KLHL3 is a BTB-BACK-Kelch family protein that serves as a substrate adapter in Cullin3 (Cul3) E3 ubiquitin ligase complexes. KLHL3 is highly expressed in distal nephron tubules where it is involved in the regulation of electrolyte homeostasis and blood pressure. Mutations in KLHL3 have been identified in patients with inherited hypertension disorders, and several of the disease-associated mutations are located in the presumed Cul3 binding region. Here, we report the crystal structure of a complex between the KLHL3 BTB-BACK domain dimer and two copies of an N terminal fragment of Cul3. We use isothermal titration calorimetry to directly demonstrate that several of the disease mutations in the KLHL3 BTB-BACK domains disrupt the association with Cul3. Both the BTB and BACK domains contribute to the Cul3 interaction surface, and an extended model of the dimeric CRL3 complex places the two E2 binding sites in a suprafacial arrangement with respect to the presumed substrate-binding sites. Targeted ubiquitination can direct substrate proteins to a variety of functional fates, including proteosomal degradation, the modulation of protein interaction networks, and altered subcellular localizations. The largest class of E3 ligases are the Cullin-RING Ligases (CRLs), which are further identified according to the type of cullin chain that constitutes the central scaffolding unit Many of the known CRL3 substrate adapters belong to the BTB-BACK-Kelch family of proteins. These proteins are made almost entirely of three concatenated structural domains: the BTB and BACK domains form the platform that engages the N-terminal region of Cul3, while the Kelch repeat domain forms a β-propeller structure for substrate binding. In humans, most of the 52 BTB-BACK-Kelch proteins can be classified into two named groups, namely the 39 KLHL proteins and the 11 KBTBD proteins KLHL3-dependent ubiquitination.PHAII, also known as familial hyperkalemic hypertension, is a rare autosomal dominant disease characterized in part by elevated electrolyte and reduced bicarbonate levels in the blood. The characterization of the molecular defect in this disease has provided key insights into the mechanisms of blood pressure regulation BTB-BACK/Cul3NTD complex and characterize the Cul3 binding properties of a series of KLHL3 PHAII mutations. We previously reported the structure of the SPOPBTB/Cul3NTD complex BTB-BACK in complex with a Cul3 N-terminal domain has been recently reported BTB-BACK/Cul3NTD complex allows an expanded comparison of the BTB-BACK/Cul3 binding interface and allows a more accurate modeling of other intensely studied BTB-BACK-Kelch proteins such as Keap1.Here, we report the crystal structure of a KLHL3BTB-BACK) was designed using the web-based Crystallization Construct Designer BTB-BACK used in crystallization was further modified by surface entropy reduction (SER). The Surface Entropy Reduction prediction (SERp) web server BTB-BACK protein used in the solution-based experiments did not include the SER mutations, and the PHAII mutations were introduced into the wild-type KLHL3BTB-BACK expression plasmid by PCR mediated site directed mutagenesis. The N-terminal domain of Cul3 comprising residues 20–381 incorporating the stabilizing mutations I342R/L346D (Cul3NTD) was cloned into a pET32a vector as described previously An expression construct for human KLHL3 comprising residues 27–276 .KLHL3BTB-BACK/Cul3NTD complex were grown by hanging drop vapor diffusion at room temperature. KLHL3BTB-BACK and Cul3NTD were mixed in a 1∶1 molar ratio at a total protein concentration of 10 mg/ml. Hanging drops were set by mixing 1 µl of the protein solution with 1 µl of a reservoir solution containing 0.1 M sodium tartrate and 17% w/v PEG 3350, and incubating against 1 mL of reservoir solution. Crystals were soaked in a reservoir solution containing 15% ethylene glycol for 30 s prior to flash freezing. Diffraction data were collected at 100 K on beamline 19-ID at the Advanced Photon Source at a wavelength of 0.98 Å using ADSC Quantum 315r detector. Data were processed with HKL3000 BTB-BACK/Cul3NTD structure was solved by molecular replacement with PHENIX AutoMR BTB-BACK and one chain of Cul3NTD in the asymmetric unit, with a solvent content of 66%. The solution generated a KLHL3 BTB-BTB homodimer via a crystallographic 2-fold symmetry operator, even though no information about this expected dimer was included in the molecular replacement procedure. As a further test of the solution, an independent molecular replacement search was carried out using the same search model, except that residues corresponding to helices H1 and H2 of Cul3 were deleted. Following rigid body refinement of this partial model, difference density maps clearly showed the density for the deleted residues (BTB/Cul3NTD (PDB ID 4EOZ) Crystals of the KLHL3residues . SimilarBTB-BACK and residues 26–379 of Cul3NTD. The quality of the final model was verified with a composite omit map calculated with PHENIX BTB-BACK/Cul3NTD interface residues and buried surface area were determined using the EMBL PISA web server Refinement and model building were performed using PHENIX Protein samples were injected onto a Superdex S75 size exclusion column equilibrated with buffer A and elution was monitored at 280 nm.BTB-BACK at 150 µM were injected into Cul3NTD solutions at 15 µM. Data were processed using Origin, and binding isotherms were calculated based on a one-site binding model. A single titration was conducted for wild type KLHL3 BTB-BACK and each mutant. Kd error values are based on the sum of square deviations between the non-linear regression curve and the experimental data.Isothermal titration calorimetry (ITC) binding experiments were performed using a VP-ITC Micro Calorimeter at 25°C. All proteins were dialyzed in buffer A for three days prior to analysis. Aliquots of KLHL3KLHL3 ubiquitin ligase complex was generated following the approach used in making the SCF3SPOP model BTB-BACK and KLHL9BTB-BACK homology models, and these were superimposed onto KLHL3BTB-BACK in the Cul3 complex structure. No insertions or deletions are present near Keap1 C151 and KLHL9 L95 relative to KLHL3, and the backbones of the three BTB-BACK domains were in excellent agreement in the areas near the mutation sites.A model of the complete SCF3BTB-BACK/Cul3NTD complex reveals a 2∶2 complex, with two chains of Cul3NTD bound independently to two equivalent and non-overlapping surfaces of a KLHL3BTB-BACK homodimer  helices α3 and α3.1 in the α3/β4 loop, ii) β4 and residues in the β4/α4 loop, iii) α5 and the α5/α6 loop, and iv) α7 and the α7/α8 loop , 3. The BTB/Cul3NTD structure A multiple sequence alignment of BTB-BACK-Kelch proteins reveals that the α3/α3.1 region is relatively well conserved, while other Cul3 binding regions have lower sequence similarity . As discThe overall shape of the Cul3-interacting surface formed by the BTB and BACK regions is fairly consistent, however, the characteristics of these surfaces are remarkably varied . For exaBTB-BACK/Cul3NTD (this work), SPOPBTB/Cul3NTDBTB-BACK/Cul3NTD (PDB ID 4AP2)) are similar. The three Cul3 chains can be superposed with an average Cα RMSD of 0.6 Å, and the three BTB domains can be superposed with an average Cα RMSD of 1.2 Å The BACK domain of KLHL3 and KLHL11 are less similar, and superpose with a Cα RMSD of 2.2 Å.Overall, the individual subunits in the three available BTB/Cul3 complexes  bound to the Kelch domain of KLHL3. In PHAII, the relevant KLHL3 substrate appears to be the NaCl cotransporter (NCC) BTB-BACK/Cul3NTD structure. Keap1 and KLHL3 are near-neighbors in sequence space and share 33% sequence identity. This makes KLHL3 a preferred template for modeling relative to KLHL11 or Gigaxonin, which only share 19% and 21% identity to Keap1, respectively. Keap1 residue C151  As a test for the molecular replacement solution, an |Fo-Fc| map was calculated from a molecular replacement model which did not include Cul3 helix H2 and contoured at 2σ. (B) An |Fo-Fc| omit map was calculated based on the final refined structure, and shows good agreement between the model and the density.(EPS)Click here for additional data file.Figure S2Size exclusion chromatography. Elution profiles are shown for KLHL3BTB-BACK, Cul3NTD and an equimolar mixture of the two proteins. Size standards are indicated. The calculated MW of a Cul3NTD monomer, a KLHL3BTB-BACK homodimer and a KLHL3BTB-BACK/Cul3NTD 2∶2 complex are 42 kDa, 63 kDa and 148 kDa, respectively. The slightly larger apparent molecular weights for KLHL3BTB-BACK, Cul3NTD and the complex are most likely due to the non-spherical shape of the proteins. Vo indicates the void volume of the column.(EPS)Click here for additional data file.Figure S3Models of Keap1 and KLHL9. The BTB domains are shown as green ribbons and Cul3 is shown as a grey surface. (A) The position of the Keap1 electrophile-sensitive residue C151 is shown in orange. (B) The position of KLHL9 L95 is shown in orange. See the main text for details.(EPS)Click here for additional data file.


