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Variegated expression of killer Ig-like receptors (KIR) in human NK cells is a stochastic process exclusive to subsets of mature NK cells and CD8+ T cells. Allele-specific KIR expression is maintained by DNA methylation within the proximal promoter regions. Because KIR genes are densely methylated in NK cell progenitors, there is an implied stage of human NK cell development in which DNA demethylation takes place to allow for active transcription. When and how this process occurs is unknown. In this study, we show that KIR proximal promoters are densely methylated in less mature CD56bright NK cells and are progressively demethylated in CD56dim NK cells as they mature and acquire KIR. We hypothesized that ten-eleven translocation (TET) enzymes, which oxidize 5mC on DNA could mediate KIR promoter demethylation. The catalytic efficiency of TET enzymes is known to be enhanced by ascorbic acid. We found that the addition of ascorbic acid to ex vivo culture of sorted CD56bright NK cells increased the frequency of KIR expression in a dose-dependent manner and facilitated demethylation of proximal promoters. A marked enrichment of the transcription factor Runx3 as well as TET2 and TET3 was observed within proximal KIR promoters in CD56bright NK cells cultured with ascorbic acid. Additionally, overexpression of TET3 and Runx3 promoted KIR expression in CD56bright NK cells and NK-92 cells. Our results show that KIR promoter demethylation can be induced in CD56bright, and this process is facilitated by ascorbic acid.

A pre-metastatic niche (PMN) facilitates cancer metastasis through mobilization and recruitment of bone marrow-derived cells (BMDCs) and associated factors. In bone marrow, hematogenous cells, including osteoclasts, macrophages and lymphocytes, and mesenchymal cells, including mesenchymal stem cells, osteoblasts and adipocytes, are involved in PMN formation. Patients with lung cancer and metastasis have a poor prognosis and shortened median survival time. Bone marrow has been considered fertile ground for dormant and proliferating tumor cells, and mobilizing and recruiting BMDCs and immune cells can establish a PMN. However, the role of BMDCs in PMN formation is not yet fully understood. The present study aimed to investigate the association between BMDCs and PMN in bone marrow tissue samples. The results demonstrated that bone marrow served an important role in lung cancer progression and that eight pathways were potentially involved, including 'T-cell receptor signaling pathway', 'osteoclast differentiation', 'MAPK signaling pathway', 'VEGF signaling pathway', 'leukocyte transendothelial migration', 'signaling pathways regulating the pluripotency of stem cells', 'oxytocin signaling pathway' and 'cell adhesion molecules (CAMs)'. In addition, the present study investigated the role of BMDCs in facilitating lung cancer metastasis. In conclusion, the results from the present study suggested that molecular alterations in gene expression may provide a novel signature in lung cancer, which may aid in the development of novel diagnostic and therapeutic strategies for patients with lung cancer and bone metastasis.

Ginseng is often prescribed together with cisplatin for treatment of cancer, but the interaction between ginseng and cisplatin is still unknown. This study employed ginsenoside Rb1 (Rb1), one of the major components in ginseng, to explore the effects and involved mechanisms of cisplatin on the pharmacokinetics of ginseng. The effects of cisplatin on the pharmacokinetics of Rb1 and its bioactive metabolites Rd, Rg3, and F2 were investigated by using A549-bearing mice with and without cisplatin intervention. Our data showed that cisplatin could significantly decrease the AUC(0-t) and Cmax of Rd, Rg3, and F2, except Rb1. To evaluate the involved mechanisms, feces and intestinal mucosa were collected to explore the effects of cisplatin on the gut metabolism of Rb1 in vitro; meanwhile, Caco-2 cell model and small intestine histological characters were examined to evaluate the effects of cisplatin on the gut absorptive areas and permeability. The mechanisms involved may be mainly related to the comprehensive contributions of inhibited intestinal bacteria and mucosa metabolisms, narrowed intestinal absorptive area, increased efflux ratio of intestinal absorption and enhanced intestinal permeability. All these findings suggested that the dosage of ginseng traditionally used for health protection should be adjusted when it was prescribed together with cisplatin in the treatment of cancer.

By a pilot trial on investigating immunomodulatory activity and target of ginsenosides, the major bioactive components of ginseng, here we report that structural analogues in herbal medicines hit a shared target to achieve cumulative bioactivity. A ginsenoside analogues combination with definite immunomodulatory activity in vivo was designed by integrating pharmacodynamics, serum pharmacochemistry and pharmacokinetics approaches. The cumulative bioactivity of the ginsenoside analogues was validated on LPS/ATP-induced RAW264.7 macrophages. The potentially shared target NLRP3 involved in this immunomodulatory activity was predicted by systems pharmacology. The steady binding affinity between each ginsenoside and NLRP3 was defined by molecular docking and bio-layer interferometry assay. The activation of NLRP3 inflammasomes in LPS/ATP-induced RAW264.7 was significantly suppressed by the combination, but not by any individual, and the overexpression of NLRP3 counteracted the immunomodulatory activity of the combination. All these results demonstrate that the ginsenoside analogues jointly hit NLRP3 to achieve cumulative immunomodulatory activity., Zhang et al. design ginsenoside structural analogues and demonstrate that their combination shows more potent immunomodulatory activities than individual ginsenosides used alone at the same dosages. They predict that these analogues act on the joint target NLRP3 and consequently suggest that structural analogues hit a shared target to achieve cumulative bioactivity.

Epigenetic landscapes can provide insight into regulation of gene expression and cellular diversity. Here, we examined the transcriptional and epigenetic profiles of seven human blood natural killer (NK) cell populations, including adaptive NK cells. The BCL11B gene, encoding a transcription factor (TF) essential for T cell development and function, was the most extensively regulated, with expression increasing throughout NK cell differentiation. Several Bcl11b-regulated genes associated with T cell signaling were specifically expressed in adaptive NK cell subsets. Regulatory networks revealed reciprocal regulation at distinct stages of NK cell differentiation, with Bcl11b repressing RUNX2 and ZBTB16 in canonical and adaptive NK cells, respectively. A critical role for Bcl11b in driving NK cell differentiation was corroborated in BCL11B-mutated patients and by ectopic Bcl11b expression. Moreover, Bcl11b was required for adaptive NK cell responses in a murine cytomegalovirus model, supporting expansion of these cells. Together, we define the TF regulatory circuitry of human NK cells and uncover a critical role for Bcl11b in promoting NK cell differentiation and function.

