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Breast cancer is a malignant tumor with a high mortality rate among women. Therefore, it is necessary to develop novel therapies to effectively treat this disease. In this study, iron selenide nanorods (FeSe2NRs) were designed for use in magnetic hyperthermic, photothermal, and chemodynamic therapy (MHT/PTT/CDT) for breast cancer. To illustrate their efficacy, FeSe2NRs were modified with the chemotherapeutic agent methotrexate (MTX). MTX-modified FeSe2(FeSe2-MTX) exhibited excellent controlled drug release properties. Fe2+released from FeSe2NRs induced the release of •OH from H2O2via a Fenton/Fenton-like reaction, enhancing the efficacy of CDT. Under alternating magnetic field (AMF) stimulation and 808 nm laser irradiation, FeSe2-MTX exerted potent hyperthermic and photothermal effects by suppressing tumor growth in a breast cancer nude mouse model. In addition, FeSe2NRs can be used for magnetic resonance imaging in vivo by incorporating their superparamagnetic characteristics into a single nanomaterial. Overall, we presented a novel technique for the precise delivery of functional nanosystems to tumors that can enhance the efficacy of breast cancer treatment.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that can bind to several receptors and mediate distinct molecular pathways in various cell settings. Changing levels of LECT2 have been implicated in multiple human disease states, including cancers. Here, we have demonstrated reduced serum levels of LECT2 in patients with epithelial ovarian cancer (EOC) and down-regulated circulating Lect2 as the disease progresses in a syngeneic mouse ID8 EOC model. Using the murine EOC model, we discovered that loss of Lect2 promotes EOC progression by modulating both tumor cells and the tumor microenvironment. Lect2 inhibited EOC cells’ invasive phenotype and suppressed EOC’s transcoelomic metastasis by targeting c-Met signaling. In addition, Lect2 downregulation induced the accumulation and activation of myeloid-derived suppressor cells (MDSCs). This fostered an immunosuppressive microenvironment in EOC by inhibiting T-cell activation and skewing macrophages toward an M2 phenotype. The therapeutic efficacy of programmed cell death-1 (PD-1)/PD-L1 pathway blockade for the ID8 model was significantly hindered. Overall, our data highlight multiple functions of Lect2 during EOC progression and reveal a rationale for synergistic immunotherapeutic strategies by targeting Lect2.

Leptomeningeal metastasis (LM) occurs when tumor cells spread to the leptomeningeal space surrounding the brain and the spinal cord, thereby causing poor clinical outcomes. The triple-negative breast cancer (TNBC) has been associated with symptoms of LM and mechanism remained unclear. Through proteomic analysis, we identified high expression of ICAM2 in leptomeningeal metastatic TNBC cells, which promoted the colonization of the spinal cord and resulted in poor survival in vivo. Two-way demonstration indicated that high levels of ICAM2 promoted blood–cerebrospinal fluid barrier (BCB) adhesion, trans-BCB migration, and stemness abilities and determined the specificity of LM in vivo. Furthermore, pull-down and antibody neutralizing assay revealed that ICAM2 determined the specificity of LM through interactions with ICAM1 in the choroid plexus epithelial cells. Therefore, neutralizing ICAM2 can attenuate the progression of LM and prolong survival in vivo. The results suggested that targeting ICAM2 is a potential therapeutic strategy for LM in TNBC.

Neuroendocrine prostate cancer (PCa) (NEPC), an aggressive subtype that is associated with poor prognosis, may arise after androgen deprivation therapy (ADT). We investigated the molecular mechanisms by which ADT induces neuroendocrine differentiation in advanced PCa. We found that transmembrane protein 1 (MCTP1), which has putative Ca2+sensing function and multiple Ca2+-binding C2 domains, was abundant in samples from patients with advanced PCa. MCTP1 was associated with the expression of the EMT-associated transcription factors ZBTB46, FOXA2, and HIF1A. The increased abundance of MCTP1 promoted PC3 prostate cancer cell migration and neuroendocrine differentiation and was associated with SNAI1-dependent EMT in C4-2 PCa cells after ADT. ZBTB46 interacted with FOXA2 and HIF1A and increased the abundance of MCTP1 in a hypoxia-dependent manner. MCTP1 stimulated Ca2+signaling and AKT activation to promote EMT and neuroendocrine differentiation by increasing the SNAI1-dependent expression of EMT and neuroendocrine markers, effects that were blocked by knockdown of MCTP1. These data suggest an oncogenic role for MCTP1 in the maintenance of a rare and aggressive prostate cancer subtype through its response to Ca2+and suggest its potential as a therapeutic target.

Currently, the survival rate for breast cancer is more than 90%, but once the cancer cells metastasize to distal organs, the survival rate is dramatically reduced, to less than 30%. Triple-negative breast cancer accounts for 15-20% of all breast cancers. Triple-negative breast cancer (TNBC) is associated with poor prognostic and diagnostic outcomes due to the limiting therapeutic strategies, relative to non-TNBC breast cancers. Therefore, the development of targeted therapy for TNBC metastasis remains an urgent issue. In this study, high Carboxyl-terminal modulator protein (CTMP) is significantly associated with recurrence and disease-free survival rate in TNBC patients. Overexpression of CTMP promotes migration and invasion abilities in BT549 cells. Down-regulating of CTMP expression inhibits migration and invasion abilities in MDA-MB-231 cells. In vivo inoculation of high-CTMP cells enhances distant metastasis in mice. The metastasis incidence rate is decreased in mice injected with CTMP-downregulating MDA-MB-231 cells. Gene expression microarray analysis indicates the Akt-dependent pathway is significantly enhanced in CTMP overexpressing cells compared to the parental cells. Blocking Akt activation via Akt inhibitor treatment or co-expression of the dominant-negative form of Akt proteins successfully abolishes the CTMP mediating invasion in TNBC cells. Our findings suggest that CTMP is a potential diagnostic marker for recurrence and poor disease-free survival in TNBC patients. CTMP promotes TNBC metastasis via the Akt-activation-dependent pathway.

Recent studies have found that exosomes or extracellular vehicles (EVs) are associated with cancer metastasis, disease progression, diagnosis, and treatment, leading to a rapidly emerging area of exocrine vesicle research. Relying on the superior targeting function and bio-compatibility of exosomes, researchers have been able to deliver drugs to cancer stem cells deep within tumors in mouse models. Despite significant efforts made in this relatively new field of exosome research, progress has been held back by challenges such as inefficient separation methods, difficulties in characterization/tracking, and a lack of specific biomarkers. Therefore, current researches are devoted to combining nanomaterials with exosomes to improve these shortcomings. Adding inorganic/organic nanoparticles such as artificial liposomes and iron oxide can bring more drug options and various fluorescent or magnetic diagnostic possibilities to the exosome system. Moreover, the applications of exosomes need to be further evaluated under actual physiological conditions. This review article highlights the potential of exosome-biomimetic nanoparticles for their use as drug carriers to improve the efficacy of anticancer therapy.

