Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue.The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin.In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.Gg-laforin is more soluble and stable than human laforin  Lafora disease (LD) is an autosomal recessive, neurodegenerative disorder resulting in myoclonus, epilepsy, dementia, and death -3. AffecEPM2A (epilepsy of progressive myoclonus type 2 gene A) gene that encodes the protein laforin [EPM2A is conserved in all vertebrate genomes, but it is absent from the genome of most non-vertebrate organisms including standard model organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster[Approximately 50% of Lafora disease cases are caused by mutations in the  laforin -6. EPM2Aanogaster,8. An exanogaster,8. Thus,http://www.cazy.org) database [in vitro and in vivo[Laforin is a bimodular protein with a carbohydrate-binding module (CBM) at its amino-terminus and a dual-specificity phosphatase (DSP) domain at its carboxy-terminus -11. CBMsdatabase . CBM20s database -15. Typidatabase ,9. Laford in vivo,9,16-19.Glycogen is an energy storage molecule synthesized by bacterial, fungal and animal species consisting of α-1,4 and α-1,6 linked residues of glucose, with 12-14 residues per branch . GlycogeE. coli is largely insoluble and must be purified from inclusion bodies [E. coli[Although the substrate and function of laforin have recently been elucidated, the structural basis for the unique glucan phosphatase activity of laforin remains unknown. Ourselves and others have experienced difficulty purifying laforin in sufficient quantities and of sufficient quality for crystallographic studies . One gron bodies . This pr [E. coli.in vitro assays [Arabidopsis that are functionally similar to laforin, and the structures of other DSP domains and CBMs are available [Gallus gallus laforin (Gg-laforin). Previously, the CBM of Gg-laforin was fused to a glutathione S-transferase (GST) tag and shown to bind glycogen [Our lab has purified enough recombinant laforin from the soluble portion of bacterial cell lysates to perform o assays ,16,29-31vailable ,33. Howeglycogen . In thisglycogen ,35. Phosglycogen ,30,36. TE. coli[in vitro assays, the protein is unstable and precipitates from solution. Thus, we sought to optimize the purification procedure using an additive. His6-tagged Hs-laforin was expressed and purified from E. coli by affinity chromatography. Approximately 5 mg of soluble Hs-laforin was obtained from 1 L of E. coli cells. In order to increase the solubility of Hs-laforin, we tested the addition of the sugars maltose and β-cyclodextrin (BCD) to the purification buffer. The addition of 15% maltose (w/v) or 10 mM BCD to the lysis and purification buffers improved the yield of soluble Hs-laforin to 8 mg and 9 mg per 1 L culture, respectively , Gallus gallus (red rooster), Xenopus tropicalis (frog), Anolis carolinensis (lizard) and Danio rerio (zebrafish) were aligned using ClustalW. Each of these orthologs contains the four invariant aromatic residues characteristic of a laforin CBM  and the signature DSP amino acid sequence, DX30CX2GX2R , Xenopus tropicalis (Xt-laforin), and Gallus gallus (Gg-laforin). Recombinant Mm-laforin was expressed with a His6-tag, and Xt-laforin was expressed as a His6-SUMO fusion protein in E. coli. Mm-laforin and Xt-laforin were purified in the absence of any sugars and these preparations yielded more soluble protein than Hs-laforin, 6 and 10 mg/L of E. coli, respectively. However, the yield for Mm-laforin was not significantly greater than Hs-laforin, and Xt-laforin exhibited the same tendency as Hs-laforin to aggregate and precipitate  over a range of pH values, from 5.0-8.0. Gg-laforin displayed similar specific activity to Hs-laforin and also, like Hs-laforin, displayed a preference for a lower pH  and Like Sex Four 2 (LSF2). SEX4 contains a CBM and DSP domain, while LSF2 lacks a CBM. The individual laforin domains are likely to resemble the domains of SEX4 and LSF2 [Human laforin has proven to be a difficult protein to express in recombinant systems. These difficulties are highlighted by previous reports that Hs-laforin must be purified from inclusion bodies in n E. coli,28. Whiln E. coli,39-46. Wand LSF2 ,33. Indeand LSF2 ,8,47-49.and LSF2 ,33. Althin vitro assays without using denaturation and refolding steps, but recombinant Hs-laforin has proved difficult to work with in experiments requiring large quantities of protein, due to low yields and the tendency to aggregate and precipitate. We sought a laforin ortholog with greater solubility and stability yet possessing similar in vitro characteristics as Hs-laforin; such an ortholog would be a more conducive target for crystallography and other biophysical techniques. The structure of this ortholog would provide insight into the mechanism of laforin function and may shed light on why mutations in certain amino acids lead to LD.Our lab has been successful in purifying sufficient amounts of Hs-laforin for 6-SUMO-Gg-laforin is expressed as a soluble protein in E. coli, Gg-laforin remains soluble after cleavage of the fusion protein during experimental manipulation, and it possesses both phosphatase and glucan binding activity. Gg-laforin can be purified without the use of denaturation and refolding steps, and the protein does not require a sugar to improve its stability. We showed that Gg-laforin is present as a multimer and monomer, it remains monomeric after size-exclusion chromatography, and it possesses phosphatase and glucan binding activity as a monomer. Monomeric Gg-laforin has robust phosphatase activity against the artificial substrate pNPP and also the more biologically relevant substrate amylopectin, similar to the activity of Hs-laforin as previously described [We have demonstrated that Hisescribed ,8,30. Co6 tag.The ppSUMO plasmid was a generous gift from Dr. Jack Dixon . The plasmid pGL-EPM2A containing the gene for Mm-laforin (NP_034276.2) was a kind gift from Dr. Kazuhiro Yamakawa . Mm-laforin was subcloned into pET21a that includes a C-terminal His6-tag to aid purification. Sequences were verified by DNA sequencing. pET21a Vaccinia H1-related phosphatase (VHR) and pET21a Hs-laforin constructs have been described previously [Expressed sequence tags (ESTs) of Xt-laforin (NP_001123695.1) and Gg-laforin (NP_001026240.1) were purchased from Open Biosystems and Delaware Biotechnology Institute, respectively, and cloned into ppSUMO according to standard protocols. ppSUMO encodes a small Ub-like modifier (SUMO) fusion tag that includes an amino-terminal Hiseviously ,38.E. coli cells (Stratagene) and purified using an IMAC column on a Profinia purification system (BioRad) followed by size exclusion chromatography. Bacterial cultures were grown in 1 L 2xYT or Terrific Broth (IBI Scientific) with 1 mM kanamyacin and 1 mM chloramphenicol at 37°C until OD600 reached ~0.8. Cultures were chilled on ice for 20 minutes, and isopropyl thio-β-D-galactopyranoside (IPTG) was added for a final concentration of 0.4 mM to induce protein expression. After growth for approximately 12-16 hours, cells were harvested by centrifugation and stored at -20°C. Bacterial pellets expressing Hs-laforin were resuspended in buffer A: 50 mM Tris/HCl (pH 7.5), 300 mM NaCl, and 2 mM dithiothreitol (DTT). Pellets expressing Mm-laforin were resuspended in buffer B: 50 mM Tris/HCl (pH 8.0), 300 mM NaCl, and 0.05% β-mercaptoethanol. Pellets expressing VHR, Xt-laforin or Gg-laforin were resuspended in buffer C: 20 mM Tris/HCl (pH 7.5), 100 mM NaCl and 2 mM DTT. 15% maltose (w/v) or 10 mM β-cyclodextrin was added to some preparations. Resuspended cells were lysed with a microfluidizer , and soluble fractions were separated by high-speed centrifugation . His6-SUMO-tagged Xt-laforin and Gg-laforin were purified using a Profinia IMAC column (Bio-Rad) with a Profinia protein purification system (Bio-Rad) and dialyzed into buffer C in the presence of the SUMO-specific protease ULP1 that also contains a His6-tag. Reverse purification over the Profinia IMAC column was used to remove ULP1-His6 and the fusion tag. Each protein was then purified using a HiLoad 16/60 Superdex 200 size exclusion column and ÄKTA FPLC . Fractions containing the Gg-laforin monomer species were collected and put back over the same column. Mm-laforin, Hs-laforin and VHR were also expressed as His6-tagged recombinant proteins and purified in a similar manner.All proteins were expressed in BL21-CodonPlus g, and protein concentration was measured using a Bradford assay. To test long-term stability, samples were concentrated to approximately 2-4 mg/ml and incubated at room temperature for eight days. Protein concentration was monitored during the course of the experiment using a Bradford assay.Protein purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with Coomassie brilliant blue to visualize proteins. To quantify stability of Hs-laforin and Gg-laforin, elution fractions were concentrated using centrifugal filter units . Volume and concentration were monitored throughout centrifugation at 3,220 × Dynamic light scattering (Protein Solutions DynaPro-99) was utilized to determine the hydrodynamic radius of particles in solution. The DLS system measures the size distribution of particles by detecting fluctuations in light intensity over time. Scattering intensity was presented as a fraction of the total protein mass; poly- or monodispersity in the sample was determined by the number of peaks on the DLS histogram. A standard curve embedded in the DLS software was used to calculate the approximate size of a globular protein with the observed hydrodynamic radius. Measurements were performed on a protein sample of 1 mg/ml at room temperature.6-tagged protein was mixed with 30 μl amylose beads in buffer C and protease inhibitor cocktail  while rotating at 4°C for 30 min. Amylose beads were pelleted by centrifugation , the supernatant was removed, proteins in the supernatant were precipitated, and proteins in the pellet and supernatant were visualized by Western analysis. Blots were probed with mouse anti-His6 1:4000 (NeuroMabs) and goat anti-mouse HRP (Invitrogen). SuperSignal West Pico (Thermo Scientific) was used to detect the HRP signal.Amylose immobilized on agarose resin (New England Biolabs) was pre-incubated with 1% BSA at room temperature for 30 min to prevent nonspecific binding. 0.25-1 μg of each recombinant Hispara-nitrophenylphosphate (pNPP) and potato amylopectin as described previously [N-ethylmaleimide and 80 μl malachite green reagent was added to quench the reaction, and absorbances were measured at 620 nm after 40 minutes. Assays were performed in triplicate for each enzyme at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0.Phosphatase activity was determined using the substrates eviously ,29,30. Thttp://projects.tcag.ca/lafora/) [Amino acid sequences of laforin orthologs were obtained from NCBI, aligned by ClustalW , and reflafora/) .para-nitrophenylphosphate; DLS: Dynamic light scattering; BCD: β-cyclodextrin; IMAC: Immobilized metal affinity chromatography; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.LD: Lafora disease; LB: Lafora body; CBM: Carbohydrate-binding module; DSP: Dual-specificity phosphatase; SUMO: Small ubiquitin-like modifier; EST: Expressed sequence tag; VHR: Vaccinia H1-related phosphatase; IPTG: Isopropyl thio-β-D-galactopyranoside; DTT: Dithiothreitol; ULP1: Ubiquitin-like specific protease-1; pNPP: The authors declare that they have no competing interests.MSG conceived the study. MKB, VVD, MBJ, and MSG carried out the cloning of laforin orthologs. VVD expressed and purified Mm-laforin. MKB and SH carried out expression, purification and characterization of Hs-laforin, Xt-laforin and Gg-laforin. MKB and MSG wrote the manuscript. All authors read and approved the final manuscript.Multimerization of Xt-laforin. Xt-laforin was purified by IMAC and passed over a HiLoad 16/60 Superdex 200 size exclusion column. The chromatogram shows a prominent peak corresponding to a multimeric species and unresolved peaks corresponding to the Xt-laforin dimer and monomer .Click here for file