Select subsets of immune effector cells have the greatest propensity to mediate antitumor responses. However, procuring these subsets is challenging, and cell-based immunotherapy is hampered by limited effector-cell persistence and lack of on-demand availability. To address these limitations, we generated a triple-gene-edited induced pluripotent stem cell (iPSC). The clonal iPSC line was engineered to express a high affinity, non-cleavable version of the Fc receptor CD16a and a membrane-bound interleukin (IL)-15/IL-15R fusion protein. The third edit was a knockout of the ecto-enzyme CD38, which hydrolyzes NAD+. Natural killer (NK) cells derived from these uniformly engineered iPSCs, termed iADAPT, displayed metabolic features and gene expression profiles mirroring those of cytomegalovirus-induced adaptive NK cells. iADAPT NK cells persisted in vivo in the absence of exogenous cytokine and elicited superior antitumor activity. Our findings suggest that unique subsets of the immune system can be modeled through iPSC technology for effective treatment of patients with advanced cancer. acceptedVersion

Variegated expression of killer immunoglobulin-like receptors (KIR) in human natural killer (NK) cells is a stochastic process exclusive to subsets of mature NK cells and CD8(+) T cells. Allele-specific KIR expression is maintained by DNA methylation within the proximal promoter regions. Because KIR genes are densely methylated in NK cell progenitors, there is an implied stage of human NK cell development where DNA demethylation takes place to allow for active transcription. When and how this process occurs is unknown. Here, we show that KIR proximal promoters are densely methylated in less mature CD56(bright) NK cells and are progressively demethylated in CD56(dim) NK cells as they mature and acquire KIR. We hypothesized that ten-eleven translocation (TET) enzymes, which oxidize 5mC on DNA could mediate KIR promoter demethylation. The catalytic efficiency of TET enzymes is known to be enhanced by ascorbic acid. We found that the addition of ascorbic acid to ex vivo culture of sorted CD56(bright) NK cells increased the frequency of KIR expression in a dose-dependent manner and facilitated demethylation of proximal promoters. A marked enrichment of the transcription factor Runx3 as well as TET2 and TET3 was observed within proximal KIR promoters in CD56(bright) NK cells cultured with ascorbic acid. Additionally, overexpression of TET3 and Runx3 promoted KIR expression in CD56(bright) NK cells and NK-92 cells. Our results show that KIR promoter demethylation can be induced in CD56(bright), and this process is facilitated by ascorbic acid.

“Adaptive” NK cells expressing the activating receptor NKG2C expand and persist in HCMV-seropositive individuals. Cichocki et al. demonstrate enhanced oxidative and glycolytic metabolism for adaptive NK cells and implicate ARID5B as an important regulator of mitochondrial metabolism, IFN-γ production, and survival., Natural killer (NK) cells with adaptive immunological properties expand and persist in response to human cytomegalovirus. Here, we explored the metabolic processes unique to these cells. Adaptive CD3−CD56dimCD57+NKG2C+ NK cells exhibited metabolic hallmarks of lymphocyte memory, including increased oxidative mitochondrial respiration, mitochondrial membrane potential, and spare respiratory capacity. Mechanistically, we found that a short isoform of the chromatin-modifying transcriptional regulator, AT-rich interaction domain 5B (ARID5B), was selectively induced through DNA hypomethylation in adaptive NK cells. Knockdown and overexpression studies demonstrated that ARID5B played a direct role in promoting mitochondrial membrane potential, expression of genes encoding electron transport chain components, oxidative metabolism, survival, and IFN-γ production. Collectively, our data demonstrate that ARID5B is a key regulator of metabolism in human adaptive NK cells, which, if targeted, may be of therapeutic value., Graphical Abstract

Hypoxia, the most commonly observed characteristic in cancers, is implicated in the establishment of an immunosuppressive niche. Recent studies have indicated that extracellular vesicle (EV)-mediated cancer-stroma interactions are considered to play a critical role in the regulation of various cellular biological functions, with phenotypic consequences in recipient cells. However, the mechanisms underlying the relationship between EVs and hypoxia during cancer progression remain largely unknown. In this study, we found that EVs derived from hypoxic lung cancers increased M2-type polarization by miR-103a transfer. Decreased PTEN levels caused by hypoxic cancer-cell-derived EV miR-103a increased activation of AKT and STAT3 as well as expression of several immunosuppressive and pro-angiogeneic factors. In contrast, inhibition of miR-103a by an miRNA inhibitor effectively decreased hypoxic cancer-mediated M2-type polarization, improving the cytokine prolife of tumor infiltration macrophages. Macrophages received cancer-cell-derived EV miR-103a feedback to further enhance cancer progression and tumor angiogenesis. Finally, circulating EV miR-103a levels were higher in patients with lung cancer and closely associated with the M2 polarization. In conclusion, our results delineate a novel mechanism by which lung cancer cells induce immunosuppressive and pro-tumoral macrophages through EVs and inspire further research into the clinical application of EV inhibition or PTEN restoration for immunotherapy., Graphical Abstract, Extracellular vesicle (EV) miR-103 can be transferred from hypoxic cancer cells to macrophages, resulting in the enhancement of M2 polarization by the downregulation of miR-103a’s direct target PTEN. EV miR-103a increases the stimulatory effects of macrophages on cancer progression and angiogenesis.