Molecular descriptors have been extensively used in the field of structure-oriented drug design and structural chemistry. They have been applied in QSPR and QSAR models to predict ADME-Tox properties, which specify essential features for drugs. Molecular descriptors capture chemical and structural information, but investigating their interpretation and meaning remains very challenging.http://commode.i-med.ac.at.This paper introduces a large-scale database of molecular descriptors called COMMODE containing more than 25 million compounds originated from PubChem. About 2500 DRAGON-descriptors have been calculated for all compounds and integrated into this database, which is accessible through a web interface at  Molecular descriptors have been proven essential in drug design, structural and mathematical chemistry, bioinformatics, and related disciplines -7. DescrPubChem-trimethyl-ammonium; methyl sulfate (id 5824)). As DRAGON fails to calculate molecular descriptors for these compounds, the relation Error with the Boolean attribute error was introduced. As 3D information of the compounds is not available using the provided SDF-files, the corresponding 3D descriptors have not been calculated. To handle the large number of result files, an additional Java routine was developed, which integrates those files into the previously generated database relations, where the first attribute for each group contains the identifier from PubChem.After the successful completion of the compound integration, 2,489 molecular descriptors have been calculated by applying DRAGON to all SDF-files. Table DescriptorNames contains attributes describing a molecular descriptor. DescriptorNames is associated with the relation GroupNames, which stores the name of all DRAGON groups. Figure Meta-data about molecular descriptors and their corresponding groups can also be found in the database scheme. Therefore, the relation PubChem identifier, exact mass, heavy atom count, different types of name, molecular formula, and ranges for various molecular descriptors. All search criteria are concatenated using a Boolean “AND”, which means that all found molecules must fulfill any given search criteria. Figure PubChem identifier. COMMODE uses this list and is able search for corresponding values of all integrated molecular descriptors. As the database contains around 25 million compounds the resulting tables can be huge, for performance reasons we limit the query result to 1,000 tuples.A php-based web application has been developed, which manages the access to the MySQL database. The user can search COMMODE in two different ways. The first way is based on providing different search criteria like the PubChem page. This view also shows a 2D and a 3D plot from the molecule derived from PubChem. The user can further see the values of all molecular descriptors for the compound of interest or the values from a single molecular descriptor for all compounds of the given query.After querying COMMODE various views displaying the results are provided. The first view shows a list with general information about the resulting compounds. Each single molecule can be selected from this list and explored. Therefore, a view was implemented showing different names and values for each molecule as well as a link to the corresponding When analyzing data of molecular descriptors on a large-scale, a statistical analysis is crucial. For example, this relates to estimating the correlations between descriptors to examine whether they capture chemical or structural information of compounds similarly. Note that this problem has been already tackled by Basak et al.  and TodeThe same reason described above applies to the analysis of the discrimination power of molecular descriptors. Figure ®; and XML format. The connection table of the SDF-file is converted from the stored SMILES code using the Chemistry Development Kit (CDK) [The usage of results within other applications is necessary for scientists, as a lot of downstream analysis can be performed on the integrated data. Therefore, the application supports the following file-formats to export: SDF, SMILES, CSV, MS-Excelit (CDK)  and openit (CDK)  in a speThis work introduces a large chemical database containing chemical compound data and their corresponding molecular descriptor values. These molecular descriptors can be used in QSPR and QSAR models to predict different chemical parameters using the structure of the compounds, and are utilized in drug design.PubChem identifiers. Afterwards, results for molecular descriptors can be exported in different file-formats. These results can further be combined with investigated attributes or properties of the given compounds. New models can be designed using these combinations, which can further be used to predict these attributes and properties for other compounds.The published database, COMMODE, includes more than 25 million compounds and about 2,500 computed descriptors. Clearly, COMMODE extends MOLEdb as this database contains only 1,124 molecular descriptors and 234,773 molecules ,40. To uAs not all molecular descriptors are necessary for the downstream analysis the introduced application is able to calculate descriptive values for each molecular descriptor representing the discrimination power or the correlation coefficient between chosen descriptors.An additional research area supported by COMMODE is the field of chemical graph theory . COMMODEOverall, this novel database provides a flexible access to compounds and their related molecular descriptors, which can be used in different research areas.Project name: COMMODE (COMpilation of MOlecular DEscriptors)• Project home page:http://commode.i-med.ac.at• Operating system(s): Platform independent• Programming language: Java, php• Other requirements: Web Browser• Any restrictions to use by non-academics: none• The authors declare that they have no competing interests.The conceptual idea for COMMODE by using molecular descriptors goes back to MD. Calculation of all molecular descriptors using DRAGON was performed by MD. Furthermore, MD, AD and FES wrote the manuscript. AD and LM performed the implementation of the database. AD and SP implemented Java routines. RG and AG reviewed the database scheme and performed tests. In summary, the project was initiated and coordinated by MD. All authors contributed to the interpreation of the results and read and approved the final manuscript.


Closteroviridae as an exciting complex virus family.Forty years ago, an unusual name—closterovirus—was coined for an unusual group of elongated plant viruses  and Citrus tristeza virus (CTV), have significant economic importance and, therefore, attracted considerable biological and epidemiological attention long before their molecular characterization. The seminal paper by Kitajima et al. , Norwich. Shortly after my arrival, I realized that, 3 years earlier, a JII Ph.D. candidate had begun working on the characterization of BYV, but that work had been discontinued due to difficulties in obtaining purified BYV preparations. I asked for permission to use the CTV purification procedure for the isolation of BYV and, to the delight of the JII director, the late Prof. Roy Markham, with a few minor modifications this method was highly successful.Working in cooperation with Roger Hull, we obtained sufficient amounts of BYV for the biophysical and molecular characterization of the virions and determined the sizes of their major coat protein subunits and RNA  to reflect the common morphological characteristic of its members, in contrast to previous groups of elongated plant viruses whose names were derived from their type members. Later analysis of Carnation necrotic fleck virus (CNFV), which shares several common biological features with BYV, further indicated the considerable degree of molecular and cytopathological similarity among closteroviruses as reported in the first review of this group (Bar-Joseph et al., However, because of the considerable variation in length, we suggested that the new group should be named Closteroviridae was instrumental for their molecular cloning and genome characterization, despite the absence of purified virions.In 1980, Dr. Allan Dodds found large amounts of distinct dsRNA molecules in CTV-infected citrus tissues. The extension of his analyses to plants infected with BYV and CNFV revealed considerable similarities in the amounts of dsRNA they contained, as well as in their dsRNA profiles (Dodds and Bar-Joseph, Closteroviridae work ended in 1986 with the realization that, despite advances in serological and molecular detection methods, the natural spread of CTV in Israel had developed into an epidemic that could not be controlled by eradication. Fortunately, most prevalent isolates induced only mild symptoms and even now, almost 30 years later, CTV remains a minor disease problem regardless of earlier projections that giving up on eradication would destroy the local citrus industry (see Bar-Joseph et al., The first phase of my Closteroviridae research mostly by new groups of molecular virologists whose excellent work is summarized in the present Frontiers series. Looking back, despite the difficulties and disappointments, I feel a great deal of satisfaction from friendships shared through these years with numerous dear colleagues and students that, unfortunately, space limits prevent listing.The other reasons for considering this period as the beginning has to do with considerable advances in


Diabetes is associated with a higher risk for adverse cardiovascular outcomes. To improve the health outcomes of patients with type 2 diabetes (T2DM), the American Diabetes Association (ADA) recommended target goals for the improvement of glycemic control and the reduction of cardiovascular risk factors associated with the disease. This retrospective analysis calculated the absolute benefit increase (ABI) of using exenatide once weekly (QW), a glucagon-like peptide-1 (GLP-1) receptor agonist, vs an oral glucose-lowering medication or insulin glargine to achieve ADA-recommended goals. The number needed to treat (NNT) to achieve these goals was also calculated and provides a useful clinical metric for comparing potential therapies from different drug classes.Patient data from three double-blind or open label, 26-week, randomized, controlled trials were retrospectively analyzed separately. ABI and NNT were calculated by comparing the percentage of patients treated with exenatide QW (N = 641) vs metformin (N = 246), sitagliptin (N = 329), pioglitazone (N = 328), or insulin glargine (N = 223), who achieved a single glycemic, weight, blood pressure, or lipid goal or a composite of these recommended goals, during the DURATION-2, -3, and -4 clinical trials.Significant ABIs favoring exenatide QW over all four glucose-lowering medications were observed for at least one HbA1c glycemic goal. NNTs of 4 and 5 were calculated when exenatide QW was compared to sitagliptin for attaining HbA1c goals of <7.0% and ≤6.5%, respectively. Additionally, significantly more patients using exenatide QW compared to sitagliptin, pioglitazone, or insulin glargine attained the composite goal of HbA1c <7% or ≤6.5%, without weight gain or hypoglycemia. Exenatide QW was also favored over sitagliptin and insulin glargine for the achievement of the composite goals of HbA1c <7% (or ≤6.5%), systolic blood pressure <130 mm Hg, and low-density lipoprotein <2.59 mmol/L. For most goals, exenatide QW and metformin