The toxicity of dysregulated Plk1 activity revealed by its suppressor mutations.

Polo-like kinase 1 (Plk1) is a mitotic kinase that has multiple functions throughout the cell cycle. Catalytic activation of Plk1 is known to be regulated by phosphorylation of the kinase domain, including Thr210, and by releasing the kinase domain from its inhibitory polo-box domain. However, how Plk1 is activated to fulfill its proper roles, in time and space, is not well understood. In this study, we unintentionally found that the expression of a constitutively active form of human Plk1 is toxic to bacterial cells, such that cells contained point mutations that alleviate the kinase activity. Structural prediction revealed that these mutations are adjacent to the amino acids supporting the kinase activity. When human cells express these mutants, we found decreased levels of Plk1's substrate phosphorylation, resulting in mitotic defects. Moreover, unlike in bacterial cells, the expression of activated Plk1 mutants did not affect cell proliferation in human cells unless localized at the right place in mitosis. Our observations identified new suppressor mutations and underscored the importance of spatiotemporal regulation in Plk1, providing a basis for how we might intervene in this kinase for therapeutic purpose in human cells.

Assessment of oral hypofunction and its association with age among Korean community-dwelling older adults.

BACKGROUND: Due to the increasing proportion of older adults in Korea and growing interest in aging, the concepts of oral aging and oral hypofunction have recently been introduced. Thus, it is necessary to investigate the age-specific oral function levels of Korean older adults and develop expert intervention methods for healthy aging. METHODS: Dysphagia, independence of daily living, and oral hypofunction were assessed in 206 older adults living in Wonju, Gangwon State, South Korea. Subjective dysphagia was assessed through self-report questionnaires using the Dysphagia Handicap Index (DHI), the Korean version of Eating Assessment Tool-10, and the Korean version of the Modified Barthel Index. In addition, the oral hypofunction assessment items included decreased chewing ability, occlusal pressure, tongue pressure, oral dryness, and oral cleanliness. RESULTS: DHI increased significantly with age, with those in their 80 s reporting the most difficulty swallowing. Oral function in terms of chewing ability (maximum occlusal pressure and number of remaining teeth), maximum occlusal pressure, and maximum tongue pressure also declined with increasing age. While there was no significant difference in oral dryness by age, those in their 80 s had dry mouth according to the criteria of the oral moisture checking device. CONCLUSIONS: In an assessment of oral function in community-dwelling, independent Korean older adults, the number of items that were assessed as oral hypofunction increased with age. The findings can be used to standardize the oral hypofunction assessment item and develop age-based individualized intervention plans for the early management of oral health and individual oral myofunctional rehabilitation in Korean community-dwelling older adults.

Profiling chromosomal-level variations in gastric malignancies.

Aneuploidy arises from persistent chromosome segregation errors, or chromosomal instability. Although it has long been known as a hallmark of cancer cells, reduced cellular fitness upon induced ploidy alterations hinders the understanding of how aneuploidy relates to cancer development in the body. In this study, we used FISH analysis targeting centromeres to indicate ploidy changes, and quantitatively evaluated the ploidy statuses of gastric tumors derived from a total of 214 patients, ranging from early to advanced disease. We found that cancer cells reveal a marked elevation of aneuploid population, increasingly in cases diagnosed in advanced stages. The expansion of the aneuploid population is well associated with p53 deficiency, consistent with its essential role in genome maintenance. Comparisons among multiple locations within the tumor, or between the primary and metastatic tumors, indicated that cancer cells mostly retain their ploidy alterations throughout primary tumors, but metastatic tumors may consist of cells with either increased or decreased levels of aneuploidy. We also found that a notable proportion of polyploid cells are often already present in chronic gastritis epithelia. These observations underscore that chromosome-level variations are widespread in gastric cancers, shaping their genetic heterogeneity and malignant properties.

Mechanisms of palmitic acid-conjugated antisense oligonucleotide distribution in mice.

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1-/-) and FcRn knockout (FcRn-/-).

Unraveling pathologies underlying chromosomal instability in cancers.

