Immune–SNS cross-talk is required to maintain homeostasis under normal conditions, to develop an immune response of appropriate magnitude after injury or immune challenge, and subsequently restore homeostasis. Typically, β2-AR-induced cAMP is immunosuppressive. However, many studies report actions of β2-AR stimulation in immune cells that are inconsistent with typical cAMP–PKA signal transduction. Research during the last decade in non-immune organs, has unveiled novel alternative signaling mechanisms induced by β2-AR activation, such as a signaling switch from cAMP–PKA to mitogen-activated protein kinase (MAPK) pathways. If alternative signaling occurs in immune cells, it may explain inconsistent findings of sympathetic regulation of immune function. Here, we review β2-AR signaling, assess the available evidence for alternative signaling in immune cells, and provide insight into the circumstances necessary for “signal switching” in immune cells.Cross-talk between the sympathetic nervous system (SNS) and immune system is vital for health and well-being. Infection, tissue injury and inflammation raise firing rates of sympathetic nerves, increasing their release of norepinephrine (NE) in lymphoid organs and tissues. NE stimulation of β Communication with immune cells occurs directly by neurotransmitter release from sympathetic nerves that bind to postsynaptic receptors expressed on immune cells. Indirect effects of sympathetic activity may occur by regulating cytokine release or stromal cell function. The purpose of SNS–immune communication is to maintain immune homeostasis under basal conditions, augment host defense to eliminate pathogens, promote healing after tissue injury, and restore homeostasis after pathogen elimination or tissue repair. The SNS-immune pathway also provides “hardwired” circuitry through which psychological and social factors can profoundly adapt host immunity and healing. Sympathetic–immune regulation is mediated largely by stimulated release of its major neurotransmitter, norepinephrine (NE) and subsequent intercellular signaling via postsynaptic adrenergic receptors (ARs) expressed in closely apposed immunocytes , and secondly, its signaling pathways must be able to intersect with the diverse signaling pathways that mediate these cellular functions. This notion is consistent with a confusing literature documenting that the SNS can increase or decrease most immune measures. Recent findings begin to unravel the cellular mechanisms that may explain this duality in function under normal and pathologic conditions. Moreover, prolonged or inappropriate activation of either the SNS or immune system can result in the failure of the immune and sympathetic nervous systems to shut-off immune responses and to re-establish immune system homeostasis within normal physiological ranges. Under such conditions the immune system and/or SNS can promote pathological and lethal effects, including chronic inflammation, toxic shock, tissue damage, immune deficiency, autoimmunity and cancer.Under normal physiological conditions, SNS–immune regulation is adaptive, influencing the normal immune response to injury or foreign antigens/toxins/infectious agents, with subsequent restoration of homeostasis and recovery to a healthy state ,2. Impor2-ARs, thus, we will focus this review on this receptor subtype. Historically, β2-ARs were thought to exert largely inhibitory signals to cells of the immune system by inducing cAMP and protein kinase A (PKA). It is now clear that signaling via β2-ARs is much more complex and can, in addition to their traditional signaling pathway, activate multiple signal transduction pathways to exert inhibitory and/or enhancing effects on cell functions. These receptors, like other GPCRs, are now viewed as complex, multidimensional activators of a variety of potential signaling cascades rather than simply activating binary inhibitory or stimulatory signaling pathways via coupling to G proteins. In this paper, we review current information on β2-AR traditional  and nontraditional  signal transduction pathways, then discuss the possibility that nontraditional signal pathways identified for β2-ARs are involved in sympathetic regulation of immune cell functions.New information has emerged regarding mechanisms through which intercellular communication occurs between sympathetic nerves and its AR-expressing target cells that may explain the ability of the SNS to both inhibit and enhance immune responses depending upon the context of the immune response. Much of the new understanding of how these G protein-coupled receptors (GPCRs) activate both inhibitory and stimulatory signaling pathways has not yet been extended to understanding SNS regulation of functions in immune cells. Most research regarding sympathetic regulation of immune cell functions has focused on regulation via βin vitro methods with various cell lines and have largely focused on β2-ARs in non-immune cells. The extent to which activation of non-canonical signaling pathways via β2-ARs is physiologic or pathologic or whether they are functional signaling pathways in immune cells is not clear. Here, we review the current information on the traditional and non-traditional mechanisms through which β2-ARs signal, how β2-AR functions are regulated by SNS nerve firing (SNS activity) and cross-talk with other signaling pathways activated by immune challenge, and the existing evidence for non-canonical signaling via β2-AR in immune cells. Recent findings from our group and others do support that a “switch” in signal from canonical to non-canonical pathways for β2-AR can occur in immune cells under inflammatory conditions. Further, the functional implications for signaling via non-canonical pathways with regard to immune functions and the clinical relevance of understanding how β2-ARs are regulated are discussed.Over the past decade, it has become clear that ARs and other GPCRs can activate non-traditional signal transduction pathways in addition to their traditional signaling pathways ,4,5,6. T2-ARs are coupled to Gαs, which activates cAMP-PKA-mediated intracellular signaling  and beta/gamma subunits, respectively) dissociation from each other  present in distinct subdomains of the plasma membrane and cytoplasm , which is bound to a catalytic subunit (C) . Two majShirshev  and will2-AR expression, respectively, which affects receptor function. Chronic stimulation or high NE concentrations activate the β2-AR and induce receptor desensitization and down-regulation in target cells, including cells of the immune system. Receptor desensitization and down-regulation result from β2-AR phosphorylation by PKA and transiently by G protein-coupled kinases (GRKs), specifically GRK2. PKA and GRK2-mediated phosphorylation target different serine sites on the carboxy tail of the β2-AR [s  comprise the currently known subtypes of this family of kinases [2-AR, which creates a binding site for β-arrestin [2-AR by GRK2 changes the conformation of the receptor, which allows β-arrestin to bind to the carboxy-terminal tail of the receptor [2-AR, which impedes Gs protein coupling to the receptor, and prevents receptor signaling via cAMP. This process is referred to as β2-AR desensitization [GRKs play a crucial role in agonist-induced desensitization of the β kinases . GRK2 pharrestin ,33,34,35receptor ,36,37. Ttization .2-AR phosphorylation and subsequent β-arrestin binding, the agonist-bound β2-ARs are internalized [2-ARs undergo one of two fates. They may be dephosphorylated and recycled to the plasma membrane  alters the receptor conformation, which impairs Gs binding to the β2-AR , and p38 MAPK—phosphorylate many transcription factors and other kinases that are important regulators for gene expression [As indicated above, βhe β2-AR , #1, but protein , #2. Bot protein , #3. In  protein , #2 indu protein , #4 [47,pression ,50,51,52pression ,54,55 anpression ,57,58,592-AR recruits cyclic nucleotide PDE to the receptor-β-arrestin complex (. PDEs are a family of enzymes that hydrolyze cAMP to nucleotide 5'-monophosphate (5'-AMP) by degrading the phosphodiester bond  levels, and therefore, the amplitude and duration of the second messenger response. PDE also establishes gradients within localized subcellular compartments or domains particularly in compartments where PDE concentrations are high [Binding of β-arrestin to the β complex , #5 [60,ter bond , #6. Thuignaling , #7. Thuare high .in vitro [.There are eleven families of PDEs, of which three specifically hydrolase cAMP–PDE4, 7 and 8 ,66. PDEsin vitro .2-ARs by GRKs and β-arrestins plays essential roles in maintaining the normal functions of cells and tissues, including the immune cells. Cells of the immune system express high levels of GRK2, exceeding levels in the heart by 4–5-fold [2-AR is observed in many diseases where net SNS firing rate (activity) is chronically elevated and robust/chronic inflammation ensues, including hypertension [2-AR density and poorer responsiveness are significant predictors of cardiovascular mortality and myocardial infarction [2-AR agonist for 2 weeks equally enhances expression of cardiac and lymphocyte expression of GRK2 mRNA in the heart and lymphocytes [The regulation of β4–5-fold . In addi4–5-fold ,69,70, brtension , sepsis rtension ,73,74, rrtension ,76,77, artension ,79,80. Ffarction . In factphocytes . This rephocytes .2-AR density and its capacity to produce cAMP in PBMCs are increased, not shut-off as in RA. These findings in MS patients strongly correlate with disease activity [2-AR up-regulation, deficient GRK2 expression, and increased cAMP production as a consequence of PKA-mediated β2-AR phosphorylation in MS. Consistent with these findings, GRK2(+/−) mice, which express reduced GRK2, do not develop relapsing and remitting EAE that is observed in the wild-type EAE mice [2-ARs and other GPCR signaling, and their regulation by GRK2 in autoimmunity.Reduced GRK2 levels are also observed in PBMCs from patients with RA  and multactivity ,86,87. MEAE mice . The abs2-AR internalization is well recognized to homeostatically attenuate receptor responsiveness, there is mounting evidence that the endocytic pathway can also generate receptor-initiated signals that are G protein-independent. These studies reveal that GRKs are involved in initiating a GPCR-mediated, β-arrestin-dependent, G protein-independent signaling pathway, including β2-ARs. This β-arrestin-dependent signaling pathway induces physiological responses that differ from the canonical G protein-mediated responses [2-AR is serine/threonine-specifically phosphorylated by different GRK subtypes. Non-canonical signaling induces the activation of alternative signaling pathways that typically oppose canonical signaling by β2-ARs. Site-specific phosphorylation by specific subtypes of GRKs determines whether the GPCR undergoes desensitization or signals via an alternative pathway [2-AR by GRK2 or 5/6 is dependent on the concentration of the agonist [2-AR.While βesponses ,89,90. I pathway . Nobles  pathway  find tha agonist ,92,93. H2-ARs can signal via G protein-independent, β-arrestin-mediated non-canonical signaling pathways. Four arrestin subtypes are identified, arrestins 1–4. Of these, β-arrestin 1 and 