had similar effects in treatment naïve patients.This analysis assessed the between-therapy differences in achieving therapeutic goals with therapies commonly used for glycemic control in patients with T2DM. In clinical trials, exenatide QW assisted more patients in reaching the majority of ADA-recommended therapeutic goals than treatment with sitagliptin, pioglitazone, or insulin glargine.NCT00637273, NCT00641056, NCT00676338 Although the primary goal in treatment of type 2 diabetes mellitus (T2DM) is the reduction of hyperglycemia, significant benefits of glycemic control on cardiovascular disease (CVD) in patients with T2DM were observed during the UK Prospective Diabetes Study (UKPDS), which showed a 14% decrease in the risk of myocardial infarction and 12% decrease in risk of stroke for each 1% decrease in glycated hemoglobin A1c (HbA1c)  significantly favored exenatide QW over metformin for the glycemic goals of HbA1c ≤6.5% and fasting blood glucose (FBG) <6.99 mmol/L (126 mg/dL) , with associated NNTs of 9 and 8, respectively . The primary outcome of the two trials showed that exenatide QW was superior to sitagliptin in reducing HbA1c, with LS mean reductions in HbA1c of -1.5% (exenatide QW) vs -0.9% (sitagliptin) in DURATION-2, and -1.5% (exenatide QW) vs -1.2% (sitagliptin) in DURATION-4 ,25.Consistent with the findings of a greater reduction in HbA1c with exenatide QW compared to sitagliptin, a larger percentage of patients treated with exenatide QW compared to sitagliptin attained the HbA1c goals of <7% or ≤6.5% Table . The larThe DURATION-2 and DURATION-4 studies also compared exenatide QW with pioglitazone. The reduction in HbA1c with exenatide QW treatment was consistent in both trials (-1.5%), while the reduction in HbA1c with pioglitazone treatment was -1.2% in DURATION-2 (on a background of metformin-treated patients) and -1.6% in DURATION-4 (in a drug-naïve patient population); resulting in exenatide QW superiority over pioglitazone in DURATION-2 and lack of noninferiority in DURATION-4.The ABI for both HbA1c single goals favored exenatide QW over pioglitazone in both studies; however, the results were not significant in the DURATION-4 study Table . The ABIThe efficacy and safety of exenatide QW vs insulin glargine was compared in the DURATION-3 study resulting in a reduction in HbA1c of -1.5% for exenatide QW and -1.3% for insulin glargine . In the The most frequent adverse events occurring in patients treated with exenatide QW, metformin, sitagliptin, pioglitazone, or insulin glargine in each of the three studies is listed in Table The selection of an optimal agent for treatment of T2DM can pose a challenge given that many therapeutic options are effective in reducing HbA1c, an indicator of blood glucose control. However, not all therapies reduce HbA1c sufficiently to achieve therapeutic goals. The recent Standards in Medical Care in Diabetes from the ADA has outlined a set of target goals believed to favorably affect the health outcomes of patients with T2DM . The guiThis analysis compared the ability of exenatide QW vs metformin, sitagliptin, pioglitazone, or insulin glargine to assist patients in achieving ADA-recommended target goals. NNTs were calculated as a metric to assess how many patients would need to be treated with one therapy rather than another to allow one additional patient to reach a target. Compared to the other therapies examined, exenatide QW assisted a larger proportion of patients who were not already at HbA1c ≤6.5% or <7% at baseline in reaching the target goal after 26 weeks of treatment. The ABI for the goal of HbA1c ≤6.5% significantly favored exenatide QW over metformin, sitagliptin, pioglitazone, or insulin glargine in all studies except for DURATION-4 where the ABI for exenatide QW vs pioglitazone favored exenatide QW, but did not reach statistical significance. Contrary to the HbA1c goals, insulin was significantly favored over exenatide QW for the FBG goal. One reason for the discrepancy between the two glycemic endpoints is that the insulin was titrated according to a FBG target. Thus, the dosage of insulin glargine was raised until the FBG target was met, thereby contributing to bias. In addition, FBG is a short-term measure of blood glucose levels while HbA1c indirectly measures an average glucose concentration over a prolonged period of time. As insulin glargine is a titrated, long-acting basal insulin, it may function well to reduce fasting glucose levels but permit higher postprandial excursions leading to a larger average glucose concentration compared to the glucose-dependent activity of exenatide QW ,33.The results of this analysis provide information to assist in personalization of therapy. The NNT for the HbA1c ≤6.5% goal was 8 when exenatide QW was compared to insulin glargine. However, for the alternate glycemic goal, FBG <6.99 mmol/L, the NNT was -8 for exenatide QW vs insulin glargine (favoring insulin glargine). This means that, in patients for whom FBG may be the more important clinical concern, treatment of 8 patients with insulin glargine rather than exenatide QW would allow one additional patient to reach a FBG goal. However, in patients wishing to achieve the HbA1c target (≤6.5%), 8 patients would need to be treated with exenatide QW rather than insulin to allow one additional patient to achieve the goal.Although the main focus of treatment is a reduction in hyperglycemia, improved glycemic control is associated with weight gain and the risk of hypoglycemia for some medications, and these CV risk factors also reduce patients’ quality of life. Unlike medications that promote weight gain or increase the risk of hypoglycemia, GLP-1 receptor agonists are generally associated with weight loss and minimal risk of hypoglycemia. In the current analysis, examination of the weight and hypoglycemia effects of exenatide QW was assessed as a composite goal of HbA1c <7% with no weight gain and no hypoglycemia. Exenatide QW provided a significant ABI for achieving this composite goal compared to sitagliptin, pioglitazone, and insulin glargine, with NNTs as low as 3 (vs pioglitazone and insulin glargine). Across the three studies, exenatide QW assisted between 47 and 48% of patients who were not already at this composite goal in achieving the goal after 26 weeks. The comparators assisted between 10% (pioglitazone) and 48% (metformin) of patients in achieving this goal. These data are consistent with an analysis pooling 804 patients treated with exenatide QW during the DURATION trials, where 50% of patients treated with exenatide QW achieved the goal of HbA1c <7% with no weight gain and no hypoglycemia after 24-30 weeks . A recenThe magnitudes of the NNT values obtained for some goals reflect key differences between therapies. To put NNT values into better perspective, an NNT of 1 would mean that every patient treated would reach the specified goal. The significant NNTs for the composite goal of HbA1c <7% with no weight gain and no hypoglycemia ranged from 3 when exenatide QW was compared to insulin glargine and pioglitazone (in DURATION-2) to 8 when exenatide QW was compared to sitagliptin in DURATION-4. However, an NNT of 250 was observed for exenatide QW compared to metformin suggesting no greater benefit of either treatment in achieving this composite goal. As commonly used, an NNT of 20 or less is generally sufficient to justify the choice of a clinical intervention in the absence of a more significant clinical harm ,36.A second composite goal, based on findings from the Steno-2 study, highlighted the importance of HbA1c, blood pressure, and lipids in the reduction of CV risk in patients with T2DM . The SteLimitations of the current analysis include its retrospective, post hoc design and the small sample size. Common NNT analyses generally examine larger populations over a longer time period to observe a particular rare but clinically important outcome in a significant number of patients ,38,39. AThe current analysis comparing glucose-lowering therapies does not take into account differential cost or convenience factors; nor does it consider patient or physician perception of the different routes of administration. In addition, although the composite goals examined provide a clinically relevant combination of treatment targets, a single component of the composite could drive the result. For example, pioglitazone and insulin glargine are often associated with weight gain and therefore would not be favored in achieving composite goals containing a weight neutral component. Likewise, since insulin glargine is a glucose-independent, titrated medication, there is a greater risk for hypoglycemia which would lead to an unfavorable comparison with a glucose-dependent therapy (such as exenatide QW) in a composite goal including no hypoglycemia.Although surrogate endpoints are not typically used in NNT assessments, the ability to achieve therapeutic goals is clinically important and relevant for short-term treatment decisions. Indeed, in a recent 1-year study with liraglutide, the NNT to achieve a loss of 10% of total body weight was calculated as 2 with liraglutide compared to 7 for placebo . While tThis analysis compares the ability of exenatide QW vs commonly used glucose-lowering therapies representative of other drug classes  to assist patients in reaching important therapeutic goals. The results from clinical trials indicate that for the majority of ADA-recommended therapeutic goals, exenatide QW assists more patients in reaching the goal than treatment with sitagliptin, pioglitazone, or insulin glargine. The NNTs provided here indicate that fewer patients need to be treated with exenatide QW than with sitagliptin, pioglitazone or insulin glargine to attain the same composite goal of glycemic control without weight gain or hypoglycemia. The attainment of ADA-recommended goals promise to provide a clinical benefit in the reduction of CV risk and ongoing CV clinical trials are expected to provide answers as to whether reaching target goals are reliable predictors of reduced morbidity and mortality.ABI: Absolute benefit increase; ADA: American Diabetes Association; ADVANCE: Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation; ACCORD: Action to Control Cardiovascular Risk in Diabetes Trial; CI: Confidence interval; CV: Cardiovascular; CVD: Cardiovascular disease; DCCT: Diabetes Control and Complications Trial; DURATION: Diabetes Therapy Utilization: Researching Changes in A1C, Weight, and Other Factors Through Intervention with Exenatide Once Weekly; EXSCEL: Exenatide Study of Cardiovascular Event Lowering; FBG: Fasting blood glucose; GLP-1: Glucagon-like peptide-1; HbA1c: Glycated hemoglobin A1c; ITT: Intent-to-treat; LDL: Low density lipoprotein; NHANES: National Health and Nutrition Examination Survey; NNT: Number needed to treat; QW: Once weekly; SBP: Systolic blood pressure; T2DM: Type 2 diabetes mellitus; UKPDS: UK Prospective Diabetes Study.During the development of this manuscript, ARM, MBD, JH, JB, and MG were employees and stock holders of Amylin Pharmaceuticals, Inc. Amylin Pharmaceuticals, LLC is now a wholly owned subsidiary of Bristol-Myers Squibb.ARM, MBD, JH, JB, and MG participated in the design of the analysis. JH performed the statistical analysis. All authors were involved in the interpretation of the analysis as well as drafting or critically revising the manuscript for important intellectual content. All authors read and approved the final manuscript.