Aneuploidy is a widespread feature of malignant tumors that arises through persistent chromosome mis-segregation in mitosis associated with a pathological condition called chromosomal instability, or CIN. Since CIN is known to have a causal relationship with poor prognosis accompanying by multi-drug resistance, tumor relapse, and metastasis, many research groups have endeavored to understand the mechanisms underlying CIN. In this review, we overview possible etiologies of CIN. The key processes to achieve faithful chromosome segregation include the regulation of sister chromatid cohesion, kinetochore-microtubule attachment, bipolar spindle formation, spindle-assembly checkpoint, and the activity of separase. Aberrant chromosome structures during DNA replication might also be a potential cause of CIN. Defective regulation in these processes can lead to chromosome mis-segregation, manifested by lagging chromosomes, and DNA bridges in anaphase, leading to gross chromosome rearrangements. Investigation into the molecular etiologies of CIN should allow us to explore novel strategies to intervene in CIN to control cancers.

Enhanced Potency of GalNAc-Conjugated Antisense Oligonucleotides in Hepatocellular Cancer Models.

Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (-) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC.

Precise tuning of the glyoxylate cycle in Escherichia coli for efficient tyrosine production from acetate.

BACKGROUND: Acetate is one of promising feedstocks owing to its cheap price and great abundance. Considering that tyrosine production is gradually shifting to microbial production method, its production from acetate can be attempted to further improve the economic feasibility of its production. RESULTS: Here, we engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux distribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield which are 1.6-fold and 1.9-fold increased values of the parental strain. CONCLUSIONS: Tyrosine production from acetate requires precise tuning of the glyoxylate cycle and we obtained substantial improvements in production titer and yield by synthetic promoters and 5' untranslated regions (UTRs). This is the first demonstration of tyrosine production from acetate. Our strategies would be widely applicable to the production of various chemicals from acetate in future.

In vitro site-specific recombination mediated by the tyrosine recombinase XerA of Thermoplasma acidophilum.

In organisms with circular chromosomes, such as bacteria and archaea, an odd number of homologous recombination events can generate a chromosome dimer. Such chromosome dimers cannot be segregated unless they are converted to monomers before cell division. In Escherichia coli, dimer-to-monomer conversion is mediated by the paralogous XerC and XerD recombinases at a specific dif site in the replication termination region. Dimer resolution requires the highly conserved cell division protein/chromosome translocase FtsK, and this site-specific chromosome resolution system is present or predicted in most bacteria. However, most archaea have only XerA, a homologue of the bacterial XerC/D proteins, but no homologues of FtsK. In addition, the molecular mechanism of XerA-mediated chromosome resolution in archaea has been less thoroughly elucidated than those of the corresponding bacterial systems. In this study, we identified two XerA-binding sites (dif1 and dif2) in the Thermoplasma acidophilum chromosome. In vitro site-specific recombination assays showed that dif2, but not dif1, serves as a target site for XerA-mediated chromosome resolution. Mutational analysis indicated that not only the core consensus sequence of dif2, but also its flanking regions play important roles in the recognition and recombination reactions mediated by XerA.

uPAR induces expression of transforming growth factor ß and interleukin-4 in cancer cells to promote tumor-permissive conditioning of macrophages.

Cancer cells condition macrophages and other inflammatory cells in the tumor microenvironment so that these cells are more permissive for cancer growth and metastasis. Conditioning of inflammatory cells reflects, at least in part, soluble mediators (such as transforming growth factor ß and IL-4) that are released by cancer cells and alter the phenotype of cells of the innate immune system. Signaling pathways in cancer cells that potentiate this activity are incompletely understood. The urokinase receptor (uPAR) is a cell-signaling receptor known to promote cancer cell survival, proliferation, metastasis, and cancer stem cell-like properties. The present findings show that uPAR expression in diverse cancer cells, including breast cancer, pancreatic cancer, and glioblastoma cells, promotes the ability of these cells to condition co-cultured bone marrow-derived macrophages so that the macrophages express significantly increased levels of arginase 1, a biomarker of the alternatively activated M2 macrophage phenotype. Expression of transforming growth factor ß was substantially increased in uPAR-expressing cancer cells via a mechanism that requires uPA-initiated cell signaling. uPAR also controlled expression of IL-4 in cancer cells via a mechanism that involves activation of ERK1/2. The ability of uPAR to induce expression of factors that condition macrophages in the tumor microenvironment may constitute an important mechanism by which uPAR promotes cancer progression.

