Besides drug transport such carrier systems are well suited for the protection of drugs against enzymatic and chemical degradation. The preparation of biocompatible and biodegradable nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) is intensively described in literature, while especially nanoparticles with cationic properties show a promising increased cellular uptake. This is due to the electrostatic interaction between the cationic surface and the negatively charged lipid membrane of the cells. Even though several studies achieved the successful preparation of nanoparticles stabilized with the cationic surfactants such as didodecyldimethylammonium bromide (DMAB), in most cases insufficient attention was paid to a precise analytical characterization of the nanoparticle system. The aim of the present work was to overcome this deficit by presenting a new perspective in the formulation and characterization of DMAB-stabilized PLGA nanoparticles. Therefore these nanoparticles were carefully examined with regard to particle diameter, zeta potential, the effect of variation in stabilizer concentration, residual DMAB content, and electrolyte stability. Without any steric stabilization, the DMAB-modified nanoparticles were sensitive to typical electrolyte concentrations of biological environments due to compression of the electrical double layer in conjunction with a decrease in zeta potential. To handle this problem, the present study proposed two modifications to enable electrolyte stability. Both polyvinyl alcohol (PVA) and polyethylene glycol (PEG) modified DMAB-PLGA-nanoparticles were stable during electrolyte addition. Furthermore, in contrast to unmodified DMAB-PLGA-nanoparticles and free DMAB, such modifications led to a lower cytotoxic activity against Caco-2 cells. In conclusion this study offers a closer and critical point of view on preparation,  One of the greatest challenges of nanotechnology is the establishment of a suitable nanoparticulate carrier system for overcoming physiological barriers like the intestine. Oral administration is the preferred route of drug delivery because it provides the highest patient convenience and compliance , 2. TherEspecially in cancer research, many drugs show low bioavailability after peroral administration due to their poor stability, solubility, and permeability. Therefore, intravenous application in many cases is unavoidable. However, the encapsulation of such pharmaceutically challenging molecules within a polymeric nanoparticle matrix results in an increased drug absorption in targeted tissues or cells and protects the drug from enzymatic and hydrolytic degradation . ConsequBecause of its proven biocompatibility and biodegradation, poly (DL-lactic-co-glycolic acid) (PLGA) is approved by the FDA for therapeutic use in humans and is one of the most successful starting materials for drug carrier preparations , 9. PLGABasically nanoparticles formed of PLGA are prepared in the presence of polyvinyl alcohol (PVA) as a widely used steric stabilizer. The most described and applied preparation method is the emulsification-diffusion method, which leads to particle diameters in the range of 150 to 300 nm .The degree of nanoparticle absorption by cells depends on diameter and surface properties such as surface charge or hydrophobicity, which in turn are associated with the strength of interaction between the nanoparticles and the cell membrane . The surNevertheless recent studies illustrated the comparable pronounced cytotoxic activity of DMAB , 19, whiThis initial study took a critical look at the characterization and stability of DMAB-stabilized nanoparticles by physico-chemical as well as cell culture analysis. Two new modifications are proposed to minimize cytotoxicity and optimize electrolyte stability and therefore to enable nanoparticle application under physiological conditions in future experiments. These modifications could be helpful in preparing DMAB-stabilized nanoparticles for therapeutic use.Poly (DL-lactide-co-glycolide) (PLGA) Resomer RG 502H and Poly(DL-lactide-co-glycolide)-co-polyethylene glycol diblock Resomer RGP d50155 were obtained from Evonik Industries AG . Polyvinyl alcohol (PVA) 30,000–70,000 Da and didodecyldimethylammonium bromide (DMAB) were purchased from Sigma Aldrich .For cell culture experiments Dulbecco’s modified Eagle’s medium (DMEM) and all used supplements were received from Biochrom AG . VECTASHIELD Mounting Media with DAPI  was purchased from Vector Laboratories Inc.  and Wheat germ agglutinin (WGA) AlexaFluor 350 from Life Technologies . All other chemicals were obtained from Roth .Nanoparticles were prepared by a literature described emulsification-diffusion