Profunda femoris artery aneurysm (PFAA) is an extremely rare entity, with most cases being asymptomatic, which makes obtaining an early diagnosis difficult. We herein report a case series of PFAA, in which more than half of the PFAAs, which presented with no clinical symptoms, were discovered incidentally. All PFAAs were treated surgically with aneurysmectomy with or without vascular reconstruction. In cases involving a patent superficial femoral artery (SFA), graft replacement of the profunda femoris artery (PFA) is not mandatory; however, preserving the blood flow of the PFA is necessary to maintain lower extremity perfusion in patients with occlusion of the SFA. Therefore, the treatment of PFAAs should include appropriate management of both the aneurysmectomy and graft replacement, if possible. Profunda femoris artery aneurysm (PFAA) is an uncommon condition, accounting for only 0.5% of peripheral aneurysms and only 1–2.6% of all femoral artery aneurysms . Based oA retrospective review was performed on all patients with a diagnosis of PFAA who underwent surgical treatment at Tokyo Medical and Dental University Hospital between January 2005 and December 2014. All subjects provided their informed consent, and approval was obtained from our Institutional Review Board for a retrospective review of the patients' medical records and images. The inclusion criterion was aneurysmal dilatation of a PFA of more than 20 mm, based on preoperative imaging findings. Cases of pseudoaneurysms of PFAA due to trauma were excluded, and only true aneurysms were included. The medical records were abstracted to include basic demographic information, preoperative symptoms, aneurysm size measurements, intraoperative findings, perioperative complications, and long-term imaging findings. The characteristic features of the patients are given in A 76-year-old asymptomatic male presented for follow-up magnetic resonance imaging (MRI) after open surgical repair of an abdominal aortic aneurysm (AAA). MRI showed a PFAA measuring 45 × 40 mm on the right side of the thigh. The aneurysm was successfully resected under general anesthesia without vascular reconstruction, as the superficial femoral artery (SFA) was patent, and the distal portion of the PFA was very small, making it unsuitable for revascularization. The patient's postoperative course was uneventful, and the postoperative ankle brachial pressure was within the normal limits without any lower limb ischemia.A 69-year-old female presented with pain and swelling of the left thigh. Computed tomography (CT) showed a left PFAA measuring 34 × 24 mm. Furthermore, CT detected a right PFAA measuring 25 × 22 mm, without clinical symptoms, and the bilateral common femoral arteries (CFAs) showed aneurysmal changes . The bilA 73-year-old asymptomatic male presented for follow-up CT after open surgical repair of AAA and bilateral common iliac artery aneurysms and an assessment of an untreated thoracic artery aneurysm measuring 40 mm in size. CT exhibited a PFAA measuring 25 × 22 mm on the right side of the thigh . The aneA 65-year-old asymptomatic female presented for the ultrasonography to evaluate the varicose vein. US showed the 25 mm sized mass on her right groin. Further contrast enhanced CT scanning showed the right PFAA measuring 26 × 25 mm. Under general anesthesia, the SFA and the proximal and distal part of PFAA were well controlled; the aneurysmectomy was successfully performed with the interposed 8 mm prosthetic graft placed between the proximal and distal PFA . PostopeA 70-year-old male presented with a palpable mass and pain in the left thigh. Contrast enhanced CT revealed a left PFAA measuring 86 × 76 mm . The aneA total of six PFAAs were resected in five patients. The mean operative time was 130 minutes (range: 81–210 minutes) and the mean amount of intraoperative blood loss was 122 mL (range: 15–594 mL); therefore, none of the patients required a blood transfusion. Four of the six PFAAs were interposed with a prosthetic graft, and, in case 2, the bilateral PFAAs and CFAAs were resected simultaneously with revascularization. Two of the six PFAAs were treated with ligation without revascularization because the distal part of each PFAA was located too far to achieve revascularization. The pathological findings of the resected aneurysms showed degenerative and atherosclerotic changes in all six PFAAs.None of the patients exhibited lower limb ischemia after the surgical procedures and all were discharged successfully. During the long-term follow-up period , no patients presented with signs of lower limb ischemia, and all of the interposed grafts remained patent.Previous reviews of published cases have indicated that patients with PFAA often have synchronous aneurysms, occurring in 65–75% of cases, including AAA and popliteal artery aneurysms . In our It has been reported that PFAAs are much more common in males (92–100%) than in females, and most PFAAs are discovered in the sixth to seventh decades of life . FurtherAlthough patients with PFAAs usually remain asymptomatic and the lesions are discovered incidentally, such patients may present with symptoms related to local compression, thrombosis, or embolism, with consequent rupture. Compression-related symptoms include groin swelling, pain, and pulsatile masses . In our Following the diagnosis of PFAA, elective surgical repair is recommended whenever the patient's general condition allows for surgical intervention . A reasoThe aim of surgical treatment for PFAAs is to eliminate the risk of complications, including distal ischemia and rupture, and maintain perfusion to the lower extremities. Therefore, surgical repair consists of aneurysmectomy with or without graft replacement , 10. WheIn conclusion, we herein reported a case series of PFAAs treated surgically with aneurysmectomy with or without graft replacement. Providing an early diagnosis and surgical treatment is necessary to prevent complications, and reconstruction of the PFA is recommended, unless the SFA is patent and performing graft replacement is technically difficult.


In a laser-induced injury mouse model of thrombosis, it appears that neutrophils are required to initiate thrombus formation and coagulation activation on inflamed arteriolar endothelia. In this model, by using P2X1−/ − mice, we recently showed that P2X1 receptors, expressed on platelets and neutrophils, play a key role in thrombus growth and fibrin generation. Intriguingly, in a model of endotoxemia, P2X1−/ − mice exhibited aggravated oxidative tissue damage, along with exacerbated thrombocytopenia and increased activation of coagulation, which translated into higher susceptibility to septic shock. Thus, besides its ability to recruit neutrophils and platelets on inflamed endothelia, the P2X1 receptor also contributes to limit the activation of circulating neutrophils under systemic inflammatory conditions. Taken together, these data suggest that P2X1 receptors are involved in the interplay between platelets, neutrophils and thrombosis. We propose that activation of these receptors by ATP on neutrophils and platelets represents a new mechanism that regulates thrombo-inflammation.Inflammation shifts the hemostatic mechanisms in favor of thrombosis. Upon tissue damage or infection, a sudden increase of extracellular ATP occurs, that might contribute to the crosstalk between inflammation and thrombosis. On platelets, P2X1 receptors act to amplify platelet activation and aggregation induced by other platelet agonists. These receptors critically contribute to thrombus stability in small arteries. Besides platelets, studies by our group indicate that these receptors are expressed by neutrophils. They promote neutrophil chemotaxis, both  Vessel wall injury and the extravasation of blood from the circulation rapidly initiate events in the vessel wall and in the blood that seal the breach. Circulating platelets are recruited to the site of injury, where they become a major component of the developing thrombus; blood coagulation is initiated by endothelium-expressed tissue factor and leads to the generation of thrombin and fibrin. Under normal conditions, regulatory mechanisms restrain thrombus formation both temporally and spatially 22 as a significant secondary platelet agonist Adenosine diphosphate (ADP) plays crucial roles in the physiological process of hemostasis and in the development and extension of arterial thrombosis. By itself ADP is a weak agonist of platelet aggregation inducing only reversible responses as compared to strong agonists such as thrombin or collagen. However, due to its presence in large amounts in the platelet dense granules and its release upon activation at sites of vascular injury, ADP is an important so-called secondary agonist amplifying most of the platelet responses, which contributes to the stabilization of the thrombus The receptors for extracellular nucleotides belong to the P2 family which consists of two classes of membrane receptors: P2X ligand-gated cation channels (P2X1–7) and G protein-coupled P2Y receptors  i.e. variability of platelet inhibitory