// Jen-Yu Hung 1, 2 , Meng-Chi Yen 3 , Shu-Fang Jian 4 , Cheng-Ying Wu 4 , Wei-An Chang 2, 4 , Kuan-Ting Liu 1, 3, 4 , Ya-Ling Hsu 5 , Inn-Wen Chong 2, 6 and Po-Lin Kuo 4, 7 1 School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 3 Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Department of Respiratory Therapy, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 7 Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan Correspondence to: Inn-Wen Chong, email: chong@kmu.edu.tw Po-Lin Kuo, email: kuopolin@seed.net.tw Keywords: SPARC, WNK1, lung cancer, EMT, migration Received: March 24, 2017 Accepted: June 20, 2017 Published: July 22, 2017 ABSTRACT The extracellular matrix is a component of physiological microenvironment and a regulator of cellular processes such as migration and proliferation. Secreted Protein Acidic and Rich in Cysteine (SPARC/osteonectin) is an extracellular matrix-associated glycoprotein involved in the regulation of cell proliferation and cell migration in several types of cancers. However, the role of SPARC in lung cancer is paradoxical and details of the regulatory mechanism are not well-known. In this study, we investigated novel SPARC-mediated signaling pathways. Treatment of SPARC increased cell proliferation, migration, and mesenchymal phenotype in two non-small cell lung cancer cell lines, CL1-5 and H1299. We found that these phenotypes were not regulated by focal adhesion kinase and Src kinase, but were mediated by with no lysine (K) kinase 1 (WNK1). Suppression of WNK1 expression decreased the expression of SPARC-induced N-cadherin and smooth muscle actin. Moreover, Snail, an important transcription factor for regulating epithelial–mesenchymal transition, is also involved in SPARC/WNK1 pathway. In a murine tumor model, SPARC treatment significantly induced phosphorylation of Akt and WNK1 in lung tumor nodules when compared to control mice. In conclusion, these data suggest that WNK1 is a novel molecule in SPARC-mediated mesenchymal signaling pathway in non-small cell lung cancer.

// Wei-An Chang 1, 2 , Jen-Yu Hung 2, 3 , Shu-Fang Jian 1 , Yi-Shiuan Lin 1 , Cheng-Ying Wu 1 , Ya-Ling Hsu 4 , Po-Lin Kuo 1, 4, 5 1 Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2 Division of Pulmonary and Critical Care Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 3 School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 4 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 5 Institute of Medical Science and Technology, National Sun Yat-Sen University, Kaohsiung, Taiwan Correspondence to: Po-Lin Kuo, email: kuopolin@seed.net.tw Ya-Ling Hsu, email: hsuyl326@gmail.com Keywords: laricitrin, dendritic cell, lung cancer, IL-10, STAT3 Received: August 20, 2016 Accepted: October 24, 2016 Published: November 09, 2016 ABSTRACT Natural polyphenolic compounds of grapes and their seeds are thought to be therapeutic adjuvants in a variety of diseases, including cancer prevention. This study was carried out to investigate the effect of grape phenolic compounds on the regulation of cancer-mediated immune suppression. Laricitrin exhibits the greatest potential to ameliorate the suppressive effects of lung cancer on dendritic cells’ (DCs’) differentiation, maturation and function. Human lung cancer A549 and CL1-5 cells change the phenotype of DCs that express to high levels of IL-10 and prime T cells towards an immune suppression type-2 response (Th2). Laricitrin treatment stimulated DC differentiation and maturation in the condition media of cancer cells, a finding supported by monocyte marker CD14’s disappearance and DC marker CD1a’s upregulation. Laricitrin decreases expression of IL-10 in cancer-conditioned DCs, and subsequently switches CD4 + T cell response from Th2 to Th1 in vitro and in vivo . Reversal of laricitrin on lung cancer-induced DCs’ paralysis was via inhibiting the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Laricitrin also potentiated the anticancer activity of cisplatin in mouse models. Thus, laricitrin could be an efficacious immunoadjuvant and have a synergistic effect when combined with chemotherapy.

Balanced Hash Tree (BHT) is a mechanism for service function (SF) chaining, service routing, and traffic steering that enables switches to select SF instances for load balancing without involving the controller. In an experimental evaluation, BHT decreased packet-in message-processing time by 92.5 percent and achieved near-perfect load-balancing performance.

All-trans Retinoic acid (RA) and its derivatives are potent therapeutics for immunological functions including wound repair. However, the molecular mechanism of RA modulation in innate immunity is poorly understood, especially in macrophages. We found that topical application of RA significantly improves wound healing and that RA and IL-4 synergistically activate Arg1, a critical gene for tissue repair, in M2 polarized macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to coordinate RAR, STAT6 and chromatin remodeler, Brg1 to remodel the +1 nucleosome of Arg1 for transcription initiation. By recruiting elongation factor TFIIS, Med25 also facilitates transcriptional initiation-elongation coupling. This study uncovers synergistic activation of Arg1 by RA and IL-4 in M2 macrophages that involves feed forward regulation of RA synthesis and dual functions of Med25 in nucleosome remodeling and transcription initiation-elongation coupling that underlies robust modulatory activity of RA in innate immunity.

Receptor-interacting protein 140 (RIP140) is a wide-spectrum coregulator for hormonal regulation of gene expression, but its activity in development/stem cell differentiation is unknown. Here, we identify RIP140 as an immediate retinoic acid (RA)-induced dual-function chaperone for LSD1 (lysine-specific demethylase 1). RIP140 protects LSD1's catalytic domain and antagonizes its Jade-2-mediated ubiquitination and degradation. In RA-induced neuronal differentiation, the increased RIP140/LSD1 complex is recruited by RA-elevated Pit-1 to specifically reduce H3K4me2 modification on the Pax6 promoter, thereby repressing RA-induction of Pax6. This study reveals a new RA-induced gene repressive mechanism that modulates the abundance, enzyme quality, and recruitment of histone modifier LSD1 to neuronal regulator Pax6, which provides a homeostatic control for RA induction of neuronal differentiation.