method . BrieflyThe nanoparticles were evaluated for mean diameter and for polydispersity index (PDI), a dimensionless indicator of size distribution, by a Zetasizer  using the dynamic light-scattering technique. The zeta potential, representing both surface charge and stability, was also obtained by means of Zetasizer measurement based on electrophoretic mobility under an electric field.The resulting particle yield after purification was determined gravimetrically. Therefore an aliquot (20.0 μl) of the respective nanoparticle sample was put in micro weighing dishes and dried for 2 h at 80°C until constant weight.When using non-PEGylated PLGA as starting material, modification with PVA was performed by resuspension of the nanoparticle pellet after the third centrifugation step in aqueous PVA solution (1% w/v) followed by shaking overnight at 550 rpm. Afterwards, the nanoparticles were washed once with deionized water by centrifugation and resuspension (referred as PLGA-DMAB-PVA-NP).PEG-modified DMAB nanoparticles were prepared similar to PLGA-DMAB-NP, but using PEGylated PLGA and emulsification for 10 min (PLGA-DMAB-PEG-NP).The nanoparticles were titrated using a titration tool  with NaCl solution (250 mol/L) to evaluate the influence of electrolyte concentration on stability. Mean diameter, PDI, and zeta potential of triplicate experiments were determined by Zetasizer  measurements.Human epithelial colorectal adenocarcinoma cells (Caco-2) were commercially purchased from Institut für angewandte Zellkultur Dr. Toni Lindl GmbH .2.The used cultivation medium of the investigated Caco-2 cells was Dulbecco’s modified Eagle’s medium (DMEM) with the addition of 1% (v/v) non-essential amino acids, 1% (v/v) penicillin/streptomycin/glutamine, and 10% fetal calf serum (FCS). The cells were subcultured twice a week with a ratio of 1:3, after reaching 80% confluence, or were used for experiments. Incubation experiments with nanoparticle formulations or free DMAB were performed using serum-free DMEM. For cultivation or incubation with substances, the cells were maintained at 37°C and 10% CO4 cells/well on Millicell EZ slides  and were allowed to grow overnight. This was followed by 4 h incubation at 37°C and 10% CO2 in serum free media with 0.02 mg/mL fluorescence dye-loaded PLGA-DMAB-NP, PLGA-DMAB-PVA-NP, and PLGA-DMAB-PEG-NP. Cells were washed once with phosphate buffered saline (PBS) and incubated with a Wheat germ agglutinin AlexaFluor 350 solution (10 μg/mL) for 10 min at room temperature for membrane staining. Cells were then washed three times with PBS, fixed in 4% para-formaldehyde for 15 min and washed again with PBS. For nuclear staining, cells were covered with VECTASHIELD Mounting Media with DAPI . Samples were analyzed using a IX81 fluorescence microscope  with filter systems including excitation at 360–370 nm, dichroic mirror at 400 nm, emission at 426–446 nm for DAPI and Alexa Fluor 350 and excitation at 535–555 nm, dichroic mirror at 565 nm, emission at 570–650 nm for Lumogen F Red 305.Fluorescent nanoparticle formulations for localization experiments were prepared by dissolving 0.4 mg/mL of the fluorescence dye Lumogen F Red 305 in the ethyl acetate solution used during the nanoparticle preparation. Caco-2 cells were seeded with a density of 5 x 10The performed assay is based on the ability of living cells to reduce a water-soluble red dye, WST-1, to a yellow-colored formazan product by cellular dehydrogenases . The nanoparticle formulations used for the study were freeze-dried and resuspended in serum-free DMEM.4 cells/well and incubated for 48 h. The cells were incubated with DMAB, PLGA-DMAB-NP, PLGA-DMAB-PVA-NP, and PLGA-DMAB-PEG-NP, respectively, in concentrations ranging between 0.5 and 2,000 μg/mL. After 24 h the cells were washed twice with PBS solution with calcium and magnesium and wells were filled with 100 μL serum-free medium.Caco-2 cells were seeded in 96-well plates with a density of 1 x 10Absorbance was measured at 460 nm using a microplate reader  to obtain the background absorbance in order to minimize the error based on unspecific light scattering of the nanoparticles. Untreated cells were taken as negative control with 100% viability, and cells treated with DMSO were used as positive control with 0% viability.50 values were calculated by curve fitting of the cell viability data finally measured at 460 nm . Measurements were performed six fold in three passages and results are expressed as mean ± standard deviation (SD).After incubation with nanoparticle formulations or free DMAB for 24 hours 10 μL WST-1 were added to each well, followed by further incubation at 37°C for 1 h. ECThe developed method to determine residual DMAB