2 are present in all mammalian cells examined [2-ARs desensitization and internalization [2-ARs through β-arrestin 2 induces sustained ERK1/2 rather than the traditional cAMP-PKA signaling [Over the last decade, it has been repeatedly demonstrated in various cell types that βexamined . Both β-lization . Howeverlization . Signaliignaling ,38,48,922-ARs, β-arrestin 2 binds to the β2-AR after its phosphorylation by GRK 5/6 , and the immune stimuli applied, the time point of evaluation after immune activation, and the environmental conditions of the study. However, most studies investigating the role of NE or β2-AR agonists on inflammation and cellular effector functions report immunosuppressive effects . Stil2-ARs is commonly studied as a co-stimulus in combination with a known immune activator. β2-AR activation of the canonical pathway can negatively or positively regulate the response of immune activators, such as concanavalin A, phytohemagglutinin, anti-CD3/anti-CD28, lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), depending on the type of immune stimulus and timing of β2-AR stimulation relative to immune activation [2-agonist, isoproterenol (ISO) suppresses TNF-α production in LPS-stimulated peritoneal macrophages [2-AR stimulation reduces ERK1/2 and p38 MAPK activity in the LPS-challenged macrophages [2-ARs in immune cells can enhance inflammation via MAPK signaling pathways   that are differentiated towards the macrophage lineage by treatment with phorbol 12-myristate 13-acetate (PMA) [2-AR-induced TNF-α production is associated with an increase in ERK1/2 and p38 MAPKs expression [2-AR signaling pathways. In both of these conditions, pretreatment with the β-AR antagonist, propranolol blocks ISO-enhanced cytokine production, verifying mediation by β-ARs [2-AR signal transduction pathway activated depends on the immune stimulus. Thus, it seems that the β2-AR-mediated direction of change in inflammatory mediators produced by immune cells depends on the nature of the inflammatory stimulus, when the SNS is activated, and the context of the stimulation [The immunomodulatory effect of βtivation ,109,110.rophages , consistrophages . In contnd JNKs) . For exate (PMA) . These fte (PMA) ). These te (PMA) . The β2-pression , supportby β-ARs . The mecmulation .2-AR functions are not known, we are hypothesizing that the different signaling pathways activated by LPS and PMA can lead to β2-AR modifications that alter β2-AR signaling  expressed on macrophages. LPS stimulation of the TLR4 complex induces activation of both nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and MAPK pathways  [2-ARs. LPS promotes adenylyl cyclase activity [2-AR signaling via the cAMP-PKA canonical pathway  . Activatactivity , suppresactivity , and redactivity . These e pathway . LPS-ind2-ARs in a manner that causes them to stimulate, rather than inhibit TNF-α production remains unclear. Szenlenyi and coworkers [2-AR stimulation on LPS and PMA -induced TNF-α production to differences in the kinetics of MAPK activation by the immune stimulus. Activation of MAPK occurs more rapidly and robustly after treatment with PMA than with LPS. In addition, LPS treatment also induces a more sustained activation of ERK and p38 than PMA. How differences in the kinetics and/or magnitude of MAPK (pERK and p38) production result in the opposing β2-AR agonist induced effects on TNF-α production, and the extent that this mechanism is responsible for the difference in receptor function, remains unclear. Alternatively PMA is also an activator of protein kinase C (PKC). In other non-immune cell types, PKC modulates β2-AR functions in several ways. Like PKA, this kinase can directly phosphorylate the β2-AR to promote receptor desensitization, albeit with less efficacy than PKA [2-AR signaling either promote or reduce β2-AR signaling via cAMP. They do not explain the β2-AR-induced increase in TNF-α production in PMA-activated macrophages.Unfortunately, how phorbol esters, such as PMA, alter βoworkers  attributthan PKA ,115,116.than PKA , an effethan PKA ,119,120,than PKA . However2-AR potentiation of PMA-induced macrophage production of TNF-α is by regulating NF-κB  0) cells can differentiate into either Th1 or Th2 cells, which drive cellular or humoral immune responses, respectively [2-AR on Th0 and Th1 cells and their absence on Th2 cells. Cellular immune responses are directed by Th1 cells through their production of IL-2, which enhance proliferation and expansion of activated naïve CD4+ T cells and their production of interferon (IFN)-γ that promote Th1 cell but suppresses Th2 cell differentiation. Treatment of cultured naïve CD4+ T lymphocytes with NE or selective β2-AR agonists reduces IL-2 and IFN-γ . This l2-AR agonist, β2-AR stimulation increases Th0 cell production of IFN-γ [2-ARs prior to antigen activation decreases IFN-γ synthesis, while β2-agonist stimulation after antigen stimulation increases IFN-γ production. Despite the fact that these findings are over a decade old, the mechanisms by which β2-ARs induce activation of signaling pathways that increase in IFN-γ remains a mystery.When naïve CD4+ Th0 cells are activated by an antigen or by anti-CD3/CD28 costimulation with or without NE or a βof IFN-γ . In thisof IFN-γ . So, sti2-AR-mediated increases in IFN-γ may be gained from the Th0 cell activation conditions in which this occurs. Antigen activation of T cell receptors (TCRs) on Th0 cells induces synthesis of IFN-γ by activating the p38 MAPK pathway [2-AR agonist after antigen challenge. If so, antigen challenge could result in an increase in GRK5 expression and subsequent GRK5 phosphorylation of β2-ARs to induce G protein-independent signaling via β-arrestin to increase production of IFN-γ. Obviously, future studies are needed to test this hypothesis.Clues to the underlying mechanisms for β pathway ,132  the arthritic joints and the spleens were harvested to assess sites where arthritogenic T cells develop. Mesenteric lymph nodes (MLNs) were examined as a site where T lymphocytes that do not transfer the disease to naïve animals reside. Interestingly, arthritic animals treated with the β2-AR agonist, terbutaline altered IFN-γ production differently in each lymphoid organ examined, increasing IFN-γ in DLN cells, having no effect on IFN-γ in splenocytes, and inhibiting IFN-γ in MLN cells [2-AR stimulation to reduce IFN-γ production by increasing cAMP levels. Findings in the spleen indicate β2-AR desensitization and down-regulation induced by chronic agonist stimulation as β-AR density and cAMP production are reduced in splenocytes from these rats [2-AR-mediated increases in IFN-γ observed in DLN cells cannot be explained by the canonical signaling pathway for β2-ARs or a loss of receptor numbers or activity.Recent findings from our laboratory provide evidence for a shift in βel of RA ,133. In ese rats   are important for GRK-mediated β2-AR phosphorylation in DLN cells.One possible explanation for the βse IFN-γ ,132 . Given iewed in ), the diDramatic changes in the expression of GRK2 and 6 in peripheral blood mononuclear cells are observed in RA patients ,84,85 an2-ARs in immune cells? The SNS regulates all aspects of innate and adaptive immune functions as part of its major role in integrating body functions to maintain homeostasis [2-ARs signal intracellularly. Data support that inflammatory cytokines, regulate both β2-AR expression and signal transduction by increasing the expression of intracellular GRKs. The exciting discovery that β2-AR can activate ERK1/2 by a Gs-independent, β-arrestin-dependent pathway raises many questions regarding SNS regulation of innate and adaptive immune functions in health and disease. Whether such signaling occurs in immune cells has not been determined. However, there are many cases in the literature where β2-ARs induce changes in cytokine responses in directions not explained by increases in cAMP, and that are blocked by β2-AR antagonists. Such responses could be explained via β2-AR-induced increases in ERK1/2. It is also unclear if this alternate signaling pathway represents normal physiological regulation of immune functions that are adaptive or are strictly pathologic. There is data to support the latter, as GRK6 expression, which would drive β2-AR signaling via ERK, is increased in MRL/lpr mice, a murine model of systemic lupus erythematosus (SLE), and in spleens upon autopsies of SLE patients [2-AR signaling, are linked to many diseases. Thus, it will be important to understand how β2-AR signaling via ERK pathways is involved in susceptibility, development and progression of diseases mediated by the immune system.What are the implications of altered SNS signaling via βeostasis . Cross-tpatients . Furtherpatients , hyperalpatients ,139, andpatients . Chronic


Increasing evidence indicates that lncRNAs play critical roles in various biological processes, but many have not been functionally characterized. Here, we report a novel radiation-inducible lncRNA, namely lnc-RI which is essential for cell survival and appropriate mitotic progression. Our data indicated that knockdown of lnc-RI resulted in spindle abnormalities and mitotic arrest simultaneously with sharply decreased mRNA and protein expression of PLK1, a key regulator of mitosis. Our data demonstrated that PLK1 is a key downstream mediator of lnc-RI in regulating mitosis, whereby lnc-RI competitively bound to the negative PLK1 regulating miRNA, miRNA-210-p3. Taken together, we have identified lnc-RI as a new regulator of mitosis which acts by releasing PLK1 mRNA activity via competition for binding to miRNA-210-3p. The mammalian genome transcribes numerous long noncoding RNAs (lncRNAs) but only a few of them have been characterized234567823467The mitotic process of mammalian cells is precisely controlled by a set of regulators which includes cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors. Polo-Like Kinase 1 (PLK1) is a key regulator of progression through mitosis from entry, spindle formation, cytokinesis to mitotic exit. In mammalian cells, dysregulation of PLK1 can result in the formation of aberrant spindles and mitotic arrest1112141517182022Lnc-RI (lncRNA Radiation Induced) was originally identified as one of several lncRNAs which could be upregulated by ionizing radiation in our labLnc-RI was recently identified by our group as an ionizing radiation-inducible transcript of a long non-coding RNA (lncRNA) with1423 nucleotides encoded by a gene located in chromosome 7p22.3 (ENST00000382528) and containing 2 exons . AlthougTo investigate the effect of lnc-RI depletion on cell growth, HEK293 and HeLa cells were transfected with lnc-RI RNAi-2 or a scrambled control sequence, Ctrl RNAi. Cell growth curves were monitored after transfection. The growth curves indicated that the knockdown of lnc-RI significantly inhibited the proliferation of both cell lines . To furtIn order to understand the critical role of Lnc-RI in cellular proliferation, the effect of lnc-RI on mitotic progression was analysed. HeLa cells were transfected with lnc-RI RNAi-1, lnc-RI RNAi-2, or Ctrl RNAi respectively. The cells were fixed and labelled with the antibody against p-H3, a marker of mitotic cells, and stained with PI at 48 h post-transfection. As