Phthalates are used as plasticizers in the manufacture of flexible vinyl, which is used in food contact applications. Phthalates have been demonstrated to have an adverse impact on human health, particularly in terms of cancer development. In the present study, we showed for the first time that benzyl butyl phthalate (BBP) potentiates the effect of tumor‑associated dendritic cells (TADCs) on the chemoresistance of breast cancer. Specific knockdown analysis revealed that S100A9 is the major factor responsible for the chemoresistance of doxorubicin/cyclophosphamide induced by BBP-stimulated TADCs in breast cancer. BBP exposure also increased tumor infiltrating myeloid-derived suppressor cell (MDSC) secretion of S100A8/A9, thereby exacerbating the resistance of breast cancer to doxorubicin with cyclophosphamide. In addition, BBP also stimulated the production of CXCL1/GROα by TADCs, which increased the angiogenesis of breast cancer in a mouse model. Inhibition of CXCL1/GROα by a neutralizing antibody, decreased the BBP-induced angiogenesis induced by BBP after chemotherapy in the mouse model. These results, for the first time, provide evidence that BBP influences the efficacy of chemotherapy by remodeling the tumor microenvironment of breast cancer.

Cytolytic effector lymphocytes must contend with unfavorable microenvironments when infiltrating sites of infection or malignancy. Tumor cells typically have high levels of oxidative stress and produce reactive oxygen species (ROS) that suppress the cytotoxic functions of both natural killer (NK) cells and CD8+ T cells. Levels of activated granulocytes that release ROS are also elevated in cancer patients. Free radicals, such as ROS, cause detrimental cellular effects including protein oxidation, lipid peroxidation and DNA damage. Chronic viral infections, including CMV, are also associated with increased oxidative stress. We hypothesized that adaptive NK cells, which arise specifically in response to CMV, could have properties that allow these cells to persist and retain function in high oxidative stress environments. Adaptive NK cells are present in the peripheral blood of many otherwise healthy CMV seropositive individuals and expand in response to CMV reactivation in hematopoietic cell transplant (HCT) patients. Mounting evidence suggests that CMV peptides presented by HLA-E on infected cells can trigger the expansion of adaptive NK cells expressing the activating receptor NKG2C. The majority of NKG2C-positive adaptive NK cells co-express the terminal maturation marker CD57. Work by our group and others has shown that adaptive NK cells exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) and interferon (IFN)-γ production relative to canonical NK cells, appear to persist long-term and have metabolic attributes similar to memory CD8+ T cells. We also reported clinical correlations between adaptive NK cell numbers and reduced relapse risk in HCT patients with hematologic malignancies. Here, we show that CD38 expression is markedly reduced on adaptive NK cells from CMV seropositive individuals. This observation was first made from analyses of RNA-seq data comparing adaptive and canonical NK cells and was validated by flow cytometry (Figure 1A). CD38 is expressed both intracellularly and on the plasma membrane and functions as an NADase, degrading nicotinamide adenine dinucleotide (NAD+) into ADP-ribose and nicotinamide. NAD+ is a necessary cofactor for the sirturin family of protein deacetylases, which protect against oxidative stress. We hypothesized that CD38 downregulation in adaptive NK cells could be associated with more resistance to oxidative stress-induced cell death through increased NAD+ levels and sirturin activity. To determine whether there was a connection between CD38 expression and resistance to oxidative stress, we isolated NK cells from the peripheral blood of CMV seropositive donors and cultured them overnight with or without 15 mM H2O2 (hydrogen peroxide), known to induce oxidative stress and cell death. We found that NKG2C+ adaptive NK cells were markedly more resistant to oxidative stress-induced cell death compared to NKG2C-negative canonical NK cells as determined by annexin V and a fixable amine-reactive dye (LIVE/DEAD) that can permeate damaged membranes of dead cells and react with interior amines (Figure 1B). Similar results were observed in assays where NK cells from CMV seropositive donors were co-cultured with neutrophils pre-activated with phorbol 12-myristate 13-acetate (PMA) to induce the release of reactive oxygen species. To determine whether CD38 expression is directly associated with the NK cell response to oxidative stress, we generated induced pluripotent stem cell (iPSC) lacking CD38 through CRISPR/Cas9 gene editing that were differentiated into NK cells and tested for their ability to resist oxidative stress-induced death. Compared to control iPSC-derived NK (iNK) cells that express high levels of CD38 (Figure 1C), a substantially larger percentage of CD38 knockout iNK cells were viable when cultured overnight with H2O2 (Figure 1D). Our results have implications for adoptive immunotherapy to treat patients with cancer where a major goal is to manufacture cytotoxic cells that can persist and function in a tumor environment that contains high levels of oxidative radicals. We are exploring other cell stressors of high translational relevance such as freeze/thaw stress in adaptive and CD38 knockout cells that will be critical for cell therapy platforms. Disclosures Cichocki: Fate Therapeutics, Inc: Research Funding. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bjordahl:Fate Therapeutics, Inc.: Employment. Valamehr:Fate Therapeutics, Inc: Employment. Miller:Fate Therapeutics, Inc: Consultancy, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: NK cells

Maintaining pluripotency and indefinite self-renewal of embryonic stem cells requires a tight control of the expression of several key stemness factors, particularly Nanog and Oct4 transcription factors. The mammalian SWItch/Sucrose NonFermentable (SWI/SNF) complex contains Brg1 or Brm as its core subunit, along with Brg1-associated factors. Our previous studies have addressed chromatin-remodeling of the Oct4 gene locus in retinoic acid (RA)-treated embryonal carcinoma cell line P19, which involves receptor-interacting protein 140 (RIP140) for heterochromatinization on the proximal promoter region of this gene locus. However, the mechanism of RIP140 action in RA-triggered repressive chromatin-remodeling is unclear. The current study examines RA repression of the Nanog gene and compares the results with RA repression of the Oct4 gene on the chromatin level. The results show a loose nucleosome array on the Nanog gene promoter in undifferentiated embryonic stem cells. On RA treatment, the Nanog gene locus remodels specifically in the CR1 region of its proximal promoter, with the insertion of a nucleosome and compaction of this region. Further, RA induces coordinated chromatin-remodeling of both Nanog and Oct4 gene loci, which requires RA receptor-α, RIP140 and Brm. Finally, in these RA-triggered repressive chromatin-remodeling processes, lysine acetylation of RIP140 is critical for its recruiting Brm.