effect) for the management of ischemic coronary syndromes Large-scale clinical trials have demonstrated the beneficial effects of thienopyridines, targeting P2Y12 receptors, in the prevention of major cardiac events after coronary artery stenting and in the secondary prevention of major vascular events in patients with a history of cerebrovascular, coronary or peripheral artery disease. More recently, new classes of P2Y12 inhibitors have been developed in order to circumvent clopidogrel limitations  that assemble to form a variety of homotrimeric and heterotrimeric receptors widely expressed in the body. Each P2X subunit contains two transmembrane domains, intracellular amino and carboxy termini and a large extracellular ligand-binding loop. P2X receptors vary in their kinetics of desensitization and pharmacology, although all are activated by the physiological ligand ATP ex vivo and on thrombosis in vivoThe P2X1 receptor plays an important role in thrombus formation especially under high-shear conditions. P2X1-deficient mice have no prolongation of bleeding time as compared to the wild-type mice, indicating that they conserve normal hemostasis 2 +2 + influx into the cell following activation, as well as causing membrane depolarization. The time course of ATP-evoked P2X1 receptor-mediated currents is concentration-dependent with low concentrations taking several seconds to reach a peak response, which can be sustained for > 30 s. In contrast, at maximal agonist concentrations, P2X1 receptor currents peak within tens of milliseconds and desensitize completely within seconds 2 + leads to the activation of ERK1/2 MAPK and MLCK that phosphorylates myosin light chain (MLC), a process accompanying platelet shape change and degranulation 2 +-mobilization following activation of a variety of major platelet receptors through both G-proteins and tyrosine kinases About 10% of current flow through the P2X1 receptor is mediated by CaThese results clearly indicate that the P2X1 receptor might be considered as a potential target for antiplatelet strategies, with the interesting feature that P2X1 antagonists should be effective only at sites of severe stenosis where shear forces are very high, without having a deleterious effect on normal hemostasis.4ex vivo stimulation of P2X1−/ − neutrophils with various stimuli, including bacterial formylated peptides, phorbol esters, and opsonized zymosan particles resulted in increased production of reactive oxygen species as compared to neutrophils isolated from wild-type mice. These results indicated that P2X1 would act to limit systemic neutrophil activation through a negative feedback loop, allowing them to migrate to the site of inflammation. In agreement with this proposition, intraperitoneal injection of a sub-lethal dose of lipopolysaccharide (LPS) in P2X1−/ − mice, led to increased release of plasma myeloperoxidase (MPO) concentration, an indicator of neutrophil systemic activation, as compared to wild type mice. In addition, peripheral P2X1−/ − neutrophils expressed higher levels of CD11b in response to LPS injection, reflecting their higher activation state. Concomitantly, we observed that the LPS-induced drop in platelet and lymphocyte counts were both worsened in the P2X1−/ − mice as compared to their wild type littermates. Immunohistochemistry and MPO activity assay revealed exaggerated neutrophil relocalization into the lungs of P2X1−/ − mice, where these cells formed large aggregates in the capillary lumen. Finally, intraperitoneal injection of a lethal dose of LPS, the P2X1−/ − mice exhibited shorter survival time than wild type mice, most likely as a consequence of enhanced neutrophil-dependent ischemic events and subsequent multiple organ failure. Notably, this phenotype was not associated with altered plasma levels of the main LPS-induced cytokines, TNF-α, IL-6, IL-1β, and INF-γ. Taken together, these findings support an important role for P2X1 receptors in the homeostatic regulation of circulating neutrophils and in their recruitment at the sites of inflammation/infection.We recently showed that P2X1 receptors are also expressed on neutrophils 5Several studies indicate that besides their ability to kill pathogens, neutrophil activation promotes coagulation in the microcirculation, trapping invading pathogens in fibrin mesh, thereby restricting microbial dissemination e.g. P2X5, to form heteromeric ion channels with distinct properties The data reported so far assumed that the effects of platelet and neutrophil P2X1 receptors are mediated by homotrimeric P2X1 receptors. It must be known that P2X1 can also interact with other P2X subunits, 6In summary, our latest findings indicate that P2X1 receptors contribute to ATP-dependent thrombosis in mouse microcirculation by promoting early neutrophil and platelet recruitment and subsequent fibrin generation, locally, at sites of endothelial injury . Upon syTargeting P2X1 receptors will not only inhibit platelets but also alter neutrophil function, and may therefore represent an innovative therapeutic strategy to prevent local thrombo-inflammation, only if neutrophil regulatory homeostasis is preserved. Future research should focus on the role of P2X1 receptors in the pathophysiology of thrombo-inflammatory disorders such as ischemic stroke. In stroke, thromboembolic occlusion of major or multiple smaller intracerebral arteries leads to focal impairment of the downstream blood flow, and to secondary thrombus formation within the cerebral microvasculature i.e., VWF-GPIb, collagen-GPVI), but not aggregation (αIIbβ3 inhibitors), reduces infarct size. Platelets serve pro-inflammatory functions that are likely involved in infarct growth.In a mouse model of acute ischemic stroke (tMCAO), it appears that platelets contribute to stroke progression by mechanisms that at least partially differ from those involved in thrombus formation However, the mechanistic links between platelets and inflammation remain largely unknown. Our recent experimental data indicate that P2X1 receptors expressed on both platelets and neutrophils may represent such a link. It would therefore be interesting to determine whether defective thrombus formation observed in the microcirculation of P2X1−/− mice would protect these mice from thrombo-inflammatory ischemic brain infarction.


Coronary computed tomography angiography (CCTA) allows for non-invasive coronaryartery disease (CAD) phenotyping. There are still some uncertainties regarding theimpact this knowledge has on the clinical care of patients.To determine whether CAD phenotyping by CCTA influences clinical decision makingby the prescription of cardiovascular drugs and their impact on non-LDLcholesterol (NLDLC) levels.We analysed consecutive patients from 2008 to 2011 submitted to CCTA withoutprevious diagnosis of CAD that had two serial measures of NLDLC, one up to 3months before CCTA and the second from 3 to 6 months after.A total of 97 patients were included, of which 69% were men, mean age 64 ±12 years. CCTA revealed that 18 (18%) patients had no CAD, 38 (39%) hadnon-obstructive (< 50%) lesions and 41 (42%) had at least one obstructive≥ 50% lesion. NLDLC was similar at baseline between the grups . We found significative reduction in NLDLC among patients with obstrctivelesions . We also found a positive relationship between clinicaltreatment intensification with aspirin and cholesterol reducing drugs and theseverity of CAD.Our data suggest that CCTA results were used for cardiovascular clinical treatmenttitration, with especial intensification seen in patients with obstructive≥50% CAD. However, the number needed to treat(NNT) varies according to the studied population and low cardiovascular risk patientsbenefit less than those at high cardiovascular risk. However, the potential for adverseeffects remains similar6.According to the variation of baseline risk, the NNT may vary from 24 to 549 treatedpatients, for the reduction of an event5.The main therapeutic intervention used in patients with atherosclerosis, as a means ofprimary prevention of ischemic events, are Cholesterol-Lowering Drugs (CLD)8 and it is known thatthese findings are associated with increased incidence of cardiovascular events,independently from and in addition to the clinical risk factors10. However, it is still uncertain how doctors use the results of CCTA inthe clinical treatment titration of their patients.Studies show that approximately 20% to 30% of asymptomatic patients considered at lowcardiovascular risk (event rate less than 10% in ten years) have atherosclerosis in thecoronary computed tomography angiography (CCTA)This study aimed to evaluate the changes in both the prescription and plasma cholesterollevels in the short term, after a CCTA assessment in patients with no prior diagnosis ofCAD, according to the severity of CAD found at the examination.A retrospective and analytical cohort was analyzed, and the project was approved by theResearch Ethics Committee of HUCFF/FM/UFRJ, under protocol number27341114.7.0000.5257.All patients (123) submitted to CCTA between the years 2008 and 2011 in a cardiacimaging laboratory in Rio de Janeiro, with no prior diagnosis of CAD  and had cholesterolmeasurement recorded at two different times: one up to three months before the CCTA(index measurement), followed by a second sample taken from three to six months afterthe CCTA (follow-up measurement) were included in this analysis. This period was chosendue to the homogeneity of image acquisition protocols used at that time.In this institution, a physician from the team performs an interview with the patientprior to the examination, in which information such as anthropometric data, indicationfor the examination, risk factors, current medications and previous examinations isrecorded. Access to the existing clinic database was requested for this study.The analyzed items were: gender, age, CCTA indication , risk factors , medications being used in the index consultationand at the second consultation, such as antiplatelet drugs and Cholesterol-LoweringDrugs (CLD) and the cholesterol levels both at the index and the second consultations.CLD were defined as any drug of the statins or fibrates classes.Patients whose medical records did not provide the necessary data for this analysis,such as current medication or present risk factors were excluded from the analysis.Additionally patients whose imaging tests had inadequate quality for analysis in threeor more coronary segments were also excluded.The primary outcome of this study was non-LDL cholesterol (NLDLC) reduction afterassessment by CCTA in the pre-specified period (three to six