human cancer cells. We seek to investigate the effect of monensin on proliferation of human colorectal cancer cells and explore whether IGF1R signaling pathway is involved in anti-cancer mechanism of monensin. METHODS: Cell proliferation and migration were assessed by crystal violet staining and cell wounding assay respectively. Cell apoptosis was analyzed by Hoechst 33258 staining and flow cytometry. Cell cycle progression was detected with the use of flow cytometry. Cancer-associated pathways were assessed with the use of pathway-specific reporters. Gene expression was detected by touchdown-quantitative real-time PCR. Inhibition of IGF1R was tested by immunofluorescence staining. Inhibition of IGF1R signaling was accomplished by adenovirus-mediated expression of IGF1. RESULTS: We found that monensin not only effectively inhibited cell proliferation, cell migration as well as cell cycle progression, but also induced apoptosis and G1 arrest in human colorectal cancer cells. Monensin was shown to target multiple cancer-related signaling pathways such as Elk1, AP1, as well as Myc/max, and suppressed IGF1R expression via increasing IGF1 in colorectal cancer cells. CONCLUSION: Monensin could suppressed IGF1R expression via increasing IGF1 in colorectal cancer cells. It has the potential to be repurposed as an anti-colorectal cancer agent, but further studies are still required to investigate the detailed mechanisms of monensin underlying its anti-cancer motion.Key MessagesMonensin inhibits the cell proliferation and the migration, induces apoptosis and inhibits cell cycle progression in human colorectal cancer cells.Monensin may exert anti-cancer activity by targeting multiple signaling pathways, including the IGF1R signaling pathway.Monensin has the potential to be repurposed as an anti-colorectal cancer agent.

Injectable, Hierarchically Degraded Bioactive Scaffold for Bone Regeneration.

Bioactive materials play vital roles in the repair of critical bone defects. However, bone tissue engineering and regenerative medicine are still challenged by the need to repair bone defects evenly and completely. In this study, we functionally simulated the natural creeping substitution process of autologous bone repair by constructing an injectable, hierarchically degradable bioactive scaffold with a composite hydrogel, decalcified bone matrix (DBM) particles, and bone morphogenetic protein 2. This composite scaffold exhibited superior mechanical properties. The scaffold promoted cell proliferation and osteogenic differentiation through multiple signaling pathways. The hierarchical degradation rates of the crosslinked hydrogel and DBM particles accelerated tissue ingrowth and bone formation with a naturally woven bone-like structure in vivo. In the rat calvarial critical defect repair model, the composite scaffold provided even and complete repair of the entire defect area while also integrating the new and host bone effectively. Our results indicate that this injectable, hierarchically degradable bioactive scaffold promotes bone regeneration and provides a promising strategy for evenly and completely repairing the bone defects.

Facile Label-Free Electrochemical DNA Biosensor for Detection of Osteosarcoma-Related Survivin Gene.

A sensitive and selective electrochemical deoxyribonucleic acid (DNA) biosensor was developed for the determination of a osteosarcoma-related survivin gene by using celestine blue (CB) as a label-free hybridization indicator. The proposed strategy adopted a facile and low-cost working electrode with no need for other substances for electrode or DNA functionalization. The interaction mode between CB and DNA was studied by electrochemical and spectroscopic approaches, illustrating that the possible mode was intercalation with a binding number of 2 and a binding constant ß of 1012.87. Moreover, the label-free electrochemical DNA biosensor exhibited a good linear relationship toward the target gene in a range from 1.00 nM to 50.00 nM with a detection limit as low as 0.046 nM using 3σ estimating system. This facile and low-cost electrochemical method realized the rapid detection and accurate quantification of the target sequence in complicated serum samples, endowing its promising potential in the diagnosis and monitoring of genetic diseases.

Correction: Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids.

[This corrects the article DOI: 10.1371/journal.pone.0093608.].

SIRT1 alleviates high-magnitude compression-induced senescence in nucleus pulposus cells via PINK1-dependent mitophagy.