Glioblastoma (GBM) is one of the deadliest and most invasive brain cancers/gliomas, and there is currently no established way to treat this disease. The treatment of GBM typically involves intracranial surgery followed by chemotherapy. However, the blood–brain barrier (BBB) impedes the delivery of the chemotherapeutic drug, making the treatment challenging. In this study, we embedded a chemotherapeutic drug and other nanomaterials into a nanobubble (NB), utilized active tracking and other guidance mechanisms to guide the nanocomposite to the tumor site, and then used high-intensity focused ultrasound oscillation to burst the nanobubbles, generating a transient cavitation impact on the BBB and allowing the drug to bypass it and reach the brain. FePt enhances the resolution of T2-weighted magnetic resonance imaging images and has magnetic properties that help guide the nanocomposite to the tumor location. FePt nanoparticles were loaded into the hydrophobic core of the NBs along with doxorubicin to form a bubble-based drug delivery system (Dox-FePt@NB). The surface of the NBs is modified with a targeting ligand, transferrin (Dox-FePt@NB-Tf), giving the nanocomposite active tracking abilities. The Dox-FePt@NB-Tf developed in the present study represents a potential breakthrough in GBM treatment through improved drug delivery and biological imaging.

Brian metastasis, which is diagnosed in 30% of triple-negative breast cancer (TNBC) patients with metastasis, causes poor survival outcomes. Growing evidence has characterized miRNAs involving in breast cancer brain metastasis; however, currently, there is a lack of prognostic plasma-based indicator for brain metastasis. In this study, high level of miR-211 can act as brain metastatic prognostic marker in vivo. High miR-211 drives early and specific brain colonization through enhancing trans-blood–brain barrier (BBB) migration, BBB adherence, and stemness properties of tumor cells and causes poor survival in vivo. SOX11 and NGN2 are the downstream targets of miR-211 and negatively regulate miR-211-mediated TNBC brain metastasis in vitro and in vivo. Most importantly, high miR-211 is correlated with poor survival and brain metastasis in TNBC patients. Our findings suggest that miR-211 may be used as an indicator for TNBC brain metastasis.

The blood–brain barrier (BBB) is a physical barrier that selectively prevents certain substances from entering the brain through the blood. The BBB protects the brain from germs and causes difficulty in intracranial treatment. The chemotherapy drug temozolomide (TMZ), embedded in nanobubbles (NBs) and combined with persistent luminescent nanoparticles (PLNs), has been used to treat glioblastoma (GBM) effectively through image tracking. Through ultrasound induction, NBs produce cavitation that temporarily opens the BBB. Additionally, the PLNs release near-infrared emission and afterglow, which can penetrate deep tissues and improve the signal-to-noise ratio of bioimages. In this work, the nanosystem crossed the BBB for drug delivery and image tracking over time, allowing the enhancement of the drug’s therapeutic effect on GBM. We hope that this nanosystem can be applied to the treatment of different brain diseases by embedding different drugs in NBs.

An increasing number of enzymes involved in serine biosynthesis have been identified and correlated with malignant evolution in various types of cancer. Here we showed that the overexpression of phosphoserine aminotransferase 1 (PSAT1) is widely found in lung cancer tissues compared with nontumor tissues and predicts a poorer prognosis in patients with lung adenocarcinoma. PSAT1 expression was examined in a tissue microarray by immunohistochemistry. The data show that the knockdown of PSAT1 dramatically inhibits the in vitro and in vivo metastatic potential of highly metastatic lung cancer cells; conversely, the enforced expression of exogenous PSAT1 predominantly enhances the metastatic potential of lung cancer cells. Importantly, manipulating PSAT1 expression regulates the in vivo tumor metastatic abilities in lung cancer cells. Adjusting the glucose and glutamine concentrations did not alter the PSAT1-driven cell invasion properties, indicating that this process might not rely on the activation of its enzymatic function. RNA microarray analysis of transcriptional profiling from PSAT1 alternation in CL1-5 and CL1-0 cells demonstrated that interferon regulatory factor 1 (IRF1) acts as a crucial regulator of PSAT1-induced gene expression upon metastatic progression. Decreasing the IRF1-IFIH1 axis compromised the PSAT1-prompted transcriptional reprogramming in cancer cells. Our results identify PSAT1 as a key regulator by a novel PSAT1/IRF1 axis in lung cancer progression, which may serve as a potential biomarker and therapeutic target for the treatment of lung cancer patients.

Hepatocellular carcinoma (HCC) is a common type of liver cancer that can be diagnosed by magnetic resonance imaging (MRI). If diagnosing HCC by basic MRI is difficult, then doctors use T1-weighted or T2-weighted imaging with a contrast agent that has long-term retention in the liver, such as Fe3O4or FePt, to aid in MRI diagnosis. One challenging goal of cancer prevention is developing a method that can further treat or inhibit HCC cells at the time of diagnosis. Functionalized porous kaolinite can serve not only as a drug delivery system for treating cancer cells but also as a scaffold to carry FePt nanoparticles (FePt NPs) and drugs such as doxorubicin (Dox). FePt NPs combined with kaolinite modified with cetyltrimethylammonium bromide (CTAB) can form FePt@Kao nanocomposites. Multifunctional FePt@Kao can serve as a magnetic fluid hyperthermia (MFH) agent that can also be used to simultaneously visualize and treat HCC cancer cells. After being loaded with the chemotherapeutic drug Dox, FePt@Kao-Dox can provide both MFH treatment and chemotherapy. From the systematic analysis results, we demonstrated that this functionalized FePt@Kao-Dox nanocomposite can be successfully used as a platform to integrate MRI, magnetically guided targeting, and therapeutic treatment into a multifunctional drug delivery system.

ABSTRACTHER2-ECD (human epidermal growth factor receptor 2 – extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer, but HER2-specific therapeutic options for treating advanced gastric cancer remain limited. We have developed antibody-drug conjugates (ADCs), comprising IgG1 linked via valine-citrulline to monomethyl auristatin E, with potential to treat HER2-positive gastric cancer in humans. The antibodies optimally selected from the ADC discovery platform, which was developed to discover antibody candidates suitable for immunoconjugates from synthetic antibody libraries designed using antibody-antigen interaction principles, were demonstrated to be superior immunoconjugate targeting modules in terms of efficacy and off-target toxicity. In comparison with the two control humanized antibodies (trastuzumab and H32) derived from murine antibody repertoires, the antibodies derived from the synthetic antibody libraries had enhanced receptor-mediated internalization rate, which could result in ADCs with optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models in vivoand to cultured cells in vitro. The in vivoexperiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies’ propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our in vivotests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics.

Triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. The major sites for TNBC metastasis include the lungs, brain, liver, and bone. Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nucleotides and have been reported as important regulators in BC metastasis. However, the underlying mechanisms for lncRNAs regulating TNBC metastasis are not fully understood. Here we found that linc-ZNF469-3was highly expressed in lung-metastatic LM2-4175 TNBC cells and overexpression of linc-ZNF469-3enhanced invasion ability and stemness properties in vitro and lung metastasis in vivo. Furthermore, we found linc-ZNF469-3physically interacted with miR-574-5pand overexpression of miR-574-5pattenuated ZEB1expression. Importantly, endogenous high expressions of linc-ZNF469-3and ZEB1were correlated with tumor recurrence in TNBC patients with lung metastasis. Taken together, our findings suggested that linc-ZNF469-3promotes lung metastasis of TNBC through miR-574-5p-ZEB1signaling axis and may be used as potential prognostic marker for TNBC patients.