This study investigated the antioxidant potential and anti-fatigue effects of phenolics extracted from the seed coat of Euryale ferox Salisb. The in vitro antioxidant potentials, including scavenging DPPH, hydroxyl radical activities and reducing power were evaluated. Antioxidant status in vivo was analyzed by SOD, CAT, GSH-Px activities and the MDA content in liver and kidneys of D-galactose-induced aging mice. The anti-fatigue effect was evaluated using an exhaustive swimming test, along with the determination of LDH, BUN and HG content. The phenolic extract possessed notable antioxidant effects on DPPH, hydroxyl radical scavenging and reducing power. The mice which received the phenolic extract showed significant increases of SOD, CAT (except for in the kidney), GSH-Px activities, and a decrease of MDA content. The average exhaustive swimming time was obviously prolonged. Meanwhile, increase of LDH content and decrease of BUN content were observed after mice had been swimming for 15 min. The HG storage of mice was improved in the high and middle dose extract groups compared with the normal group. The contents of total phenols and gallic acid of the extract were determined. Three compounds in the extract were identified as 5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-chroman-4-one, 5,7,4-trihydroxyflavanone and buddlenol E. These results suggest that the extract of E. ferox is a promising source of natural antioxidants and anti-fatigue material for use in functional foods and medicines.

The interaction between tumors and their microenvironments leads to a vicious cycle, which strengthens both immune suppression and cancer progression. The present study demonstrates for the first time that tumor-associated dendritic cells (TADCs) are a source of resistin, which is responsible for increasing lung cancer epithelial-to-mesenchymal transition. In addition, large amounts of resistin in the condition medium (CM) of TADCs increase cell migration and invasion, as well as the osteolytic bone metastatic properties of lung cancer cells. Neutralization of resistin from TADC-CM prevents the advanced malignancy-inducing features of TADC-CM. Significantly elevated levels of resistin have been observed in mice transplanted with lung cancer cells, tumor-infiltrating CD11c(+) DCs in human lung cancer samples and lung cancer patients' sera. Induction of lung cancer progression by TADC-derived resistin is associated with increased expression of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), a histone methyltransferase. Resistin-induced WHSC1 increases the dimethylation of histone 3 at lysine 36 and decreases the trimethylation of histone 3 at lysine 27 on the promoter of Twist, resulting in an enhancement of the expression of Twist. Knockdown of WHSC1 by small interfering RNA transfection significantly decreases resistin-mediated cancer progression by decreasing the upregulation of Twist, suggesting that WHSC1 plays a critical role in the regulation of Twist by epigenetic modification. Furthermore, mice that received antiresistin antibodies showed a decreased incidence of cancer development and metastasis. These findings suggest that TADC-derived resistin may be a novel candidate in promoting the development of lung cancer.

All-trans retinoic acid (atRA), one of the active ingredients of vitamin A, exerts canonical activities to regulate gene expression mediated by nuclear RA receptors (RARs). AtRA could also elicit certain non-canonical activities including, mostly, rapid activation of extracellular signal regulated kinase 1/2 (ERK1/2); but the mechanism was unclear. In this study, we have found that cellular retinoic acid binding protein I (CRABPI) mediates the non-canonical, RAR- and membrane signal-independent activation of ERK1/2 by atRA in various cellular backgrounds. In the context of embryonic stem cells (ESCs), atRA/CRABPI-dependent ERK1/2 activation rapidly affects ESC cell cycle, specifically to expand the G1 phase. This is mediated by ERK stimulation resulting in dephosphorylation of nuclear p27, which elevates nuclear p27 protein levels to block G1 progression to S phase. This is the first study to identify CRABPI as the mediator for non-canonical activation of ERK1/2 by atRA, and demonstrate a new functional role for CRABPI in modulating ESC cell cycle progression.

Hypoxia plays a critical role during the evolution of malignant cells and tumour microenvironment (TME).Tumour-derived exosomes contain informative microRNAs involved in the interaction of cancer and stromal cells, thus contributing to tissue remodelling of tumour microenvironment. This study aims to clarify how hypoxia affects tumour angiogenesis through exosomes shed from lung cancer cells. Lung cancer cells produce more exosomes under hypoxic conditions than do parental cells under normoxic conditions. miR-23a was significantly upregulated in exosomes from lung cancer under hypoxic conditions. Exosomal miR-23a directly suppressed its target prolyl hydroxylase 1 and 2 (PHD1 and 2), leading to the accumulation of hypoxia-inducible factor-1 α (HIF-1 α) in endothelial cells. Consequently, hypoxic lung cancer cells enhanced angiogenesis by exosomes derived from hypoxic cancer under both normoxic and hypoxic conditions. In addition, exosomal miR-23a also inhibits tight junction protein ZO-1, thereby increasing vascular permeability and cancer transendothelial migration. Inhibition of miR-23a by inhibitor administration decreased angiogenesis and tumour growth in a mouse model. Furthermore, elevated levels of circulating miR-23a are found in the sera of lung cancer patients, and miR-23a levels are positively correlated with proangiogenic activities. Taken together, our study reveals the clinical relevance and prognostic value of cancer-derived exosomal miR-23a under hypoxic conditions, and investigates a unique intercellular communication, mediated by cancer-derived exosomes, which modulates tumour vasculature.

A reversed-phase high-performance liquid chromatographic method was developed for the simultaneous quantification of seven phenolic compounds from Rhizoma Sparganii. The samples were separated on a Waters SunFire C18 column with a temperature of 3088C. Gradient elution was applied, using 0.8% acetic acid (solvent A) and methanol (solvent B) with a flow rate of 1.0 mL/min, and detection wavelength was 280 nm. The validation of the method included linearity, precision, repeatability, stability and recovery. The calibration curves showed good linear regression (R 2 > 0.9996) within the test range. The developed method indicated good precision and accuracy, with the overall intra-day and inter-day variation at less than 3%. The range of recoveries for the seven analytes was 95.34‐100.06% with relative standard deviation < 3.08 %. The established method was successfully applied for the determination of seven phenolic compounds in 12 batches of Rhizoma Sparganii. The study may be useful in the quality evaluation of Rhizoma Sparganii, and can provide technical support for pharmacological and clinical research of related drugs.