months). The NLDLC wasconsidered as the sum of VLDL cholesterol and LDL cholesterol. It was decided torestrict the follow-up to such short period in order to minimize the influence offactors rather than the CCTA outcome on therapeutic decision-making.As a secondary outcome, the change in medications prescription after the CCTA outcome.was assessed.CCTA images were acquired using 256-channel devices  or one of the two 64-channel scanners. For CCTA acquisition, venous and oral beta-blockers wereused aiming to reduce HR to less than 60 bpm. Isosorbide dinitrate, 0.4 mg, sublinguallywas also administered to all patients without contraindications, 3 to 5 minutes prior toimage acquisition.2 compatible with coronary atheromatosis, whereas thedegree of luminal stenosis was defined as the ratio between the smallest luminaldiameter at the lesion and the reference diameter closest to the lesion.Image analysis was performed by a single expert that had broad experience with themethod. The coronary plaques were defined as the presence of image with soft tissuedensity ≥ 1 mmPatients were classified according to the highest degree of coronary stenosis found,considering: I - no plaque; II - non-obstructive plaques only (< 50% in stenosis);III - at least one obstructive plaque (≥ 50% in stenosis). To measure thecholesterol-lowering effects, a positive CCTA was considered the one with any evidenceof coronary atherosclerosis.The following software programs were used for data processing: SPSS version 19.0 andMicrosoft Excel 2000© (9.0.2812).To calculate sample size, we considered a difference of 30% in LDL cholesterolbetween patients with positive (estimated at 30% of the sample) and negative CCTA(70% of the sample). The estimated "n" was 90 patients for an alpha error of 0.05 andbeta of 0.25.t test, as appropriate. Categorical variables were expressed asamounts and proportions and were compared using the Chi-square and Fisher tests, whenappropriate. p values <0.05 were considered significant.Quantitative data showed normal distribution through the Kolmogorov-Smirnov test witha significance level of 5%. Continuous variables were expressed as mean ±standard deviation and were compared using paired and unpaired Student’sPatients were divided into three groups: without CAD, non-obstructive CAD (< 50%in stenosis) and obstructive CAD (≥ 50% in stenosis). Statistical analysis wascarried out for each group separately and the two periods were compared by paired-ttest.Of a total of 123 patients that had two cholesterol measurements recorded in theproposed time period, 24 were excluded due to incomplete filling out of medical recordsand two due to poor quality of the images, resulting in 97 patients included in theanalysis, of which 67 (69%) were men. The mean age was 64 ± 12 years. Nineteenpercent had no risk factor; 15% had only one risk factor; 35% had two or three riskfactors; and 31% had four or more risk factors for CAD.CCTA was performed to assess pain with angina characteristics in 10% of patients;atypical pain in 16%; dyspnea or decreased functional capacity of 23%; and 51% ofpatients were asymptomatic. The clinical characteristics are described in NLDLC significantly decreased from 136 ± 44 mg/dL in the first to 117 ±38 mg/dL in the follow-up visit (p = 0.007), a 14% decrease in the average of thegeneral population .Between the index and the follow-up visit, the variation in NLDLC in the group withnegative CCTA was 4% , inthe group with non-obstructive CCTA was 10%  and the in group in which CCTA showed obstructive lesions was 18%. There was no significantdifference in NLDLC values between the different groups of CCTA in the index visit (p= 0.3).Of the 42 individuals that had lesions ≥ 50% in stenosis, 32 (76%) showed LDLvalues < 100 mg/dL at the second visit .versus 2 (3%) patients withnormal CCTA (p < 0.05). Considering only the 34 patients that used CLD in theindex visit, 8 (23%) had negative CCTA (of which 3 discontinued use of CLD) and 5 hadat least one coronary segment with lesion (obstructive or not) and discontinued thesedrugs. Considering only the 64 (65%) patients that did not use the CLD in the index visit,28 (43%) started using them after CCTA, when the latter showed at least one coronarysegment with lesion (obstructive or not) Aspirin use was started after the normal CCTA result in 2 (11%) patients and in 17(21%) patients with positive CCTA .ImportaThe combined use of aspirin and CLD was started at 0%, 2% and 19% of patients withnegative CCTA, with non-obstructive lesions and lesions ≥ 50%, respectively (p= 0.004), and only one of the two drugs alone in 18%, 24% and 38% respectively (p =0.006).14.CAD phenotyping by CCTA has a relevant prognostic impact and improves riskclassification for cardiovascular events when compared to the classic riskfactorsThis study allowed to evaluate, in the real world and in the short term, the impact ofCCTA results on drug therapy, evaluating trends in cholesterol levels and the use ofmedications shortly after the CCTA results.15. A cost-effectivenessanalysis showed that the aspirin is only cost-effective in men with clinical risk in tenyears greater than 10% and in women when the risk is > 15%16. In our study, aspirin was started in 5% of patientsafter a completely normal CCTA result, which probably would not be indicated,considering that the annual risk is < 0.1% of combined coronary events17. Conversely, we observed that 22% ofpatients with positive CCTA (obstructive or not) in our study started aspirintherapy.The analysis of this impact on clinical therapy is relevant, as the benefit ofantiplatelet agents and CLD is directly associated with the patient’s cardiovascularrisk. It has been demonstrated that in an asymptomatic population with zero calciumscore, the NNT with aspirin is approximately two thousand individuals to prevent onemajor cardiovascular event, while the number needed to harm (NNH) is 442 individuals,demonstrating a much greater risk in the prescription of aspirin than itsbenefits19. Among the42 patients with obstructive CAD ≥ 50%, 12 (29%) maintained the prescription ofstatin and/or aspirin and 24 (57%) started the use of the two drugs or added one of thetwo to previous therapy, after the CCTA result, corroborating the results of otherpublished studies23.The proportion of patients that initiated therapy with CLD has increased significantlyas the severity of CAD increased , demonstrating that in the real world there is an agreementbetween therapy intensity and the severity of CAD lesions. There was no reduction inNLDLC in patients with normal CCTA, which occurred in patients with obstructive lesions≥ 50%. There was a NLDLC decrease trend in patients with non-obstructive lesions,and these data are corroborated by other studies 24 involving 170,000patients showed that a 1-mmol/L decrease in LDL cholesterol was able to reduce the riskof cardiovascular events by 20%. We observed in this study that 28 (29%) showed LDLdecrease > 1 mmol/L, which implies a reduction of cardiovascular risk in thisgroup.A meta-analysis19.The reduction in all lipid fractions, except HDL, demonstrates not only the potentLDL-lowering effect of the CLD, as well as reductions more related to changes in dietaryhabits, as demonstrated by the reduction in triglycerides 25in a Brazilian population, inwhich it was found that only 30% of patients had LDL values within the Guideline’stargets.Of all patients considered at high risk by CCTA (lesion ≥ 50%), 32 (76%) reachedthe LDL target <100 mg/dL. This result contrasts with the data collected by Vacantiet al.29. The results of this study indirectlycorroborate that, showing patients with higher atherosclerotic disease severity havehigher reduction in cholesterol levels.The stratification of cardiovascular risk made by visualization of coronaryatherosclerosis has previously demonstrated to have greater impact on medicationadherence and change in clinical management than the risk stratification by clinicalscores26 recently addressed theimpact of CCTA findings on drug therapy in a study that evaluated 2,839 patients with amean follow-up of 3.6 years, and found that after the CCTA examination there was anincrease in the prescription of aspirin even in the group without CAD . This study also found statin prescriptionintensification after the CCTA results. After the CCTA results, 36% of patients withoutCAD were using CLD and 18% had been prescribed therapy intensification; in patients withnon-obstructive lesions < 50%, 72% were using CLD and in 42%, prescriptionintensified occurred; in patients with lesions > 50% in stenosis, of the total 90% inuse of statin, there was an intensification of 63%.Hulten et al.Among the assessed patients, two of them are especially illustrative for the analysis ofthe results. The first one, aged 54, had fatigue on moderate exertion, hypertension anddid not use statins or aspirin. He had NLDLC of 201 mg/dL in the index visit. The CCTAdisclosed 50% -70% lesion in the middle third of the Anterior Descending Artery (ADA).In the follow-up assessment, the patient was using aspirin and CLD and the NLDLC haddecreased to 140 mg/dL. The second patient, aged 66 years, had atypical chest pain,diabetes, dyslipidemia, was hypertensive and sedentary, was using aspirin and CLD in theindex visit had a NLDLC of 120 mg/dL. The CCTA disclosed a 50% -70% lesion in theproximal third of the ADA. At the second assessment, the NLDLC had decreased to 100mg/dL with medication maintenance. The two patients were at high risk for cardiovascularevents, but in the second case, there was not so marked decrease in NLDLC values,probably because the patient was already undergoing medical treatment and the medicationwas only optimized after CCTA the result.Some limitations should be considered in our study, which was observational andretrospective, performed at a single diagnostic imaging center, with two CTs ofdifferent manufacturers, with a relatively small sample. Because we selected patientswith two sequential cholesterol measurements, there may have been some selectionbias, preferably excluding patients that had not started treatment with CLD andtherefore had no clinical need to collect a new blood sample for lipid levelmeasurement. Therefore, the results are more relevant for the analysis of factorsinvolved in the prescription of antiplatelet agents and CLD than for the lack oftheir indication.The severity of CAD positively correlated with the decrease in cholesterol levels andmore frequent prescription of antiplatelet agents and CLD. These data suggest that theCCTA result can influence drug treatment approach in the short term in the realworld.