Pyruvate kinase muscle isozymes (PKMs) have crucial roles in regulating metabolic changes during carcinogenesis. A switch from PKM1 to PKM2 isoform was thought to lead to aerobic glycolysis promoting carcinogenesis, and was considered as one of the cancer signatures. However, recent evidence has argued against the existence of PKM isoform switch and related metabolic effects during cancer progression. We compared the effects of PKM1 and PKM2 in cell invasiveness and metastasis of pancreatic ductal adenocarcinoma (PDAC). Both PKM1 and PKM2 expression affected cell migration and invasion abilities of PDAC cells, but only knockdown of PKM2 suppressed metastasis in a xenograft model. By comparing the established PKM2 mutants in the regulation of cell invasion, we found that PKM2 may control cell mobility through its protein kinase and phopho-peptide binding abilities. Further survey for PKM2-associated proteins identified PAK2 as a possible phosphorylation target in PDAC. In vitro binding and kinase assays revealed that PKM2 directly phosphorylated PAK2 at Ser20, Ser141, and Ser192/197. Knockdown of PKM2 decreased PAK2 protein half-life by increasing ubiquitin-dependent proteasomal degradation. Moreover, we identified PAK2 as an HSP90 client protein and the mutation at Ser192/197 of PAK2 reduced PAK2–HSP90 association. Knockdown of PAK2 diminished in vitro cell mobility and in vivo metastatic ability of PKM2 overexpressed PDAC cells. PKM2 and PAK2 protein expression also positively correlated with each other in PDAC tissues. Our findings indicate that PKM2–PAK2 regulation is critical for developing metastasis in PDAC, and suggest that targeting the PKM2/HSP90/PAK2 complex has a potential therapeutic value in this deadly disease.

Light therapy has become the subject of research on cancer treatment because of its selectivity, low invasive damage, and side effects. Photothermal therapy (PTT) and photodynamic therapy (PDT) are prevalent treatments used to induce cancer cell apoptosis by generating heat and reactive oxygen species (ROS). In this study, mesoporous silica shell-coated gold nanorods (AuNR@mS) are synthesized by seed crystal growth method. AuNR@mS are assembled into nanocomposites through electrostatic adsorption with lanthanide-doped upconversion nanoparticles (UCNP). When controlling the aspect ratio of gold nanorods (AuNRs), the surface plasmon resonance peaks of the short-axis and the long-axis match the maximum absorption cross section at 520 and 660 nm of the fluorescence light released by the UCNPs. The converted fluorescence stimulates AuNRs to generate heat through energy transfer. ROS production is induced by loading the photosensitizer Merocyanine 540 (MC540) in the mesoporous silica layer and is further enhanced through the surface plasma resonance effect of the AuNRs. This novel nanoplatform combines PTT and PDT in a single 808 nm near-infrared synergistic light therapy.

Photodynamic therapy (PDT) based on photosensitizers (PSs) constructed with nanomaterials has been widely applied to treat cancer. This therapy is characterized by an improved PS accumulation in tumor regions. However, challenges, such as short penetration depth of light and low extinction coefficient of PSs, limit PDT applications. In this study, a nanocomposite consisting of NaYF4:Yb/Er upconversion nanoparticles (UCPs) conjugated with gold nanorods (Au NRs) was developed to improve the therapeutic efficiency of PDT. Methylene blue (MB) was embedded in a silica shell for plasmon-enhanced PDT. UCPs served as a light converter from near-infrared (NIR) to visible light to excite MB to generate reactive oxygen species (ROS). Au NRs could effectively enhance upconversion efficiency and ROS content through a localized surface plasmon resonance (SPR) effect. Silica shell thickness was adjusted to investigate the optimized MB loading amount, ROS production capability, and efficient distance for plasmon-enhanced ROS production. The mechanism of plasmon-enhanced PDT was verified by enhancing UC luminescence intensity through the plasmonic field and by increasing the light-harvesting capability and absorption cross section of the system. This process improved the ROS generation by comparing the exchange of Au NRs to Au nanoparticles with different SPR bands. NIR-triggered nanocomposites of UCP@SiO2:MB-NRs were significantly confirmed by improving ROS generation and further modifying folic acid (FA) to develop an active component targeting OECM-1 oral cancer cells. Consequently, UCP@SiO2:MB-NRs-FA could highly produce ROS and undergo efficient PDT in vitro and in vivo. The mechanism of PDT treatment by UCP@SiO2:MB-NRs-FA was evaluated via the cell apoptosis pathway. The proposed process is a promising strategy to enhance ROS production through plasmonic field enhancement and thus achieve high PDT therapeutic efficacy.

Photodynamic therapy (PDT) is a promising antitumor treatment that is based on photosensitizers. This therapy kills cancer cells by generating reactive oxygen species (ROS) after irradiation with specific laser wavelengths. Being a potential photosensitizer, graphitic carbon nitride (g-C3N4) quantum dots (QDs) are noncytotoxic. Although the use of g-C3N4QDs is challenged by the limited tissue penetration of UV light, g-C3N4QDs display excellent ultraviolet (UV) light-triggered cytotoxicity. The g-C3N4QDs were synthesized using a solid-phase hydrothermal method. The well-distributed hydrophilic g-C3N4can be combined with NaYF4:Yb3+/Tm3+upconversion nanoparticles via the positive ligand poly(l-lysine) to produce the final nanocomposite, NaYF4:Yb/Tm-PLL@g-C3N4. Upconversion nanoparticles can transfer IR light into UV light and promote g-C3N4to release blue-to-green visible light to generate different images. Moreover, g-C3N4is a promising photosensitizer in PDT because g-C3N4can transfer oxygen into toxic ROS. The singlet oxygen formed by g-C3N4displays great potential for use in the treatment of cancer.

ABSTRACTThe functional interleukin 6 (IL-6) signaling complex is a hexameric structure composed of IL-6, IL-6Rα, and the signaling receptor gp130. There are three different modes of IL-6 signaling, classic signaling, trans-signaling, and trans-presentation, which are not functionally redundant and mediate pleiotropic effects on both physiological and pathophysiological states. Monoclonal antibodies against IL-6 or IL-6Rα have been successfully developed for clinical application. However, designing therapeutic interventions that block specific modes of IL-6 signaling in a pathologically relevant manner remains a great challenge. Here, we constructed a fusion protein Hyper-IL-6 (HyIL-6) composed of human IL-6 and IL-6Rα to develop specific blocking antibodies against the IL-6/IL-6Rα complex. We successfully screened the monoclonal antibody C14mab, which can bind to HyIL-6 with the binding constant 2.86 × 10−10and significantly inhibit IL-6/IL-6Rα/gp130 complex formation. In vitro, C14mab effectively inhibited HyIL-6-stimulated signal transducer and activator of transcription 3 (STAT3) activation and related vascular endothelial growth factor (VEGF) induction. Moreover, C14mab efficaciously suppressed HyIL-6-induced acute phase response in vivo. Our data from hydrogen-deuterium exchange mass spectrometry demonstrate that C14mab mainly binds to site IIIa of IL-6 and blocks the final step in the interaction between gp130 and IL-6/IL-6Rα complex. Additionally, data from enzyme-linked immunosorbent assays and kinetics assays indicate that C14mab interacts simultaneously with IL-6 and IL-6Rα, while it does not interact with IL-6Rα alone. The unique features of C14mab may offer a novel alternative for IL-6 blockade and illuminate a better therapeutic intervention targeting IL-6.