content in the nanoparticle suspension takes advantage of a direct spectrophotometric assay of quaternary ammonium compounds and is based on the ionic interaction between the cationic quaternary ammonium ion and the anionic dye bromothymol blue buffered at pH 7.5 . BromothThe assay was performed by mixing 160 μL of the nanoparticle dispersion with 25 μL NaOH (1 N). The mixture was incubated for 15 min at 60°C under shaking. Then 25 μL HCl, 140 μL bromothymol blue solution, and sufficient phosphate buffer pH 7.5 were added to a volume of 2.0 mL. The samples were shaken for 1 h at room temperature. After centrifugation at 20,000 g for 10 min to separate the supernatant containing excess bromothymol blue from the precipitated green DMAB-bromothymol blue complex, the supernatant was diluted with water at a ratio of 1:1 and measured photometrically at 610 nm. For linear assay calibration 160 μL DMAB working solutions (0 to 300 μg/mL DMAB) instead of the nanoparticle dispersion were used.Validation of this method was performed according to the ICH harmonized tripartite guideline “Validation of analytical procedures: Text and Methodology Q2(R1)”. Therefore the method was tested with pure DMAB and DMAB plus hydrolyzed PLGA in three different DMAB concentrations covering the specified range for the procedure. The samples were measured six fold and analyzed as described above.All investigations were done in triplicate. The results are presented as average value with standard deviation. In order to compare independent groups, one way ANOVA was performed using the software SigmaPlot version 11 . A significance level of ≤ 5% (p ≤ 0.05) between the groups was declared as a significant difference (*). With a significant level of ≤ 1% (p ≤ 0.01) the difference was defined as highly significant (**). For a most highly significant difference (***) the significance level was ≤ 0.1% (p ≤ 0.001).For the preparation of DMAB-stabilized nanoparticles the method described by Bhardwaj et al. was adopted with suitable modifications . In compAs seen in In order to increase particle stability two different modifications of the preparation process were investigated: I. Resuspension of nanoparticles in PVA solution or II. The use of PLGA-PEG copolymer instead of the established PLGA polymer.The analysis of PLGA-DMAB-PVA-NP showed, that adsorptive bound nonionic PVA led to a significant increase in particle diameter. However with a PDI below 0.1 the particle system remained monodisperse. The coating with PVA also resulted in a decrease of the zeta potential .At comparable DMAB concentrations the use of PLGA-PEG block copolymer as starting material led to an increasing diameter of about 100 nm and a PDI higher than 0.1. Only the application of 10 mg/mL DMAB in the aqueous phase resulted in acceptable nanoparticles with a diameter of 103.7 ± 2.4 nm and a PDI of 0.13. In contrast to nanoparticles based on PLGA the usage of the block copolymer led to a decreased zeta potential of +14 mV.As an electrolyte (NaCl) was added to the nanoparticle suspension, the magnitude of the zeta potential decreased progressively . This caEven though the unmodified PLGA-DMAB-NP were unstable in electrolyte solutions, both the PLGA-DMAB-PVA-NP and PLGA-DMAB-PEG-NP remained stable under electrolyte influence. Although the zeta potential decreased rapidly to ±0 mV a constant diameter of about 110 nm was measured over the whole titration experiment. Taking a closer look at the diameter curve progression as a function of electrolyte concentration, it was striking to note that a small increase by about 10 nm occurred for both particle modifications .In the next step the ability of the nanocarriers to enter Caco-2 cells was examined by incubation with Wheat germ agglutinin AlexaFluor 350 and DAPI in the presence of the various dye-loaded NP formulations. To evaluate the cytotoxic effect of the nanoparticles on human cells, Caco-2 cells were used as a model cell line. The effect of different nanoparticle formulations and free DMAB on cell viability was evaluated in a concentration range from 0.5–2000 μg/mL using the WST-1 assay.50 value of 10 μg/mL. The PLGA-DMAB-NP exhibited a EC50 value of 50 μg/mL and an initial cytotoxicity at about 10 μg/mL  the proposed method was validated with regard to linearity and accuracy of pure DMAB and DMAB in combination with hydrolyzed PLGA. To prove linearity a series of DMAB dilutions within the range of 0 and 300 μg/mL were analyzed as described above and a calibration curve was calculated with a correlation coefficient of 0.9984 .For verification of precision and accuracy test solutions containing DMAB and DMAB plus hydrolyzed PLGA at three different concentrations  