Of mechanistic importance, laforin monomer and dimer possess equal phosphatase activity, and they both associate with malin and bind glucans to a similar extent. However, we found differences between the two states' ability to interact simultaneously with malin and carbohydrates. Furthermore, we tested other members of the glucan phosphatase family. Cumulatively, our data suggest that laforin monomer is the dominant form of the protein and that it contains phosphatase activity.Lafora Disease (LD) is a fatal neurodegenerative epileptic disorder that presents as a neurological deterioration with the accumulation of insoluble, intracellular, hyperphosphorylated carbohydrates called Lafora bodies (LBs). LD is caused by mutations in either the gene encoding laforin or malin. Laforin contains a dual specificity phosphatase domain and a carbohydrate-binding module, and is a member of the recently described family of glucan phosphatases. In the current study, we investigated the functional and physiological relevance of laforin dimerization. We purified recombinant human laforin and subjected the monomer and dimer fractions to denaturing gel electrophoresis, mass spectrometry, phosphatase assays, protein-protein interaction assays, and glucan binding assays. Our results demonstrate that laforin prevalently exists as a monomer with a small dimer fraction both  EPM2A (epilepsy of progressive myoclonus type 2 A) gene EPM2A cause Lafora disease (LD) Laforin is a dual specificity phosphatase encoded by the in vitroLaforin contains an amino-terminal carbohydrate-binding module (CBM) belonging to the CBM20 family and a carboxy-terminal dual specificity phosphatase (DSP) domain EPM2B gene that encodes the protein malin also cause LD Autosomal recessive mutations in the in vitro and in vivoA recent study reported that laforin forms SDS-resistant dimers both Laforin is a cytoplasmic phosphatase and therefore the occurrence of laforin dimerization is both intriguing and applicable in determining the molecular etiology of Lafora disease. One could envision dimerization affecting glucan-binding, protein-protein interactions, and/or phosphatase activity of laforin. In addition, laforin oligomerization could be involved in the formation of LBs. Proteinaceous accumulations are a common theme in neurological disorders. Even though LBs are mainly made up of insoluble glucans, unlike the protein deposits seen in Alzheimer's and Parkinson's disease, it has been suggested that LD pathology may have a component linked to mis-regulation of the proteasome 6, pET21a At-SEX4-HIS6(Δ81), pET21a Cm-laforin-HIS6, pCDNA3.1NF-malin, and pET-GST-malin-HIS6 are described in refs pET21a Hs-laforin-HISDenatured gel electrophoresis was carried out using NuPAGE 10% Bis-Tris gels. Coomassie-stained bands were cut from gels, digested with trypsin, desalted, and analyzed by MALDI TOF/TOF. The peptides were searched with Protein Pilot against Swiss-Prot database. The mass spectrometric analysis was performed at the University of Kentucky, Center for Structural Biology Protein Core Facility.para-nitrophenylphosphate /3-O-methyl fluorescein phosphate  and amylopectin (0.9 µg/µl) were used to determine phosphatase activity of Hs-laforin, Cm-laforin, and SEX4. Assays were performed as previously described N-ethylmaleimide prior to the addition of malachite green reagent.6 cells were plated onto 60 mm culture dishes the day before transfection. Cells were transfected with 1 µg of pCMVmyc-laforin plasmid using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were scraped on ice in lysis buffer . Cells were lysed by repeated passage through a 25-gauge needle. To analyze proteins under non-reducing conditions, cell extracts (25 µg) were diluted in SDS- and DTT-free loading buffer  and analyzed by regular SDS-PAGE and immunoblotting using an anti-myc (Sigma) antibody.Human embryonic kidney (HEK293) cells were grown in DMEM (Lonza) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 10% inactivated fetal bovine serum (GIBCO). 1.5×106 antibody. For testing the effect of malin on glucan-binding ability of laforin, purified HIS6-GST-malin was added to laforin monomer and dimer along with amylopectin and same method was followed with use of mouse monoclonal antibody to detect laforin (Abnova).Glucan-binding assays were performed as previously described HEK293 cells were transfected with FLAG-tagged malin using Lipofectamine 2000 (Invitrogen) as above. Cells were lysed using modified RIPA buffer . FLAG-malin was immunoprecipitated with anti-FLAG M2 agarose beads (Sigma), the beads were washed twice with modified RIPA buffer, and then incubated with laforin monomer or dimer for 1 hr at 4° C. Following this incubation, the beads were washed once with modified RIPA buffer and proteins were eluted with 50 µl of 2× NuPage sample buffer (Invitrogen) at 95°C. Western analysis was used to detect laforin with a rabbit polyclonal antibody (GeneTex) and an anti-FLAG-HRP antibody (Sigma) to detect malin.Values are given as means ± SEM of at least three independent experiments. Differences between groups are analyzed by two-tailed student's t-tests or by one-way analysis of variances. The significance has been considered at * p<0.05 and ** p<0.001, as indicated in each case. Data for protein purification, gel electrophoresis, glucan-binding, and immunoprecipitation is representative of al least three independent determinations.6 expressed in bacteria results in two prominent peaks, laforin dimer (peak A) and laforin monomer (peak B)  . To eval(peak B) . These rE. coli proteins arnA, Hsp70, and GroEL as well as laforin at around 75 kDa, of which arnA was the predominant band  is highly pure and present at the expected 37 kDa band. However, the dimeric peak (peak A) showed a single band at 37 kDa and three defined bands around 75 kDa . To deteant band . AlthougE. coli arnA, and that the vast majority of laforin in peak A is denatured and runs as a 37 kDa species. Importantly, these results confirm that laforin does form dimers, but only a minor fraction of recombinant laforin dimerizes. In addition, the dimer fraction (peak A) is contaminated with E. coli proteins.Our mass spectrometry results identified arnA as the major contaminant in the dimeric fraction A . arnA isE. coli proteins, and thus phosphatase activity normalized for total protein content does not truly represent the phosphatase activity of the dimer form.A previous study reported that monomeric laforin lacks phosphatase activity and that only laforin dimers possesses phosphatase activity E. coli proteins in the dimer fraction, we normalized both monomer and dimer fractions for laforin content using a laforin-specific antibody and Western analysis , one cannot simply use total protein as a means to assess the amount of laforin in the dimer fraction. Therefore, we normalized the dimer fraction so that we analyzed equal amounts of laforin in the pNPP assay and all subsequent assays. After adjusting for total laforin amount instead of total protein, we found that the pNPP phosphatase activity for the laforin monomer and dimer fractions was equal , a DSP domain, and a CBM SEX4Cyanidioschyzon merolae, Cm-laforin, which is 25% identical to human laforin Kingdom Plantae/Archaeplastida genomes lack a true ortholog of laforin, but their genomes do encode for a functional equivalent of laforin We purified SEX4 and Cm-laforin using a similar two-step purification method that included size exclusion chromatography. For each protein, we observed that multiple peaks were eluted from the column, similar as we observed for human laforin . We analHs-laforin, SEX4, and Cm-laforin all belong to glucan phosphatase family Given the difference between our data and a previous report suggesting that laforin monomer is inactive, we decided to determine a possible cause for this discrepancy. We found an interesting result when laforin was stored at −20°C in the absence of reducing agent. We purified monomeric laforin using Ni-NTA resin and size-exclusion chromatography by collecting peak B , and theThe finding that storage of laforin in low levels of DTT is necessary prompted us to further examine the effect of reducing agents on laforin oligomerization and phosphatase activity. When we analyzed the non-reducing peak of laforin  by gel eDual specificity phosphatases employ a two-step catalytic mechanism. After nucleophilic attack of the substrate phosphorus atom, a phosphoryl-cysteine intermediate is formed before hydrolysis of the intermediate and release of phosphate In order to further probe the effect of reducing conditions on laforin dimerization, we analyzed the oligomeric status of laforin in mammalian HEK293 cells lysed in the presence and absence of reducing agent. When the cell extracts were prepared in the absence of DTT, clear monomeric, dimeric, and multimeric species were resolved by non-reducing gel electrophoresis . HoweverThe dimer interface of laforin could involve the carbohydrate-binding module (CBM), and if so dimerization could provide a mechanism to modulate glucan-binding. To test whether dimerization of laforin impacts its ability to bind glucans, we utilized a glucan-binding assay. In this assay, proteins are added to an amylopectin solution and the mixture undergoes ultracentrifugation. Proteins in the pellet and supernatant fractions are then assessed by Western analysis. Proteins with glucan-binding ability are retained in the pellet fraction and proteins lacking this ability are observed in the supernatant fraction. Immunoblotting of the pellet and supernatant fractions from the glucan-binding assay showed that both laforin monomer and dimer bind to amylopectin and are enriched in the pellet fraction . TherefoMultiple groups have reported that glycogen inhibits laforin phosphatase activity As discussed in the introduction, laforin forms a functional complex by associating with malin and this complex is involved in ubiquitination and proteasomal degradation of multiple proteins involved in glycogen metabolism Given that malin and laforin form a complex, we decided to test the activity of monomeric and dimeric laforin in the presence of malin using the malachite green assay. The presence of malin did not affect the phosphatase activity of either monomeric or dimeric laforin . Thus, b6 with both forms of laforin and then performed the glucan-binding assay as described above. The presence of malin decreased the binding of monomeric laforin to glucans as indicated by the presence of monomeric laforin in both the pellet and supernatant fractions  monomeric laforin purified via Ni-NTA resin and resolved using size exclusion chromatography possesses pNPP activity (2) the same monomeric laforin also possesses activity against the phospho-glucans, a biologically relevant substrate (3) monomeric SEX4 from Arabidopsis possesses phosphatase activity against pNPP and a phospho-glucan (4) monomeric laforin from the red algae C. merolae possesses phosphatase activity against pNPP and a phospho-glucan (5) when DTT levels are increased to ≥10 mM DTT only monomeric laforin exists  the ubstrate ; 3) monoo-glucan ; 4) monoo-glucan ; and 5) n exists  and FiguHs-laforin, Cm-laforin, and SEX4 all contain a CBM and DSP domain and all belong to the newly discovered class of glucan phosphatases in vivo as a combination of monomeric and oligomeric forms, and changes in the cellular reducing conditions may regulate the transition from one state to the other. In support of this hypothesis, a recent paper found that a laforin mutation, laforin-Ser25Ala, is unable to interact with itself in both a yeast two-hybrid system and in mammalian cell culture experiments Our results from cell culture suggest that laforin dimerization may be a dynamic process. The sensitivity of the oligomeric-monomeric transition to the presence of reducing agents indicates that inter-molecular Cys-Cys bridges play a key role in oligomer formation. These data suggests that laforin is present We found that both monomeric and dimeric laforin bind glucans with equal affinity. This finding suggests that sites involved in laforin dimerization do not affect the conformation of essential CBM residues involved in glucan-binding. Next, we analyzed the inhibitory role of glycogen on laforin phosphatase activity. We observed that this inhibitory role is not due to alterations in the oligomeric-monomeric transition, as the presence of glycogen did not affect oligomer formation. Moreover, a dual specificity phosphatase lacking a carbohydrate-binding domain (VHR) was resistant to glycogen inhibition. Therefore, our results suggest that glycogen either induces a conformational change in laforin structure or inhibits phosphatase activity because it blocks the entry of substrates to the phosphatase catalytic site.In addition to phosphatase activity, we analyzed laforin monomer and dimer for their ability to interact with malin. The equal association of monomeric and dimeric laforin with malin suggests that both forms possess active scaffolding function and form laforin-malin complexes. Phosphatase activity of laforin monomer and dimer was not affected by presence of malin, which suggests that laforin-malin complex is functionally active. While we did not observe a difference in glucan-binding between the two forms, we did observe a difference when malin was present. Malin affected the glucan-binding of only monomeric laforin and did not affect glucan-binding of the dimer form.in vitro results and previous in vivo data clearly demonstrate that monomeric laforin is far more abundant than the dimer and that changes in the cellular reducing conditions may regulate the transition from one state to the other. These results are especially germane in terms of defining the form(s) of laforin that is most relevant to the etiology of Lafora disease.In conclusion, our findings establish that monomeric laforin is an active enzyme, and that laforin dimerization is not essential for its physiological activity. In addition, we found that monomer and dimer possess equal specific activity in removing phosphate from both generic and biologically relevant substrates. Our Figure S1Purification of Arabidopsis Starch EXcess4 (SEX4) and C. merolae laforin (Cm-laforin). (A) The 32 kDa SEX4 protein was purified in a similar manner as Hs-laforin. The chromatogram is of SEX4-HIS6 purified using a Superdex-75 and contains three distinct peaks. This is a representative of 5 purifications. (B) The 74 kDa Cm-laforin protein was purified in a similar manner as Hs-laforin. The chromatogram is of Cm-laforin-HIS6 purified using a Superdex-200 and contains three distinct peaks. This is a representative of 4 purifications.(EPS)Click here for additional data file.Figure S2Quantification of monomer versus dimer of SEX4 and Cm-laforin. (A) A representative immunoblotting image of varying concentration of SEX4 monomer and dimer fractions detected using anti-HIS6 monoclonal antibody. (B) A representative immunoblotting image of varying concentration of Cm-laforin monomer and dimer fractions detected using anti-HIS6 monoclonal antibody.(EPS)Click here for additional data file.