Mechanical overloading-induced nucleus pulposus (NP) cells senescence plays an important role in the pathogenesis of intervertebral disc degeneration (IVDD). The silent mating type information regulator 2 homolog-1 (SIRT1)-mediated pathway preserves the normal NP cell phenotype and mitochondrial homeostasis under multiple stresses. We aimed to investigate the role of SIRT1 in IVDD by assessing the effects of SIRT1 overexpression on high-magnitude compression-induced senescence in NP cells. High-magnitude compression induced cellular senescence and mitochondrial dysfunction in human NP cells. Moreover, SIRT1 overexpression tended to alleviate NP cell senescence and mitochondrial dysfunction under compressive stress. Given the mitophagy-inducing property of SIRT1, activity of mitophagy was evaluated in NP cells to further demonstrate the underlying mechanism. The results showed that SIRT1-overexpression attenuated senescence and mitochondrial injury in NP cells subjected to high-magnitude compression. However, depletion of PINK1, a key mitophagic regulator, impaired mitophagy and blocked the protective role of SIRT1 against compression induced senescence in NP cells. In summary, these results suggest that SIRT1 plays a protective role in alleviating NP cell senescence and mitochondrial dysfunction under high-magnitude compression, the mechanism of which is associated with the regulation of PINK1-dependent mitophagy. Our findings may provide a potential therapeutic approach for IVDD treatment.

Low Magnitude of Compression Enhances Biosynthesis of Mesenchymal Stem Cells towards Nucleus Pulposus Cells via the TRPV4-Dependent Pathway.

Mesenchymal stem cell- (MSC-) based therapy is regarded as a promising tissue engineering strategy to achieve nucleus pulposus (NP) regeneration for the treatment of intervertebral disc degeneration (IDD). However, it is still a challenge to promote the biosynthesis of MSC to meet the requirement of NP regeneration. The purpose of this study was to optimize the compressive magnitude to enhance the extracellular matrix (ECM) deposition towards discogenesis of MSCs. Thus, we constructed a 3D culture model for MSCs to bear different magnitudes of compression for 7 days (5%, 10%, and 20% at the frequency of 1.0 Hz for 8 hours/day) using an intelligent and mechanically active bioreactor. Then, the underlying mechanotransduction mechanism of transient receptor potential vanilloid 4 (TRPV4) was further explored. The MSC-encapsulated hybrids were evaluated by Live/Dead staining, biochemical content assay, real-time PCR, Western blot, histological, and immunohistochemical analysis. The results showed that low-magnitude compression promoted anabolic response where high-magnitude compression induced the catabolic response for the 3D-cultured MSCs. The anabolic effect of low-magnitude compression could be inhibited by inhibiting TRPV4. Meanwhile, the activation of TRPV4 enhanced the biosynthesis analogous to low-magnitude compression. These findings demonstrate that low-magnitude compression promoted the anabolic response of ECM deposition towards discogenesis for the 3D-cultured MSCs and the TRPV4 channel plays a key role on mechanical signal transduction for low-magnitude compressive loading. Further understanding of this mechanism may provide insights into the development of new therapies for MSC-based NP regeneration.

Whole-Proteome Analysis of Human Craniosynostotic Tissue Suggests a Link between Inflammatory Signaling and Osteoclast Activation in Human Cranial Suture Patency.

BACKGROUND: The pathophysiology of nonsyndromic craniosynostosis remains poorly understood. The authors seek to understand the cause of this condition with a specific focus on how osteoclasts may contribute to craniosynostosis. Here, the authors characterize proteins differentially expressed in patent and fused cranial sutures by comparing their respective proteomes. METHODS: Fused and patent suture samples were obtained from craniosynostotic patients undergoing surgery at a single academic medical center. Extracted protein from samples was interrogated using mass spectrometry. Differential protein expression was determined using maximum likelihood-based G-test with a q-value cutoffs of 0.5 after correction for multiple hypothesis testing. Immunolocalization of lead protein candidates was performed to validate proteomic findings. In addition, quantitative polymerase chain reaction analysis of corresponding gene expression of proteins of interest was performed. RESULTS: Proteins differentially expressed in patent versus fused sutures included collagen 6A1 (Col6A1), fibromodulin, periostin, aggrecan, adipocyte enhancer-binding protein 1, and osteomodulin (OMD). Maximum likelihood-based G-test suggested that Col6A1, fibromodulin, and adipocyte enhancer-binding protein 1 are highly expressed in patent sutures compared with fused sutures, whereas OMD is up-regulated in fused sutures compared with patent sutures. These results were corroborated by immunohistochemistry. Quantitative polymerase chain reaction data point to an inverse relationship in proteins of interest to RNA transcript levels, in prematurely fused and patent sutures that potentially describes a feedback loop mechanism. CONCLUSIONS: Proteome analysis validated by immunohistochemistry may provide insight into the mechanism of cranial suture patency and disease from an osteoclast perspective. The authors results suggest a role of inflammatory mediators in nonsyndromic craniosynostosis. Col6A1 may aid in the regulation of suture patency, and OMD may be involved in premature fusion. Additional validation studies are required.

BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients.

The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long-term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long-term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.

Repair of critical sized cranial defects with BMP9-transduced calvarial cells delivered in a thermoresponsive scaffold.