were prepared and measured in triplicate. Both the percentage of DMAB recovery and the confidential interval at a probability value of 5% were calculated . At mediBased on the validated method the different nanoparticle formulations were analyzed for DMAB content after particle purification. The more DMAB was used for particle preparation the more DMAB remained in the particle suspension after purification, which is shown by The present study was designed to reveal and overcome several problems in the preparation of DMAB-stabilized PLGA nanoparticles associated with agglomeration under raising electrolyte concentration. In principle, PLGA nanoparticles for drug delivery are routinely prepared with PVA as a stabilizer . The resIn a first step of the present study the influence of the used DMAB concentration on the physico-chemical parameters of the resulting nanoparticles was evaluated. During particle preparation the decreased interfacial tension as a function of the initial DMAB concentration led to smaller emulsion droplets in the primary emulsion during the homogenization step and to smaller nanoparticles after nanoprecipitation. The quantification of the residual DMAB content after purification showed that the more DMAB was initially used for particle preparation the more residual DMAB was quantified in the nanoparticles. Thus more DMAB remained at the interface between PLGA and water, which correlates with a higher surface charge and a higher zeta potential .One of our aims in preparing DMAB-stabilized PLGA nanoparticles was to establish a nanoparticulate drug carrier system for future oral drug delivery. Therefore the application of a colloidal system in physiological media for use in cell culture studies or an in vivo set up requires electrolyte stability. Thus the smallest nanoparticles of about 70 nm in diameter, prepared with an initial DMAB concentration of 10 mg/mL in the aqueous phase, were titrated with an NaCl solution up to a final salt concentration of 150 mmol/L NaCl to evaluate the influence of electrolyte content on stability. For unmodified PLGA-DMAB-NP a rapid agglomeration even after the addition of minor NaCl concentrations was observed. The stability of the nanoparticle dispersions and their tendency to agglomerate below a zeta potential of +40 mV under electrolyte influence can be described within the context of attractive  and repulsive forces (such as electrostatic or steric forces) between particles by using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory . The smaConsequently a steric stabilization of the particles besides pure electrostatic interaction is a rational approach. For this reason two modifications of the prepared PLGA-DMAB-NP were established to overcome the detected stability issues under electrolyte influence. On the one hand a PVA coating strategy was pursued and on the other hand the starting material PLGA was exchanged against a PLGA-PEG block copolymer. Both used hydrophilic compounds PEG and PVA provide steric stabilization of particle dispersions by a repulsion effect caused by a steric mechanism of stabilization involving both enthalpic and entropic contributions . PEG, a The results of the electrolyte stability studies were supported by the cellular localization of the nanoparticles . Hence a50 = 10 μg/mL). Both free DMAB and PLGA-DMAB-NP showed a high cytotoxic activity against Caco-2 cells, as a common used model cell line to analyze gastrointestinal absorption. The parameter of cytotoxicity is crucial for further experiments regarding permeability of nanoparticles and therefore has to be screened for all preparations. The fact that PLGA-DMAB-NP showed high cytotoxic potential (EC50 = 50 μg/mL) though DMAB is not freely available may be attributed to the strong cationic charge on the particle surface. As previously described, cationic nanoparticles engage in strong ionic interactions with the negatively charged cell membranes [Furthermore in previous studies a high cytotoxic activity of DMAB for diverse cell types was demonstrated , 19. Thiembranes . In contThese results of cell-viability testing clarify the need for a quantitation method for the surfactant DMAB in particle systems. Described methods are not feasible without high technical complexity , 32. ThuDMAB is an appropriate surfactant for the preparation of small PLGA nanoparticles with a positive surface charge. However, upon closer examination of the stability under electrolyte influence, it becomes apparent that PLGA-DMAB-NP are possibly not qualified for application in physiological media. We have demonstrated that modification with hydrophilic polymers such as PVA or PEG leads to electrolyte stability and a decreased cytotoxicity against Caco-2 cells. Thus, modified DMAB nanoparticles could be useful in developing a suitable nanoparticulate drug carrier for biomedical applications in contrast to solely DMAB stabilized nanoparticle preparations.This study also proposes a simple, suitable and convenient method for DMAB quantification that could be helpful in further studies for a comprehensive characterization of DMAB containing nanoparticle systems.