Perivascular macrophages and microglia are critical to CNS function. Drugs of abuse increase extracellular dopamine in the CNS, exposing these cells to elevated levels of dopamine. In rodent macrophages and human T-cells, dopamine was shown to modulate cellular functions through activation of dopamine receptors and other dopaminergic proteins. The expression of these proteins and the effects of dopamine on human macrophage functions had not been studied.To study dopaminergic gene expression, qRT-PCR was performed on mRNA from primary human monocyte derived macrophages (MDM). Expression and localization of dopaminergic proteins was examined by immunoblotting isolated plasma membrane, total membrane and cytosolic proteins from MDM. To characterize dopamine-mediated changes in cytokine production in basal and inflammatory conditions, macrophages were treated with different concentrations of dopamine in the presence or absence of LPS and cytokine production was assayed by ELISA. Statistical significance was determined using two-tailed Students’ T-tests or Wilcoxen Signed Rank tests.These data show that MDM express mRNA for all five subtypes of dopamine receptors, and that dopamine receptors 3 and 4 are expressed on the plasma membrane. MDM also express mRNA for the dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC). DAT is expressed on the plasma membrane, VMAT2 on cellular membranes and TH and AADC are in the cytosol. Dopamine also alters macrophage cytokine production in both untreated and LPS-treated cells. Untreated macrophages show dopamine mediated increases IL-6 and CCL2. Macrophages treated with LPS show increased IL-6, CCL2, CXCL8 and IL-10 and decreased TNF-α.Monocyte derived macrophages express dopamine receptors and other dopaminergic proteins through which dopamine may modulate macrophage functions. Thus, increased CNS dopamine levels due to drug abuse may exacerbate the development of neurological diseases including Alzheimer’s disease and HIV associated neurological disorders. In individuals who abuse drugs such as cocaine and methamphetamine ,2, extra. Dopamine acts through activation of dopamine receptors (DR), which are divided into two sub-classes, D1-like dopamine receptors, D1R and D5R, and D2-like dopamine receptors, D2R, D3R and D4R [Dopamine is synthesized by conversion of tyrosine to L-DOPA by the enzyme tyrosine hydroxylase (TH), and L-DOPA is then converted to dopamine by aromatic amino acid decarboxylase (AADC) ,29. Dopa and D4R . Dopamin and D4R -34. Reca and D4R . Thus, tDopamine receptors are expressed on human T-cells, neutrophils, monocytes and B-cells . T-cellsTo examine the response of human macrophages to the increased dopamine levels induced by drug abuse, we characterized gene and protein expression of DR, DAT, VMAT2, TH and AADC in MDM. We also studied dopaminergic effects on cytokine production in both basal and inflammatory conditions by using untreated and LPS-treated macrophages. Macrophages have been shown to express mRNA for all subtypes of DR and have D3R and D4R on the plasma membrane. Macrophages also expressed mRNA and protein for DAT, VMAT2, TH and AADC, with DAT on the plasma membrane and VMAT2 in cellular membranes. Our data demonstrated that dopamine treatment significantly increased IL-6 and CCL2 in both untreated and lipopolysaccharide (LPS)-treated MDM, increased CXCL8 and IL-10 and decreased TNF-α in LPS-treated MDM. These data indicate that dopamine is an important mediator of both macrophage homeostasis and of the response of macrophages to injury and infection, and that drug-induced changes in CNS dopamine may alter the development of neurological disease.+/K+ ATPase . Other reagents were anti-fade with 4',6-diamidino-2-phenylindole (DAPI), TRIzol reagent and phalloidin conjugated to Texas red, obtained from Molecular Probes (Invitrogen).RPMI-1640 medium and penicillin/streptomycin (P/S) were from Invitrogen . LPS from E.Coli 055:B5, hydroxyethyl piperazineethanesulfonic acid (HEPES), fish gelatin, β-mercaptoethanol, IgG-free bovine serum albumin (BSA), horse serum, Tween 20 and dopamine hydrochloride (DA) were obtained from Sigma-Aldritch . Dopamine was resuspended at 20 mM in dH2O, aliquoted and stored at −80°C for up to 2 months before use. Fetal calf serum (FCS) and human AB serum were from Lonza . Macrophage colony stimulating factor (M-CSF) was from Peprotech . Antibodies used were rabbit polyclonal anti-DR3 (1:250), rabbit polyclonal anti-DR4 (1:250), rabbit polyclonal anti-VMAT2 (1:250) and rabbit polyclonal anti-AADC (1:200) , rabbit polyclonal anti-TH (1:500) , rabbit polyclonal anti-DAT , anti α-tubulin , fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibodies (Sigma), anti-rabbit IgG and anti-rabbit NaHuman peripheral blood mononuclear cells (PBMC) were separated from blood obtained from de-identified healthy donors  by Ficoll-Paque  gradient centrifugation. After isolation of PBMC, MDM were obtained either by adherence over 3 days or by using CD14 magnetic beads (EasySep Human CD14 Positive Selection kit), according to the manufacturer’s protocol . MDM were cultured in RPMI-1640 with 10% FCS, 5% human AB serum, 10 mM HEPES, 1% P/S, and M-CSF (10 ng/mL) for 3 days, washed twice with fresh media, and cultured for another 3 days in fresh media containing M-CSF. After 6 days in culture cells were washed again and considered to be mature MDM.Total RNA was extracted from MDM using TRIzol (Invitrogen). Purity and concentration were determined using a Nanodrop spectrophotometer . On the same day of the extraction, 2 μg of RNA were used to synthesize cDNA from each donor using the iScript cDNA synthesis kit , generating random cDNA fragments using both oligo-dT and random primers. FirstChoice Human Brain Total RNA was used as a positive control  and samples containing no cDNA served as the negative controls. Target mRNA was quantified with the Absolute Blue QPCR SYBR low ROX Mix (Thermo Scientific). Single-product amplification was confirmed by melting-curve analysis. Reactions in which the negative control showed amplification curves that were detected before 37 cycles were not used. Specific dopaminergic and housekeeping genes were amplified from cDNA by quantitative PCR (qPCR) from 1 μL of cDNA reaction using gene-specific primers designed using Primer3  and syntMDM cultured on 35-mm Mat Tek dishes  were fixed and permeabilized in 70% ethanol for 30 minutes at 4°C. Cells were washed in PBS, incubated in block solution  for 30 minutes at room temperature (RT) and then in primary antibodies  overnight at 4°C. Cells were washed with PBS and incubated for 1 hr at RT with the appropriate secondary antibody conjugated to FITC  as well as phalloidin-conjugated Texas Red . Cover slips were mounted on Mat Tek dishes using Prolong Gold Antifade reagent with DAPI (Invitrogen). MDM were examined by confocal microscopy using a Leica microscope .6 MDM cultured at 10 × 106 MDM per dish were used to generate either a single plasma membrane or total membrane and cytoplasmic lysate.Isolation of MDM plasma membrane, total membrane and cytosolic fractions was performed using a polyethylene glycol and dextran T-500-based centrifugal gradient kit, according to the manufacturer's protocol . For each lysate, 20 to 40 × 10Human MDM were lysed with mammalian protein extraction reagent  with added protease  and phosphatase inhibitors . For detection of TH and AADC, lysates were sonicated, centrifuged at 14,000 × g, and stored at −80°C until used. Loading buffer containing β-mercaptoethanol was added to lysates that were then heated to 100°C for 5 minutes. Western blot analyses were performed using 4 to 12% Nupage polyacrylamide gels (Invitrogen). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen) and blots were blocked in Tris-buffered saline (TBS-T) (1×TBS with 1.2% Tween 20) containing 3% BSA/5% non-fat powdered milk. Signal was detected using Supersignal WestPico Chemiluminescent substrate for TH or Supersignal WestFemto Chemiluminescent substrate for AADC (Pierce). After probing for TH or AADC, blots were stripped using Restore PLUS Western Stripping Buffer (Pierce) and probed for α-tubulin as a loading control.+/K+ ATPase and α-tubulin to confirm the specificity of the membrane isolations.Plasma membrane and cytosolic lysates from the same donor were probed for either D3R, D4R or DAT, while total membrane and cytosolic fractions from the same donor were probed for VMAT2. Denaturing lysates by heating altered the mobility of the DR, DAT and VMAT2. Therefore, in place of heating, lysates were incubated in loading buffer containing β-mercaptoethanol for 45 minutes at RT. Western blots were run as described above, and after probing for DR or DAT, blots were stripped and reprobed for Na5 per well in 48-well plates  were incubated for 24 hrs with different concentrations of dopamine  in the presence or absence of LPS (1 ng/mL). LPS treatment was used to model inflammation. Controls were treated with 1 ng/mL LPS without dopamine, or were untreated. After 24 hours, supernatants were collected, aliquoted and stored at −80°C. Supernatants were analyzed for IL-6, CCL2, TNF-α, CXCL8 and IL-10 using sandwich capture ELISAs was performed according to the manufacturer's protocol . The limits of detection for ELISAs were IL-6, 23.44 pg/mL, CCL2, 31.25 pg/mL, TNF-α, 15.625 pg/mL, CXCL-8, 31.25 pg/mL and IL-10, 62.5 pg/mL.MDM cultured at 5 × 10Statistical analyses were performed using Prism 5.0 , using either a Wilcoxen Signed Rank Test (for experiments with N ≥10) or a two-tailed Student’s T-test . P <0.05 was considered significant. To evaluate dopamine mediated changes in each cytokine, the fold-changes in the amount of each factor upon dopamine treatment from all donors were pooled and compared to the combined mean fold-change in the control condition (either untreated MDM or MDM treated with 1 ng/mL LPS), which was set to one.T relative to β-actin for D1R was 12 +/− 2.4 (mean +/− SD), for D2R was 16.6 +/− 4.2, for D3R was 12.6 +/− 3.7, for D4R was 14.7 +/− 3.7 and for D5R was 9.6 +/− 3.5. The negative control did not show amplification curves before 37 cycles in any amplification reaction.We previously showed that dopamine treatment of human macrophages significantly increases HIV replication , and oth+/K+ ATPase, which was enriched in the PM fraction, and the cytosolic protein α-tubulin, which was not found in the PM fraction, confirming the localization of the DR in the PM. These data demonstrate that human MDM express mRNA for all types of DR, and that D3R and D4R are present in the plasma membrane.To demonstrate that D3R and D4R are expressed on the plasma membrane of MDM, plasma membrane (PM) proteins were examined by western blotting. Representative Western blots show both D3R  from a single donor are shown in Figure T relative to β-actin was 11.2 +/− 5.7 for DAT and 8.5 +/− 3 for VMAT2. The negative control did not show amplification detected before 37 cycles in any reaction.To determine whether MDM have the capacity to take up dopamine from the extracellular space to the cytoplasm and transport it into storage vesicles, we analyzed expression of DAT and VMAT2 mRNA and protein from MDM derived from three different donors. Dopamine transporter was also examined by immunofluoresence. Using qRT-PCR, DAT and VMAT2 mRNA were detected in all donors examined, and representative amplification curves of these genes (red curves) and the housekeeping gene β-actin (red curve) are shown in Figure +/K+ ATPase and the absence of α-tubulin in the membrane fractions. Dopamine transporter expression on MDM was also demonstrated by immunofluorescence. Figure Western blot analysis of plasma membrane proteins isolated from three different donors shows DAT . Control cells were treated with media alone (Untx) or LPS in the absence of dopamine. LPS was used to model the effect of dopamine on macrophage cytokine secretion in an inflammatory environment. Supernatants from each treatment condition were analyzed by ELISA for secretion of IL-6, CCL2, TNF-α, CXCL8 or IL-10. These cytokines were selected because of their important roles in the neuroinflammation and in the development of neurological diseases such as HAND and Alzheimer’s Disease. Cytokine secretion in control MDM was compared to cytokine secretion in cells treated with different concentrations of dopamine. It is important to note that the variability inherent in studying primary human cells resulted in large inter-donor differences in cytokine secretion. In MDM not treated with LPS, dopamine had the most pronounced effect on IL-6, significantly increasing its elaboration by 2 and 3 fold at 2 and 20 μM dopamine respectively  only altered TNF-α, IL-6 and CCL2 production by LPS-treated MDM. Lower concentrations of dopamine also showed a trend towards modulation of both