•Glycosaminoglycan biosynthesis pathway and CHST11, a key chondroitin sulfate biosynthetic enzyme, were up-regulated in NSCLC metastasis.•The enzymatic activity of CHST11 confers NSCLC metastasis in vitroand in vivo.•CHST11 and its downstream effector, CP facilities NSCLC metastasis in vitroand in vivo.•CHST11 promotes NSCLC metastasis via CP-iron metabolism.•The CHST11-CP-iron axis may serve as a new therapeutic target against NSCLC metastasis.

Graphical AbstractTitanium dioxide (TiO2) nanomaterials (NMs) have been widely used to develop commercial products such as sunscreen cosmetics because of their unique optical properties to provide complete protection from ultraviolet (UV) light.Titanium dioxide (TiO2) nanomaterials (NMs) have been widely used to develop commercial products such as sunscreen cosmetics because of their unique optical properties to provide complete protection from ultraviolet (UV) light. The most dangerous type of UV radiation is UVA, which comprises nearly 97% of the UV radiation that reaches the Earth. This type of radiation is also the major cause of skin damage. As the most beneficial content of sunscreen cosmetics, TiO2NMs exhibit immense capability to protect the human skin from UVA exposure through their scattering and reflecting physical properties. Therefore, investigating the factors involved in using TiO2NMs in cosmetics is necessary. In this study, various human oral and lung cell lines were selected to evaluate the cytotoxicity of treatment using different sizes and shapes of TiO2NMs, including spheres (AFDC and AFDC300) and rods (M212 and cNRs). The morphology, size, and crystalline phase of the selected TiO2NMs were studied to characterize each physical property. Based on cell viability and endocytic behavior results, treatment with all the selected TiO2NMs were nearly non-toxic to the oral cell lines. However, high cytotoxicity was obviously observed in lung cells with M212 and AFDC treatments at 50 μg mL−1, which was larger by approximately 20% than with ADC300 and cNRs treatments because the smaller the TiO2NMs, the larger their specific surface area. This condition resulted in the progress of apoptosis from the considerable aggregation of TiO2NMs in the cytoplasm. Moreover, compared with those of TiO2NMs with a similar structure (e.g., cNRs) and size (e.g., M212), the cellular uptake of AFDC was evidently low, which resulted in the approximated non-toxicity. Moreover, the similar sizes and different shapes of AFDC and cNRs were considered to treat lung cells to investigate further the influence of morphology on the cell cycle and the apoptosis effect. Consequently, AFDC and cNRs could inhibit the growth of lung cells and allow a considerable proportion of the cells to remain in the G1/G0 phase. Furthermore, a high-dose treatment would directly induce the apoptosis pathway, whereas a low-dose treatment might decrease cell regeneration.

Mouse embryonic stem cells (ES cells) can proliferate indefinitely. To identify potential signals involved in suppression of self-renewal, we previously screened a kinase/phosphatase expression library in ES cells, and observed that inhibition of Dual Leucine zipper-bearing Kinase (DLK) increased relative cell numbers. DLK protein was detected in both the pluripotent and differentiated states of mouse ES cells while DLK kinase activity increased upon differentiation. Overexpression of DLK in mouse ES cells displayed reductions in relative cell/colony numbers and Nanog expression, suggesting a suppressive role of DLK in self-renewal. By examining protein sequences of DLK, we identified 2 putative Akt phosphorylation sites at S584 and T659. Blocking PI3K/Akt signaling with LY-294002 enhanced DLK kinase activity dramatically. We found that Akt interacts with and phosphorylates DLK. Mutations of DLK amino acid residues at putative Akt phosphorylation sites (S584A, T659A, or S584A and T659A) diminished the level of DLK phosphorylation. While the mutated DLKs (S584A, T659A, or S584A and T659A) were expressed, a further reduction in cell/colony numbers and Nanog expression appeared in mouse ES cells. In addition, these mutant DLKs (S584A, T659A, or S584A and T659A) exhibited more robust kinase activity and cell death compared to wild type DLK or green fluorescence (GFP) controls. In summary, our results show that DLK functions to suppress self-renewal of mouse ES cells and is restrained by Akt phosphorylation.

Ribosome biogenesis governs protein synthesis. NIFK is transactivated by c-Myc, the key regulator of ribosome biogenesis. The biological function of human NIFK is not well established, except that it has been shown to interact with Ki67 and NPM1. Here we report that NIFK is required for cell cycle progression and participates in the ribosome biogenesis via its RNA recognition motif (RRM). We show that silencing of NIFK inhibits cell proliferation through a reversible p53-dependent G1 arrest, possibly by induction of the RPL5/RPL11-mediated nucleolar stress. Mechanistically it is the consequence of impaired maturation of 28S and 5.8S rRNA resulting from inefficient cleavage of internal transcribed spacer (ITS) 1, a critical step in the separation of pre-ribosome to small and large subunits. Complementation of NIFK silencing by mutants shows that RNA-binding ability of RRM is essential for the pre-rRNA processing and G1 progression. More specifically, we validate that the RRM of NIFK preferentially binds to the 5′-region of ITS2 rRNA likely in both sequence specific and secondary structure dependent manners. Our results show how NIFK is involved in cell cycle progression through RRM-dependent pre-rRNA maturation, which could enhance our understanding of the function of NIFK in cell proliferation, and potentially also cancer and ribosomopathies.

Recently, CuInS2quantum dots (CIS QDs) are extensively applied in biological applications because of their distinctive optical property. These novel ternary semiconductor CIS QDs can be developed into good biomarkers or trackers because they do not contain cadmium, unlike CdTe and CdSe QDs with high risk for cytotoxicity. However, reports on toxicity and effective factors affecting CIS QDs are seldom developed, and in vivo chemical stability has not been clearly investigated. In this study, we focused on the fate, degradation, and exposure time of CIS QDs in Caenorhabditis elegans(C. elegans), which is used as a model organism in biology. Moreover, X-ray absorption near-edge structure (XANES) is used to identify the oxidation state of CIS and CIS/ZnS QDs in various exposure times. The purpose was to use different oxidation states of copper and zinc ions of QDs to achieve chemical stability in C. elegans. CIS and CIS/ZnS QDs were synthesized by hydrothermal method, and QDs were transferred to aqueous solution by coating with O-carboxymethylchitosan (OCMCS). Moreover, intracellular uptake and cell viability tests were estimated as preliminary experiments for in vitro cytotoxicity testing. Our results showed that the supported QD materials can be applied in biological systems. Consequently, we further considered the function of QD materials in C. elegans. The QD materials of coating OCMCS could be successfully delivered to the interior of the C. elegansthrough the alimentary system in a manner dependent on the exposure time. Most importantly, XANES results revealed that the oxidative state of CIS QDs did not change without an outer layer after treatment for 96 h in C. elegans. Therefore, the extreme chemical stability of CIS QDs may explain the low cytotoxicity in the organism and thus has potential biomedical applications.