Pharmacists’ role may be ideal for improving rationality of drug prescribing practice. We aimed to study the impact of multifaceted pharmacist interventions on antibiotic prophylaxis in patients undergoing clean or clean-contaminated operations in cardiothoracic department.A pre-test–post-test quasiexperimental study was conducted in a cardiothoracic ward at a tertiary teaching hospital in Suzhou, China. Patients admitted to the ward were collected as baseline group (2011.7–2012.12) and intervention group (2013.7–2014.12), respectively. The criteria of prophylaxis antibiotic utilization were established on the basis of the published guidelines and official documents. During the intervention phase, a dedicated pharmacist was assigned and multifaceted interventions were implemented in the ward. Then we compared the differences in antibiotic utilization, bacterial resistance, clinical and economic outcomes between the 2 groups. Furthermore, patients were collected after the intervention (2015.1–2015.6) to evaluate the sustained effects of pharmacist interventions.P < 0.001). Meanwhile, a significant reduction was seen in the rate of unnecessary replacement of antibiotics and the rate of unnecessary combinations (P < 0.001). Besides, pharmacist intervention resulted in favorable outcomes with significantly decreased rates of surgical site infections, prophylactic antibiotic cost, and significantly shortened length of stay (P < 0.05). Furthermore, there were also significant decreases of the rates of antibiotic resistant enterobacter cloacae, klebsiella pneumonia, and staphylococcus aureus (P < 0.05). Moreover, the effects were sustained after discontinuation of the active interventions, as shown in prophylactic antibiotic utilization data.412 and 551 patients were included in the baseline and intervention groups, while 156 patients in postintervention group, respectively. Compared with baseline group, a significant increase was found in the proportion of antibiotic prophylaxis, the proportion of rational antibiotic selection, the proportion of suitable prophylactic antibiotic duration, and the proportion of suitable timing of administration of the first preoperative dose (Pharmacist interventions in cardiothoracic surgery result in a high adherence to evidence-based treatment guidelines and a profound culture change in drug prescribing with favorable outcomes. The effects of pharmacist intervention are sustained and the role of pharmacists is emphasized for rational medication and optimal outcomes in clinical treatment. More than 50% of pathogens implicated in infections are the coagulase-positive Staphylococcus aureus or the coagulase-negative Staphylococcus epidermidis.2 Among different approaches to prevent SSIs, antibiotic prophylaxis is substantially important. In 2006 and 2007, the Society of Thoracic Surgeons Practice released evidence-based practice guidelines on antibiotic prophylaxis in cardiac surgery.3 Furthermore, based on the national circumstance, the National Health and Family Planning Commission (NHFPC) of China has incorporated the guidelines into the national drug policy. The official document for rational use and standard management of antibiotics,4 and the notice regarding national special measure scheme on clinical use of antibiotics,5 were issued in 2009 and 2011, respectively. The main recommendations of prophylactic antibiotic use for cardiothoracic surgery are based on the above guidelines and official documents.Cardiac surgery and cardiopulmonary bypass are potent inducers of a systemic inflammatory response.6 With respect to antibiotic resistance that may be caused by irrational antibiotic use, they still attributed responsibility to patients, other countries, and healthcare settings. Meanwhile, they regarded antibiotic resistance as a low priority and a distant consequence of antibiotic prescribing.7 As for cardiovascular surgery, the antimicrobial prophylaxis is discordance with practice standards, leading to inappropriate administration of many antibiotics.8 Analogously, the irrational use of antibiotics in the perioperative period of surgical procedures was ubiquitous in the department of cardiothoracic surgery of the first affiliated hospital of Soochow University, located in Suzhou, China. To