Large skeletal defects caused by trauma, congenital malformations, and post-oncologic resections of the calvarium present major challenges to the reconstructive surgeon. We previously identified BMP-9 as the most osteogenic BMP in vitro and in vivo. Here we sought to investigate the bone regenerative capacity of murine-derived calvarial mesenchymal progenitor cells (iCALs) transduced by BMP-9 in the context of healing critical-sized calvarial defects. To accomplish this, the transduced cells were delivered to the defect site within a thermoresponsive biodegradable scaffold consisting of poly(polyethylene glycol citrate-co-N-isopropylacrylamide mixed with gelatin (PPCN-g). A total of three treatment arms were evaluated: PPCN-g alone, PPCN-g seeded with iCALs expressing GFP, and PPCN-g seeded with iCALs expressing BMP-9. Defects treated only with PPCN-g scaffold did not statistically change in size when evaluated at eight weeks postoperatively (p = 0.72). Conversely, both animal groups treated with iCALs showed significant reductions in defect size after 12 weeks of follow-up (BMP9-treated: p = 0.0025; GFP-treated: p = 0.0042). However, H&E and trichrome staining revealed more complete osseointegration and mature bone formation only in the BMP9-treated group. These results suggest that BMP9-transduced iCALs seeded in a PPCN-g thermoresponsive scaffold is capable of inducing bone formation in vivo and is an effective means of creating tissue engineered bone for critical sized defects.

Bone morphogenetic protein 9 (BMP9) induces effective bone formation from reversibly immortalized multipotent adipose-derived (iMAD) mesenchymal stem cells.

Regenerative medicine and bone tissue engineering using mesenchymal stem cells (MSCs) hold great promise as an effective approach to bone and skeletal reconstruction. While adipose tissue harbors MSC-like progenitors, or multipotent adipose-derived cells (MADs), it is important to identify and characterize potential biological factors that can effectively induce osteogenic differentiation of MADs. To overcome the time-consuming and technically challenging process of isolating and culturing primary MADs, here we establish and characterize the reversibly immortalized mouse multipotent adipose-derived cells (iMADs). The isolated mouse primary inguinal MAD cells are reversibly immortalized via the retrovirus-mediated expression of SV40 T antigen flanked with FRT sites. The iMADs are shown to express most common MSC markers. FLP-mediated removal of SV40 T antigen effectively reduces the proliferative activity and cell survival of iMADs, indicating the immortalization is reversible. Using the highly osteogenic BMP9, we find that the iMADs are highly responsive to BMP9 stimulation, express multiple lineage regulators, and undergo osteogenic differentiation in vitro upon BMP9 stimulation. Furthermore, we demonstrate that BMP9-stimulated iMADs form robust ectopic bone with a thermoresponsive biodegradable scaffold material. Collectively, our results demonstrate that the reversibly immortalized iMADs exhibit the characteristics of multipotent MSCs and are highly responsive to BMP9-induced osteogenic differentiation. Thus, the iMADs should provide a valuable resource for the study of MAD biology, which would ultimately enable us to develop novel and efficacious strategies for MAD-based bone tissue engineering.

A thermoresponsive polydiolcitrate-gelatin scaffold and delivery system mediates effective bone formation from BMP9-transduced mesenchymal stem cells.

Successful bone tissue engineering requires at the minimum sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we investigated the potential use of a biodegradable citrate-based thermosensitive macromolecule, poly(polyethyleneglycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin (PPCNG) as a scaffold for the delivery of BMP9-stimulated MSCs to promote localized bone formation. The addition of gelatin to PPCN effectively enhanced the cell adhesion and survival properties of MSCs entrapped within the gel in 3D culture. Using the BMP9-transduced MSC line immortalized mouse embryonic fibroblasts (iMEFs), we found that PPCNG facilitated BMP9-induced osteogenic differentiation of iMEFs in vivo and promoted the formation of well-ossified and vascularized trabecular bone-like structures in a mouse model of ectopic bone formation. Histologic evaluation revealed that vascularization of the bony masses retrieved from the iMEFs + PPCNG group was significantly more pronounced than that of the direct cell injection group. Accordingly, vascular endothelial growth factor (VEGF) expression was shown to be significantly higher in the bony masses recovered from the iMEFs + PPCNG group. Taken together, our results suggest that PPCNG may serve as a novel biodegradable and injectable scaffold and carrier for gene and cell-based bone tissue engineering.

Immortalized Mouse Achilles Tenocytes Demonstrate Long-Term Proliferative Capacity While Retaining Tenogenic Properties.