Objection— Thrombin upregulates expression of several proteins in vascular smooth muscle cells (VSMCs) which may contribute to atherosclerosis. Here, we investigated the mechanisms underlying thrombin-induced EGF receptor (EGFR) expression and its effect on VSMCs. Methods and Results— Normal rat VSMCs were used. First, Western blotting and RT-PCR analyses showed that thrombin induces the expression of EGFR at transcription and translation levels in VSMCs. Second, pharmacological inhibitors, dominant negative mutants, and short hairpin RNA interference (shRNA) technology enabled us to demonstrate that thrombin-induced EGFR expression is mediated through PKC(δ)/c-Src-dependent transactivation of EGFR linking to PI3K/Akt and ERK1/2. We further investigated whether the transcription factors AP-1 and NF-κB are involved in this response by a promoter assay. Finally, data obtained by using EGFR shRNA technology and XTT assay demonstrated that thrombin-enhanced VSMC proliferation was mediated through upregulation of EGFR. Conclusions— Our results demonstrate that thrombin-enhanced VSMC proliferation was mediated through upregulation of EGFR via a PKC(δ)/c-Src-dependent transactivation of EGFR, PI3K-Akt, and ERK, and AP-1/NF-κB pathway.

The use of hydrogen peroxide (H 2 O 2 ) for improved photocatalytic degradation of phenol in aqueous suspension of commercial TiO 2 powders (Degussa P-25) was investigated. Photodegradation was compared using direct photolysis (UV alone), H 2 O 2 /UV, TiO 2 /UV, and H 2 O 2 /TiO 2 /UV processes in a batch reactor with high-pressure mercury lamp irradiation. The effects of operating parameters such as catalyst dosage, light intensity, pH of the solution, the initial phenol, and H 2 O 2 concentrations on photodegradation process were examined. It was shown that photodegradation using H 2 O 2 /TiO 2 /UV process was much more effective than using either H 2 O 2 /UV or TiO 2 /UV process. The effect of the initial phenol concentration on TOC removal was also studied, demonstrating that more than 8 h was required to completely mineralize phenol into water and carbon dioxide. For all the four oxidation processes studied, photodegradation followed the first-order kinetics. The apparent rate constants with 400-W UV ranged from 5.0 × 10 −4 min −1 by direct photolysis to 1.4 × 10 −2 min −1 using H 2 O 2 /TiO 2 /UV process. The role of H 2 O 2 on such enhanced photodegradation of phenol in aqueous solution was finally discussed.

In a previous study, interleukin-1beta (IL-1beta) has been shown to induce matrix metalloproteinases (MMPs) expression through mitogen-activated protein kinases and nuclear factor-kappaB pathways in rat brain astrocytes. Moreover, transactivation of growth factor receptors and phosphatidylinositol 3-kinase (PI3K)/Akt cascade has been mentioned in the expression of several inflammatory genes. Here, we first report that IL-1beta-induced up-regulation of proMMP-9 was inhibited by genistein. IL-1beta also stimulated phosphorylation of several protein tyrosine kinases such as c-Src and platelet-derived growth factor receptor (PDGFR), which was further confirmed by western blotting using an anti-phospho-c-Src or anti-phospho-PDGFR antibody, respectively. IL-1beta-stimulated c-Src, PDGFR, and Akt phosphorylation and proMMP-9 expression were attenuated by the inhibitors of c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), respectively, or transfection with dominant negative plasmid of c-Src or short hairpin RNAs of PDGFR and Akt. Moreover, IL-1beta-induced proMMP-9 expression was blocked by pre-treatment with curcumin (a p300 inhibitor). We further confirmed that IL-1beta stimulated p300 recruitment to MMP-9 promoter, and then acetylated histone H4 by immunoprecipitation and chromatin immunoprecipitation-PCR assays. The recruitment and activation of p300 in MMP-9 promoter were inhibited by pre-treatment with PP1, AG1296, and LY294002, respectively. Moreover, IL-1beta stimulated the c-Src-dependent transactivation of PDGFR/PI3K/Akt cascade is independent of nuclear factor-kappaB pathway. These results indicated that in rat brain astrocytes cells, PI3K/Akt activation was mediated through c-Src-dependent transactivation of PDGFR promoted transcriptional co-factor p300 recruitment and activation and eventually led to increased proMMP-9 expression by IL-1beta.

Matrix metalloproteinase (MMP)-9 expression induced by interleukin-1beta (IL-1beta) was investigated in rat brain astrocyte-1 (RBA-1). Here we report that the mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) pathways participate in the induction of MMP-9 expression by IL-1beta. Zymographic, western blotting, and RT-PCR analyses showed that IL-1beta increased expression of MMP-9 mRNA and protein, which were inhibited by inhibitors of MEK1/2 (U0126), p38 (SB202190), and JNK (SP600125). In accordance with these findings, IL-1beta stimulated phosphorylation of p42/p44 MAPK, p38, and c-Jun N-terminal kinase (JNK), which was attenuated by U0126, SB202190, or SP600125, respectively. Furthermore, this up-regulation of MMP-9 mRNA and protein was blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of inhibitory kappa B-alpha (IkappaB-alpha) was revealed by western blotting and immunofluorescence staining, which was blocked by helenalin, but not by U0126, SB202190, or SP600125. Taken together, these results suggest that in RBA-1 cells, activation of p42/p44 MAPK, p38, JNK and NF-kappaB pathways is essential for IL-1beta-induced MMP-9 gene expression via transcription and translation processes. An increased understanding of the signal transduction pathways involved in IL-1beta-induced MMP-9 expression on RBA-1 may be of potential therapeutic value in the treatment of inflammatory disease.