prevent the complications of inappropriate administration of antimicrobials, it is quite necessary to implement interventions to improve the rationality of antibiotic prophylactic utilization.Despite the availability of these guidelines, the practice of antibiotic prophylaxis is still far from optimal. A considerable portion of surgeons do not adhere to the basic principles suggested by issued guidelines.9 Discharge counseling sessions implemented by pharmacists were essential to improve outpatients’ primary medication adherence.10 Pharmacist interventions can facilitate knowledge translation of new evidence into practice. It has been reported that the implementation of pharmacist intervention in stress ulcer prophylaxis was associated with a decrease in inappropriate acid suppression rates during hospitalization and upon discharge, as well as significant cost savings.11 Furthermore, pharmacist involvement on the intensive care unit team was associated with a substantial increase in therapeutic optimization and a clinically notable reduction in preventable adverse drug events, as well as an estimated 30% increase in associated cost savings.12 As for antibiotic prophylaxis, pharmacist intervention promoted rational use of antibiotic with significant reduction in antibiotic costs.14 Until now, there have not been literatures about pharmacist interventions in cardiothoracic surgery wards, especially in improving rational drug utilization.Special pharmacists have become an established feature of the medical stewardship landscape in hospitals. As key members of the medical team, they fulfilled a vital function of overall responsibility for initiatives to promote rational drug prescribing.In this single-group pre-test–post–test quasiexperimental study, we compared hospital antibiotic prescribing practices pre- and during intervention. We found that the implementation of pharmacist intervention resulted in a high adherence to evidence-based treatment guidelines, with a favorable clinical and economic outcome and improved bacterial resistance. Our results highlight the role of pharmacists in rational drug prescribing practices. We believe this newly reported antibiotic pharmacist intervention may play a key role in a culture change in antimicrobial prescribing, improving the current practice and leading to favorable clinical outcomes.Patients admitted to a cardiothoracic surgical ward in the first affiliated hospital of Soochow University, which is a high-volume tertiary hospital in Suzhou, China, between July 1, 2011 and December 30, 2012 (baseline group), between July 1, 2013 and December 30, 2014 (intervention group) and between January 1, 2015 and June 30, 2015 (postintervention group) were screened for eligibility. Patients were included if they had undergone cardiothoracic surgery and the wound class of the surgical operation was clean or clean-contaminated. Patients were excluded if they had undergone emergency operation or salvage operation, which was performed less than 0.5 hour after admission or decision to avoid unnecessary morbidity and death. Patients were also excluded if they were infected before the surgery.This study was approved by the ethical committee of the first affiliated hospital of Soochow University. Written contents were obtained from all study participants or their legal representative for the patients under guardianship.15 The indicators assessed as rational use of prophylactic antibiotics are judged against these criteria.The criteria of prophylactic antibiotic use for cardiothoracic surgery were established on the basis of the published guidelines and official documents as follows: evidence-based practice guideline on antibiotic prophylaxis in cardiac surgery issued by the Society of Thoracic Surgeons Practice, the official document for rational use and standard management of antibiotics issued by NHFPC, the notice regarding national special measure scheme on clinical use of antibiotics issued by NHFPC. The main criteria of prophylaxis antibiotic use for cardiothoracic surgery are shown as follows: antimicrobial prophylaxis should be given to all patients undergoing cardiothoracic surgeries; the first- or second-generation cephalosporins (cefazolin or cefuroxime) are the antibiotics of choice. Clindamycin is reserved for cases of allergy to beta-lactams and vancomycin is recommended if presumed or known methicillin-resistant staphylococcus aureus colonization is present; the duration of antimicrobial prophylactic use should not be longer than 48 hours; the timing of the first dose should be within 0.5 to 2 h prior to the skin incision. However, the timing of the first dose is at variance in China, compared with