Dynamic phosphorylation of tyrosine 665 in pseudopodium-enriched atypical kinase 1 (PEAK1) is essential for the regulation of cell migration and focal adhesion turnover.

Pseudopodium-enriched atypical kinase 1 (PEAK1) is a recently described tyrosine kinase that associates with the actin cytoskeleton and focal adhesion (FA) in migrating cells. PEAK1 is known to promote cell migration, but the responsible mechanisms remain unclear. Here, we show that PEAK1 controls FA assembly and disassembly in a dynamic pathway controlled by PEAK1 phosphorylation at Tyr-665. Knockdown of endogenous PEAK1 inhibits random cell migration. In PEAK1-deficient cells, FA lifetimes are decreased, FA assembly times are shortened, and FA disassembly times are extended. Phosphorylation of Tyr-665 in PEAK1 is essential for normal PEAK1 localization and its function in the regulation of FAs; however, constitutive phosphorylation of PEAK1 Tyr-665 is also disruptive of its function, indicating a requirement for precise spatiotemporal regulation of PEAK1. Src family kinases are required for normal PEAK1 localization and function. Finally, we provide evidence that PEAK1 promotes cancer cell invasion through Matrigel by a mechanism that requires dynamic regulation of Tyr-665 phosphorylation.

Crosstalk between the urokinase-type plasminogen activator receptor and EGF receptor variant III supports survival and growth of glioblastoma cells.

A truncated and constitutively active form of the EGF receptor, variant III (EGFRvIII), is a major determinant of tumor growth and progression in glioblastoma multiforme (GBM). Extensive bidirectional crosstalk occurs in the cell-signaling pathways downstream of the EGFR and the urokinase-type plasminogen activator receptor (uPAR); however, crosstalk between EGFRvIII and uPAR has not been examined. Here, we show that uPAR does not regulate ERK activation in EGFRvIII-expressing GBM cells; however, in GBM cells isolated from four separate xenografts in which EGFRvIII expression was down-regulated in vivo, uPAR assumed a major role in sustaining ERK activation. Phosphorylation of Tyr-845 in the EGFR, which is mediated by Src family kinases, depended on uPAR in EGFRvIII-expressing GBM cells. Activation of the mitogenic and prosurvival transcription factor, STAT5b, downstream of EGFRvIII, also required uPAR. The EGFR-selective tyrosine kinase inhibitors, erlotinib and gefitinib, blocked not only EGFRvIII signaling to ERK but also uPAR-dependent STAT5b activation. uPAR gene silencing in EGFRvIII-expressing GBM cells and in cells from tumors that escaped dependency on EGFRvIII decreased cell survival and proliferation. Xenografts of EGFRvIII-expressing cancer cell lines and a human GBM, which was propagated as a xenograft, were robustly immunopositive for uPAR and phospho-Tyr-845 by immunohistochemistry. A human GBM in which the EGFR gene was amplified without truncation was immunonegative for both uPAR and phospho-Tyr-845. These studies identify distinct cell-signaling activities for uPAR in GBM cells that express EGFRvIII and in cells released from dormancy when EGFRvIII is neutralized. uPAR and its crosstalk pathways with EGFRvIII emerge as logical targets for therapeutics development in GBM.

Validation of the Korean Academy of Geriatric Dentistry screening questionnaire and oral frailty diagnostic criteria in community-dwelling older adults.

OBJECTIVES: This study aimed to establish the validity-specifically, the sensitivity and specificity-of the screening questionnaire and diagnostic criteria for oral frailty proposed by the Korean Academy of Geriatric Dentistry (KAGD) among community-dwelling older adults. METHODS: This study enrolled 100 participants. Among various definitions of oral frailty, this study used the criteria proposed by Tanaka as the reference test. The screening questionnaire consisted of 11 items for screening physical frailty, chewing ability, swallowing difficulties, oral dryness, and tongue and lip motor function. Each question had a different scoring weight, and if the total score was 1 or higher, an oral frailty diagnostic examination proposed by the KAGD would be recommended. The diagnostic test was the oral frailty diagnostic criteria proposed by the KAGD including 6 measures: chewing ability, occlusal force, tongue pressure, oral dryness, swallowing difficulty, and oral hygiene. If a participant exhibited 2 or more positive measures, this participant was classified as "oral frail." The screening questionnaire was analyzed using a cut-off value of 1 or higher, while the diagnostic criteria utilized a cut-off of 2 or more positive measures. Sensitivity and specificity were calculated. RESULTS: The screening questionnaire showed significant power for screening oral frailty (area under the receiver operating characteristic curve, 0.783; sensitivity, 87.8%; specificity, 52.5%). The diagnostic accuracy of the newly proposed diagnostic criteria was acceptable (sensitivity, 95.1%; specificity, 42.4%). CONCLUSIONS: The newly proposed screening questionnaire and diagnostic criteria in Korea appear to be a useful tool to identify oral frailty in community-dwelling older adults.