shown in To further investigate the effects of knockdown of lnc-RI on mitosis, HeLa cells (HeLa cells is suitable for mitosis study) transfected with lnc-RI RNAi-2 or Ctrl RNAi were treated with nocodazole for 16 h. Synchronized mitotic cells were obtained using a shake-off method and were released after switching to nocodazole-free fresh medium. Cell cycle profiles were examined at various time points after release. As shown in The formation of a symmetrical bipolar spindle is one of the most important events in mitosis. Aberrant spindle formation could disturb mitotic progression, and induces mitotic arrest and genetic instability. To investigate the role of lnc-RI in regulating spindle formation, HeLa cells transfected with lnc-RI RNAi-1, lnc-RI RNAi-2, or Ctrl RNAi were stained with anti-β-tubulin antibody and DAPI at 48 h after transfection. Aberrant spindles were induced by transfection with lnc-RI RNAi and exhibited multiple morphological characteristics including asymmetrical spindle, displaced spindle, tripolar spindle, monopolar spindle, and disorganized spindle . A totalhttp://string-db.org/)(STRING), a database of protein interactions, was used to investigate functional associations among the target genes of lnc-RI and networks involved in the biological function of lnc-RI. All of the genes with altered expression were uploaded into STRING 10 (except 8 genes not present in the database), which identified 3 gene networks. As shown in To explain the mechanism underlying the effects of lnc-RI in regulating spindle formation and mitotic progression, we screened for downstream genes regulated by lnc-RI. HeLa cells were transfected with lnc-RI RNAi-1, lnc-RI RNAi-2 or Ctrl RNAi, and alterations in gene expression were monitored using a Human Genome U133 Plus 2.0 expression profile cDNA microarray. The expression profile analysis identified 153 genes with altered expression, including 118 genes that were up-regulated greater than 1.5-fold in both lnc-RI RNAi-1 and lnc-RI RNAi-2-transfected cells, compared with Ctrl RNAi-transfected cells, and 35 genes that were downregulated . Search Among the three gene networks, we were most interested in PLK1, a well-known key regulator of mitosis from mitotic entry to exit. The cellular phenotypes by depleting lnc-RI observed in this study was very similar to those induced by PLK1 suppression as reported previously131415To investigate the mechanism of lnc-RI to regulate the PLK1 expression, we first examined the effect of lnc-RI knockdown on the activity of the PLK1 promoter. As shown in et al.One of the major mechanisms of miRNA regulation of gene expression is to bind to the 3′UTR of target mRNA and promote mRNA degradation. It was reported that lncRNAs may affect mRNA stability by competing for binding to miRNAsTo further examine whether lnc-RI regulates PLK1 expression by competing for miRNA-210-3p, HeLa infected with LV-shRNA-lnc-RI was transfected with miRNA-210-3p inhibitor and PLK1 mRNA and protein expression were analysed 24 h after transfection. As shown in As described in Increasing evidence indicate that lncRNAs play critical roles in multiple processes. Thus, lncRNAs are attracting much interest. To our knowledge, this study is the first report of clear evidence demonstrating the biological function of the newly identified long noncoding RNA, lnc-RI. In the present study, cell proliferation and colony formation assays demonstrated that lnc-RI is necessary for cell survival. Knockdown of lnc-RI induced aberrant spindles, mitotic arrest, and apoptosis, and mitotic regulator PLK1 was a mediator of knockdown of lnc-RI. Lastly, we found lnc-RI is a target of miRNA-210-3p and lnc-RI regulates PLK1 mRNA stability via competing for binding to miRNA-210-3p.et al. found that a long noncoding RNA named SAT III RNA binds to the kinetochore component CENP-C and is required for correct localization of the centromere-defining proteins CENP-A and CENP-C, as well as outer kinetochore proteins12131415Spindle formation is a critical event during mitosis, and aberrant spindles activate mitotic arrest. Spindle formation is controlled by multiple regulators involved in centrosome formation, microtubule regulation, and chromosome segregation. Only one lncRNA has previously been found to be involved in spindle formation. Rošić S. et al. reported that miR-210-3p could regulate PLK1 by binding PLK1 mRNA 3′UTR and thus disturb spindle formation and mitotic progress, which are similar to the phenotypes found in the present study.LncRNAs regulate targets by different modes, including as competing endogenous RNA (ceRNA)In summary, this is the first study to demonstrate the function of lnc-RI. The data in this study indicate that lnc-RI is a novel regulator of mitosis via competing for miR-210-3p as a ceRNA to release PLK1 activity, thus playing a key role in proper spindle formation and mitotic progression.HeLa and HEK293 cells (Kept in our lab) were cultured in PRIM-1640(Sigma) or DMEM(Gibco) containing 10% fetal bovine serum (Hyclone), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37 °C in 5% CO2.MiRNA mimics, miRNA inhibitors, and small interfering RNA (siRNA) duplexes were synthesized and purified by Shanghai GenePharma Co. . The plasmids expressing shRNAs targeting lnc-RI were constructed by Shanghai GenePharma Co. The sequences of miRNAs, miRNA inhibitors, siRNAs, and shRNAs are listed in To synchronize ce lls in the cell cycle, cells were treated with nocodazole (100 ng/mL) for 16 h. Floating, mitotic cells were then harvested by a shake-off method, washed twice, and reseeded into fresh medium.The following antibodies were used: rabbit anti-phospho-histone H3 (S10) polyclonal antibody ; mouse anti-β-tubulin (D-10) monoclonal antibody ; mouse anti-PLK1 (F-8) monoclonal antibody ; mouse anti-β-actin monoclonal antibody ; HRP-conjugated anti-mouse IgG ; FITC-conjugated anti-rabbit IgG ; and FITC-conjugated anti-mouse IgG .Cells were harvested and washed twice with ice-cold PBS. Cell pellets were lysed in lysis buffer  with protease inhibitors. Cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes for western blot analysis.Total RNA was extracted using RNA TRIzol  according to the manufacturer’s instructions. PrimeScript RT reagent kit  was used to synthesize cDNAaccording to the manufacturer’s instructions. RT-PCR experiments were performed using PCR buffer . Real-time PCR experiments were performed by the Taqman probe method. The primers and probes for PCR are listed in Cells were placed in 24-well plates 1d before transfection. On the next day, 100 ng of reporter plasmids and 1 ng of pRL-CMV internal control plasmid were co-transfected with siRNAs or miRNA mimics into HeLa cells. Cells were harvested 48 h after transfection. Luciferase activity was measured using a Dual Reporter assay system . Firefly luciferase activityof each reporter was normalized to Renilla luciferase activity.Cultures with 30% confluency were seeded in a 96-well plate 24 h before transfection. The number of cells transfected with siRNA for lnc-RI or negative control was counted using Cell Counting Kit-8  at 0, 1, 2, 3, and 4 d after transfection. For colony formation assays, plasmids expressing shRNA to lnc-RI  were transfected into HeLa cells. One day after transfection, 500 cells were reseeded into 6-well plates and cultured with G418 (1000 ng/mL) for 14 days and the number of resulting colonies was analysed.For cell cycle analysis, the cells were harvested and fixed with 75% ethanol. The fixed cells were washed with PBS twice and permeabilised with 0.25% Triton X-100 in PBS for 15 min at room temperature. Cell pellets were incubated with the primary antibody  at room temperature for 30 min and with the secondary antibody  in the dark for 30 min. The cells were re-suspended in PBS plus 1 μg/mL RNase A, incubated for 20 min at 37 °C and stained with 25 μg/mL propidium iodide (PI). Apoptosis assays were performed using an Annexin-V-FITC apoptosis detection kit  according to the manufacturer’s protocol. Cell cycle and apoptosis assays were performed using a flow cytometer .Cells were grown on polylysine-coated glass coverslips. The cells were then fixed in PBS containing 4% formaldehyde for 15 min and permeabilised in Triton buffer (0.5% Triton X-100 in PBS). The fixed cells were blocked with blocking buffer  in a humidified chamber for 30 min at 37 °C. Staining was performed using an anti-β-tubulin antibody (1:100) overnight at 4 °C in a humidified chamber and washed twice in a blocking buffer. The cells were incubated with FITC-conjugated anti-mouse (1:400) secondary antibodies. DNAs were stained with DAPI in a mounting solution. Images were obtained using a Zeiss Observer A1 microscope equipped with a Zeiss camera and software at magnification of 40×.HeLa cells were harvested 48 h after transfection with Ctrl RNAi, lnc-RI RNAi-1, or lnc-RI RNAi-2. Total RNA was extracted and Human Genome U133 Plus 2.0 expressing profile chips  were used to screen the targets of lnc-RI by Capitalbio Technology Corporation .The lentivirus expressing shRNA-lnc-RI-2 (LV-shRNA-lnc-RI) or negative control (LV-NC) were purchased from GenePharma . HeLa cells were infected with lentivirus (20MOI) according to the GenePharma Recombinant Lentivirus Operation Manual. After infection, puromycin (2 μg/ml) was added and cultured for 3 days. Real Time PCR was employed to analyze the expression levels of lnc-RI.Experiments were performed with at least three replicates in each assay or three independent experiments. Data is presented as mean ± S.D. Statistical analysis was performed using Student’s t-test. Significance was set at a probability value less than 0.05.How to cite this article: Wang, Z.-D. et al. Long noncoding RNA lnc-RI is a new regulator of mitosis via targeting miRNA-210-3p to release PLK1 mRNA activity. Sci. Rep.6, 25385; doi: 10.1038/srep25385 (2016).


The rehabilitation is part of a drug free therapy management of peripheral polyneuropathy. The Transthyretin Familial Amyloid Polyneuropathy this illness entails deficiencies that do impact on the day to day physical comfort and everyday life of patients. They have motor function and sensory consequences.To be able to offer a well-adapted rehabilitation program, the rehabilitation therapists have put into place assessments in order to estimate the different clinical manifestations described by the patients. The pain, the paresthesia and strength deficiency will be evaluated in this way, and their evolution followed up, thanks to comparative test. We have been looking for tools to be able to evaluate the clinical manifestations and their evolution.For that purpose, we have chosen comparative tests that allow to measure quantitative and qualitative results. The pain and the tiredness are evaluated with visual analogue scales, the strength with muscular testing and dynamometer, the functional aspect is tested with an evaluation of: the standing upright; the handicap situation and the 6 minute walking test. The assessments' have improved in recent years with more precise tools.It is essential to stay as close as possible to the felt effects of the patients to deal with unbiased variations and put into place an adapted rehabilitation program.