Investigating the cellular processes underlying tendon healing can allow researchers to improve long-term outcomes after injury. However, conducting meaningful studies to uncover the injury healing mechanism at cellular and molecular levels remains challenging. This is due to the inherent difficulty in isolating, culturing, and expanding sufficient primary tenocytes, due to their limited proliferative capacity and short lifespan. In this study, we sought to establish a novel line of immortalized mouse Achilles tenocytes (iMATs) with primary tenocyte properties, but increased proliferative capacity suitable for extensive in vitro experimentation. We show that isolated primary mouse Achilles tenocytes (pMATs) can be effectively immortalized using a piggyBac transposon expressing SV40 large T antigen flanked by FLP recombination target site (FRT). The resulting iMATs exhibit markedly greater proliferation and survival, which can be reversed with FLP recombinase. Furthermore, iMATs express the same set of tendon-specific markers as that of primary cells, although in lower levels, and respond similarly to exogenous stimulation with bone morphogenetic protein 13 (BMP13) as has been previously reported with pMATs. Taken together, our results suggest that iMATs acquire long-term proliferative capacity while maintaining tenogenic properties. We believe that iMATs are a suitable model for studying not only the native cellular processes involved in injury and healing, but also potential therapeutic agents that may augment the stability of tendon repair.

The Prodomain-Containing BMP9 Produced from a Stable Line Effectively Regulates the Differentiation of Mesenchymal Stem Cells.

BACKGROUND: BMPs play important roles in regulating stem cell proliferation and differentiation. Using adenovirus-mediated expression of the 14 types of BMPs we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs), which was undetected in the early studies using recombinant BMP9 proteins. Endogenous BMPs are expressed as a precursor protein that contains an N-terminal signal peptide, a prodomain and a C-terminal mature peptide. Most commercially available recombinant BMP9 proteins are purified from the cells expressing the mature peptide. It is unclear how effectively these recombinant BMP9 proteins functionally recapitulate endogenous BMP9. METHODS: A stable cell line expressing the full coding region of mouse BMP9 was established in HEK-293 cells by using the piggyBac transposon system. The biological activities and stability of the conditioned medium generated from the stable line were analyzed. RESULTS: The stable HEK-293 line expresses a high level of mouse BMP9. BMP9 conditioned medium (BMP9-cm) was shown to effectively induce osteogenic differentiation of MSCs, to activate BMP-R specific Smad signaling, and to up-regulate downstream target genes in MSCs. The biological activity of BMP9-cm is at least comparable with that induced by AdBMP9 in vitro. Furthermore, BMP9-cm exhibits an excellent stability profile as its biological activity is not affected by long-term storage at -80ºC, repeated thawing cycles, and extended storage at 4ºC. CONCLUSIONS: We have established a producer line that stably expresses a high level of active BMP9 protein. Such producer line should be a valuable resource for generating biologically active BMP9 protein for studying BMP9 signaling mechanism and functions.

A Novel Organ Culture Model of Mouse Intervertebral Disc Tissues.

The intervertebral disc (IVD) is a fibrocartilaginous joint between two vertebral bodies. An IVD unit consists of a gelatinous central nucleus pulposus, encased by the annulus fibrosus, which is sandwiched between cartilaginous endplates (EPs). The IVD homeostasis can be disrupted by injuries, ageing and/or genetic predispositions, leading to degenerative disc disorders and subsequent lower back pain. The complex structure and distinct characteristics of IVDs warrant the establishment of robust in vitro IVD organ culture for studying the etiology and treatment of disc degeneration. Here, we isolate mouse lumbar IVDs and culture the minimal IVD units in submersion or suspension medium supplemented with 2% bovine serum or 10% fetal bovine serum (FBS). We find the minimal IVD units remain healthy for up to 14 days when cultured in submersion culture supplemented with 10% FBS. New bone formation in the EPs of the cultured IVDs can be assessed with calcein labeling. Furthermore, the cultured IVDs can be effectively transduced by recombinant adenovirus, and transgene expression lasts for 2 weeks. Thus, our findings demonstrate that the optimized IVD organ culture system can be used to study IVD biology and screen for biological factors that may prevent, alleviate and/or treat disc degeneration.

Antibiotic monensin synergizes with EGFR inhibitors and oxaliplatin to suppress the proliferation of human ovarian cancer cells.