IL-6 in untreated MDM and IL-10 in LPS treated MDM, but these effects were not significant. Dopaminergic modulation of cytokines has been shown in both rodent macrophages ,22 and hTreatment of macrophages with LPS was used to model inflammatory conditions within the CNS. The effects of dopamine on LPS-treated macrophages indicate that macrophages in an inflammatory environment may respond differently to dopamine than do macrophages in homeostatic conditions. Elevated levels of LPS during HIV infection , and in Changes in cytokine production may also be beneficial, as decreases in TNF-α and increases in both IL-10 and CCL2 could limit neuroinflammation or be neuroprotective, depending on the kinetics and localization of their expression. For example, in Parkinson’s disease, in which TNF-α plays a central role in the loss of dopaminergic neurons , decreasMacrophages are central to CNS homeostasis, immune and inflammatory response. Thus, the effects of elevated dopamine on macrophages may have a profound impact on neuroinflammation and the CNS response to insult as well as on the development of neurological diseases including Alzheimer’s disease and HAND. The use of illegal drugs such as cocaine and methamphetamine increases CNS dopamine, and could therefore alter the development of neuroinflammation and modulate macrophage functions. Several therapeutic drugs also increase extracellular dopamine in the CNS, and may therefore have broader effects than treatment of a specific disease or condition. More detailed analyses of the actions of dopamine on macrophages are important for our understanding of the effects of legal and illegal dopaminergic drugs on the CNS. These studies will address the efficacy and safety of long-term treatment regimens as well as facilitate the development of novel interventional strategies for neurologic diseases in the context of drug abuse.AADC: aromatic amino acid decarboxylase; BSA: bovine serum albumin; CNS: Central nervous system; DA: dopamine hydrochloride; DAPI: 4',6-diamidino-2-phenylindole; DAT: dopamine transporter; DR: dopamine receptor; D1R: dopamine receptor 1; ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothiocyanate; HAND: HIV-associated neurocognitive disorders; HEPES: hydroxyethyl piperazineethanesulfonic acid; LPS: lipopolysaccharide; M-CSF: macrophage colony stimulating factor; MDM: Monocyte-derived macrophage; PBMC: peripheral blood mononuclear cells; PM: plasma membrane; PVDF: polyvinylidene fluoride; TBS: Tris-buffered saline; TH: Tyrosine hydroxylase; TNF: Tumor necrosis factor; VMAT2: vesicular monoamine transporter 2.The authors declare that they have no competing interests.PJG designed the study with JWB, performed the immunoassays, subcellular fractionation and immunoblotting, and participated in the qRT-PCR with LC and the confocal microscopy with EAE, and drafted the manuscript. LC participated in the qRT-PCR with PJG and helped edit the manuscript. EAE performed the confocal microscopy with PJG and helped edit the manuscript. JWB participated in the design of the experiments with PJG, coordinated and helped to draft and edit the manuscript. All authors have read and approved the final version of this manuscript.


Molecular Cancer for the inconvenience caused.The authors would like to retract the article "Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions" . After p


Curiosity about the numerical abilities of non-human animals has been a topic in experimental psychology for about as long as such a science has existed. Some of the earliest comparative work looked at how well animals could use number as a cue in making different kinds of judgments. Early results were mixed, and in some cases were incredibly controversial . After the “fall” of behaviorism and the “rise” of cognitive psychology, the question of numerical competence became one of the dominant research areas in the new field of comparative cognition. The present series of papers in this special topic represents the newest additions to that research area. This special topic also serves as an anniversary of sorts, as it comes 25 years after one of the most influential papers on the subject of numerical competence in animals . Subitizing remains an interesting phenomenon, sometimes evident in animal research but other times not evident (see Murofushi, Davis and Perusse  and the This special topic encompasses 16 novel studies, including mammals (e.g., Beran et al., The special topic also includes theoretical and research studies on human infants. In particular, attention has been focused on number-space interaction (de Hevia et al.,


The World Health Organization (WHO) selected antimicrobial resistance (AMR) as the theme for World Health Day 2011. The slogan was “Combat Drug Resistance – No action today, no cure tomorrow” A six-point policy package was launched as a core product for World Health Day. It aimed to stimulate extensive and coherent action to overcome the many challenges presented by antimicrobial resistance.As a preparation for World Health Day, interviews were conducted with a series of key informants, mainly senior government staff, to assess their awareness of the topic and the interventions proposed in the policy package. Since the key informant interview methodology was used with a small number of interviewees, it may be difficult to demonstrate the validity of the findings.Key informants from twelve out of fifteen countries responded, which included Fiji (n = 5), Kiribati (n = 1), Lao PDR (n = 2), Malaysia (n = 6), Micronesia (n = 3), Mongolia (n = 5), the Philippines (n = 5), Vietnam (n = 6), Vanuatu (n = 1), Solomon Islands (n = 3), Cambodia (n = 5) and Brunei (n = 1). There was a total of forty-three respondents (n = 43). AMR was widely recognized as a problem. Lack of a coherent, comprehensive and national plan or strategy was noted. Surveillance was often seen as weak and fragmented even where presented. Laboratory capacity was felt to be insufficient across all countries interviewed. The majority of respondents stressed the need for national and local plans to combat AMR including reliable estimates of the financial cost of combating and managing AMR, the need for legislation to control inappropriate use of antimicrobials in food animals and more serious efforts to promote Standard Treatment Guidelines (STGs) and Rational Prescription. Also, importance was highlighted of the need to include infection prevention and control (IPC) as a part of accreditation and registration of health institutions and programs to promote IPC to the general population.A coalition of interested parties at the local, national and international levels need to generate and sustain the political will to organize a more comprehensive, sustainable, and coherent approach to AMR. Among the most important medicines ever discovered, antimicrobial agents have saved millions of lives and improved the outcomes for countless patients since these drugs were introduced in the early 1930s .Antimicrobial resistance (AMR) is defined as the resistance of a microorganism to an antimicrobial medicine to which it was previously sensitive ,4. ResisWHO GLOBAL STRATEGY FOR CONTAINMENT OF ANTIMICROBIAL RESISTANCE along with a series of recommendations aimed at enabling countries to define and implement national policies in response to antimicrobial resistance [The world has recognized for many years that antimicrobial resistance is a major problem and that comprehensive and coordinated action is desirable . In 2001sistance .In 2002, the Regional Committee for the Western Pacific passed a resolution on antimicrobial resistance that recognized the WHO Global Strategy for Containment of Antimicrobial Resistance . The resThe World Health Assembly resolutions related to AMR in 1998, 2005 and 2009 and from various WHO Regional Committee resolutions have been passed, but the consensus is that national and global responses have been inadequate . StrategThe six-point policy package was proposed as a core product for World Health Day. It was developed from the WHO Global Strategy for Containment of AMR . The pacThe six items in the six-point policy package were: (1) to commit to a comprehensive, financed national plan with accountability and civil society engagement, (2) to strengthen surveillance and laboratory capacity, (3) to ensure medicines of good quality and of regular supply, (4) to regulate and promote the rational use of medicines including those in animal husbandry and ensure proper patient care, (5) to enhance infection prevention and control, and (6) to foster innovations and research and development of new tools .The Western Pacific Regional Office (WPRO) of WHO, as a part of its preparation for World Health Day, commissioned key informant interviews with key policy makers in the area of AMR in individual member states. The purpose of the interview was for the key informants to assess the relevance and suitability of the proposed policy package. It also provided an opportunity to see if senior decision makers had knowledge about whether or not the proposed key aspects of the AMR policy package were in place and to sensitize them on the need of the AMR policy package to guide actions against AMR at the country level.The Health Services Development unit developed an instrument for use in conducting key informant interviews. The questionnaire for structure interview was commented upon and refined by members of the AMR Working Group of the WPRO.Key informant interviews (KII) involve interviewing a select group of individuals who are likely to provide needed information, ideas, and insights on a particular subject. Because information comes directly from knowledgeable people, KII often provide data and insight that cannot be obtained by other methods. But, because KII provide only a very limited basis for quantification, they are rarely appropriate when quantitative data are needed. Moreover, having a small sample pool may prove difficult to demonstrate the validity of the findings .The instrument followed the format of the six-point policy package and consisted of a quantitative and a qualitative part. Each of the six-points had 5–6 closed-ended questions which required a “Yes”, “No”, or “Do Not Know (DNK)” answer. These were then linked to an open-ended question on the same topic that was used to explore the respondent’s answer further. The questionnaire was an applied mixture of both self-reporting and face-to-face interviews conducted by the Health Services Development unit of WPRO, WHO.The interviewees were identified by the WHO country offices in their respective regions in cooperation with the focal point in the Ministry of Health (MoH). The interviewees were typically a senior researcher from a local research institute or an official from the MoH. The task involved identifying the officials from the MoH in different departments. Given the time constraints in accessing senior policy makers, the focal point persons from the MoH and WHO were asked to help make arrangements for the interviews. Due to the time limits and budget constraints, it was not possible to carry out interviews with the private sector and professional associations.The questionnaire could be completed by the key informant independently or through a face-to-face interview. An Interview took about 30 minutes. Key informants felt comfortable with the “Yes”, “No”, and “DNK” questions, but for the more detailed comment questions, some felt unease as it required more time and a more thorough discussion. The interviews were conducted only in the Member States of the Western Pacific Region where the WHO had an office, which meant that the majority of the responses gathered were from low and middle-income countries. The plan was to have three to five interviews per country, with five from the larger countries and three from the smaller.Interviewees from twelve out of fifteen countries responded, which included Fiji (n = 5), Kiribati (n = 1), Lao PDR (n = 2), Malaysia (n = 6), Micronesia (n = 3), Mongolia (n = 5), the Philippines (n = 5), Vietnam (n = 6), Vanuatu (n = 1), Solomon Islands (n = 3), Cambodia (n = 5), and Brunei (n = 1). The total number of respondents was forty-three (n = 43). Respondents identified themselves from a wide variety of professions including pharmacists, medical doctors, physicians, pathologists, medical microbiologists, bacteriologists, public health specialists, health administrators, family medical consultants, laboratory scientists and epidemiologists.Because small samples were chosen and random sampling methods were not used, it would not be safe to make generalizations about the results. However, the KII provides valuable information for programs, particularly when following the general process and steps. In this study, it first identified stakeholders and