Medicine in low socio-economic countries is primarily disease-oriented; prevention and rehabilitative care are secondary concerns. Hence, curative care erodes the few resources allocated to health. Despite the well-documented benefits of community-rehabilitation in the management of chronic conditions, little is known about common conditions present in the community in Butembo.The objective of this study was to determine the conditions encountered during rehabilitation in Butembo and to identify the trends of the five most common conditions during the study period.Data were extracted from a rehabilitation programme connect to one centre in Butembo. A descriptive retrospective medical study was performed for the period between 2004 and 2007. Descriptive statistics with percentages were computed. The Chi-square test was used to evaluate the differences with a probability of 5%.p = 0.0290), clubfoot (p < 0.0100) and glaucoma (p < 0.0100) than female patients. Children aged five or younger had a higher incidence of cerebral palsy  cataract (p = 0.0170), clubfoot (p < 0.0010), and glaucoma (p = 0.0010). Additionally, the overall comparisons by gender and age demonstrated differences for the five most common conditions .Cerebral palsy (46.9%), cataract (17.3%), clubfoot (11.8%), glaucoma (6.8%), and cleft lip (4.5%) were the most commonly encountered conditions, with cerebral palsy the most common condition throughout the study period. With regard to gender, male patients were significantly more affected by cataracts (Common conditions and associated factors were identified that will add to the effectiveness of the programme in terms of materials needed, staff skills, and programming. Special skills are still needed to help treat some acute conditions that can be handled at the rehabilitation centre, and a triage of attending rehabilitation centres could improve the effectiveness of the programme and lower the possibility of missed opportunities for acute stage patients. This study will be in aid for planning a rehabilitation service in Butembo and similar settings in the allocation of resources, the deployment of staff and in the composition of the kits.1Medical interventions in many developing countries, including Congo, are completely cure-oriented. Patients as well as providers are mostly oriented towards the treatment of diseases rather than the prevention of diseases or the rehabilitation of so-called ‘chronic disability diseases’. It follows that all efforts are focused on expensive medication and investigations, and little resources are allocated for the prevention of disease and the improvement of quality of life. As a result the health system is struggling to treat conditions that do not respond to curative measures, eroding the already scarce allocated resources. It was felt that without rehabilitation, people with disabilities and chronic illnesses may be forced to live in institutional settings to access health care, and those who stay in the community may either have their lives at risk or be highly dependent on family members.2 Hence, patients can have family members with whom they share joys and achievements, decision making and assistance when required, all of which leads to more satisfaction.3The role that patients’ families play in rehabilitation programmes cannot be overemphasised. Whilst providing care in the community or family context, community rehabilitation allows injured people, for example, to remain part of the family and community life, participate in normal activities and responsibilities, and become an active and contributing member of his or her social groups.4 and helps with carrier decisions5 once the patient is socially integrated. To provide help in performing everyday activities, to facilitate interactions with people with disabilities or sick people, and to provide assistance in treatment prior to any contact with a point of care,6 are additional roles fulfilled by families in rehabilitation programmes. Finally, family caregivers play a role in the medication adherence of their family members.7 However, from the caregiver's perspective, the role of family members in rehabilitation is not usually positive. The role of family members was, for example, perceived as restrictive for patients in rehabilitation programmes for stroke8 when they were discouraging patients from travelling outdoors for fear of recurrence, falling, losing their way, or an inability to manage the distance.The family also increases the assistance provided to older persons in rehabilitation centers9Several attempts to redress the situation have been undertaken with little success. With the spread of several non-government organisations, a focus on rehabilitation in the community using different approaches was suggested, one of them being a community-based approach (CBR). CBR is a strategy for rehabilitation, equalisation of opportunities, and social inclusion of all children and adults with disabilities.11 by making it easier to integrate people with disabilities through education programmes12 and by making it possible to train generic community workers in the delivery of rehabilitation and prevention services to people with disabilities and their families.14 Congenital malformations account for a large proportion of chronic disabilities.15 The morbidity and disability experienced by surviving children with disabilities, who comprise a large portion of rehabilitation services users, also have a major public health impact.16This approach has shown several advantages such as high effectiveness and value for people with disabilities in the communityThe management itself of such clients is time-consuming, and requires sufficient resources for ‘few direct results’ in the end. All of this requires good planning in terms of staff, equipment, time, resources, and even prior knowledge of common conditions encountered in the programme.We decided therefore firstly, to identify conditions encountered by rehabilitative teams in the Butembo zone and surrounding areas and to correlate them to the gender and age of the participants. Secondly, we sought to identify the trends of the five most common conditions during the study period.This study describes conditions encountered by the centre, which will serve in planning rehabilitation services in Butembo and similar settings regarding the allocation of resources, deployment of staff, and the composition of the kits.There were no direct potential physical, psychological, or disclosure danger that could be anticipated for the study. However, similar to several other studies, this study provides benefits for the participants and for society in general by providing data currently unavailable on common conditions of patients consulting rehabilitation services that could be used to improve service delivery. The minor risk of discrimination that could arise from the disclosure of participants was minimised by removing all participant identifiers and keeping them locked in a file with a password. We obtained approval to conduct the study from the rehabilitation service. No additional measure was necessary to protect data, as the data were collected from a place where all universal data protections regulations were already applied in the database. Informed consent was not sought for this paper for several reasons: its retrospective nature with no blanket informed consent; furthermore, this paper does not report on primary data. In addition, patients have been treated in line with the national guidelines, and the paper does not report on experimental data or new guidelines. It should also be kept in mind that this programme was not designed as a research project or a new protocol but as a treatment programme, and finally that the study was carried out as an audit or evaluation of the programme of rehabilitation.The population from which the sample was taken included all clients or patients attended to on site or receiving outreach at a rehabilitation centre called ‘Heshima Letu’ in Butembo.Successive participants attending the centre were included in the study irrespective of the nature and acuity of their conditions. Some participants travelled from their homes to consult a rehabilitation team directly at the centre, but most participants visited outreach sites during community visits. Patients of all ages and both sexes were included.2 in size and is located at an altitude between 1630 m to 2000 m. Its latitudinal and longitudinal location is between 29° and 29°30′ E and 0° and 0°16′ N, respectively. Heshima Letu is the centre where the study was conducted.Our study was performed in Butembo, Province of Nord-Kivu in the Eastern Congo. It is a city inhabited by 700 000 people subdivided in four parts. Butembo is 19.034 kmThis programme is an outreach programme that started as an orthopaedics programme before expanding its activities to common chronic conditions. It was not intended as a substitute or an alternative to mainstream medical services, and government officials continued to be in charge of the hospitalisation of severe cases, the treatment of patients with complications or acute episodes, and the referral of some of the clients who were not in the project.The team was composed of medical doctors , community nurses, physiotherapists, patient attendants, and community volunteers. Patients or clients were coming for assessment, follow-up, referral, massage or treatment, and those with operable or severe conditions were referred to the district hospital for further investigation and management.This study was a descriptive retrospective study of a rehabilitation centre.After obtaining approval from the management team, registers of patients or clients who consulted between 2004 and 2009 were reviewed by the first-mentioned author. Data were collected after checking for completeness of data regarding variables of interest in this study, including age, gender, period of consultation, and type of abnormality. All recorded patients with incomplete data in the registers were excluded from the study .p < 0.0500.All compiled data were screened for completeness and accuracy. A reviewed diagnosis were made during the visits made by a multidisciplinary team composed of an orthopaedic surgeon, an eye specialist, community nurses, a rehabilitation technician. This was recorded in the data collection spreadsheet for cases that were selected using a simple random sampling method. Thereafter, data were transferred to the Statistical Package for Social Science (SPSS version 16) for descriptive statistical analysis. Percentages were calculated, and a graph of the trends of common diseases was plotted for the period covered by the study. Descriptive statistics were computed. Chi-square tests were used for categorical data to compare the five most common conditions regarding age and gender. The one-way Chi-squared test with Yates correction for continuity  was used to verify whether a frequency distribution fits a specific pattern for each of the five common conditions by gender and age range. Significance was set at In total, 638 participants were enrolled in the study. Amongst these participants, 375 (58.8%) were male, and 263 (41.2%) were female. The distribution of the ages of male participants were as follow, 324 (86.4%) participants were aged five or younger, whilst 51 (13.6%) were between 6 and 18 years old. In regard to female participants, 228 (86.6%) were under 5 years old, whilst 35 (13.3%) were between 6 and 18 years old. The mean age of participants was 10 years (with the standard deviation [s.d.] approximately 2 years). Cataracts, retinoblastoma, glaucoma, and blindness were the most common eyes conditions; whilst amongst orthopaedic conditions, cerebral palsy, hydrocephalus, and clubfoot were predominant . Most paχ2 = 15.300, df = 4, p < 0.0040) and age . With regard to gender, a statistical difference between male participants and female participants was found for incidence of cataract , clubfoot  and glaucoma . A statistical difference was found between participants aged less than 5 years and those aged 6–14 years, for the incidence of cerebral palsy , for cataracts, for clubfoot , and for glaucoma .There were statistically significant differences amongst the five groups of common conditions  regardinThe trends of five different common malformations during the study period are most effectively comprehended by viewing p = 0.0040) and age (p = 0.0000) of participants.Statistically significant differences were found for the distributions of the five aforementioned conditions regarding gender (p < 0.0100), clubfoot (p = 0.0100), and cataract (p = 0.0290) for both male and female participants (p = 0.0000), of clubfoot (p = 0.0000), and of glaucoma (p = 0.0010) were statistically different between the two age ranges.Differences were observed between male participants and female participants regarding the incidence of glaucom  are significantly predominant amongst children less than 5 years old. This is probably because of high early mortality rates linked to conditions such as cerebral palsy and also the social role associated with clubfoot, which plays a role in early care-seeking behaviours.We are most concerned by the high rate of cerebral palsy in our study (81.5%). Cerebral palsy is either isolated or associated with other deformities in complex syndromes such as developmental dysplasia of the hip and neuromuscular disorders including The mixed morbidity pattern observed in this study is more complicated. Some minor deformities such as polydactyly and syndactyly have been reported without leading to either disability or chronic conditions. Those minor conditions may just translate to either late care-seeking behaviour or misinformation of parents regarding rehabilitation centres in Africa. In addition, the frequent home deliveries in our settings is denying new-born babies access to a full neonatal assessment after birth by trained personnel during which such small deformities could be detected, corrected, or referred promptly for appropriate management.spina bifida. This mixture goes beyond the condition's type encountered and includes conditions in need of curative measures together with those in need of pure rehabilitation. It translates probably either the label given to health providers as primarily ‘curative guys’ by the community or the misinformation of the population about the potential and scope of rehabilitative activities. More cases brought to attention were congenital in nature, and perhaps their high proportion could explain the possible association made previously in the neighbouring region of Ituri regarding possible correlations between the high occurrences of congenital malformations with the on-going armed conflicts.19Patients with other severe complications such as an imperforated anus are seeking rehabilitation in an ordinary manner. Unlike those seeking consultation for minor ailments as described earlier, this second group underscores the gravity of imperforated anus. It appears that in addition to the high level of ignorance, to complain about a condition is a socially constructed concept that can vary with several parameters, including culture. If a condition is well accepted in a setting, it can prevent consultation with a professional or result in late consultation, with consequent poor or limited results, as illustrated by neglected conditions such as 3 in elderly integration,20 quality of life,22 cerebral palsy,23 mental health,25 leprosy,26 and stroke.27 Other studies have examined the attitude of the community toward people with disabilities.28 A study focused exclusively on describing conditions observed in a rehabilitation unit was not identified as such to our knowledge; many studies dealt with either one condition, the setup of the rehabilitation, the process of CBR, or the outcomes of community based-rehabilitation programmes.This study is included in the large spectrum of evidence published by various scholars. Globally, in the literature, community-based rehabilitation is not a scarce concept per se, as it has been widely used in different countries and for different purposes. Many conditions within it have been addressed such as rehabilitation in spinal injury,This study is important because it provides a list of conditions with their distribution that are observed in the rehabilitation centre in Butembo. With this information, providers can now tailor their kits accordingly and their teams can be adjusted to meet the needs expressed by patients in a satisfactory manner. Programmes can also project expenses in terms of training, direct cost, and fund raising.The trend of the conditions as a whole was not recorded for practical reasons because of the high number of conditions, most of them with small frequencies and/or being present for a year or two. We decided that the five most common conditions that were present during the study period could portray the trends effectively.We could not generalise the results of the current study to the community because the data were from an outreach facility, although it was based on the results of clients seen in the community. That is to say, we cannot extrapolate the findings and apply them to the whole community of Butembo.More light will be shed in this field by researching the workload and contribution of each member of the team in the management of different patients. Simple descriptive and/or case-control studies followed by analytical or experimental studies on cerebral palsy, the most common condition, would help to identify the magnitude of the problem, the risk factors, and probably the causes.A community survey of the cases in need of rehabilitation could fill in this picture and reveal the real needs of the community served.In light of the results, we recommend the implementation of a triaging service to screen clients before they see the consulting team in order to minimise the waiting time of clients in need of urgent curative care. A multidisciplinary team could help to provide an entire package of comprehensive care and follow-up consultations after contact with the rehabilitation unit. This could also help to reach more people in need who are affected by medical conditions.The main conditions managed in CBR in Butembo, their trends throughout the years, and their gender distributions have been elucidated. There remain uncertainties with regard to the mandate of the rehabilitative team, which results in an assortment of cases presented to the team when they visit the centre. Further studies aimed at exploring some frequent conditions such as cerebral palsy would be of significance. Sensitisation of the community for early consultation and triage of clients in need of rehabilitation services are warranted.