Furthermore, the in vivo image experimental results indicated that a larger amount of DOX was accumulated in the bone metastatic tumor tissue after DOX loaded DOX-hyd-PEG-ALN micelle was intravenously administered, which was confirmed by histological analysis. Finally, DOX loaded DOX-hyd-PEG-ALN micelle effectively delayed the tumor growth, decreased the bone loss and reduced the cardiac toxicity in tumor-bearing nude mice as compared with free DOX. In conclusion, DOX loaded DOX-hyd-PEG-ALN micelle had potential in treating bone metastatic tumor.In order to increase the therapeutic effect of doxorubicin (DOX) on bone metastases, a multifunctional micelle was developed by combining pH-sensitive characteristics with bone active targeting capacity. The DOX loaded micelle was self-assembled by using doxorubicin-poly (ethylene glycol)-alendronate (DOX-hyd-PEG-ALN) as an amphiphilic material. The size and drug loading of DOX loaded DOX-hyd-PEG-ALN micelle was 114 nm and 24.3%. In pH 5.0 phosphate buffer solution (PBS), the micelle released DOX significantly faster than in pH 7.4 PBS. In addition, with the increase of incubation time, more red DOX fluorescence was observed in tumor cells and trafficked from cytoplasm to nucleus. The IC Some kinds of human cancers such as lung cancer, prostate cancer and breast cancer almost invariably metastasize to bone in their advanced stage. This is because the physiological microenvironment of bone is very suitable for tumor cells to adhere and proliferateet al. found that docetaxel did not inhibit the growth of bone metastases tumor at the dose that was effective for subcutaneous implanted tumorThe methods to treat bone metastases are radiotherapy, surgery and chemotherapy. However, their efficacies are limited due to the low selectivity to the multiple bone metastatic nodules and poor permeability in the bone tumor tissues3et al. found that the conjugation of cisplatin with BPs did not significantly improve the therapeutic efficacy of cisplatin in bone metastatic tumor model even at very high doseAdult skeleton is composed of 50–70% mineral, 20–40% organic matrix, 5–10% water and 1–5% lipids. The main mineral component of bone is hydroxyapatite (HA). Bisphosphonates (BPs) show strong affinity with hydroxyapatite and can selectively accumulate in bone and bone metastases tumor1011Polymer-drug conjugate is one of the most promising approaches for cancer therapy1417et al. used CHOL-TOE-BPs as the targeting moiety to modify liposome and the results suggested that the CHOL-TOE-BPs modified liposome was mainly localized at the bone site21et al. prepared DOX-loaded H40-star-PEG/ALE micelles. The affinity of H40-star-PEG/ALE micelle to bone was confirmed by the hydroxyapatite (HA) binding assay. The results indicated that the H40-star-PEG/ALE micelle was a promising bone-targeted drug carrier for bone metastases tumorBecause of the poor permeability, specific physiological and biochemical processes of bone tissue, it is difficult for drug to effectively accumulate in bone metastases tumor. Therefore, active bone-targeted drug delivery systems are needed. Hengst in vitro cytotoxicity and in vivo anti-tumor activity of DOX loaded DOX-hyd-PEG-ALN micelle were further studied.In this paper, in order to increase the therapeutic effect of DOX on bone metastases, doxorubicin-poly (ethylene glycol)-alendronate (DOX-hyd-PEG-ALN) copolymer was synthesized. The hydrophilic and hydrophobic segment was conjugated by the hydrazone bond which was responsive to the acidic microenvironment of endolysosome in tumor cellAlendronate (ALN), hydroxyapatite (HA), N-hydroxysuccinimide (NHS), 3--2,5-diphenyl tetrazolium bromide (MTT), 1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDCI) and trifluoroacetic acid (TFA) were the product of Sigma-Aldrich company . α,ω-dicarboxyl poly(ethylene glycol)  was obtained from Shanghai Yare Biotech Inc.. Doxorubicin·HCl (DOX·HCl) was the product of Hisun Pharmaceutical Co. . 4′,6-diamidino-2-phenylindole (DAPI) was the product of Invitrogen Technologies Company . All other analytical grade chemicals were purchased from J&K company . A549 cell was purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. Primary antibodies rabbit anti-cyclin A and mouse anti-cyclin D1, and the secondary antibodies goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Proteintech (USA).Female athymic nude mice were supplied by the Experimental Animal Center, Fourth Military Medical University. All animal experimental procedures were executed in accordance with protocols that were authorized by the Animal Care and Use Committee, Fourth Military Medical University .2NH2·H2O was added into the solution and the reaction mixture was stirred for 24 h to obtain HOOC-PEG-CONHNH2. Finally, 80 mg HOOC-PEG-CONHNH2 was dissolved in 20 mL DMSO, and then 20 mg DOX and 10 μL trifluoroacetic (TFA) were added into the solution and stirred for more 48 h in darkness. The reaction mixture was completely dialyzed in deionized water and lyophilized to get DOX-hyd-PEG-COOH conjugate.The DOX-hyd-PEG-COOH was synthesized according to our previously reported study2O and dropped into the above reaction mixture. During the reaction process, the pH value of reaction mixture was maintained at 7 by dropwisely adding 0.2 mol/L NaOH solution. After 2 h, the pH value of reaction mixture was quickly adjusted to 9 to stop the reaction by using 0.2 mol/L NaOH solution. Finally, the reaction mixture was dialyzed in deionized water and lyophilized to obtain DOX-hyd-PEG-ALN conjugate. DOX-hyd-PEG was synthesized as a control conjugate by using methoxy-PEG-COOH to replace HOOC-PEG-COOH.ALN was conjugated with DOX-hyd-PEG-COOH by amino bondThe micelle was prepared by the dialysis methodThe micelle size, poly dispersity index and zeta potential were determined by using Beckman Coulter Particle Analyzer . All measurements were performed at room temperature and the data were obtained from the average of three measurements. The morphology of micelle was examined by using transmission electron microscopy To determine the drug loading and entrapment efficiency, the freeze-dried micelles were dispersed in 4 mL of 1 mol/L HCl and shook in water bath at 50 °C for 6 h to produce DOXTo investigate the pH-responsive characteristic of the DOX-loaded DOX-hyd-PEG-ALN micelle, the size change was evaluated by DLSThe release of DOX from DOX loaded DOX-hyd-PEG-ALN micelle was investigated by using dialysis methodThe binding kinetics of the DOX, DOX loaded DOX-hyd-PEG micelle and DOX loaded DOX-hyd-PEG-ALN micelle with HA was assessed according to a previously reported procedure312 humidified atmosphere.Human lung cancer cell line (A549), which is easy to metastasize to bone5 cells/mL and were cultured overnight at 37 °C. The cell culture medium then was replaced with fresh cell culture medium containing DOX loaded DOX-hyd-PEG-ALN micelle  and was incubated for 5 min, 30 min or 3 h at 37 °C. Then, the cells were slightly rinsed with PBS for three times and treated with 4′,6-diamidino-2-phenylindole  for 15 min to stain nucleus. Finally, the cells were slightly rinsed with PBS for three times and fixed with 4% paraformaldehyde. Cover slip was placed on glass slide and the uptake of DOX was observed by 80i fluorescence microscope . By using ImageJ software, the mean fluorescence intensity (MFI) of DOX in the nucleus or cytoplasm was calculated in an area of 4 μM2 for each sample.The A549 cells were planted into coverslip-containing 24-well plate at a density of 1.5 × 106 cells/mL and cultured with DOX loaded DOX-hyd-PEG-ALN micelle  for 5 min, 30 min and 3 h at 37 °C. The cells were then washed, re-suspended in 0.2 mL PBS, and quantitatively determined by flow cytometry .Flow cytometry was also used to monitor the cellular uptake of micelle by A549 cells. Firstly, the A549 cells were planted into 6-well plates with a density of 2 × 103 cells per well and cultured for 24 h. After the medium was removed from the well, and a series of concentration of free DOX or DOX loaded DOX-hyd-PEG-ALN micelle dissolved in fresh cell culture medium were added. The cells were cultured for 24 h or 48 h. The 5 mg/mL of MTT solution was added and the cells were incubated for more 4 h. After the culture medium was removed, DMSO (150 μL) was added. By using a CODA Automated EIA Analyzer , the absorbance of DMSO solution in each well was determined at 570 nm.The toxicity of free DOX and DOX loaded DOX-hyd-PEG-ALN micelle on A549 cells was evaluated by using MTT method. The cells were planted into 96-well plates with a density of 6 × 106 cells per well and cultured for overnight to allow cells attachment. The cell culture medium was then replaced with fresh cell culture medium that contained free DOX or DOX loaded DOX-hyd-PEG-ALN micelle , and the cells were cultured for 24 h or 48 h at 37 °C. The cells were harvested and then re-suspended in 70% ice cold ethanol and frozen overnight. After being centrifugated at 800 × g for 5 minutes, the cells were washed with cold PBS (pH 7.4) and resuspended in PBS containing PI , Triton X-100 , and DNase-free RNase (1 μg/mL) for 1 h at 37 °C. Finally, the cells were analyzed by flow cytometry . For each sample, a total of 10,000 events were acquired. Based on cell size, cell debris and cellular aggregates were gated and excluded. In addition, the effect of free DOX or DOX loaded DOX-hyd-PEG-ALN micelle on cell cycle protein cyclin A and cyclin D1 was determined by western blot according to the literatureCell cycle was analyzed according to the previous literature7 cells/animal) were injected into tibia of the male nude mice (23 ± 2 g) to induce the bone metastases. When the tumor volume reached about 300 mm3, 200 μL of normal saline, free DOX (5 mg/kg) and DOX loaded DOX-hyd-PEG-ALN micelle (5 mg DOX/kg) were respectively administered to mice by tail vein injection. Twenty four hours after the administration of micelle, the major organs, such as heart, liver, spleen, kidney, lung and bone with tumor were removed and analyzed by using Caliper IVIS Lumina II in vivo image . The fluorescence intensity in organs and bone tumor tissues was quantitatively analyzed by using Living Image 4.2 software. To investigate the details of DOX distribution in tumor site, the tumor tissues were sectioned into 5 μm thickness and the nucleus was stained with DAPI. Then the tissue section was observed by using confocal laser scanning microscopy. The mean fluorescence intensity (MFI) of DOX in the nucleus or cytoplasm was calculated by using aforementioned method.A549 cells  were injected into the tibia of the male nude mice to induce the bone metastases. After 10 days of tumor cells inoculation, mice were randomized in three different groups with 5 in each group: (i) normal saline treated group, (ii) DOX (5 mg/kg) treated group, (iii) DOX loaded DOX-hyd-PEG-ALN micelle (5 mg DOX/kg) treated group. Treatment was carried out once in a week by tail vein injection for 35 days. During the treatment, the body weight of tumor-bearing mice was recorded as index of toxicity. The long diameter (L) and the short diameter (W) of a tumor were measured with a caliper every 3 days. The tumor volume was calculated by using the formula: volume = LW2/2. At the end of the experiment, the tumor and heart tissue of tumor-bearing mice were removed and stained by hematoxylin and eosin (H & E) to observe tissue injury.Five no-tumor male nude mice were used as normal control (no tumor). A549 cells (1 × 10ex vivo micro-computed tomography (micro-CT) analysis of mouse leg bones was performed to validate the bone injury. Siemens Inveon Micro-CT  was used. Experimental parameters were as follows: X-ray: 50 keV, 500 μA; resolution: 111.25 μm. Micro-CT leg bones 3D reconstruction was performed by the open-source software Osirix. 