uPAR induces epithelial-mesenchymal transition in hypoxic breast cancer cells.

Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. In this study, we show that breast cancer cells cultured in 1.0% O(2) demonstrate changes consistent with epithelial-mesenchymal transition (EMT). Snail translocates to the nucleus, and E-cadherin is lost from plasma membranes. Vimentin expression, cell migration, Matrigel invasion, and collagen remodeling are increased. Hypoxia-induced EMT is accompanied by increased expression of the urokinase-type plasminogen activator receptor (uPAR) and activation of cell signaling factors downstream of uPAR, including Akt and Rac1. Glycogen synthase kinase-3beta is phosphorylated, and Snail expression is increased. Hypoxia-induced EMT is blocked by uPAR gene silencing and mimicked by uPAR overexpression in normoxia. Antagonizing Rac1 or phosphatidylinositol 3-kinase also inhibits development of cellular properties associated with EMT in hypoxia. Breast cancer cells implanted on chick chorioallantoic membranes and treated with CoCl(2), to model hypoxia, demonstrate increased dissemination. We conclude that in hypoxia, uPAR activates diverse cell signaling pathways that cooperatively induce EMT and may promote cancer metastasis.

Low density lipoprotein receptor-related protein (LRP1) regulates Rac1 and RhoA reciprocally to control Schwann cell adhesion and migration.

LDL receptor-related protein (LRP1) is expressed by Schwann cells in vivo mainly after injury to the peripheral nervous system (PNS). Schwann cells in primary culture, which provide a model of Schwann cells in the injured PNS, also express abundant LRP1. Herein, we show that LRP1 gene-silencing or treatment with receptor-associated protein (RAP) promotes Schwann cell adhesion and inhibits cell migration on fibronectin. LRP1 gene-silencing also resulted in the formation of prominent focal adhesions and actin stress fibers. These changes, which were induced by loss of LRP1 expression or activity, were explained mechanistically by an increase in activated RhoA, coupled with a decrease in activated Rac1. Known LRP1 ligands, including matrix metalloprotease-9, tissue-type plasminogen activator, and alpha(2)-macroglobulin activated Rac1 in LRP1-expressing Schwann cells. An inhibitor of Rac1 activation promoted Schwann cell adhesion. Conversely, in cells in which LRP1 was silenced, a Rho kinase inhibitor promoted migration and inhibited adhesion. These results demonstrate that direct binding of ligands to LRP1 controls activation of small Rho family GTPases. The effects of LRP1 gene-silencing and RAP implicate autocrine pathways involving endogenously produced LRP1 ligands. Regulation of Schwann cell migration by LRP1 may be important in PNS injury.

Kinetochore stretching-mediated rapid silencing of the spindle-assembly checkpoint required for failsafe chromosome segregation.

The spindle-assembly checkpoint facilitates mitotic fidelity by delaying anaphase onset in response to microtubule vacancy at kinetochores. Following microtubule attachment, kinetochores receive microtubule-derived force, which causes kinetochores to undergo repetitive cycles of deformation; this phenomenon is referred to as kinetochore stretching. The nature of the forces and the relevance relating this deformation are not well understood. Here, we show that kinetochore stretching occurs within a framework of single end-on attached kinetochores, irrespective of microtubule poleward pulling force. An experimental method to conditionally interfere with the stretching allowed us to determine that kinetochore stretching comprises an essential process of checkpoint silencing by promoting PP1 phosphatase recruitment after the establishment of end-on attachments and removal of the majority of checkpoint-activating kinase Mps1 from kinetochores. Remarkably, we found that a lower frequency of kinetochore stretching largely correlates with a prolonged metaphase in cancer cell lines with chromosomal instability. Perturbation of kinetochore stretching and checkpoint silencing in chromosomally stable cells produced anaphase bridges, which can be alleviated by reducing chromosome-loaded cohesin. These observations indicate that kinetochore stretching-mediated checkpoint silencing provides an unanticipated etiology underlying chromosomal instability and underscores the importance of a rapid metaphase-to-anaphase transition in sustaining mitotic fidelity.