The phosphoprotein phosphatases are emerging as important androgen receptor (AR) regulators in prostate cancer (PCa). We reported previously that the protein phosphatase 1 catalytic subunit (PP1α) can enhance AR activity by dephosphorylating a site in the AR hinge region (Ser650) and thereby decrease AR nuclear export. In this study we show that PP1α increases the expression of wildtype as well as an S650A mutant AR, indicating that it is acting through one or more additional mechanisms. We next show that PP1α binds primarily to the AR ligand binding domain and decreases its ubiquitylation and degradation. Moreover, we find that the PP1α inhibitor tautomycin increases phosphorylation of AR ubiquitin ligases including SKP2 and MDM2 at sites that enhance their activity, providing a mechanism by which PP1α may suppress AR degradation. Significantly, the tautomycin mediated decrease in AR expression was most pronounced at low androgen levels or in the presence of the AR antagonist enzalutamide. Consistent with this finding, the sensitivity of LNCaP and C4–2 PCa cells to tautomycin, as assessed by PSA synthesis and proliferation, was enhanced at low androgen levels or by treatment with enzalutamide. Together these results indicate that PP1α may contribute to stabilizing AR protein after androgen deprivation therapies, and that targeting PP1α or the AR-PP1α interaction may be effective in castration-resistant prostate cancer (CRPC). Most patients initially respond to androgen deprivation therapy , but the tumors inevitably recur despite castrate levels of androgen . AR expression is generally increased in CRPC, and one mechanism driving its activity is intratumoral synthesis of androgens from adrenal gland derived precursors or lesterol –4. This lesterol , 6. Howelesterol , 8. TherAR mRNA is highly expressed in CRPC with the AR gene being amplified in many cases , 10, whiIn this study we show that PP1α can also increase AR expression independently of S650 dephosphorylation. We find that PP1α binds to the AR ligand binding domain and decreases AR ubiquitination and degradation, particularly at low androgen levels or in the presence of AR antagonists. Mechanistically, we show that PP1α can dephosphorylate and inactivate ubiquitin ligases that target AR for ubiquitylation and degradation. Together these findings show that PP1α can contribute to maintaining AR protein expression and activity in CRPC, and that targeting PP1α or the AR-PP1α interaction may be a novel therapeutic approach.Phosphorylation of AR at S650 enhances its nuclear export and subsequent degradation . We repoTogether these results indicated that PP1α may regulate AR activity by one or more additional S650 independent mechanisms. Consistent with our previous report , PP1α ovAs PP1α and AR can form a complex , we nextWe then took a coimmunoprecipitation approach to assess for a role of this candidate motif in the AR-PP1α interaction. As shown in figure We next systematically assessed the association between PP1α and a panel of constructs spanning the AR N-terminal domain (NTD) that bears the major transcriptional activation function, the central DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD). Analysis in androgen-containing medium demonstrated that PP1α could associate with the isolated NTD and LBD, with the inclusion of the DBD not causing an appreciable increase in binding Figure . CompariNotably, the PP1α-associated AR migrated on the gels as a major band with a series of distinct minor bands separated by ∼8 KDa, consistent with ubiquitylation. To confirm that these bands were ubiquitylated AR, we co-expressed histidine-tagged ubiquitin (his-Ub) together with PP1α and AR, and then used Nickel(Ni)-NTA resin under denaturing conditions to pull-down ubiquitylated proteins. Immunoblotting for the HA-tagged AR then revealed higher molecular weight bands indicative of ubiquitylation associated primarily with constructs containing the LBD Figure . It shouThe above findings suggested that PP1α may stabilize AR protein by decreasing ubiquitylation of the LBD. To examine this further, we assessed effects of PP1α overexpression and proteasome inhibitor (MG132) treatment on ubiquitylation of the AR LBD. In the absence of transfected PP1α, treatment with MG132 increased both the levels of oligo- and poly-ubiquitylated AR LBD Figure . In contTo determine whether this PP1α mediated decrease in AR polyubiquitylation resulted in decreased AR degradation, we next used cycloheximide to block new protein synthesis and thereby assess AR protein stability. PP1α cotransfection markedly decreased degradation of full length AR Figure . MoreoveSeveral ubiquitin ligases, including MDM2 and SKP2, have been implicated as mediators of AR ubiquitylation and degradation –26. MDM2Consistent with previous reports, AR protein expression was decreased by cotransfection of MDM2 or SKP2 Figure . CotransTo determine whether endogenous PP1α may regulate the phosphorylation and activity of these ubiquitin ligases, we treated HeLa and LNCaP cells with tautomycin and assessed for effects on endogenous MDM2 and SKP2. We did not observe clear increases in MDM2 or SKP2 in either cell line Figure . HoweverTaken together these findings indicated that PP1α inhibition may increase AR ubiquitylation and degradation by increasing the activity of one or more AR degrading ubiquitin ligases. However, it was not clear whether or how these effects on MDM2 of SKP2 were related to the PP1α-AR interaction. Therefore, we next asked whether AR could function as a scaffold to bridge PP1α and MDM2 or SKP2. HeLa cells were transfected with Flag-tagged PP1α and MDM2 or SKP2, with or without cotransfected AR. Anti-Flag immunoprecipitation then indicated that the PP1α-SKP2 association was substantially increased by cotransfected AR, consistent with a scaffold function Figure . AR cotrAs noted in Figure Significantly, effects of tautomycin on AR protein expression in LNCaP and C4–2 cells were also enhanced by treatment with the AR antagonist enzalutamide Figure . TherefoAR undergoes basal and ligand stimulated phosphorylation at multiple sites, and these modifications can modulate it stability, cellular localization and transcriptional activity. AR phosphorylation may also be regulated by protein phosphatases, including PP2A and PP1. PP2A can dephosphorylate multiple sites in the AR N-terminal domain, including S81, S94, S258, S308, and S424, that are normally increased in response to androgen and are associated with increased AR transcriptional activity . SignifiIn contrast to PP2A, we had previously shown that PP1α could bind to AR and dephosphorylate AR at S650 in the hinge region and thereby suppress AR nuclear export, providing a mechanism by which PP1α can enhance AR activity . In the This study also found that PP1α binding to AR is mediated primarily by the AR LBD. Although the precise site on the LBD remains to be determined, it does not appear to be the coactivator binding site as binding is not ligand dependent. We also cannot rule out that binding is indirect and mediated by a not yet identified PP1 regulatory protein. An attractive hypothesis is that PP1α associated with the AR LBD directly protects AR from ubiquitylation by locally inactivating ubiquitin ligases. Alternatively, PP1α may more globally suppress a subset of ubiquitin ligases, with the AR associated PP1α having distinct functions including the dephosphorylation of S650. Further studies are underway to test these hypotheses.In any case, these findings indicate that therapies targeting PP1α, or possibly the AR-PP1α interaction, may have efficacy in CRPC. Significantly, the decrease in AR protein in response to tautomycin was most dramatic under androgen depleted conditions and in cells treated with the AR antagonist enzalutamide. Therefore, PP1α activity may play a particularly important role in maintaining AR expression in CRPC and in resistance to enzalutamide.Anti-Flag M2 affinity gel was from Sigma-Aldrich, tautomycin and fostriecin were from Calbiochem and Enzo Life Science, Ni-NTA agarose was from Qiagen, and microcystin-agarose was from EMD Millipore. Anti-AR, anti-PP1, anti-HA, anti-p27, and anti-p53 antibodies were from Cell Signaling, anti-β-Actin was from Abcam, anti-β-tubulin was from EMD Millipore, and anti-flag-M2 was from Sigma-Aldrich. The anti-SKP2 (Santa Cruz) and anti-p-SKP2-S72  antibodies were kindly provided by Dr. Inuzuka Hiroyuki, and the anti-MDM2 and anti-p-MDM2-S166 (Santa Cruz) antibodies were kindly provided by Dr. Ming Chen . FBS and CDS  were from Hyclone.For coimmunoprecipitation (co-IP), cells were harvested in triton lysis buffer (TLB) containing protease inhibitors. The lysates were incubated on ice for 20 min and centrifuged for 5 min at maximum speed. 