3D microstructural properties of leg bone including bone volume fraction (BV/TV) and bone mineral density (BMD) were calculated by using software of Siemens Inveon Micro-CT.Additionally, leg bones  were dissected from the killed mice and stored in 4% paraformaldehyde solution. An Experiments were done in triplicates. The results are expressed as mean ± SD. Statistical analyses were performed with Graph Pad Prism.1H NMR spectrum of DOX-hyd-PEG-ALN is showed in 36The et al. prepared DOX loaded DOX-Hyd-PLA-PEG-FOL micelle with DOX and PLA as hydrophobic core. The size and PDI of DOX-Hyd-PLA-PEG-FOL micelle were 182 nm and 0.28 ± 0.04, respectivelyet al. prepared beclomethasone dipropionate loaded PHEA-PEG2000-DSPE micelles. The size and PDI of this micelle were 200 nm and 0.4, respectivelyThe particle size and size distribution of the DOX loaded DOX-hyd-PEG-ALN micelle and DOX loaded DOX-hyd-PEG micelle are showed in 42et al. prepared the bortezomib loaded ALN-PEG-PLGA nanoparticle to deliver bortezomib to bone. The drug loading was 0.04% and 0.74% respectivelyet al. prepared the PTX-PEG-ALN micelle to delver paclitaxel (PTX) to bone to treat tumor bone metastases. The drug loading of PTX-PEG-ALN micelle was 4.7%Zeta potential is an important index of micelle. As displayed in et al. found that the micelle breakup rate was similar in plasma and bovine serum albumin (BSA) solutions. The adsorption with albumin was the main process involving in micelle breakup in blood53The adsorption capacity of nanoparticle with plasma opsonin is closely related to the uptake of nanoparticle by MPSIn pH 7.4 PBS, the size of DOX loaded DOX-hyd-PEG-ALN micelle did not obviously change in 4 h . HoweverThe drug release behavior of the DOX loaded DOX-hyd-PEG-ALN micelle was investigated under a simulated physiological condition (pH 7.4) and an acidic microenvironment (pH 6.5 and 5.0) at 37 °C. As showed in in vitro. The binding kinetic is showed in For tumor metastasized bone tissue, the main constituent of skeleton HA was exposed in lysis sites. It is well-documented that ALN has a strong affinity with bone tissue because of its hydroxyl group and phosphonate groupIn this experiment, DOX itself was used as a fluorescence markeret al. reported that the cellular uptake of 50 nm nanoparticle was faster than that of 250 nm particleThe cellular uptake of DOX loaded DOX-hyd-PEG-ALN micelle and DOX loaded DOX-hyd-PEG micelle by A549 cells were further evaluated by flow cytometry. The results are showed in 5950 of free DOX and DOX-loaded DOX-hyd-PEG-ALN micelle is showed in The cytotoxicity of DOX loaded DOX-hyd-PEG-ALN micelle on A549 cells is showed in 2/M phase arrest2 phase arrest, and the extent of arrest augmented with the increase of incubation time. When free DOX and DOX loaded DOX-hyd-PEG-ALN micelle were respectively incubated with A549 cells for 24 h, the percentage of cells in G2 phase exhibited no significant difference between free DOX treated cells and DOX loaded DOX-hyd-PEG-ALN micelle treated cells. Compared with free DOX treated cells, the percentage of cells in S phase significantly increased in DOX loaded DOX-hyd-PEG-ALN micelle treated cells in 24 h. When DOX loaded DOX-hyd-PEG-ALN micelle and free DOX were respectively incubated with A549 cells for 48 h, the percentage of cells in G2 phase significantly increased both in DOX loaded DOX-hyd-PEG-ALN micelle treated cells and free DOX treated cells. Compared with free DOX, DOX loaded DOX-hyd-PEG-ALN micelles showed obviously higher cell cycle arrest capability in G2 phase in 48 h.DOX is reported to induce G2/M transition of the cell cycle. Cyclin D1 is an important regulator of G1 to S phase progressionThe cell cycle is regulated by several cyclins that drive cells in different phases. Cyclin A is involved in both S phase and Gin vivo. Meanwhile, the red DOX fluorescence was also found in high blood perfused organs such as liver and lung after DOX loaded DOX-hyd-PEG-ALN micelle was intravenously administered. This was due to much blood passing through these organs and non specific uptake by MPS in these organs.To further verify the active targeting capability of the DOX loaded DOX-hyd-PEG-ALN micelle, the distribution of DOX in different organs was observed and quantitatively analyzed by using the living image system. As showed in To observe whether DOX loaded DOX-hyd-PEG-ALN micelle could deliver DOX deeper in tumor tissue, the leg bone metastatic tumor tissue was sectioned and stained with DAPI, and red DOX fluorescence was quantitatively analyzed by using confocal laser scanning microscopy. As showed in et al. found that compared with nanoparticle with zeta potential of −26.9 mV, nanoparticle with zeta potential of −17.5 mV exhibited higher uptake by tumor but lower uptake by liver and lungin vivo distribution and blood circulation time of nanoparticles are dependent on its size after being systemically administered. Small nanoparticles (10–20 nm) tend to widely distribute in various organs. Large particles (>1 μm) tend to aggregate under blood physiological conditions, be rapidly uptaken by MPS and accumulate mostly in the liver and spleenet al. reported that TPGS coated nanoparticle of 100 and 200 nm sizes could escape from MPS and thus prolong the half-life of the nanoparticle in the blood system. Small size (<50 nm) nanopartical usually accumulated in the liver and kidney4964In order to deliver enough amount of antitumor drug to tumor tissue, the drug loaded nanoparticle should be able to avoid the uptake by MPS. The absorption of plasma opsonin plays an important role in the process of macrophage recognition to nanoparticle. Much evidence indicated that nanoparticle with neutral or slight charge had lower opsonization rate than the same size nanoparticle with high chargein vivo antitumor experiment was performed in male nude mice bearing A549 cell tumor. As showed in in vivo as compared with control group. When the tumor-bearing nude mice were treated with DOX loaded DOX-hyd-PEG-ALN micelle, tumor growth was significantly delayed as compared with free DOX treated mice. The percentage survival curve is showed in The Histological analysis of tumor tissue section was carried out to further evaluate the antitumor effect of DOX loaded DOX-hyd-PEG-ALN micelle, and the results are showed in The therapeutic efficacy of DOX loaded DOX-hyd-PEG-ALN micelle on tumor-bearing nude mice was further evaluated by micro-CT, the results are showed in The side effect is one of the major concerns in the development of the novel drug delivery system. The change in body weight of the tumor-bearing nude mice is an important index to evaluate systemic adverse effects. As showed in 29in vivo antitumor activity and reduced bone destruction with less systemic toxicity. DOX loaded DOX-hyd-PEG-ALN micelle had potential in the treatment of bone metastases cancer.DOX loaded DOX-hyd-PEG-ALN micelle showed high drug loading, encapsulation efficiency, and binding affinity with HA. DOX loaded DOX-hyd-PEG-ALN micelle delivered more DOX into bone metastatic tumor tissues, which greatly improved the How to cite this article: Ye, W.-l. et al. Doxorubicin-poly (ethylene glycol)-alendronate self-assembled micelles for targeted therapy of bone metastatic cancer. Sci. Rep.5, 14614; doi: 10.1038/srep14614 (2015).


Maladaptive use of the Internet results in Internet addiction (IA), which is associated with various negative consequences. Molecular and functional imaging techniques have been increasingly used for analysis of neurobiological changes and neurochemical correlates of IA. This review summarizes molecular and functional imaging findings on neurobiological mechanisms of IA, focusing on magnetic resonance imaging (MRI) and nuclear imaging modalities including positron emission tomography (PET) and single photon emission computed tomography (SPECT). MRI studies demonstrate that structural changes in frontal cortex are associated with functional abnormalities in Internet addicted subjects. Nuclear imaging findings indicate that IA is associated with dysfunction of the brain dopaminergic systems. Abnormal dopamine regulation of the prefrontal cortex (PFC) could underlie the enhanced motivational value and uncontrolled behavior over Internet overuse in addicted subjects. Further investigations are needed to determine specific changes in the Internet addictive brain, as well as their implications for behavior and cognition. Addiction to substances or activities can profoundly affect people's health and sometimes lead to serious social problems –3. For eWhile IA has drawn growing attention from scientific world, there are currently no standard diagnostic criteria. Several diagnostic criteria have been proposed to quantify IA. The most widely used diagnostic criterion is Young's Diagnostic Questionnaire –10. BaseAs an important subtype of IA, IGD has gained more and more attention from the whole world. IGD has been included in the appendix of the DSM-V, with a goal of encouraging additional studies . The DSMIn the past few years, molecular and functional imaging techniques have been increasingly used to study the neurobiological mechanism underlying IA. Molecular imaging is a rapidly developing field aimed to provide disease-specific molecular information through diagnostic imaging studies . The terMRI is a highly versatile imaging modality which uses magnet and radiofrequency energy to visualize the internal structure and soft tissue morphology of the body . The priUsing MRI, some studies have shown that brain structural changes are associated with IA. Using the Stroop color-word test , which hVoxel-based morphometry is an unbiased technique for characterizing regional cerebral volume and tissue concentration differences in structural magnetic resonance images , 31. VoxDiffusion tensor imaging (DTI) is an approach available to track brain white matter fibers noninvasively. Water molecules' diffusion was found to be much faster along the white matter fibers than perpendicular to them. The difference between these two motions is the basis of DTI , 44. DTIUsing arterial spin-labeling perfusion fMRI, Feng et al. investigated the effects of IGD on resting cerebral blood flow in adolescents . CompareFunctional connectivity impairments are also observed in individuals with IA. A recent study showed that subjects with IGD exhibited increased functional connectivity in the bilateral cerebellum posterior lobe and middle temporal gyrus compared with the control group . The bilIt has been reported that impulsivity is associated with IA . The abiRegional homogeneity is a widely used method in fMRI studies that measures the functional coherence of a given voxel with its nearest neighbors, and it can be used to evaluate resting-state brain activities based on the hypothesis that spatially neighboring voxels should have similar temporal patterns . IGD subSeveral studies investigated brain areas associated with cue-induced gaming urges –65. The An interesting study compared IGD subjects with subjects in remission from IGD and controls in cue-induced craving to play online games . BilaterMRI has also been used to assess therapeutic effects of specific pharmacological treatment on IA. Bupropion is a norepinephrine/dopamine reuptake inhibitor, which has been used in the treatment of patients with substance abuse. A study explored the possible effectiveness of bupropion, assessed brain activity in response to game cues using fMRI . IGD shoNuclear imaging approaches, which include SPECT and PET, have the advantages of high intrinsic sensitivity, unlimited depth penetration, and a broad range of clinically available molecular imaging agents . SPECT a18F-fluoro-deoxyglucose (18F-FDG) PET imaging, a study investigated the differences of cerebral glucose metabolism at resting state between young individuals with IGD and those with normal use [Using rmal use . Imaging11C-labelled raclopride and PET scans to investigate endogenous dopamine release in the human striatum during a video game [11C-raclopride to dopamine D2 receptors is sensitive to levels of endogenous dopamine, which can be detected as changes in binding potential of the radioligand. The authors reported that binding of 11C-raclopride to dopamine receptors in the striatum was significantly reduced during the video game compared with baseline levels of binding, which suggested increased release and binding of dopamine to its receptors. Moreover, they showed that there is a significant correlation between performance level during the task and reduced 11C-raclopride binding potential in the striatum. Similar results have been reported in people with IA [Emerging evidence has shown that the dopaminergic system is involved in drug addiction , 75. A pdeo game . Binding with IA . Individ with IA . They re99mTc-TRODAT-1, our group investigated