Titanium dioxide (TiO2) nanomaterials (NMs) have been widely used to develop commercial products such as sunscreen cosmetics because of their unique optical properties to provide complete protection from ultraviolet (UV) light. The most dangerous type of UV radiation is UVA, which comprises nearly 97% of the UV radiation that reaches the Earth. This type of radiation is also the major cause of skin damage. As the most beneficial content of sunscreen cosmetics, TiO2NMs exhibit immense capability to protect the human skin from UVA exposure through their scattering and reflecting physical properties. Therefore, investigating the factors involved in using TiO2NMs in cosmetics is necessary. In this study, various human oral and lung cell lines were selected to evaluate the cytotoxicity of treatment using different sizes and shapes of TiO2NMs, including spheres (AFDC and AFDC300) and rods (M212 and cNRs). The morphology, size, and crystalline phase of the selected TiO2NMs were studied to characterize each physical property. Based on cell viability and endocytic behavior results, treatment with all the selected TiO2NMs were nearly non-toxic to the oral cell lines. However, high cytotoxicity was obviously observed in lung cells with M212 and AFDC treatments at 50 μg mL−1, which was larger by approximately 20% than with ADC300 and cNRs treatments because the smaller the TiO2NMs, the larger their specific surface area. This condition resulted in the progress of apoptosis from the considerable aggregation of TiO2NMs in the cytoplasm. Moreover, compared with those of TiO2NMs with a similar structure (e.g., cNRs) and size (e.g., M212), the cellular uptake of AFDC was evidently low, which resulted in the approximated non-toxicity. Moreover, the similar sizes and different shapes of AFDC and cNRs were considered to treat lung cells to investigate further the influence of morphology on the cell cycle and the apoptosis effect. Consequently, AFDC and cNRs could inhibit the growth of lung cells and allow a considerable proportion of the cells to remain in the G1/G0 phase. Furthermore, a high-dose treatment would directly induce the apoptosis pathway, whereas a low-dose treatment might decrease cell regeneration.

Recent studies have indicated that cancer stem-like cells (CSCs) exhibit a high resistance to current therapeutic strategies, including photodynamic therapy (PDT), leading to the recurrence and progression of colorectal cancer (CRC). In cancer, autophagy acts as both a tumor suppressor and a tumor promoter. However, the role of autophagy in the resistance of CSCs to PDT has not been reported. In this study, CSCs were isolated from colorectal cancer cells using PROM1/CD133 (prominin 1) expression, which is a surface marker commonly found on stem cells of various tissues. We demonstrated that PpIX-mediated PDT induced the formation of autophagosomes in PROM1/CD133+cells, accompanied by the upregulation of autophagy-related proteins ATG3, ATG5, ATG7, and ATG12. The inhibition of PDT-induced autophagy by pharmacological inhibitors and silencing of the ATG5gene substantially triggered apoptosis of PROM1/CD133+cells and decreased the ability of colonosphere formation in vitro and tumorigenicity in vivo. In conclusion, our results revealed a protective role played by autophagy against PDT in CSCs and indicated that targeting autophagy could be used to elevate the PDT sensitivity of CSCs. These findings would aid in the development of novel therapeutic approaches for CSC treatment.

SOCS1 inactivation may be caused by Gfi‐1 expression in AML cells; inhibition of H3K9 methyltransferase can rescue SOCS1 expression. Silencing of SOCS1, a TSG, has been detected in various malignancies, including AML. However, the underlying mechanism of SOCS1inactivation remains elusive. In this study, we explored the role of histone methylation in SOCS1expression in AML cells. By ChIP assay, we demonstrated that G9a and SUV39H1, two enzymes catalyzing H3K9 methylation, were physically associated with the SOCS1promoter, and treatment with chaetocin, a histone methyltransferase inhibitor, suppressed H3K9 methylation on the SOCS1promoter and enhanced SOCS1expression. Furthermore, knockdown of G9aand SUV39H1by siRNA could also induce SOCS1expression. On the other hand, SOCS1 knockdown by shRNA eliminated chaetocin‐induced cell apoptosis. To investigate further whether any transcription factor was involved in H3K9 methylation‐related SOCS1repression, we scanned the sequences of the SOCS1gene promoter and found two binding sites for Gfi‐1, a transcription repressor. By DNA pull‐down and ChIP assays, we showed that Gfi‐1 directly bound the SOCS1promoter, and ectopic Gfi‐1 expression suppressed STAT5‐induced SOCS1promoter activation. In contrast, Gfi‐1knockdown by shRNA enhanced SOCS1 expression and inhibited STAT5 expression. Moreover, the knockdown of G9acompletely rescued the repressive effect of Gfi‐1 on STAT5A‐induced SOCS1promoter activation. Collectively, our study indicates that the expression of Gfi‐1 contributes to SOCS1silencing in AML cells through epigenetic modification, and suppression of histone methyltransferase can provide new insight in AML therapy.

High‐throughput short‐hairpin RNA (shRNA) lentivirus screening is a powerful tool for identifying multiple functional regulators in embryonic stem cells (ESCs). shRNA libraries can efficiently down‐regulate target genes persistently with high efficiency. The concurrent measurement of relative cell number by alamarBlue (AB) assay and undifferentiated ESC markers via an alkaline phosphatase (ALP) activity assay in the same cell culture well provides an efficient and economical way to pinpoint factors crucial for ESC pluripotency and/or expansion. Most of the renewal pathways affect ALP activity. Thus, multiple positive and negative regulators can be identified by this method. In addition, morphological changes and/or the expression levels of specific pluripotency or differentiation markers examined by immunofluorescence can be used as secondary screens for target‐gene selection. In summary, we describe an efficient way to identify multiple regulators of ESC renewal using shRNAs. Curr. Protoc. Stem Cell Biol. 26:5C.3.1‐5C.3.19. © 2013 by John Wiley & Sons, Inc.

Deciphering the network of signaling pathways in cancer via protein-protein interactions (PPIs) at the cellular level is a promising approach but remains incomplete. We used an in situproximity ligation assay to identify and quantify 67 endogenous PPIs among 21 interlinked pathways in two hepatocellular carcinoma (HCC) cells, Huh7 (minimally migratory cells) and Mahlavu (highly migratory cells). We then applied a differential network biology analysis and determined that the novel interaction, CRKL-FLT1, has a high centrality ranking, and the expression of this interaction is strongly correlated with the migratory ability of HCC and other cancer cell lines. Knockdown of CRKL and FLT1 in HCC cells leads to a decrease in cell migration via ERK signaling and the epithelial-mesenchymal transition process. Our immunohistochemical analysis shows high expression levels of the CRKL and CRKL-FLT1 pair that strongly correlate with reduced disease-free and overall survival in HCC patient samples, and a multivariate analysis further established CRKL and the CRKL-FLT1 as novel prognosis markers. This study demonstrated that functional exploration of a disease network with interlinked pathways via PPIs can be used to discover novel biomarkers.