20 μl of the supernatant was saved as input and the rest was incubated with anti-Flag beads, followed by incubation at 4°C on a rotator for 2 hr. Then the input and washed (4x) IP beads were boiled in 60 μl of Laemmli buffer containing 5% β-mercaptoethanol. For immunoprecipitation (IP) under denatured conditions, cells were harvested by boiling in 2% SDS followed by 20x dilution the TLB. The test then proceeded as the above regular IP. For the ubiquitylation and Ni(nickel)-NTA-IP assay, the cells were washed in PBS and an aliquot was lysed in RIPA buffer as input. The rest was lysed in buffer A , followed by sonication and centrifugation. The supernatants were incubated with Ni-NTA beads at room temperature for 3 hrs, followed by wash 2x with buffer A, 2x with buffer A/buffer TI (1:3), and 1x with buffer TI . Then both input and the IP yields were boiled in 60 μl of Laemmli buffer containing 5% β-mercaptoethanol. Microcystin-IP was as previously described .RNA isolation was carried out using the TriZOL reagent and the qRT-PCR analysis on gene expression was performed with the TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems). The TaqMan primer and probe sets for gene expression assay: PSA: Forward, 5′- GATGAAACAGGCTGTGCCG-3′; Reverse, 5′- CCTCACAGCTACCCACTGCA-3′; Probe, 5′- FAM-CAGGAACAAAAGCGTGATCTTGCTGGG-3′. The TaqMan primer-probe set for GAPDH transcripts  was purchased as inventoried mix from Applied Biosystems.LNCaP cells were grown in the RPMI-1640 medium and C4–2 and VCaP cells in DMEM medium containing 10% FBS. PC3, 293T, HeLa, and Cos1 cells were grown in DMEM medium with 5% FBS. For androgen-starved conditions, cells were grown in medium containing 5% CDS. Cellular fractionation assay were carried out with the NE-PER kit (Pierce), following the manufacture's direction. For transfection, cells were grown in normal growth medium to ∼80% confluence and plasmid DNA was transfected with LipofectAMINE 2000. Empty relevant vectors  were supplemented to equalize the total amount of plasmids in the transfections. After overnight transfection, the cultures were refreshed with indicated medium for treatments. The pCMV-pRL-Renilla and androgen-responsive reporter plasmids have been described previously [Cells were cultured in 96-well plates for 2 days and then treated for 3 days as indicated. The cell proliferation analysis was performed with the CellTiter-Glo assay kit (Promega), following the manufacture's manual.The Western blotting was developed based on X-Ray film  and the western lightning Plus-ECL reagent (PerkinElmer). The images were acquired using the CanoScan LiDE 210 scanner and processed using the Adobe Photoshop 7.0 software. Relevant images were quantified by using the ImageJ software. All gels shown are representative of results from at least three experiments.The AR constructs were generated based on the pcDNA3.1(+)-Kozak-HA-HA vector and cover the following regions: HA-HA-AR full-length (aa 1–919); HA-HA-AR-NTD (aa 1–539); HA-HA-AR-NTD-DBD (aa 1–628); HA-HA-AR-DBD (aa 538–628); and HA-HA-AR-DBD-LBD (aa 538–919). The HA-AR-LBD (aa 662–919) vector was from our Lab. The untagged PP1α plasmid was from Origene and then cloned into the 3x-Flag-CMV-10 vector (Sigma-Aldrich) to generate the Flag-PP1α expression plasmid. The his-Ub, Flag-Ub, Flag-Sox9, HA-MDM2 and HA-SKP2 expression plasmids were kindly provided by Drs. Pengda Liu, Wenjian Gan, Xin Yuan, and Inuzuka Hiroyuki (BIDMC), respectively. DNA mutagenesis on AR and PP1α was performed with the Quick-change site-directed mutagenesis Kit (Stratagene). Details for plasmids and mutants generation are available upon request.


Castration-resistant prostate cancers still depend on nuclear androgen receptor (AR) function despite their lack of dependence on exogenous androgen. Second generation anti-androgen therapies are more efficient at blocking nuclear AR; however resistant tumors still develop. Recent studies indicate Src is highly active in these resistant tumors. By manipulating AR activity in several different prostate cancer cell lines through RNAi, drug treatment, and the use of a nuclear-deficient AR mutant, we demonstrate that androgen acting on cytoplasmic AR rapidly stimulates Src tyrosine kinase via a non-genomic mechanism. Cytoplasmic AR, acting through Src enhances laminin integrin-dependent invasion. Active Matriptase, which cleaves laminin, is elevated within minutes after androgen stimulation, and is subsequently shed into the medium. Matriptase activation and shedding induced by cytoplasmic AR is dependent on Src. Concomitantly, CDCP1/gp140, a Matriptase and Src substrate that controls integrin-based migration, is activated. However, only inhibition of Matriptase, but not CDCP1, suppresses the AR/Src-dependent increase in invasion. Matriptase, present in conditioned medium from AR-stimulated cells, is sufficient to enhance invasion in the absence of androgen. Thus, invasion is stimulated by a rapid but sustained increase in Src activity, mediated non-genomically by cytoplasmic AR, leading to rapid activation and shedding of the laminin protease Matriptase. The survival of malignant tumors arising from the prostate gland is dependent on the androgen receptor (AR), a classical nuclear steroid receptor that binds androgen and activates gene transcription , 2. ThisStudies with the new anti-androgen agent MDV3100  indicate one of the ways it inactivates AR nuclear activity in the tumor cells is by preventing nuclear translocation, thus retaining a significant amount of AR the cytoplasm , 7. SeveIn vitro, Lyn was required primarily for tumor cell growth, while Src was responsible for promoting cell migration. The specific effect of Src on migration is not surprising given that many Src substrates, such as FAK, Cas, and cortactin, are intimately involved in promoting cell motility through integrin-mediated adhesion to extracellular matrices -Src antibody  is a transmembrane glycoprotein that facilitates integrin-dependent migration and invasion and whose elevated expression in primary tumors correlates with metastasis in several cancers . CDCP1 i/Trask isTo assess whether Matriptase is required for increased invasion induced by AR signaling, its expression was suppressed by siRNA in PC3-AR cells and the effect on Matrigel invasion evaluated. Inhibition of Matriptase expression Figure  decreaseIn addition to its role in regulating gene transcription in the nucleus, AR like other steroid receptors rapidly activates signal transduction pathways in the cytoplasm. Pathways reported to be activated by nongenomic AR signaling include MAPK, PI-3K, EGFR, and Src , 43, 44.We employed several different prostate cancer cell lines to demonstrate the universality of the nongenomic AR/Src invasion pathway. The first model made use of PC3 cells engineered to express AR (PC3-AR). We previously demonstrated that PC3-AR cells display constitutive AR transcriptional activity as measured by nuclear AR localization and elevated PSA and TMPRSS2 expression in the absence of androgen . Cells wDepending on the model being examined, the ability of steroid receptors to activate nongenomic signaling may or may not be ligand dependent. Our data demonstrating that Casodex and RU486, ligand antagonists, block invasion and Src activation support the conclusion that the AR/Src invasion pathway is ligand dependent. Therefore, we expected the cells expressing the AR ligand binding mutant (LBD) to be neither invasive nor to activate Src. Contrary to expectations, Src was constitutively activated and the cells were highly invasive. However, the PC3AR-LBD cells did not behave like any of the other cell lines in that their invasive activity was not dependent on Src, even though Src activity was elevated; nor did they induce Matriptase activation. Our finding that addition of cleaved and thus activated Matriptase, generated by AR/Src signaling, exogenously to non-stimulated cells was sufficient to increase invasion, indicates that the primary target of AR/Src signaling in invasion is Matriptase. Thus in the PC3AR-LBD cells, the failure to activate Matriptase in spite of active Src, suggests it is Matriptase activation that is sensitive to ligand binding. It further suggests that depending on whether Src is activated through a ligand-dependent or -independent mechanism may determine which downstream targets are affected and the biological outcome.Our data strongly support a nongenomic mechanism for Matriptase activation. It occurs in the absence of nuclear function and in the absence of new mRNA synthesis. A previous study demonstrated that androgen induces Matriptase activation and shedding in LNCaP cells 6–24 hours after stimulation . In thatin vivo for initial metastatic dissemination -Src polyclonal antibody (Invitrogen); anti-Src monoclonal antibody (Src 327) -Src, cell lysates were prepared as described above. Protein samples (500 μg), in a volume of 500 μL of 1X MAPK buffer, were incubated with 1 μg of mouse monoclonal anti-Src antibody [416]-Src antibody (Invitrogen) or mouse 4G10 anti-phospho-tyrosine antibody (Millipore) as described above.For detection of phosphorylated CDCP1 and phospho-[Yantibody  or anti-Cells were fixed with 4% paraformaldehyde in PBS at 4°C for twenty minutes and permeabilized for ten minutes with TBS  + 0.5% TritonX-100 at room temperature. Cells were then blocked with 2% BSA in TBS + 0.1% Triton X-100 for twenty minutes at room temperature before incubation with vinculin antibody (Sigma) and Alexafluor 546-Phalloidin  for one hour. Cells were incubated with goat anti-mouse Alexa Flour-488 secondary antibody for one hour at room temperature. DNA was visualized by staining with Hoechst 33258 for 10 minutes at room temperature. Cells were washed four times with TBS + 0.1% TritonX-100 over ten minutes between all steps. Epifluorescent images were acquired on a Nikon Eclipse TE300 fluorescence microscope using OpenLab v5.5.0 image analysis software (Improvision).4) were suspended in the upper well of the 8.0 μm pore size polyethylene terephthalate membrane culture inserts for (BD Biosciences) in which the bottom membrane was pre-coated with 1 μM laminin. The lower and upper chambers were filled with 400 μL phenol-red and serum-free DMEM. After 6 hours of incubation, the culture inserts were removed and washed with 1X PBS. Cells that had migrated to the lower membrane surface were stained with Crystal Violet (Millipore) and counted under a microscope in five random fields per insert in triplicate.Cell migration was measured using the modified Boyden chamber assay. Cells (5 × 104) were suspended in the upper well of the 8.0 μm pore size Matrigel membrane culture inserts (BD Biosciences) in which the bottom membrane was pre-coated with 1 μM laminin. The upper and lower chambers were filled with 400 μL phenol-red and serum-free DMEM. After 72 hour incubation, the culture inserts were removed and washed once with 1X PBS. Cells that invaded through Matrigel to the lower membrane surface were stained with Crystal Violet (Millipore) and counted under a microscope in five random fields per insert in triplicate.Serum-starved sub-confluent cells . The Amicon filter tubes were centrifuged at 4000 × g for 30 minutes at 4°C. Concentrated medium was collected and subjected to SDS PAGE and transferred to PVDF membrane for immunoblotting. Protein loading was normalized to total cell number.