striatal dopamine transporter density in IA subjects to identify potential presynaptic abnormalities [99mTc-TRODAT-1 uptake ratio of corpus striatum were greatly reduced in individuals with IA compared with controls. Taken together, these results suggest that IA is associated with dysfunction of the brain dopaminergic systems.Dopamine transporter is a plasma membrane protein that actively translocates released dopamine from the extracellular space into the presynaptic neurons . Alteredmalities . We show11C-N-methylspiperone (11C-NMSP) and 18F-FDG, in both states of resting and internet gaming task [11C-NMSP binding was found in the right inferior temporal gyrus in the IGD subjects compared to normal controls  and found that dopamine D2 receptor mediated dysregulation of the OFC could underlie a mechanism for loss of control and compulsive behavior in IGD subjects.Emerging evidence has shown that changes in brain structure and activity related to IA are relevant to brain regions involved in reward, motivation, and memory, as well as cognitive control. Molecular and functional imaging techniques have been increasingly applied to IA research, contributing significantly to our understanding of the neurobiological mechanism. Most of the previous literatures have studied IA individuals only under resting state, verified structural and functional abnormalities in the OFC, dorsolateral PFC, ACC, and PCC. Those regions may play crucial roles in salience attribution, inhibitory control, and decision making. So far, only one PET study with 11C-carfentanil to study the mu-opioid receptor availability and predict treatment outcomes of opioid receptor antagonists and help clinicians determine the most appropriate treatment for individual patients.As IA has become a serious problem worldwide, a need for effective treatment is becoming increasingly urgent. Both psychological and pharmacological treatment approaches have been applied to treat IA. Several drugs have shown to be promising in treating IA, such as antidepressants, antipsychotics, and opioid receptor antagonists . Cogniti


In-vivo voltammetry has successfully been used to detect dopamine release in rodent brains, but its application to monkeys has been limited. We have previously detected dopamine release in the caudate of behaving Japanese monkeys using diamond microelectrodes (Yoshimi 2011); however it is not known whether the release pattern is the same in various areas of the forebrain. Recent studies have suggested variations in the dopaminergic projections to forebrain areas. In the present study, we attempted simultaneous recording at two locations in the striatum, using fast-scan cyclic voltammetry (FSCV) on carbon fibers, which has been widely used in rodents. Responses to unpredicted food and liquid rewards were detected repeatedly. The response to the liquid reward after conditioned stimuli was enhanced after switching the prediction cue. These characteristics were generally similar between the ventral striatum and the putamen. Overall, the technical application of FSCV recording in multiple locations was successful in behaving primates, and further voltammetric recordings in multiple locations will expand our knowledge of dopamine reward responses. In the current study we report our efforts to record dopaminergic reward responses in multiple locations in the primate striatum using fast-scan cyclic voltammetry (FSCV) with carbon fiber microelectrodes.Voltammetric detection of behaviorally-correlated dopamine release in the non-human primate brain would serve as a valuable experimental model of drug addiction and Parkinson’s disease. However, to date, successful systematic detection of cue- and reward-induced phasic dopamine release in behaving primates has been limited , 7 and 8. These differences may be derived from the topographical organization of projections from differentially-responsive dopamine neurons, or local variations in release regulation by differential transporter distribution , 10. Apparent differences in response kinetics between ventral striatum and putamen suggest selective sensitivity to particular components of reward stimuli, such as reward value and reward timing  and 13. Also, local differences in striatal cholinergic tonically active neurons (TAN) can cause variance in dopamine neurotransmission  and 16.Voltammetric studies in rodents have revealed regional variation in reward-related dopamine responses . However, unlike rodents, the projection distribution of primate midbrain dopamine neurons is not as clearly segregated within projection areas , 10. Therefore, without local recordings of phasic dopamine release, it is difficult to identify which parts of the primate brain receive these differential dopamine signals that are recorded in the midbrain.Studies in midbrain dopaminergic unit firing in primates have suggested variable characteristics of dopamine release throughout the primate striatum  we developed a physically durable diamond microelectrode for voltammetric recording in large brains. However, there were two limitations for the use of diamond microelectrodes. First, recording with constant-potential amperometry is vulnerable to noise, and second, the sensitivity was often lost in the brain tissue. For simultaneous multiple recordings, we decided to use carbon fibers because diamond microelectrodes had a high risk of sensitivity loss for unknown reasons. The high sensitivity of carbon fiber electrodes to dopamine, and their capacity for electrical recordings of impulse activity  make them useful for combined studies with conventional electrophysiology and electrochemistry.In our previous study In this article, we share our recent technical challenges and successes in monkey voltammetry. Because our initial attempts failed to detect relevant phasic changes in our first animal, several modifications in electrodes, behavioral procedures, and voltammetric techniques were tried to find the best experimental condition to detect reliable reward responses. While our report represents primarily technical advancement, it now permits the detection of statistically significant event-related phasic dopamine changes in more than half of the recordings. We believe that this technique is now sufficiently advanced to undertake an analysis of actual dopamine release by behavioral events at multiple locations in the monkey brain.Three Japanese monkeys  participated in the voltammetry experiments. Monkey U and S were used for MFB stimulation, and S and C were for behavioral tasks  and received daily sliced sweet potato. Animals also had access to primate toys and biting wood , which wOn weekday mornings, general behavioral responses of the animals were observed while offering small pieces of soft biscuit. At least three times a week, the animal was moved to the monkey chair to inspect and clean the head chamber with saline and treated with antibiotic ointment (gentacin or ofloxacin). The ointment was also spread on the border of the skin and dental cement, and the body weight was recorded. Water restriction was minimized for just two days immediately before behavioral voltammetric recordings (150ml/day). On the other days, a 500ml water bottle was given at meal-time or after the training. Water and monkey food chow  was given around noon, unless no other treatments or trainings were made. Thus, animals are monitored at least twice daily by lab personnel on weekdays.All surgical procedures were performed under general anesthesia using aseptic techniques. A chronic recording chamber and a head holder were implanted in the monkey skull . The chaCoronal MRI images of the head and brain were taken prior to the experiment with a 0.3-T magnetic resonance scanner  to determine the position of implantation for each monkey. For MRI scanning, animals were anesthetized with ketamine-HCl  and medetomidine . The chamber was visualized on MRI image by filling either with 2% agar or ointment with paraffin liquid. The relative position of the chamber was determined from the MRI images. Unit recordings were also used to verify the depth from the chamber.During electrical stimulation of MFB, anesthesia was induced with ketamine-HCl  and medetomidine  and maintained under general inhalational anesthesia with 0.5–2% isoflurane in 30% O2 and 70% N2O. Heart rate and SPO2 was monitored until starting the recordings. N2O gas was turned off more than 10 min before releasing the animal., 20, except the shaft was elongated with a silica tube. These elongated electrodes are now commercially available . Individual carbon fibers  were connected to a urethane-coated thin wire (0.08 mm diameter) with silver glue , and the connection was sealed in pulled glass capillary tubes of 0.35 mm diameter with epoxy-resin , such that 300 μm of the carbon fibers protruded from the capillary tubes. The relatively long carbon fiber protrusion allowed for improved sensitivity. The glass capillary was elongated with a silica tube . As the insulation of the electrode rod was essential, a thin wire insulated with urethane was used to connect the carbon fiber to the potentiostat though the silica tube. The end of the thin wire was soldered to a short thicker wire to make a reliable connection with the potentiostat. The potentiostat was clamped to a micromanipulator   , which aThe position of the electrode tips was determined using a rectangular grid system . The plaThe electrode was placed into the brain through a steel guide cannula . The cannula with the protection inset core inside was placed on a particular hole on the grid, and the tip of the cannula needle placed 2–5 mm above the recording target. Then, the inset was replaced by the elongated carbon fiber microelectrode under a surgical microscope.The elongated carbon fiber was clamped by the micro-manipulator and advanced until a 30 mm mark on the shaft met the top of the known length of the cannula, then the depth on the manipulator was checked. When the carbon fiber depth reached the bottom of the cannula, neuronal impulse activity was recorded using a typical amplifier . The position was further adjusted until impulse activity adjacent to the carbon fiber was identified. The cannula and electrode were supported with glue to minimize the physical vibration of the electrodes.Two Ag/AgCl wires were placed above the dura mater and used as reference and counter electrodes. After the in vivo recordings, the used carbon fiber microelectrode tips were immediately rinsed, kept in saline, and calibrated with dopamine solutions.. The recording positions were anterior to interaural center +25.0 to 26.5, right 6.0, and 17 to 19 mm below the dural surface in the ventral striatum of monkey S; +22.5 anterior, left 2.5 to 4.0, and 18 to 26 mm below the dural surface in the ventral striatum of monkey C; +22.0 anterior, right 12.0, and 18 to 20 mm below the dural surface in the putamen of monkey S; and +18.0 to 19.5, left 10.0, and 20 to 22 mm below the dural surface in the putamen of monkey C.Two locations were selected for the voltammetric recordings in each monkey. The positions of the ventral striatum and putamen were determined using MRI images. The anterior positions of coronal slices were adjusted to a brain atlas MFB stimulation was attempted in two monkeys. The monkey was anaesthetized with ketamine and medetomidine and maintained with 1% isoflurane on a monkey chair. A stainless steel needle or tungsten electrode  was used to penetrate vertically, aiming at the medial edge of subthalamic nucleus (STN). The electrode was placed at the position of the thick bundle of tyrosine-hydroxylase-positive axons observed histologically at the medio-dorsal edge of the STN , and ele. For simultaneous voltammetric recording at multiple locations, a counter electrode grounded type three-channel potentiostat was used . Because simple potentiostat circuits set the working electrode potential at the ground level, they are not suitable for simultaneous multiple voltammetric recordings. Data acquisition was performed by a commercial control/recording system  with two multifunction boards  implemented on a Windows PC . The simultaneous 4-channel recording was made as channel 0: ventral striatum; channel 1: putamen; channel 2: timing signal of CS and juice onset and channel 3: IR sensor of lip movement. Typically, the