what information is needed and from whom. Second, developing an interview protocol, which is the rule that guides the administration and implementation of the interview, was done. Third, interviews with stakeholders were set up after explaining the purpose of the interview, why he/she has been chosen, the expected duration of the interview, while an informed consent was asked of the interviewee.The answers to the closed-end questions are presented in detail in Table The questions focused on the national planning processes indicate that most informants were not aware of a comprehensive national plan and that there was not an estimate available as to the costs of combating AMR. Also, only 40 % of the respondents answered they were aware of which individual or organizational body was responsible for AMR in their country.The second set of questions revealed a lack of confidence in the existence of comprehensive surveillance systems for AMR, although there was a better response in regards to some disease specific systems being in place. Only 50 % thought they were aware of who is responsible for AMR surveillance while only 28 % felt that laboratory capacity was adequate for AMR surveillance.A majority of the respondents answered positively to the third set of questions aimed at assessing the ability to ensure medicines of good quality and regular supply. The fourth set of questions dealt with the rational use of medicines to which the informants were less positive. It is of particular interest that only 7 % of respondents felt there was sufficient legislation to control the use of antibiotics in animal feed.The responses to fifth set of questions dealt with infection prevention and control (IPC) seemed to indicate that there was considerable activity in institutions although IPC was not a part of the health institution accreditation. Informants also felt there was not an adequate program to promote IPC in the general population. The final set of questions dealt with fostering innovation and research. About forty per cent of respondents felt that operational research was being conducted relevant to AMR and that a process existed for the assessment of new antimicrobials and diagnostics. Not surprising for low and middle income countries, the informants had a relatively low-awareness of basic research in respect to AMR.The first item in the six-point policy package concerns the commitment to a comprehensive, financed national plan with accountability and civil society engagement. The key informants agreed that a comprehensive national plan for AMR is needed and there is a need for developing plans to strengthen existing related activities in order to combat AMR. One respondent was not sure about the feasibility of such plan, perhaps discouraged by the existing fragmentation. The majority said that the local AMR plans should be developed, although one informant felt that given the national guidelines are present, there is no need for local plans. Another informant said that a local plan should be demanded by the national plan. The majority responded that costing is definitely needed and useful. The majority of the sample felt that the MoH or its equivalent within the government should be responsible for AMR, although there is recognition that there are many other stakeholders concerned.The second item is about strengthening surveillance and laboratory capacity. Most respondents said a comprehensive AMR surveillance system needs to be established and there is a need to develop an institution or department for the improvement of technical capacity for AMR surveillance. Some specific diseases, such as TB, HIV, Influenza, MRSA, VRE, and gonococcus, are established as reportable diseases and have ongoing surveillance. Some of the respondents mentioned surveillance should be expanded to other diseases and be better linked with each other. Many different organizations and departments play a role in surveillance, but some countries have no specific person taking responsibility. In most countries the National Laboratory coordinator in the MoH is responsible for AMR laboratory capacity. However, improving skills and equipment, training, budget, human resources and capacity of all related resources and facilities as well as premises are needed. Moreover some countries have no database for regulation at the national level, but many countries prepare annual reports published by the public sector.The third item is about ensuring medicines of good quality and regular supply. Many respondents said the national standards on prescribing medications exist, but related law and standards enforcement and their implementation are inadequate. Lack of laboratory capacity, illegal drug use and supply, and procurement systems that fail to comply with standard procedures contribute to AMR. While control measures by regulations and enforcement by the government officials exist for counterfeit and substandard medicines, the implementation of these control measures is imperfect. National drug committees such as the drug and therapeutic committees and disease program managers in the MoH should regularly review essential drug lists (EDLs) and standard treatment guidelines (STGs), but these mechanisms are often not functioning well at the moment. With regard to expired drugs, the majority of the countries’ respondents felt expired drugs are being destroyed appropriately by legal regulations or protocols.The fourth item concerns the regulation and promotion of the rational use of medicines, including in the area of animal husbandry, and ensuring proper patient care. STGs related to combating AMR are not a part of institutional accreditation or health worker registration for the most part. Most informants agreed that policy development is needed in these areas. Many of the countries have laws for the accessibility of antimicrobials only by prescription, but are not enforced properly, although many informants felt this law was necessary, enforcing this law has been difficult. Most of the countries also do not have legislation to control the inappropriate use of antimicrobials in food animals. The general response from some countries is that this control is partially met by the Ministry of Agriculture (MoA), however key informants from the health sector seemed to have little detail or knowledge in this area. With regard to the general public, key informants believed the public had little knowledge of AMR and had very little access to public education of AMR. They believe public education on AMR through all appropriate mediums is needed.The fifth item in the AMR policy package is enhancing infection prevention and control (IPC). Most countries have departments in charge of IPC. Some countries, namely Malaysia and the Philippines, have made IPC a part of their institutional accreditation system. While most of the other countries do not have such a system, there is a consensus for the need for its development. Some countries have a system for continuing education for health workers, while others do not. All informants agreed to its need. Most countries do not have IPC programs for the general public, although educational programs on hand-washing are carried out during outbreaks. There seems to be less incentive to continue such programs indefinitely, which would be needed for ongoing issues such as combating AMR. Some countries have specific departments for IPC, while others do not.The sixth item is on fostering innovations and research as well as the development of new tools. Most of the country informants felt the need for operational research in the use of antimicrobials and diagnostics. Most informants felt that there was not an adequate process to assess whether new antimicrobials and diagnostics should be introduced. The informants felt that basic research to develop new diagnostics and antimicrobials is needed, although those of many countries acknowledged a limited capacity to do such research.The key informants seemed to uniformly feel that AMR is an important issue and there was a need to develop information systems and infection control policy. AMR was identified as a major challenge to the respective MoH and it was acknowledged that they were not yet operating properly to meet this challenge. The informants identified the need to understand the roles and responsibilities of various parties and to develop sufficient knowledge and strong systems. Many acknowledged the need to make a comprehensive management team and task force responsible for AMR. They also expressed the need to develop a stronger political will to confront the issues.To the question on what should be expected from the WHO , the responses included: 1. technical support ; 2. facilitation of information sharing; 3. advocacy on the international level; 4. capacity building and coordinating in planning country wide training or education programs; and 5. developing instruments, guidelines and policy.The key informants recognized AMR as a problem and that the response to date has been inadequate. They felt that there has been a lack of coherent, comprehensive national planning and strategic processes. Where there has been a response to AMR, it has often been fragmented. AMR surveillance is often shown to be weak, and even where surveillance is stronger, it is often disease-specific and fragmented. Laboratory capacity was felt to be insufficient in all countries interviewed. The majority of respondents stressed the need for national and local plans to combat AMR including reliable estimates of the financial cost to combat and manage AMR. AMR surveillance in the Region will be strengthened by increasing the number of monitoring laboratories in different locations. They also expressed a need for legislation to control the inappropriate use of antimicrobials in food animals, although it was also recognized that the political will needed to be generated for this step to occur.More serious efforts to promote the STGs and rational prescription were also deemed desirable. The STGs exists in most settings but lacks a strong connection between the STGs and accreditation or licensing. Antibiotics being available without a prescription from a medical practitioner were a problem in most of the countries surveyed. Infection prevention and control activities exist, but many were related to outbreaks. It calls for a movement move to make these activities a part of the daily routine, as AMR is a problem that will continue indefinitely. The strengthening of the capacity and capability of drug regulatory authorities through direct technical support to selected Member States and sharing information through the network and will continue to be an area of priority.AMR requires a multi-faceted approach that will be sustainable. It is not a problem that can be confronted, defeated and forgotten. It requires action from a wide variety of players from multiple sectors in society. These key informant interviews show that the response to AMR to date is felt to have been inadequate in most countries.However, this study also indicates that these key informants are aware of most of the related issues and that they have a willingness to tackle them. They accept as clear and relevant the six-point policy package being promoted by the WHO as clear and relevant. However, there is some skepticism as to whether the necessary political commitment on the part of both national and international decision makers needs to be present to actually implement the package. A coalition of interested parties at the local, national and international levels need to generate and sustain the political will to organize and sustain a more comprehensive and coherent approach to AMR.It is troubling that AMR has been recognized as an important issue for decades ,18, and •Antimicrobial Resistance (AMR) is widely recognized as a problem and the need for a comprehensive national plan or strategy was noted.•Surveillance is often seen as weak and fragmented even where present, and laboratory capacity is felt to be insufficient almost everywhere.•The majority of respondents stressed the need for national and local plans to combat AMR including reliable estimates of the financial cost of combating and managing AMR, the need for legislation to control inappropriate use of antimicrobials in food animals and more serious efforts to promote the Standard Treatment Guidelines (STGs) and Rational Prescription.•Also, the importance is highlighted of the need to include infection prevention and control (IPC) as a part of accreditation and registration of health institutions and programs to promote IPC to the general population.Both authors declare having no competing interests.YL carried out the data analysis and summarization and drafted the manuscript. Both authors read and approved the final manuscript.