Interleukin (IL)-20 is a proinflammatory cytokine in the IL–10 family. IL–20 is associated with tumor promotion in the pathogenesis of oral, bladder, and breast cancer. However, little is known about the role of IL–20 in prostate cancer. We hypothesize that IL–20 promotes the growth of prostate cancer cells. Immunohistochemical staining showed that IL–20 and its receptors were expressed in human PC–3 and LNCaP prostate cancer cell lines and in prostate tumor tissue from 40 patients. In vitro, IL–20 upregulated N-cadherin, STAT3, vimentin, fibronectin, RANKL, cathepsin G, and cathepsin K, and increased the migration and colony formation of prostate cancer cells via activated p38, ERK1/2, AKT, and NF-κB signals in PC–3 cells. We investigated the effects of anti-IL–20 monoclonal antibody 7E on prostate tumor growth in vivo using SCID mouse subcutaneous and intratibial xenograft tumor models. In vivo, 7E reduced tumor growth, suppressed tumor-mediated osteolysis, and protected bone mineral density after intratibial injection of prostate cancer cells. We conclude that IL–20 is involved in the cell migration, colony formation, and tumor-induced osteolysis of prostate cancer. Therefore, IL–20 might be a novel target for treating prostate cancer.

Developing novel chemo-prevention techniques and advancing treatment are key elements to beating lung cancer, the most common cause of cancer mortality worldwide. Our previous cohort study showed that cysteinyl leukotriene receptor antagonists, mainly montelukast, decreased the lung cancer risk in asthma patients. In the current study, we conducted in vivo and in vitro experiments to demonstrate the inhibiting effect of montelukast on lung cancer and to investigate the underlying mechanisms. Using Lewis lung carcinoma-bearing mice, we showed that feeding montelukast significantly delayed the tumor growth in mice (p < 0.0001). Montelukast inhibited cell proliferation and colony formation and induced the cell death of lung cancer cells. Further investigation showed the down-regulation of B-cell lymphoma 2 (Bcl-2), up-regulation of Bcl-2 homologous antagonist/killer (Bak), and nuclear translocation of apoptosis-inducing factor (AIF) in montelukast-treated lung cancer cells. Montelukast also markedly decreased the phosphorylation of several proteins, such as with no lysine 1 (WNK1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (Erk1/2), MAPK/Erk kinase (MEK), and proline-rich Akt substrate of 40-kDa (PRAS40), which might contribute to cell death. In conclusion, montelukast induced lung cancer cell death via the nuclear translocation of AIF. This study confirmed the chemo-preventive effect of montelukast shown in our previous cohort study. The utility of montelukast in cancer prevention and treatment thus deserves further studies.

Inositol 1, 4, 5-trisphosphate receptor (IP3R)-mediated Ca(2+) release from the endoplasmic reticulum (ER) triggers many physiological responses in neurons, and when uncontrolled can cause ER stress that contributes to neurological disease. Here we show that the unfolded protein response (UPR) in neurons induces rapid translocation of nuclear receptor-interacting protein 140 (RIP140) to the cytoplasm. In the cytoplasm, RIP140 localizes to the ER by binding to the IP3R. The carboxyl-terminal RD4 domain of RIP140 interacts with the carboxyl-terminal gate-keeping domain of the IP3R. This molecular interaction disrupts the IP3R's 'head-tail' interaction, thereby suppressing channel opening and attenuating IP3R-mediated Ca(2+) release. This contributes to a rapid suppression of the ER stress response and provides protection from apoptosis in both hippocampal neurons in vitro and in an animal model of ER stress. Thus, RIP140 translocation to the cytoplasm is an early response to ER stress and provides protection against neuronal death.

Lung cancer is the leading cause of cancer death worldwide, with metastasis underlying majority of related deaths. Angiomotin (AMOT), a scaffold protein, has been shown to interact with oncogenic Yes-associated protein/transcriptional co-activator with a PDZ-binding motif (YAP/TAZ) proteins, suggesting a potential role in tumor progression. However, the functional role of AMOT in lung cancer remains unknown. This study aimed to identify the patho-physiological characteristics of AMOT in lung cancer progression. Results revealed that AMOT expression was significantly decreased in clinical lung cancer specimens. Knockdown of AMOT in a low metastatic CL1-0 lung cancer cell line initiated cancer proliferation, migration, invasion and epithelial-mesenchymal transition. The trigger of cancer progression caused by AMOT loss was transduced by decreased cytoplasmic sequestration and increased nuclear translocation of oncogenic co-activators YAP/TAZ, leading to increased expression of the growth factor, Cyr61. Tumor promotion by AMOT knockdown was reversed when YAP/TAZ or Cyr61 was absent. Further, AMOT knockdown increased the growth and spread of Lewis lung carcinoma in vivo. These findings suggest that AMOT is a crucial suppressor of lung cancer metastasis and highlight its critical role as a tumor suppressor and its potential as a prognostic biomarker and therapeutic target for lung cancer.

Microglial activation worsens neuronal loss and contributes to progressive neurological diseases like Parkinson's disease (PD). This inflammatory progression is countered by dynorphin (Dyn), the endogenous ligand of the kappa-opioid receptor (KOR). We show that microglial β-arrestin mediates the ability of Dyn/KOR to limit endotoxin-elicited production of pro-inflammatory effectors and cytokines, subsequently protecting neurons from inflammation-induced neurotoxicity. Agonist-activated KOR enhances the interaction of β-arrestin2 with transforming growth factor-beta-activated kinase 1 (TAK1)-binding protein 1 (TAB1), disrupting TAK1-TAB1 mediated pro-inflammatory gene expression. We reveal a new physiological role for β-arrestin in neuroprotection via receptor internalization-triggered blockade of signal effectors of microglial inflammatory neurotoxicity. This result offers novel drug targets in the convergent KOR/β-arrestin2 and inflammatory pathways for treating microglial inflammatory neuropathologies like PD.

Lung cancer is a major cancer, leading in both incidence and mortality in the world, and metastasis underlies the majority of lung cancer-related deaths. Galectin-1, a glycan-binding protein, has been shown to be overexpressed in lung cancer and involved in tumor-mediated immune suppression. However, the functional role of galectin-1 in lung cancer per se remains unknown. We demonstrate that ectopic expression of galectin-1 in a low-metastatic CL1-0 lung cancer cell line promotes its migration, invasion and epithelial-mesenchymal transition. Conversely, we also show that suppression of galectin-1 expression in highly invasive CL1-5 and A549 cells inhibits migration and invasion of lung cancer cell and causes a mesenchymal-epithelial transition. These effects may be transduced by increasing the expression of integrin α6β4 and Notch1/Jagged2, which in turn co-operates in the phosphorylation of AKT. The effects of galectin-1 on cancer progression are reduced when integrin β4 and Notch1 are absent. Further study has indicated that galectin-1 knockdown prevents the spread of highly metastatic Lewis lung carcinoma in vivo. Our study suggests that galectin-1 represents a crucial regulator of lung cancer metastasis. Thus, the detection and targeted treatment of galectin-1-expressing cancer serves as a new therapeutic target for lung cancer.