gain of the amplifier was 500 nA/V for FSCV and 50 nA/V for RPV. The time constant of the low-pass filter was 0.2 ms for FSCV and 2.0 ms for rectangular pulses. Voltage pulses were applied at 10 Hz. For in vitro evaluation of the current responses of chemicals, carbon fiber microelectrodes were placed in a flow of PBS, and the flow was switched to the test solutions , 22.FSCV with a triangular waveform from -0.4 to 1.3V was mainly applied for electrochemical recordings in this study. In a part of sessions, rectangular-pulse voltammetry (RPV) was performed as described previously For FSCV, a triangle waveform lasting for 8.5 ms was applied. The potential was ramped from -0.4 V to 1.3 V vs. the Ag/AgCl reference and back from 1.3 V to -0.4 V at 400 V/s. This triangle-positive wave gives the oxidation peak of dopamine at 2.5 ms from the beginning of the positive slope (appearing at the 600 mV position). For RPV, the potential was stepped from 0.0 V to 0.2 V for 30 ms. Dopamine detection was represented by averaged current value of 5 to 25 ms from the pulse onset (t5-25). The electrode was held at 0.0 V between the pulses. When a new waveform started for each method, the waveforms were applied for at least 3 min at 10Hz (1800 cycles) before starting the next measurement.The animals were seated in a primate chair and well-habituated to the task in advance, with their heads held by the head holder. A tube was placed in front of the mouth for juice delivery (0.6 ml/0.6 s), which was electrically connected to ground at the same potential with the counter electrode of the animal. A 23-inch computer screen was placed 90 cm from the animal to deliver visual conditioning stimuli (CS). During the recordings, their behavior was videotaped. Trials were excluded from further analysis when the animal did not stay calm (described in detail below). Trials were delivered at randomized intervals of 10–20 s. The monkeys received 50 juice deliveries per day for 2–3 days per week, and voltammetric recording was performed once or twice a week. Water was restricted to 150 ml/day for two days before the recording and available ad libitum on the other days. .The Pavlovian procedure was basically the same as that published by Matsumoto and Hikosaka in 2009  . At firsSmall 0.5 g biscuits  were given to the animals restricted in the monkey-chair. The examiner (K.Y.) sat in front of the monkey face to face and took the pellets one by one from the backside table, approximately every 20 s. The delivery motion  occurred within 2 seconds. The body of the examiner was electrically connected to the common ground of the potentiostat. The food reward trials were effective to detect significant responses in our first animal (monkey S), instead of the typical Pavlovian liquid reward task.Chemical reagents, including dopamine HCl (DA), serotonin HCl (5-HT), and adenosine were purchased from Sigma-Aldrich . Isoflurane was purchased from Abbott. The other special-grade reagents were purchased from WAKO ..A formaldehyde-fixed Japanese monkey brain was sliced into 40 μm coronal sections in a cryostat, and the sections were immunostained with an anti-TH rabbit antibody  and visualized using the elite-ABC kit (Vector) and DAB as described previously . The FSCV data were analyzed for each 10–20 min block of trials. First, using the timing file (channel 2), each trial was extracted as an 8-s time window . The background waveform, averaging -5.5 to -2.5 s, was defined for each trial to prepare background-subtracted waveform data. Principal component regression was performed using an in-vivo dopamine-like and pH-like teaching template pooled from the MFB stimulation responses  esponses .. A cut off criteria of 5 nA was determined empirically to exclude all of the jump trials. The current at the dopamine oxidation peak (2.4 ms from the waveform onset) was extracted, and then the difference between the highest and lowest current during the 8-second window was calculated  for each trial, and the trials with a max-min value exceeding 5nA were excluded  and also the innate robustness of FSCV recording, such current jump was rare compared to our previous study using constant-potential amperometry on diamond microelectrodes excluded . The 5nAexcluded . BecauseIn most cases, the background of FSCV data is temporally sloped, which makes the analysis of temporal change difficult. This is derived from a continuous gradual maturation of the carbon fiber, which is most evident soon after starting to apply the waveform. More than 10 min of maturation period was taken before the first recordings of the day. After the background subtraction, exclusion of extreme data and PCR, the slope was adjusted so that the linear regression of the averaged pre-event background (-5.5 to -2.5 s) of the adjacent 40–80 trials in the session became temporally flat.For the ANOVA analysis of the temporal changes , data frTo validate the dopamine detection in the monkey brain, the evoked dopamine release in the striatum was recorded for monkeys U and S. First, to identify the location of the dopamine fiber bundle, frozen sections of Japanese monkey brain were immunostained with tyrosine hydroxylase (TH). The TH-positive medial forebrain bundle (MFB) was found at the medial border of the subthalamic nucleus . ElectriThe principal procedure was the CS-juice task with FSCV recording, which is summarized in this manuscript Tables  and 2. T, 14, 17. As is the nature of this type of trial and error for technical development, the task schedule was modified continuously and was thus partially inconsistent between experimental days, particularly for the first monkey, S. The licking responses, detected by an infra-red motion sensor beside the juice spout revealed clearly differentiated licking responses between CS+ and CS- trials in monkey C, but not in monkey S , 33 elongated carbon fibers were used, 8 were snapped (4 above the guide-cannula), 4 were not sensitive to dopamine after recovery from the brain, and 3 were noisy for connection problems. One carbon fiber that half-snapped in the ventral striatum of C4 was included in the data for high remaining sensitivity to dopamine. In total, recordings by 19 electrodes in two female monkeys were analyzed, as shown below .During the juice-reward sessions, juice delivery without a prediction cue (free juice) induced marked responses in many of the successful electrodes 12/19; . On the In addition to the visual impression on the color plot, there are four good reasons supporting chemical identity of dopamine. First, the cyclic voltammograms at the juice response was roughly similar to dopamine Figs , calcula. Unique adenosine-like response was detected following CS+  = 9.88 p<0.001). Comparison at each time point revealed differences (p<0.01 with t-test) from 0.7 to 1.0s after juice onset . The reward omission consistently induced a negative response of dopamine-like component at the ventral striatum while it was occasionally positive in a part of the recordings in the putamen. This contrast might be consistent with the positive spike response to the aversive event of dorso-lateral nigral dopamine neurons  and 27 and our training may not have been sufficient for mature conditioned responses. Clear CS-induced midbrain dopamine responses need relatively extended training sessions, whereas the responses to a fixation cue can be observed early in training. The consistent response to the timing cue and poor CS responses in our results seem to be consistent with this observation of the early-to-middle stage of training [26]. On the other hand, the clear effect of CS reversal indicates some degree of discrimination learning between CS+ and CS-.Differential responses to the positive and negative cues were not detected in most cases. There are two possible reasons for these ambiguous cue responses. First, the response can be changed by training , but the number of reversed sessions in our present study was too small for further statistical analysis. However, a sharp drop in the dopamine-like component was observed after the juice and food delivery. Recently, we have shown that the optogenetic suppression of dopamine release can cause a quick drop in the dopamine signal in mice . The drop appears to be a reasonable response to a brief pause of dopaminergic activity.An evident effect of reward omission might be expected in the putamen after CS+ during the reversal session  succeeded in detecting dopamine release by RPV (chronoamperometry) in anesthetized macaques by local KCl injection in Parkinsonian monkeys. Shon  and Gale  detected electrically evoked dopamine release in anesthetized pig and monkey brains, respectively. Ariansen (2012)  attempted to detect reward responses in the striatum of awake macaque monkeys using FSCV on carbon fibers but reward-induced dopamine responses were found only occasionally and marked pH responses were observed instead. Schulter (2014)  carefully studied the stimulation positions for evoked dopamine release in macaque monkeys and showed an example of a trial with reward response but did not perform statistical analyses of the behavioral effect.For large experimental animals, there have been at least four voltammetric studies using carbon fiber. The pioneering work by Gerhardt Three technical reasons can be noted for our success in the detection of reward responses in the monkey brains: 1. statistical analysis, 2. animal dependency, 3. electrode sensitivity. First, since the electrochemical signals were small, statistical averaging is essential. We found that analysis of each individual noisy data was not very reliable, either by examination of the color plot matrix or PCR analysis. Therefore, we first applied PCR to individual recordings from the same trial type and averaged the dopamine-like components for further comparison between trial types . A similar result would also be available through averaging background-subtracted current first Figs  and 5C aSecond, in primate behavioral studies it is not unusual that the result can depend highly on the individual animal and their behavioral training. This may have contributed to the patterns of dopamine signal during juice reward task of monkey S that differed from expected outcomes. Therefore, we took steps in our behavioral procedures to further ensure the dopaminergic nature of the detected signal. First, free reward (no CS), regularly used in finding reward-responding nigral neurons in unit recording studies, and was effective to induce the response in dopamine-like component. Similarly, reversal of CS+ and CS- was also effective in enhancing the reward response., 26 and voltammetry . This was the reason we did not train 50% reward trials in advance, but the sudden introduction of 50% trials probably confused the conditioned behavior of monkey S . Although we performed the customary analysis for validation of dopamine-derived signals, it remains possible that insufficient dopamine identification by PCR is the reason for these atypical responses. Validation of the dopamine template on each experiment by inserting a stimulation electrode for every experiment or implanting a chronic stimulation electrode would improve the reliability of the teaching template. and 26. Voltammetric procedures with less temporal delay, such as RPV , would help address this issue  is identical to dopamine, the reduction peak is different , so the nsistent , and chansistent . Additionsistent  and S3A.istent Click here for additional data file.S1 FigBiting wood, plumber’s materials and kitchen tools are placed in the home cage as toys.(TIF)Click here for additional data file.S2 FigTwo examples of excluded trial data during the juice-reward sessions are presented. Adjacent trials, two previous and two following, are also shown for comparison.(TIF)Click here for additional data file.S3 FigThe same data shown in (TIF)Click here for additional data file.S4 FigRPV recording at putamen (A and B) was switched to FSCV (C and D) during C4. Responses to the CS trials (A and C) and free juice (B and D). The absolute current was lower in RPV, but sharp responses to timing cue (A) and free juice delivery (B) were observed. N = 9 to 19. Vertical lines in B and D show s.e.m.(TIF)Click here for additional data file.S1 TableAs shown in (DOC)Click here for additional data file.