Reversibility of epithelial-mesenchymal transition (EMT) induced in breast cancer cells by activation of urokinase receptor-dependent cell signaling.

Hypoxia induces expression of the urokinase receptor (uPAR) and activates uPAR-dependent cell signaling in cancer cells. This process promotes epithelial-mesenchymal transition (EMT). uPAR overexpression in cancer cells also promotes EMT. In this study, we tested whether uPAR may be targeted to reverse cancer cell EMT. When MDA-MB 468 breast cancer cells were cultured in 1% O(2), uPAR expression increased, as anticipated. Cell-cell junctions were disrupted, vimentin expression increased, and E-cadherin was lost from cell surfaces, indicating EMT. Transferring these cells back to 21% O(2) decreased uPAR expression and reversed the signs of EMT. In uPAR-overexpressing MDA-MB 468 cells, EMT was reversed by silencing expression of endogenously produced urokinase-type plasminogen activator (uPA), which is necessary for uPAR-dependent cell signaling, or by targeting uPAR-activated cell signaling factors, including phosphatidylinositol 3-kinase, Src family kinases, and extracellular signal-regulated kinase. MDA-MB 231 breast cancer cells express high levels of uPA and uPAR and demonstrate mesenchymal cell morphology under normoxic culture conditions (21% O(2)). Silencing uPA expression in MDA-MB-231 cells decreased expression of vimentin and Snail, and induced changes in morphology characteristic of epithelial cells. These results demonstrate that uPAR-initiated cell signaling may be targeted to reverse EMT in cancer.

The urokinase receptor promotes cancer metastasis independently of urokinase-type plasminogen activator in mice.

The urokinase receptor (uPAR) promotes metastasis of human malignancies; however, its mechanism of action remains incompletely understood. Established models focus on the ability of uPAR to bind urokinase-type plasminogen activator (uPA) and promote protease activation in the tumor cell microenvironment; however, uPAR also regulates cell signaling and migration by both uPA-dependent and -independent mechanisms in vitro. The significance of uPAR as a cell-signaling receptor in vivo remains unclear. In this study, we expressed either human or mouse uPAR in human embryonic kidney (HEK-293) cells. We selected HEK-293 cells because, unlike most cancer cells, they do not express uPA or uPAR endogenously. Both mouse and human uPAR increased cell adhesion and migration on vitronectin. Rac1 was activated and responsible for the increase in cell migration. HEK-293 cells that did not express uPAR formed palpable tumors in severe combined immunodeficient mice; however, metastases were exceedingly rare. The xenografts contained abundant mouse uPA, produced by infiltrating mouse cells, but no human uPA. Mouse uPA bound only to mouse uPAR and not human uPAR and, thus, could not interact with human uPAR-expressing HEK-293 cells in xenografts. Nevertheless, both mouse and human uPAR significantly increased HEK-293 cell metastasis into the lungs. The activity of human uPAR suggests that uPAR may promote cancer metastasis independent of uPA. Candidate mechanisms include its effects on adhesion, migration, and Rac1 activation.

Utility of Squaraine Dyes for Dye-Sensitized Photocatalysis on Water or Carbon Dioxide Reduction.