The synthesis and degradation of hBora is important for the regulation of mitotic entry and exist. In G2phase, hBora can complex with Aurora A to activate Plk1 and control mitotic entry. However, whether the post-translational modification of hBora is relevant to the mitotic entry still unclear. Here, we used the LC-MS/MS phosphopeptide mapping assay to identify 13 in vivo hBora phosphorylation sites and characterized that GSK3β can interact with hBora and phosphorylate hBora at Ser274 and Ser278. Pharmacological inhibitors of GSK3β reduced the retarded migrating band of hBora in cells and diminished the phosphorylation of hBora by in vitro kinase assay. Moreover, as well as in GSK3β activity-inhibited cells, specific knockdown of GSK3β by shRNA and S274A/S278 hBora mutant-expressing cells also exhibited the reduced Plk1 activation and a delay in mitotic entry. It suggests that GSK3β activity is required for hBora-mediated mitotic entry through Ser274 and Ser278 phosphorylation.

The preparation and properties of plasmonic gold nanostars (Au NSs) modified with a biopolymer chitosan are reported. The colloidal stability of Au NSs at the physiological pH of 7.5 and their performance in the photothermolysis of cancer cells in vitro were compared with those of gold nanorods (Au NRs). The optical characteristics of chitosan-modified Au NSs dispersed in a medium with pH = 7.5 had higher stability than those of chitosan-capped NRs because of the slower aggregation of NSs. At pH = 7.5, the chitosan-modified Au NRs formed aggregates with highly nonuniform sizes. On the other hand, Au NSs formed small chain-like clusters, in which individual NSs were connected to one another, preferably via association of branches with central cores. It is possible that the difference in areal charge density at these parts of NSs is responsible for their preferred association. Flow cytometry analysis showed the relatively nonequivalent distribution of the chitosan-capped Au NRs across the cell line compared with that of Au NSs because of the fast and nonuniform aggregation of NRs. An in-vitro photothermolysis experiment on J5 cancer cells showed that energy fluences of 23 and 33 mJ/cm2are necessary to cause complete death of J5 cells incubated with 4 μg/mL chitosan-capped Au NSs and NRs, respectively. When chitosan was used as a surface-capping agent, the Au NSs exhibited higher colloidal stability at the physiological pH than the NRs and lower energy fluence necessary for cell photothermolysis because of more uniform cellular uptake.

In contrast to the somatic cells, embryonic stem cells (ESCs) are characterized by its immortalization ability, pluripotency, and oncogenicity. Revealing the underlying mechanism of ESC characteristics is important for the application of ESCs in clinical medicine. We performed systematic functional screen in mouse ESCs with 4,801 shRNAs that target 929 kinases and phosphatases. One hundred and thirty‐two candidate genes that regulate both ESC expansion and stem cell marker expression were identified. Twenty‐seven out of the 132 genes were regarded as most important since knockdown of each gene induces morphological changes from undifferentiated to differentiated state. Among the 27 genes, we chose nonmetastatic cell 6 (Nme6, also named as Nm23‐H6) and nonmetastatic cell 7 (Nme7, also designated as Nm23‐H7) to study first. Nme6 and Nme7 both belong to the members of nucleoside diphosphate kinase family. We demonstrate that Nme6 and Nme7 are important for the regulation of Oct4, Nanog, Klf4, c‐Myc, telomerase, Dnmt3B, Sox2, and ERas expression. Either knockdown of Nme6 or Nme7 reduces the formation of embryoid body (EB) and teratoma. The overexpression of either Nme6 or Nme7 can rescue the stem cell marker expression and the EB formation in the absence of leukemia inhibiting factor. This implies the importance of Nme6 and Nme7 in ESC renewal. This finding not only pinpoints Nme6 or Nme7 can regulate several critical regulators in ESC renewal but also increases our understanding of the ESC renewal and oncogenesis. STEMCells2012;30:2199–2211

Although the overproduction of immunoglobulins by short-lived plasma cells accompanying an immune response links with their apoptosis, how long-lived plasma cells adapt to ensure their longevity in this context is obscure. Here, we show that apoptosis signal–regulating kinase 1 (ASK1) contributes to apoptosis of plasma cells because ASK1 activity was induced during differentiation of short-lived plasma cells, and, when produced by ASK1-deficient mice, these cells survived better than those of control mice. Moreover, antigen-specific long-lived plasma cells generated by immunization accumulated in ASK1-deficient mice, suggesting ASK1 also plays a negative role in survival of long-lived plasma cells. In malignant plasma cells, ASK1 transcription was directly suppressed by B lymphocyte–induced maturation protein-1 (Blimp-1). The expression of ASK1 and Blimp-1 showed an inverse correlation between normal human mature B cells and bone marrow plasma cells from patients with multiple myeloma (MM). Suppression of ASK1 is crucial for cell survival because its enforced expression in MM cells caused apoptosis in vitro and lowered MM load in a xenograft animal model; furthermore, alteration of ASK1 activity affected MM cell survival. Our findings indicate a novel mechanism underlying the regulation of survival in normal and malignant plasma cells by ASK1.

Abstract: With the advent of advanced program analysis and constraint solving techniques, several test generation tools use variants of symbolic execution. Symbolic techniques have been shown to be very effective in path-based test generation; however, they fail to scale to large programs due to the exponential number of paths to be explored. In this paper, we focus on tackling this path explosion problem and propose search strategies to achieve quick branch coverage under symbolic execution, while exploring only a fraction of paths in the program. We present a reachability-guided strategy that makes use of the reachability graph of the program to explore unvisited portions of the program and a conflict-driven backtracking strategy that utilizes conflict analysis to perform nonchronological backtracking. We present experimental evidence that these strategies can significantly reduce the search space and improve the speed of test generation for programs.

Minocycline has been shown to alleviate several neurological disorders. Unexpectedly, we found that minocycline had opposite effects on glioma cells: minocycline induced nonapoptotic cell death in glioma cells. The glioma cell death was associated with the presence of autophagic vacuoles in the cytoplasm. Minocycline induced autophagy was confirmed by acridine orange, monodansylcadaverine (MDC) stainings of vesicle formation and the conversion of microtubule-associated proteins light chain 3 (LC3-I) to LC3-II. Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) suppressed the induction of acidic vesicular organelles and the accumulation of LC3-II to the autophagosome membrane in glioma cells treated with minocycline. Despite the pretreatment of 3-MA, minocycline induced cell death which could result from the activation of caspase-3. Minocycline effectively inhibited tumor growth and induced autophagy in the xenograft tumor model of C6 glioma cells. These results suggest that minocycline may kill glioma cells by inducing autophagic cell death. When autophagy was inhibited, minocycline still induced cell death through the activation of caspase-3. Thus, minocycline is a promising agent in the treatment of malignant gliomas.