Objective. To investigate whether early rehabilitation reduces the occurrence of posttotal hip arthroplasty (THA) complications, adverse events, and medical expenses within one postoperative year. Method. We retrospectively retrieve data from Taiwan's National Health Insurance Research Database. Patients who had undergone THA during the period from 1998 to 2010 were recruited, matched for propensity scores, and divided into 2 groups: early rehabilitation (Early Rehab) and delayed rehabilitation (Delayed Rehab). Results. Eight hundred twenty of 999 THA patients given early rehabilitation treatments were matched to 205 of 233 THA patients given delayed rehabilitation treatments. The Delayed Rehab group had significantly  higher medical and rehabilitation expenses and more outpatient department (OPD) visits than the Early Rehab group. In addition, the Delayed Rehab group was associated with more prosthetic infection (odds ratio (OR): 3.152; 95% confidence interval (CI): 1.211–8.203; p < 0.05) than the Early Rehab group. Conclusions. Early rehabilitation can significantly reduce the incidence of prosthetic infection, total rehabilitation expense, total medical expenses, and number of OPD visits within the first year after THA. A disabled hip joint is a major inconvenience because it reduces one's functional ability and secondarily increases comorbidities caused by immobility. In severe cases, a total hip arthroplasty (THA) is mandated. One review  showed tThe success of THA is its predictable pain relief, improvements in quality of life, and restoration of normal function . BranderTo achieve better outcomes after joint replacement, recent consensus statements have advocated research on the timing of rehabilitation intervention. Chen et al.  reported Clinical Orthopaedic Rehabilitation textbook [In Taiwan, there is still no routine rehabilitation intervention after THA. There are a few protocols taken from Brotzman'stextbook . There iIn addition, Husby et al.  point ouTo the best of our knowledge, there is no large scale study focus on the proper time of rehabilitation intervention following THA and how the different timing of rehabilitation may impact on patient's outcome including medical expenses in the long run. We wanted to clarify whether early post-THA rehabilitation intervention of rehabilitation attenuates complications and comorbidities. We also hypothesized that early post-THA rehabilitation intervention reduces the need for postoperative medical services and the number of outpatient visits.p value = 0.187 [Our data were obtained from Taiwan's National Health Insurance Research Database (NHIRD), which is maintained by the National Health Research Institutes (NHRI) specifically for research. It is an administrative database that contains all medical care claims for outpatient, inpatient, and emergency room services of all NHI patients, which is approximately 99.5% of Taiwan's 23 million people. The data we used are a representative sample of the NHIRD, which contains all original claim data ), medical expenditures, rehabilitation expenditures, treatment during admission and after discharge, prescriptions, hospital levels, and each enrollee's age and gender of 1 million people randomly sampled from the 23 million beneficiaries in the NHIRD. No significant differences exist in the age, gender, or insured amount distributions between patients in our data and the original NHIRD with  = 0.187 . The NHI = 0.187  for dozeWe identified 2325 patients who had been discharged from their initial THA (ICD-9-CM procedure code: 81.51) and had undergone rehabilitation within the first postoperative year between January 1998 and December 2010. Exclusion criteria included predischarge prosthetic infection (PI) or DVT (84 patients); this was done to minimize the risk of over- or underestimating medical expenditures. Another 1009 patients were excluded because they were missing data for one of the studied variables. We finally enrolled 1232 patients in the study .n = 999) and Delayed Rehab  groups. The comorbidities looked at in this study had to be present before the date of the initial THA; they were osteoarthritis (OA) ; avascular necrosis (AN) (ICD-9-CM: 733.34); hypertension (HTN) (ICD-9-CM: 401-405); diabetes mellitus (DM) (ICD-9-CM: 250); and poor renal function (PRF) (ICD-9-CM: 585).We then subgrouped the patients, based on when they began rehabilitation , into the Early Rehab  (ICD-9-CM: 996.66), deep vein thrombosis (DVT) (ICD-9-CM: 453), and revision of hip arthroplasty (RHA) (ICD-9-CM procedure code: 81.53) within 1 year after discharge were used as the primary outcome measures. We also recorded, as one of the outcome measures, the number of visits to the OPD (regardless of the reason for the visit) within the first year after being discharged with a diagnosis of THA. Medical expenses, including total medical expenses and expenses for rehabilitation exclusively, were calculated for the first postdischarge year.χ2 tests for categorical variables and independent sample t-tests for continuous variables. Because the Early and Delayed Rehab groups may differ substantially in a number of ways, propensity-score matching was used to reduce the selection bias in our hypothesis: many confounding covariates may be present in an observational study with this number of variables. Score matching identified the predicted probability of obtaining 1 Early Rehab patient versus 4 Delayed Rehab patients from the logistic regression model based on gender, age, length of stay, Charlson Comorbidity Score (CCS), trauma code, and the comorbidities of OA, AN, HTN, DM, and PRF.Initial comparisons of baseline demographic and clinical characteristics for the early and delayed rehabilitation groups were made using Pearson p < 0.05 (2-tailed).Moreover, based on propensity-score matching, a linear regression model was used to examine how the different timing of rehabilitation influenced total medical expenses, total rehabilitation expenses, and the number of OPD visits while controlling for gender, age, group, length of stay, CCS, trauma code, and comorbidity. Finally, a logistic regression model was used to assess the risk of post-THA-associated complications  for the two rehabilitation groups, after controlling for the same confounding variables. All of the analyses were performed using SAS 9.3.1 for Windows . Significance was set at Based on propensity matching, 820 of 999 THA patients given early rehabilitation treatments matched to 205 of 233 THA patients given delayed rehabilitation treatments. Both groups were well balanced, after they had been given a propensity score, in these demographic and clinical variables: gender, age, age group, length of stay, OPD visits, total medical expenses, total rehabilitation expenses, CCS, E code, and a series of comorbidities  .p < 0.001), higher total rehabilitation expenses (p < 0.001), and more postoperative OPD visits (p < 0.001) than the early rehabilitation groups  when compared with early rehabilitation group. There was no significant difference between the incidence rates of DVT  or revision of hip replacement  between the early and delayed rehabilitation groups : 3.152; 95% confidence interval (CI): 1.211–8.203; n groups .p = 0.5) with equal sample frequencies. Moreover, the power of the significant outcome  of this study was also more than 0.95.The power of the primary outcome (prosthetic infection) of this study was more than 0.95, calculated using software Gpower for logistic regression with odds ratio = 3.152, prosthetic infection rate under null hypothesis is 0.039, and alpha error probability = 0.05, assuming the distribution of dependent variable was binomial with a balanced design  and infection (24%) . MoreoveThirdly, we found that when comparing the total medical expenses or the total rehabilitation expenses in the first year after THA, the costs for the early rehabilitation group were much less than those for the delayed rehabilitation group. In our study, total medical expense included the patient's total medical expense for the first year after THA, which can be used to interpret the patient's general medical condition after THA, that there is no significant difference in the CCS or comorbidities between the two study groups. This result is especially important now because most of the health insurance policies around the world emphasize the cost efficiency of each therapy and have implemented policies such as diagnosis-related groups (DRGs) . In TaiwFinally, Taiwan's NHI does not restrict accessibility to medical services or frequency of use. Moreover, because medical copayments are relatively low in Taiwan, patients are free to choose where and when to seek medical help . MedicalStudy Limitations. This study has several limitations. Firstly, it used NHIRD claims data for all analyses; these data are used primarily for administrative rather than clinical purposes. Therefore, detailed clinical information is often lacking in the database: the clinical presentation of the patients, the content of rehabilitation they underwent, the surgeons' techniques, and the exact surgical approach used, for example, anterolateral or posterolateral, and cemented or cementless THA. Secondly, we investigated whether early rehabilitation had a positive impact on medical expense and complications. Because of the limitations of the information in the NHIRD, we cannot know the exact date of the operation or what rehabilitation the patient underwent between admission and discharge. Thus, we can only set the time as, for example, “one week after discharge” as the cut-point and compare outcome differences. This shortcoming suggests that, in the future, prospective studies to verify the association between the timing of rehabilitation and post-THA prognosis are needed.The present study sends an important message to health policy makers around the world that intentional cost cutting might cause adverse events for patients and significantly increase postoperative expenditures. We found that early rehabilitation significantly reduced the rate of prosthetic infection, total rehabilitation costs, total medical expenses, and the number of THA-related OPD visits during the first year after a THA.