Red light-sensitized squaraine (SQ) dyes were developed and incorporated into dye-sensitized catalysts (DSCs) with the formula of SQ/TiO2/Cat, and their efficacies were evaluated in terms of performance on either water or carbon dioxide reduction. Pt nanoparticles or fac-[Re(4,4'-bis-(diethoxyphosphorylmethyl)-2,2'-bipyridine)(CO)3Cl] were used as each catalytic center within the DSC frame of SQ/TiO2/Pt (Type I) or SQ/TiO2/Re(I) (Type II). In order to convey the potential utility of SQ in low energy sensitization, the following catalytic reductions were carried out under selective lower energy irradiation (>500 nm). Type I and II showed different catalytic performances, primarily due to the choice of solvent for each catalytic condition: hydrogenation was carried out in H2O, but CO2 reduction in dimethylformamide (DMF), and SQ was more stable in aqueous acid conditions for hydrogen generation than CO2 reduction in DMF. A suspension of Type I in 3 mL water containing 0.1 M ascorbic acid (pH = 2.66) resulted in efficient photocatalytic hydrogen evolution, producing 37 µmol of H2 for 4 h. However, in photocatalysis of Type II (SQ/TiO2/Re(I)) in 3 mL DMF containing 0.1 M 1,3-dimethyl-2-phenyl-1,3-dihydrobenzimidazole, the TiO2-bound SQ dyes were not capable of working as a low energy sensitizer because SQ was susceptible to dye decomposition in nucleophilic DMF conditions, resulting in DSC deactivation for the CO2 reduction. Even with the limitation of solvent, the DSC conditions for the utility of SQ have been established: the anchoring group effect of SQ with either phosphonic acid or carboxylic acid onto the TiO2 surface; energy alignment of SQ with the flat band potentials (E fb) of TiO2 semiconductors and the reduction power of electron donors; and the wavelength range of the light source used, particularly when >500 nm.

Antioxidant, ACE inhibitory, and acetylcholinesterase inhibitory activities of subcritical water extract of blue mussel.

Subcritical water (SCW) extract of blue mussel was prepared at 100, 200, and 300 °C for 10, 30, and 60 min, respectively, and its effect on the activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, angiotensin-converting enzyme (ACE), and acetylcholinesterase (AChE) was evaluated. We found that DPPH radical scavenging, ACE inhibitory, and AChE inhibitory activities significantly increased with increasing extraction temperature and duration. For example, AChE inhibitory activity of the extract at 300 °C for 60 min increased to 63.1 ± 0.3%, while that at 100 °C for 10 min was 5.6 ± 0.3%. The results suggested that SCW extraction is attractive processing methods for obtaining high valued extract from blue mussel.

STAT3 antisense oligonucleotide AZD9150 in a subset of patients with heavily pretreated lymphoma: results of a phase 1b trial.

BACKGROUND: The Janus kinase (JAK) and signal transduction and activation of transcription (STAT) signaling pathway is an attractive target in multiple cancers. Activation of the JAK-STAT pathway is important in both tumorigenesis and activation of immune responses. In diffuse large B-cell lymphoma (DLBCL), the transcription factor STAT3 has been associated with aggressive disease phenotype and worse overall survival. While multiple therapies inhibit upstream signaling, there has been limited success in selectively targeting STAT3 in patients. Antisense oligonucleotides (ASOs) represent a compelling therapeutic approach to target difficult to drug proteins such as STAT3 through of mRNA targeting. We report the evaluation of a next generation STAT3 ASO (AZD9150) in a non-Hodgkin's lymphoma population, primarily consisting of patients with DLBCL. METHODS: Patients with relapsed or treatment refractory lymphoma were enrolled in this expansion cohort. AZD9150 was administered at 2 mg/kg and the 3 mg/kg (MTD determined by escalation cohort) dose levels with initial loading doses in the first week on days 1, 3, and 5 followed by weekly dosing. Patients were eligible to remain on therapy until unacceptable toxicity or progression. Blood was collected pre- and post-treatment for analysis of peripheral immune cells. RESULTS: Thirty patients were enrolled, 10 at 2 mg/kg and 20 at 3 mg/kg dose levels. Twenty-seven patients had DLBCL. AZD9150 was safe and well tolerated at both doses. Common drug-related adverse events included transaminitis, fatigue, and thrombocytopenia. The 3 mg/kg dose level is the recommended phase 2 dose. All responses were seen among DLBCL patients, including 2 complete responses with median duration of response 10.7 months and 2 partial responses. Peripheral blood cell analysis of three patients without a clinical response to therapy revealed a relative increase in proportion of macrophages, CD4+, and CD8+ T cells; this trend did not reach statistical significance. CONCLUSIONS: AZD9150 was well tolerated and demonstrated efficacy in a subset of heavily pretreated patients with DLBCL. Studies in combination with checkpoint immunotherapies are ongoing. TRIAL REGISTRATION: Registered at ClinicalTrials.gov: NCT01563302 . First submitted 2/13/2012.