Herein we report biorecognition studies of protein IgG using biocomapatible gold nanorods as molecular probes. Surface modification of cetyltrimethylammonium bromide (CTAB)-stabilized gold nanorods was carried out by using poly(styrenesulfonate) (PSS) to reduce the toxicity of as-synthesized gold nanorods caused by free CTAB. ζ potential analysis confirmed charge reversal on the surface of gold nanorods caused by the PSS coating. Surface plasmon resonance exhibited by gold nanorods has been employed as a tool for analyzing the binding events for biomolecules. TEM results, showing the aggregation of gold nanorods, in addition to the shift in surface plasmon resonance peak in UV−vis absorption measurements, upon the interaction of biomolecules with gold nanorods, confirmed molecular binding. Morphological changes caused by the cellular uptake of gold nanorods before and after PSS modification have been observed. Cell viability studies using gold nanorods were performed to study the cytotoxic effects of these molecular probes.

We report here the development of a seed-mediated growth method for the synthesis of iron oxide nanoparticles with tunable size distribution and magnetic properties. We have investigated the size distribution of seed as well as iron oxide nanoparticles during the growth process using transmission electron microscopy (TEM). It has been observed that the distribution of size gradually becomes narrow with time via the intraparticle ripening process and Oswald ripening process. The magnetization measurements were performed using a superconducting quantum interference device (SQUID) from room temperature to 5 K to determine the effect of size distribution on the blocking temperature ( Tb) and splitting temperature ( Tsplitting). The thermogravimetric analyses exhibited the size-dependent weight loss of the magnetic nanoparticles. The in vitro cytotoxicity tests were also performed to determine the cell viability as a function of size and concentration of the magnetic nanoparticles.

Due to the widespread demands for efficient SAT solvers in Electronic Design Automation applications, methods to boost the performance of the SAT solver are highly desired. We propose a Hybrid Solution to boost SAT solver performance in this paper, via an integration of local and DPLL-based search approaches. A local search is used to identify a subset of clauses from the original formula to be passed to a DPLL SAT solver incrementally until all the clauses have been passed. In addition, the solution obtained by the DPLL solver on the subset of clauses is fed back to the local search solver to jump over any locally optimal points. The proposed solution is highly portable to the existing SAT solvers. For satisfiable instances, up to an order of magnitude speedup was obtained via the proposed hybrid solver. For unsatisfiable instances, the speedup was smaller due to the overhead.

Carbon monoxide (CO) has been identified as an endogenous biological messenger in the brain. Heme oxygenase (HO) catalyzes the metabolism of heme to CO and biliverdin. Previously, we have shown the involvement of CO in central cardiovascular regulation, baroreflex modulation, and glutaminergic neuro-transmission in the nucleus tractus solitarii (NTS) of rats. In this study, we examined which HO isoform could be induced after hemin injection in the NTS. We also investigated their in situ distributions in the NTS after induction. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure was monitored intra-arterially. Unilateral microinjection of hemin (1 nmol), a heme molecule cleaved by HO to yield CO, produced significant decrease in blood pressure and heart rate. These cardiovascular effects of hemin were attenuated by prior administration of HO inhibitor zinc protoporphyrin IX (ZnPPIX). Microinjection of hemin into NTS resulted in significant induction of HO-1 protein expression in situ. Pretreatment of ZnPPIX significantly inhibited the HO-1 induction after hemin injection. No significant changes of HO-2 expression were found after hemin injection and ZnPPIX pretreatment. The in situ inductions of the HO-1 protein expression were further confirmed to be in glial cells and neurons after hemin injections into the NTS. These results indicated HO-1 but not HO-2 might be responsible for the generation of CO and contribute to central control of cardiovascular effects.

Scan-based transition tests are added to improve the detection of speed failures in sequential circuits. Empirical data suggests that both data volume and application time will increase dramatically for such transition testing. Techniques to address the above problem for a class of transition tests, called enhanced transition tests, are proposed in this article.The first technique, which combines the proposed transition test chains with the ATE repeat capability, reduces test data volume by 46.5&percent; when compared with transition tests computed by a commercial transition test ATPG tool. However, the test application time may sometimes increase. To address the test time issue, a new DFT technique, Exchange Scan, is proposed. Exchange scan reduces both data volume and application time by 46.5&percent;. These techniques rely on the use of hold-scan cells and highlight the effectiveness of hold-scan design to address test time and test data volume issues. In addition, we address the problem of yield loss due to incidental overtesting of functionally-untestable transition faults, and we formulate an efficient adjustment to the algorithm to keep the overtest ratio low. Our experimental results show that up to 14.5&percent; reduction in overtest ratio can be achieved, with an average overtest reduction of 4.68&percent;.

Abstract Algorithms to locate multiple design errors using region-based model are studied for both combinational and sequential circuits. The model takes locality aspect of errors and is based on a 3-value, non-enumerative analysis technique. Studies show the effectiveness of the region based model for single and multiple stuck faults and gate connection errors. For sequential circuits, we try to locate the time frame at which the error was first excited, by re-simulating as few vectors as possible preceding the erroneous vector in a fully initialized circuit to carry out the diagnosis. Experimental results on benchmark circuits are used to demonstrate rapid and accurate locating of multiple errors.

ObjectiveNitric oxide (NO) and carbon monoxide (CO) are endogenously synthesized gaseous molecules that act as neurotransmitters in both central and peripheral nervous systems. Previously, we have shown the involvement of NO and CO in central cardiovascular regulation and baroreflex modulation. In this study we investigated the possible interaction of NO and CO in the nucleus tractus solitarii (NTS) on cardiovascular effects in rats.

This paper proposes novel algorithms for computing test patterns for transition faults in combinational circuits and fully scanned sequential circuits. The algorithms are based on the principle that s@ vectors can be effectively used to construct good quality transition test sets. Several algorithms are discussed. Experimental results obtained using the new algorithms show that there is a 20% reduction in test set size, test data volume and test application time compared to a state-of-the-art native transition test ATPG tool, without any reduction in fault coverage. Other benefits of our approach, viz. productivity improvement, constraint handling and design data compression are highlighted.