Objective. To evaluate the effectiveness of massage therapy (MT) for neck and shoulder pain. Methods. Seven English and Chinese databases were searched until December 2011 for randomized controlled trials (RCTs) of MT for neck and shoulder pain. The methodological quality of RCTs was assessed based on PEDro scale. The meta-analyses of MT for neck and shoulder pain were performed. Results. Twelve high-quality studies were included. In immediate effects, the meta-analyses showed significant effects of MT for neck pain (standardised mean difference, SMD, 1.79; 95% confidence intervals, CI, 1.01 to 2.57; P

Objective. To evaluate the effectiveness of Tuina-focused integrative Chinese medical therapies (TICMT) on inpatients with low back pain (LBP). Methods. 6 English and Chinese databases were searched for randomized controlled trials (RCTs) of TICMT for in-patients with LBP. The methodological quality of the included RCTs was assessed based on PEDro scale. And the meta-analyses of TICMT for LBP on pain and functional status were conducted. Results. 20 RCTs were included. The methodological quality of the included RCTs was poor. The meta-analyses' results showed that TICMT had statistically significant effects on pain and functional status, especially Tuina plus Chinese herbal medicine (standardised mean difference, SMD: 1.17; 95% CI 0.75 to 1.60 on pain; SMD: 1.31; 95% CI 0.49 to 2.14 on functional status) and Tuina plus acupuncture (SMD: 0.94; 95% CI 0.38 to 1.50 on pain; SMD: 0.53; 95% CI 0.21 to 0.85 on functional status). But Tuina plus moxibustion or hot pack did not show significant improvements on pain. And the long-term evidence of TICMT was far from sufficient. Conclusions. The preliminary evidence from current studies suggests that TICMT might be effective complementary and alternative treatments for in-patients with LBP. However, the poor methodological quality of the included RCTs means that high-quality RCTs with long follow-up are warranted.

The bioactivity of endothelin-1 (ET-1) has been suggested in the development of CNS diseases, including disturbance of water homeostasis and blood-brain barrier integrity. Recent studies suggest that hypoxic/ischemic injury of the brain induces release of ET-1, behaving through a G-protein coupled ET receptor family. The deleterious effects of ET-1 on astrocytes may aggravate brain inflammation. Increased plasma levels of matrix metalloproteinases (MMPs), in particular MMP-9, have been observed in patients with neuroinflammatory disorders. However, the detailed mechanisms underlying ET-1-induced MMP-9 expression remain unknown. In this study, the data obtained with zymographic, western blotting, real-time PCR, and immunofluorescent staining analyses showed that ET-1-induced MMP-9 expression was mediated through an ET(B)-dependent transcriptional activation. Engagement of G(i/o)- and G(q)-coupled ET(B) receptor by ET-1 led to activation of p42/p44 MAPK and then activated transcription factors including Ets-like kinase, nuclear factor-kappa B, and activator protein-1 (c-Jun/c-Fos). These activated transcription factors translocated into nucleus and bound to their corresponding binding sites in MMP-9 promoter, thereby turning on MMP-9 gene transcription. Eventually, up-regulation of MMP-9 by ET-1 enhanced the migration of astrocytes. Taken together, these results suggested that in astrocytes, activation of Ets-like kinase, nuclear factor-kappa B, and activator protein-1 by ET(B)-dependent p42/p44 MAPK signaling is necessary for ET-1-induced MMP-9 gene up-regulation. Understanding the mechanisms of MMP-9 expression and functional changes regulated by ET-1/ET(B) system on astrocytes may provide rational therapeutic interventions for brain injury associated with increased MMP-9 expression.

[1] The close decadal relationship between the North Atlantic Oscillation (NAO) and the cold surge frequency in Northern Taiwan is identified. The cold surge frequency was higher (lower) in 1957–1977 (1981–2001), which corresponded to the negative (positive) phase of the NAO. The teleconnection pattern associated with the NAO suggests that the Siberian high, the ridge upstream of Lake Baikal and the East Asian trough, which are well-known circulation characteristics favorable for cold surge occurrences, were enhanced in the negative NAO phase. A weakened upper-level convergence over the Mediterranean-Sahara occurring in the negative NAO phase was associated with a wave-like pattern spreading toward East Asia along the subtropical jet stream. Barotropic modeling results suggest that the suppressed upper-level convergence in the Mediterranean-Sahara region may force a wave train emanating toward East Asia along the subtropical jet stream, which acts as a Rossby waveguide. The barotropic wave pattern may result in a stronger low-level northerly over subtropical East Asia and create a favorable background for the further southward penetration of cold air, and therefore more frequent cold surge occurrence. The same process may be applied to the period when the NAO is in the positive phase.

A microporous polypropylene (PP) hollow fiber membrane contactor was used as a bioreactor to degrade phenol in aqueous solutions by Pseudomonas putida BCRC 14365 at 30 degrees C. The fibers were pre-wetted by ethanol to make them more hydrophilic. The initial cell density was fixed at 0.025 gl(-1). The effects of added NaCl concentration (0-1.78 M) and pH (3-8) in substrate solution on the biodegradation were studied. The experimental results by suspended cells were discussed. It was shown that the cells in microporous hollow fibers were unable to tolerate substrate solution pH to a larger range than those in suspensions. The suspended cells grew well on 100 mg l(-1) of phenol only at NaCl concentrations below 0.44 M. However, the cells in microporous hollow fibers could completely degrade 500 mg l(-1) of phenol in solutions containing NaCl concentration up to 1.52 M, which was due to the enhanced tolerance limit to salinity effect by the membrane-attached biofilms and the sufficiently slow mass transfer of NaCl through the membrane pores.