By using transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, energy-dispersive X-ray spectrometry, and vibrating sample magnetometry, the dependence of the synthesis on the amount of additional NaOH was studied.During liquid-phase synthesis of γ-Fe2O3 nanoparticles could be modified by adding ZnCl2 to form composite nanoparticles with γ-Fe2O3/ZnFe2O4 ferrite core coated with Zn(OH)2 and adsorbed FeCl3, and that modification could be enhanced by adding NaOH.The experimental results show that the surface of the γ-Fe2O4 and Zn(OH)2 phases increased slightly and that of adsorbed FeCl3 was unchanged. When the concentration of NaOH exceeded 0.70 M, the amount of FeCl3, ZnFe2O4, and Zn(OH)2 increased.In the experimental conditions, when the concentration of additional NaOH was below 0.70 M, the amounts of ZnFe Many physical phenomena in both organic and inorganic materials have natural length scales between 1 and 100 nm (107 atoms) . For exa7 atoms) .2O3-based nanoparticles are prepared by processing a precursor based on iron oxide hydroxide and/or metal hydroxide in FeCl2 solution [2O3 nanoparticles can be prepared by using an amorphous coating of FeOOH and Mg(OH)2, as described in the following equation [Magnetic nanoparticles are an important class of functional materials that have attracted increasing interest in terms of their science and their technological applications . Studiessolution -10. In t2 to the FeCl2 solution to prepare γ-Fe2O3/ZnFe2O4 composite nanoparticles [2 in solution did not exceed 2 M (50 mL), γ-Fe2O3/ZnFe2O4 bioxide nanoparticles coated with FeCl3 · 6H2O could be prepared. Generally, alkaline solution could assist the precipitation reaction. In the present work, we attempted to enhance the surface modification by adding NaOH to the processing solution. The morphology, crystal structure, surface and bulk chemical composition, and magnetization of the as-prepared products were characterized. The structure of the particles was proposed and the role of NaOH was revealed.In another study, surface modification of the particles was undertaken during synthesis by adding ZnClarticles . Experim2 was synthesized by coprecipitation of FeCl3 and Mg(NO3)2, as described in detail elsewhere [2 solution, and the resulting mixture was heated to boiling for 20 min. Afterward, a mixture of 50 mL of 1 M ZnCl2 solution and 20 mL NaOH solution at a specific concentration was added to the boiling FeCl2 solution, and the resulting mixture was boiled continuously for 10 min. Subsequently, the mixture was allowed to cool to room temperature, and the as-prepared particles were allowed to settle. The NaOH concentrations used for the preparation were 0.35, 0.70, 1.40, and 2.10 M, corresponding to the as-prepared samples (1), (2), (3), and (4), respectively. For comparison, modified particles were prepared without adding NaOH (sample (0)).Preparation of the nanoparticles could be divided into two steps. First, the precursor based on amorphous FeOOH and Mg(OH)lsewhere . In the The morphology of the particles in the samples was observed by transmission electron microscopy , and their crystal structure was analyzed by X-ray diffraction . The chemical species were measured using X-ray photoelectron spectroscopy , and energy-dispersive X-ray spectroscopy  equipped in scanning electron microscopy . The magnetization was measured by using a vibrating sample magnetometer .g about 9.78 nm and the standard deviation lnσg = 0.28. Samples (1) – (4) images indicated that these samples consisted of irregular flake particles and nearly spherical particles, whose sizes are clearly larger than size of sample (0). This shows that NaOH can stimulate aggregation of the initial particles to grow into larger particles via oriented attachment [TEM images of the samples are shown in Figure tachment .2O3 and traces of ZnFe2O4 and Zn(OH)2, but no ZnCl2.XRD patterns Figure  reveal t2O3, ZnFe2O4, Zn(OH)2, and FeCl3. Binding energy data are listed in Table 2[2. Quantitative analysis, whose relative error are less that 1 %, shows that relative to sample (0) this ratio of Fe to Cl keeps almost invariant for low NaOH concentrations (up to 0.70 M) – samples (1) and (2), but it increase with NaOH concentrations above that level – sample (3) and (4). Alternatively, to use the same criteria used for the ratio of Fe to Zn of samples, (1) ≈ (2) < (3) < (4). Complete data are listed in Table XPS measurements show that O, Fe, Zn, and Cl but no Mg and Na species were present in the samples, as illustrated in Figure n Table 2 are listThe results of EDX measurements of all samples confirm the same chemical species as those detected by XPS. Results of quantitative analysis, whose relative error are less in 2 %, are listed in Table s were deduced by plotting σ vs. 1/H in the high-field region [Figure d region . These v2O3, ZnFe2O4, Zn(OH)2 and FeCl3, and no ZnCl2. The experimental results show that the ratios of Fe to Cl and Fe to Zn obtained by XPS were less than those obtained by EDX spectrometry, and the ratio of Cl to Zn obtained by XPS agree with that obtained by EDX spectrometry 2 phases in addition to the γ-Fe2O3 phase, but no clear FeCl3 crystal phase. These results therefore suggest that the layer of adsorbed FeCl3 was very thin and amorphous. Therefore, the structure of the composite nanoparticles consisted of four parts, namely, a γ-Fe2O3 core, external shells of ZnFe2O4 and Zn(OH)2, and an outermost layer of adsorbed FeCl3. A schematic model of this structure is shown in Figure XRD results show the presence of ZnFe2 precursor was thermally treated with FeCl2 solution, Mg(OH)2 was dissolved, an amorphous FeOOH species was transformed into γ-Fe2O3 crystallites, and Fe2+ was oxidized to Fe3+. While adding ZnCl2 to the solution, a precipitation reaction took place on the γ-Fe2O3 crystallites, which can be described as follows:When the FeOOH/Mg(OH)y/x > 1/2, thenIf 2O4 grew epitaxially on the γ-Fe2O3 crystallites and some Zn(OH)2 outside of the ZnFe2O4 layer was preserved. Clearly, additional NaOH enhanced the reaction so that x and y increased with increasing NaOH content. In addition, Fe3+ and Cl- in the liquid phase were adsorbed and were subsequently converted to composite nanoparticles coated with FeCl3. Experimental results show that with increasing NaOH content, the amount of phases based on Zn increased. When the NaOH concentration was lower than 0.70 M, the FeCl3 phase was nearly unchanged since the Fe: Cl ratio is almost invariant  increased with NaOH concentration at low NaOH concentrations, i.e., the amount of ZnFe2O4 phase increased clearly with NaOH concentration when the NaOH concentration did not exceed 0.70 M, but it increased slightly with NaOH concentration when the NaOH concentration was > 0.70 M. As the results in Table 2 increased slightly under low NaOH concentration (<0.70 M) and did clearly under high NaOH consentration (>0.70 M). According to the relation between the specific magnetization and NaOH content, the action of additional NaOH can be discussed further as follows.Thus, ZnFe2/FeCl3 < ZnFe2O4 < γ-Fe2O3. The specific magnetization of the composite nanoparticle system σ can be described as follows:The magnetization exhibited stepwise distribution, being strongest in the innermost region of the composite nanoparticle: Zn(OH)γ-Fe, ϕZn-Fe, ϕZn, and ϕCl are mass fractions; σγ-Fe, σZn-Fe, σZn, and σCl are the specific magnetizations for the γ-Fe2O3, ZnFe2O4, Zn(OH)2, and FeCl3 phases, respectively. σγ-Fe > σZn-Fe > σZn (≈ σCl) since nanoscale ZnFe2O4 could be weakly ferromagnetic [2 and FeCl3 are paramagnetic. Considering as well that ϕγ-Fe + ϕZn-Fe + ϕZn + ϕCl = 1, equation (4) can be written aswhere ϕmagnetic , and Zn2, and FeCl3 phases in the sample, respectively. In the modification to this equation, the mass of the γ-Fe2O3 phase mγ-Fe may be treated as constant. Thus, when mCl was nearly constant under low amounts of additional 0.70 M NaOH, ϕCl decreased as mZn-Fe and mZn increased. As a consequence, when ∑ mi increased at low amounts of NaOH, ΔϕCl was negative since mCl was unchanged, i.e., -ΔϕCl > 0; thus, equation (6) can be written asThe mass fraction of every phase should be directly proportional to the molar ratio. The mass fraction is defined as 2O4 and Zn(OH)2 phases lowered the value of σ, whereas a decrement in the mass fraction of the FeCl3 phase increased it. For sample (1), x and y in the precipitation reaction described by equation (2) increased slightly compared with those for sample (0); hence, ΔϕZn-Fe, ΔϕZn, and ΔϕCl were very small compared with their counterparts for sample (0). Therefore, σ of sample (1) was about the same as that of sample (0). For sample (2), x and y in the precipitation reaction increased, but the increment of x could be larger than y. Thus, it can be judged from equation (3) that the increment in molar content x/2 of the ZnFe2O4 phase would be larger than that of the Zn(OH)2 phase (y - x/2), i.e., ΔϕZn-Fe > ΔϕZn. Since σγ-Fe - σZn-Fe < σγ-Fe - σZn (≈ σγ-Fe - σCl) and |ΔϕCl| is proportional to Δϕγ-Fe + ΔϕZn, |ΔϕCl| (σγ-Fe - σCl) > ΔϕZn-Fe(σγ-Fe - σZn-Fe) + ΔϕZn(σγ-Fe - σZn). Consequently, the σ value of sample (2) was greater than those for samples (0) and (1).Equation (7) shows that increment of the mass fraction of the ZnFe3 phase increased with the NaOH content. Therefore, the variation of σ should be described as follows:When the concentration of additional NaOH exceeded 0.70 M, the amount of FeClTherefore, the σ weakened in the order of samples (2) to (4).2O3 nanoparticles from precursor composed of amorphous FeOOH and Mg(OH)2 by chemically induced transition in FeCl2 solution, Mg(OH)2 dissolved, FeOOH transformed into γ-Fe2O3 nanocrystallites, and Fe2+ was oxidized partially into Fe3+. The surface of the particles could be modified by adding ZnCl2 to form γ-Fe2O3/ZnFe2O4 composite nanoparticles coated with Zn(OH)2 and adsorbed FeCl3. Such composite nanoparticles exhibited stepwise distribution of magnetization from inner to outer regions. Thus, they could be easily dispersed in carrier liquid to form excellent ferrofluids [2 solution was constant , the modification could be enhanced by addition of NaOH. When the concentration of additional NaOH was below 0.70 M, the amount of FeCl3 adsorbed was unchanged, but that of ZnFe2O4 and Zn(OH)2 increased slightly, increasing the magnetization of the products. When the concentration of additional NaOH exceeded 0.70 M, the amount of adsorbed FeCl3 and ZnFe2O4 and Zn(OH)2 phases increased, and the specific magnetization of the as-prepared products weakened with increasing amount of NaOH. These results show that surface modification during synthesis of the composite nanoparticles γ-Fe2O3/ZnFe2O4 coated with Zn(OH)2 and FeCl3 could be enhanced by additional NaOH to obtain various proportions of phases in the composite particles. This route could be an interesting route for preparing magnetic composite nanoparticles with novel properties. It could potentially be used to prepare other composite nanoparticles based on γ-Fe2O3. In this regard, it will be investigated further.During liquid-phase synthesis of γ-Ferofluids . ExperimThe authors declare that they have no competing interests.LC carried out characteristical studies of the nanoparticles, participated in the sequence alignment and drafted the manuscript. JL conceived of the study, and participated in its design and coordination and help to draft the manuscript. YL carried out the preparation of samples. XL carried out the measurements of both VSM and XRD. JL carried out the analysis of both EDX and XPS results. XG performed the analysis of TEM results. DL participated in the design of the study. All authors read and approval the final manuscript.