This experiment was undertaken to determine the role of macrophage‐derived nitric oxide (NO) in mediating lipopolysaccharide (LPS)‐induced bone resorption by using an in vitro co‐culture system and an in vivo model of infectious bone resorption. Our results demonstrated that LPS stimulated the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)‐α mRNAs and nitrite synthesis in the J774 mouse macrophage cell line but not in the UMR‐106 (rat) and MC3T3‐E1 (mouse) osteoblast cell lines. Conditioned media (CM) from LPS‐stimulated J774 triggered only low to moderate levels of iNOS mRNAs in MC3T3‐E1 and a trivial effect in UMR‐106. On the other hand, CM induced matrix metalloproteinase‐1 (MMP‐1) gene expression in both osteoblast cell lines. The NOS inhibitor NG‐monomethyl‐L‐arginine (L‐NMMA) did not alter this effect in MC3T3‐E1 and UMR‐106, whereas TNF‐α antibody diminished the CM‐induced MMP‐1 gene expression in both cell lines. Interestingly, SNAP, a NO donor, although by itself is not a MMP‐1 stimulator for UMR‐106, augmented the TNF‐α‐stimulated MMP‐1 mRNA production in UMR‐106. In a J774/UMR‐106 co‐culture system, LPS stimulated significant MMP‐1 gene expression in UMR‐106, and this upregulation was abolished by L‐NMMA and TNF‐α antibodies. Immunohistochemical analysis in a rat model of infectious bone resorption (periapical lesion) showed co‐distributions of iNOS+macrophages and MMP‐1+osteoblasts around the osteolytic areas. Administration of L‐NMMA markedly reduced the extent of bone loss and the percentage of MMP‐1‐synthesizing osteoblasts. These data suggest that NO derived from macrophages after LPS stimulation may enhance bone loss by augmenting the cytokine‐induced MMP‐1 production in osteoblasts.

The focus of this research is on the testability analysis of the operators in the behavioral description prior to synthesis. The controllabilities of the inputs to an operator and the observabilities of the outputs of the operation are computed from the value ranges of the variables that serve as the inputs and outputs. Candidate hard-to-test operations are selected for testability enhancement based on the computed testability measures for all the involved operations in the behavioral description. While value ranges have been applied to compute variable testability measures, this paper addresses computation of the operator testability. Experimental results show that circuits synthesized using our technique exhibit higher testability than variable selection while keeping the area-performance overhead to a minimum.

We present a new test generation procedure for sequential circuits using newly traversed state and newly detected fault information obtained between successive iterations of vector compaction. Two types of techniques are considered. One is based on the new states a sequential circuit is driven into, and the other is based on the new faults that are detected between consecutive iterations of vector compaction. These data modify an otherwise random selection of vectors, to bias vector sequences that cause the circuit to reach new states, and cause previously undetected faults to be detected. The biased vectors, when used to extend the compacted test set, provide a more intelligent selection of vectors. The extended test set is then compacted. Repeated applications of state and fault analysis, vector generation and compaction produce significantly high fault coverage using relatively small computing resources. We obtained improvements in terms of higher fault coverage, fewer vectors for the same coverage, or smaller number of iterations and time required, consistently for several benchmark circuits.

Background:Double pylorus can occur as either a congenital abnormality or an acquired complication of peptic ulcer disease. There has been no report of long-term follow-up of a large series of patients treated for double pylorus. The objective of this study was to determine the course of double pylorus in 20 patients by serial endoscopic examination. Methods:Among 102,958 endoscopic examinations conducted from 1987 to 1999, a diagnosis of double pylorus was made in 20 patients, 18 of whom were followed from 2 months to 10 years. The demographics, clinical presentations, and outcome, as well as endoscopic patterns of double pylorus, were retrospectively reviewed. The rates of ulcer recurrence and symptoms were estimated and compared. Results:Most fistulous rings were located on the lesser curve of the gastric antrum (75%). Evolution from an original ulcer to fistula was observed in 9 patients. The fistula disappeared in 1 patient, remained open in 12, and converged with the normal pyloric ring in 5 patients. One or more associated systemic diseases and extensive treatment with various drugs were noted in 12 patients. Eradication of Helicobacter pyloriin infected patients resulted in a lower percentage of patients with symptoms (36% vs. 100%) and ulcer recurrence (55% vs. 100%) compared with uninfected patients, but the differences were not statistically significant. Conclusion:In this study, fistula closure did not occur in the majority of patients. Associated systemic diseases and extensive use of medications might be important factors in persistence of double pylorus. Eradication of Helicobacter pyloriwas not beneficial in terms of relief of symptoms, prevention of ulcer recurrence, and fistula closure. Surgical intervention should be considered for patients with refractory symptoms, recurrent ulcers, and other complications. (Gastrointest Endosc 2001;54:464-70.)

The mutation and/or deletion of tumor suppressor genes have been postulated to play a major role in the genesis and the progression of gliomas. In this study, the functional expression and efficacy in tumor suppression of 3 tumor suppressor genes (p53, p21, and p16) were tested and compared in a rat GBM cell line (RT-2) after retrovirus mediated gene delivery in vitroand in vivo.Significant reductions in tumor cell growth rate were found in p16 and p21 infected cells (60 ± 12% vs 66 ± 15%) compared to p53 (35 ± 9%). In vitrocolony formation assay also showed significant reductions after p16 and p21 gene delivery (98 ± 5% vs 91 ± 10%) compared to p53 (50 ± 18%). In addition, the tumor suppression efficacy were investigated and compared in vivo.Retroviral mediated p16 and p21 gene deliveries in glioblastomas resulted in more than 90% reductions in tumor growth (92 ± 26% vs 90 ± 22%) compared to p53 (62 ± 18%). Tumor suppressor gene insertions in situfurther prolonged animal survival. Overall p16 and p21 genes showed more powerful tumor suppressor effects than p53. The results were not surprising, as p16 and p21 are more downstream in the cell cycle regulatory pathway compared to p53. Moreover, the mechanism involved in each of their suppressor effects is different. This study demonstrates the feasibility of using tumor suppressor genes in regulating the growth of glioma in vitroand in situ.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to reduce the risk and mortality of colorectal cancer (CRC). Although the exact mechanisms remain unclear, the inhibition of cyclooxygenase (COX) by NSAIDs appears to abort, if not prevent, CRC carcinogenesis or metastatic tumor progression. The aim of our study was to investigate the association between COX-2 expression and CRC tumor cell invasiveness. The differences in immunoblot-detectable COX-2 protein contents in primary CRCs, metastatic hepatic lesions and corresponding normal mucosa from the same individual were evaluated in 17 patients. Three different colon cancer cell lines, SW620, Lovo, HT-29 and a metastatic variant of HT-29, HT-29/Inv3, were employed to evaluate COX-2 expression and prostaglandin E2 (PGE2) production in relation to their invasive abilities in vitro. The effects of a COX-2–selective inhibitor, etodolac, on cell proliferation and invasive activity were also determined. The results showed that 15 of 17 (88%) metastatic CRC cells from the liver and 14 of 17 (82%) primary CRC tissue exhibited much higher levels of COX-2 than corresponding adjacent normal mucosa from the same patient. Among those patients with relatively high COX-2 expression in the primary tumors, almost all exhibited even higher levels of COX-2 in their hepatic metastases. Among the 4 colon cancer cell lines, HT-29/Inv3 manifested the highest COX-2 expression, PGE2 production and in vitro invasive activity. The selective COX-2 inhibitor, etodolac, could especially exert cytotoxicity and markedly suppress the invasive property and PGE2 production, although not the COX-2 protein level, in HT-29/Inv3 cells. Our results imply that COX-2 expression may be associated with the invasive and metastatic properties of CRC tumor cells. © 2001 Wiley-Liss, Inc.