Stephen Harbarth and colleagues argue that the International Health Regulations(IHR) should be applied to the global health threat of antimicrobialresistance. The public health threat of antimicrobial resistance (AMR) is growing andneeds to be addressed urgently.The International Health Regulations (IHR), a legally binding agreementbetween 194 States Parties, whose aim is to prevent, protect against,control, and provide a public health response to the internationalspread of disease, deserve critical examination with regard to theirapplicability to AMR.We argue that the emergence and spread of antimicrobial-resistantbacteria, especially those involving new pan-resistant strains for whichthere are no suitable treatments, may constitute a public healthemergency of international concern (PHEIC) and are notifiable to theWorld Health Organization under the IHR notification requirement.The use of the IHR framework could considerably improve our response toemerging AMR threats like carbapenem-resistant Enterobacteriaceae(CRE).As more governments start to take the threat of pan-resistant bacteriaseriously, there is a window of opportunity for having a healthy debateabout the applicability of the IHR to AMR.Staphylococcus aureus,extensively drug-resistant (XDR)-tuberculosis, and carbapenem-resistantEnterobacteriaceae (CRE).The unrelenting rise of antimicrobial resistance (AMR) constitutes a serious threatto health worldwide. In the last decade, challenging multi-resistant bacteria haveexpanded while new antimicrobial drug development has lagged AMR in both humans and animals represents a complex global concern that must beaddressed “urgently and aggressively” Enterobacteriaceae, a family that includes common pathogens responsible for a largespectrum of disease, have been sensitive to many antibiotics in the past. Since the1980s, the global spread of extended-spectrum β-lactamase (ESBL)-producingEnterobacteriaceae has limited therapeutic options, but until recently, carbapenemswere still a reliable treatment. The recent emergence of CRE, resistant to mostclasses of antibiotics, has necessitated the use of third-line agents andcombination therapy with doubtful therapeutic efficacy and increased toxicity Klebsiella pneumoniae harboring KPC (KPC-Kp) have become endemic inparts of the United States, China, Israel, and Greece CRE have been associated with increased mortality and morbidity, and higher treatmentcosts, when compared to infections caused by susceptible strains The alarming spread of CRE is juxtaposed against our failure to develop new effectiveantimicrobials. The utility of tigecycline is marred by high rates of resistanceamong CRE Surveillance of AMR-pathogens such as CRE is patchy and limited by financial andtechnical constraints in large parts of the world. In some high-income countries,AMR data are compiled by publicly funded surveillance networks such as EARS-Net, anetwork of national surveillance systems in Europe, or by pharmaceuticalcompany-sponsored surveys. Informal networks, such as ProMED, also collectinformation, although selectively and with a considerable time lag. This holds eventruer for the scientific literature.Improving AMR surveillance is one of the key recommendations in a recent report The IHR provide a legal framework for international efforts to contain the risk frompublic health threats that may spread between countries, including surveillance andglobal alerts (Articles 5–11), definition of core public health capacities forsurveillance and response in all countries , and World HealthOrganization (WHO) guidance through “standing recommendations”  ” (PHEIC) and “potentially requires acoordinated international response” In order to identify events that have the “potential to cause internationaldisease spread”, WHO is bound to collect epidemiologic information“through its surveillance activities” (Article 5), notifications fromaffected countries (Article 6), and reports from third parties (Article 9) Plasmodium falciparum. “New or emerging antibioticresistance” is one of the examples listed in Annex 2 for application of thefirst criterion.We argue that certain events marking the emergence and international spread of KPCand NDM-1-producing CRE, especially those involving new pan-resistant strains forwhich there are no suitable treatments and which are of major public healthimportance, can be considered to fulfil at least two Annex 2 criteria, in particular“serious public health impact” and “international spread”, and shoK. pneumoniae was considerednotifiable by just over half of respondents Still, due to the nonspecific nature of Annex 2 and limited WHO guidance, some maycounter that CRE (and other AMR) events are irrelevant to the IHR. In a recentsurvey among National IHR Focal Points, a scenario describing a fatal hospitaloutbreak caused by pan-resistant The global threat posed by the spread of AMR cannot be addressed by individualcountries alone, but requires a coordinated international response. Recognizingthe applicability of the IHR to AMR will serve as a “wake-up call”and strengthen global AMR surveillance and response, which could in turncontribute to containing the spread of AMR. While WHO has initiated severalnetworks and provides guidance for reporting AMR, including WHONET, nonefunction as an early warning system. Although very few AMR events would bedetermined a PHEIC by the Director-General, notifications of events that fulfilthe Annex 2 criteria could serve as alerts and could be an important instrumentin the chain of “the global early warning function, the purpose of whichis to provide international support to affected countries and information toother countries if needed” Importantly, the IHR focuses on a societal investment in core surveillance andresponse capacities at different levels by setting minimum standards. WHOpledges to collaborate with the States Parties concerned “by providingtechnical guidance and assistance and by assessing the effectiveness of thecontrol measure in place, including the mobilization of international teams ofexperts for on-site assistance, when necessary”. This is relevant for thespread of AMR given the importance of appropriate infection control measures.While details of these measures need to be more closely defined, it is clearthat the application of the IHR framework is invaluable for a coordinated globalapproach to AMR.Even if WHO and a majority of States Parties considered that AMR should beaddressed under the IHR, technical, financial, and political obstacles mightinterfere. Notification of an event to WHO depends on it being detected, andreported to the National IHR Focal Point. There is concern that many StatesParties are far from being compliant with the IHR's minimum core capacityrequirements for surveillance and response. Even if relevant information filtersthrough to the national level, notification decisions may be under politicalcontrol. The fierce reaction of the Indian government to claims thatNDM-1-producing CRE isolated in the UK originated in India casts doubt on thewillingness of governments to report the existence of such events, in particularif economic interests  are at stake.These obstacles are not specific to AMR-related events, and cannot serve as anargument against the application of the IHR in this context.The final obstacles are a lack of expertise and capacities within WHO. AlthoughWHO vertical programs have successfully focused on drug resistance in selectedareas, including malaria and tuberculosis, WHO arguably does not have the meansto comply with its IHR mandate of offering assistance to States Parties affectedby the spread of multi-resistant bacteria. The dearth of leadership in this areawas the object of a WHO resolution in 2005, but it has been commented that“very little has taken place to implement the resolution WHA 58.27 sinceits passage” The IHR do not provide a panacea for the problem of AMR. However, this frameworkprovides a global surveillance infrastructure and orchestrates an appropriatepublic health response. The IHR are ultimately “owned” by the StatesParties, some of whom increasingly understand the extent and urgency of thethreat posed by AMR. However, it is up to WHO to provide leadership on the roleof the IHR in this matter. Further guidance on the application of Annex 2 tothis issue is required. With the IHR in place, increasing the capacities of thisframework at all levels to address AMR, rather than investing in new verticalprograms, seems logical. The revival of the implementation of the WHO 2001Global Strategy for the containment of AMR with incorporation of the IHRframework into the strategy is required. Although this paradigm shift eventuallyrests on the World Health Assembly and States Parties' willingness to adoptit, WHO must demonstrate leadership in this regard.The international dissemination of AMR, typified by CRE, is a serious threat forglobal health. Although the spread of AMR is less dramatic than many acute diseaseoutbreaks, it significantly reduces our therapeutic options and adds significantlyto the health care burden. A global mechanism incorporating both systematicsurveillance and effective public health response is urgently required. We wouldargue that the IHR provide an appropriate framework to coordinate efforts forcontrolling the international spread of AMR. Several obstacles need attention beforethe full potential of the IHR may be realized, but there is a window of opportunityfor having a healthy debate about the applicability of the IHR to AMR. While StatesParties and WHO share a collective responsibility in the process, WHO must clearlydelineate its position with regard to AMR and the intended role of the IHR in thiscontext.Figure S1Klebsiella pneumoniaefrom Greece to other European countries, 2007–2010Transmission of carbapenem-resistant (TIF)Click here for additional data file.