Ovarian cancer is the most lethal gynecologic malignancy with an overall cure rate of merely 30%. Most patients experience recurrence within 12-24 months of cure and die of progressively chemotherapy-resistant disease. Thus, more effective anti-ovarian cancer therapies are needed. Here, we investigate the possibility of repurposing antibiotic monensin as an anti-ovarian cancer agent. We demonstrate that monensin effectively inhibits cell proliferation, migration and cell cycle progression, and induces apoptosis of human ovarian cancer cells. Monensin suppresses multiple cancer-related pathways including Elk1/SRF, AP1, NFκB and STAT, and reduces EGFR expression in ovarian cancer cells. Monensin acts synergistically with EGFR inhibitors and oxaliplatin to inhibit cell proliferation and induce apoptosis of ovarian cancer cells. Xenograft studies confirm that monensin effectively inhibits tumor growth by suppressing cell proliferation through targeting EGFR signaling. Our results suggest monensin may be repurposed as an anti-ovarian cancer agent although further preclinical and clinical studies are needed.

The Calcium-Binding Protein S100A6 Accelerates Human Osteosarcoma Growth by Promoting Cell Proliferation and Inhibiting Osteogenic Differentiation.

BACKGROUND/AIMS: Although osteosarcoma (OS) is the most common primary malignancy of bone, its molecular pathogenesis remains to be fully understood. We previously found the calcium-binding protein S100A6 was expressed in ∼80% of the analyzed OS primary and/or metastatic tumor samples. Here, we investigate the role of S100A6 in OS growth and progression. METHODS: S100A6 expression was assessed by qPCR and Western blotting. Overexpression or knockdown of S100A6 was carried out to determine S100A6's effect on proliferation, cell cycle, apoptosis, tumor growth, and osteogenic differentiation. RESULTS: S100A6 expression was readily detected in human OS cell lines. Exogenous S100A6 expression promoted cell proliferation in vitro and tumor growth in an orthotopic xenograft model of human OS. S100A6 overexpression reduced the numbers of OS cells in G1 phase and increased viable cells under serum starvation condition. Conversely, silencing S100A6 expression induced the production of cleaved caspase 3, and increased early stage apoptosis. S100A6 knockdown increased osteogenic differentiation activity of mesenchymal stem cells, while S100A6 overexpression inhibited osteogenic differentiation. BMP9-induced bone formation was augmented by S100A6 knockdown. CONCLUSION: Our findings strongly suggest that S100A6 may promote OS cell proliferation and OS tumor growth at least in part by facilitating cell cycle progression, preventing apoptosis, and inhibiting osteogenic differentiation. Thus, it is conceivable that targeting S100A6 may be exploited as a novel anti-OS therapy.

Insider information: Testing cancer drug sensitivity for personalized therapy.

Cancer death is usually caused by incurable drug-resistant and metastatic cancers. Although tremendous progress has been made in anticancer drug development during the past two decades, cancer medicine still faces unprecedented challenges associated with choosing effective treatments for individual patients. Three recent reports have offered encouraging approaches towards potentially personalized cancer drug selection.

TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR.

The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited.

The Anthelmintic Drug Niclosamide Inhibits the Proliferative Activity of Human Osteosarcoma Cells by Targeting Multiple Signal Pathways.

Osteosarcoma (OS) is the most common primary malignant tumor of bone with a high propensity for lung metastasis. Despite significant advances in surgical techniques and chemotherapeutic regimens over the past few decades, there has been minimal improvement in OS patient survival. There is an urgent need to identify novel antitumor agents to treat human OS. Repurposing the clinically-used drugs represents a rapid and effective approach to the development of new anticancer agents. The anthelmintic drug niclosamide has recently been identified as a potential anticancer agent in human cancers. Here, we investigate if niclosamide can be developed as an anti-OS drug. We find that niclosamide can effectively inhibit OS cell proliferation and survival at low micromolar concentrations. Cell migration and wounding closure are significantly inhibited by niclosamide. Niclosamide induces cell apoptosis and inhibits cell cycle progression in OS cells. Analysis of niclosamide's effect on 11 cancer-related signal pathway reporters reveals that three of them, the E2F1, AP1, and c-Myc-responsive reporters, are significantly inhibited. To a lesser extent, the HIF1α, TCF/LEF, CREB, NFκB, Smad/TGFß, and Rbpj/Notch pathway reporters are also inhibited, while the NFAT and Wnt/ß-catenin reporters are not significantly affected by niclosamide treatment. We demonstrate that the expression of c-Fos, c-Jun. E2F1, and c-Myc in OS cells is effectively inhibited by niclosamide. Furthermore, niclosamide is shown to effectively inhibit tumor growth in a mouse xenograft tumor model of human osteosarcoma cells. Taken together, these results strongly suggest that niclosamide may exert its anticancer activity in OS cells by targeting multiple signaling pathways. Future investigations should be directed to exploring the antitumor activity in clinically relevant OS models and ultimately in clinical trials.