other countries, which is within 1 hour before the surgical incision.During the intervention period, there was a dedicated clinical pharmacist (LZ) in the cardiothoracic surgery ward. The interventions were endorsed by hospital and the leadership of cardiothoracic surgery department implemented from July 2013 through December 2014. The interventions consist of: participating in ward rounds and making drug treatment plans; communicating immediately with surgeons when irrational antibiotics were prescribed; providing educational sessions and handouts about antibiotic prophylaxis for medical teams, especially the surgical residents who prescribed antibiotics and the nurses who executed prescriptions; extracting the medical records and assessing their responsible use with the help of electronic auditing system; reporting the categorized data on irrational use of prophylactic antibiotics to leadership of cardiothoracic surgery department every week.16 as surgical site associated infections occurred within 30 days for nonimplantable surgery or within 1 year for implantable surgery and one of the following criteria was satisfied: pus punctured or drained from surgical site, except drainage fluid after contaminated surgeries; purulent secretion in surgical site or fever with temperature ≥38°C, locally pain or tenderness; evidences of infection by operational, pathological, or imageological examinations; infections diagnosed by surgeons.The surgical site infections for cardiothoracic surgery were defined The data were collected from patients’ medical records, containing patients’ demographics, antibiotic selection, antibiotic utilization, and patients’ cost. The data of bacterial resistance were obtained from bacterial testing department and SSI data were obtained from the department of infection management. The data collection was conducted by a clinical pharmacist (JJM) who was blinded to the patients’ allocation status.2 and Fisher exact tests for categorical data , t tests and Mann–Whitney U tests for continuous data  to assess statistical significance respectively, with P < 0.05 considered statistically significant. Statistical analyses were performed with SPSS 18.0 .For comparison between the 2 phases , data were analyzed using χWe recorded 412 patients admitted to the cardiothoracic department from July 2011 through December 2012, including 292 (70.9%) patients who underwent cardiac operations, such as cardiac valve replacement (35.0%), bentall operation (1.9%), coronary artery bypass grafting (9.2%), endovascular repair (8.0%), and atrial septal defect repair (15.1%). Ninety-nine (24.0%) patients underwent thoracic operations, such as radical esophageal cancer surgery (7.8%), radical pulmonary tumor surgery (12.8%), and bullae resection (3.4%). The remaining patients (5.1%) underwent radical resection of cardiac carcinoma, exploratory thoracotomy, and so on   in intervention phase compared with 83.0% in baseline phase P < 0.00  compared with the baseline group  Table . The incP < 0.001). In baseline group, only 12 patients (3.5%) were given prophylactic antibiotics not more than 48 hours, while the proportion in intervention group was 36.0%. Although the improvement was obvious, the proportion was not satisfied in both groups.Likewise, there was a significant increase in the proportion of patients receiving antibiotic prophylaxis no more than 48 hours (P = 0.01), cefonicid (P < 0.001), and clindamycin (P = 0.002) were notably improved in intervention phase  than baseline group . Moreover, significant decrease was observed in intervention group for cefepime and ceftriaxone prescription (P < 0.001). As for postoperative maintenance of prophylactic antibiotics, a high rate of cefuroxime prescription (44.7%) was observed, followed by ceftriaxone (17.8%), imipenem (9.6%), ciprofloxacin (5.8%), cefonicid (5.3%), and cefepime (5.0%). Cefuroxime, cefonicid, clindamycin, and vancomycin accounted for 51.8% of the prophylactic antibiotics used. In intervention group, there was a significant increase in cefuroxime (P < 0.001), cefonicid (P = 0.006), clindamycin (P = 0.03) prescription, which accounted for 97.3% of the prophylactic antibiotics used. Other irrational antibiotics were all significantly decreased in intervention group (P < 0.01), compared with the baseline group.As shown in Table We found some patients took 2 antibiotic prescriptions at the same time, which were obviously irrational utilization. In baseline group, there were 80 (23.4%) patients took 2 prophylactic antibiotics. For example, one patient was given ceftriaxone and ciprofloxacin at the same time for prophylactic purpose. During the intervention phase, this antibiotic combination was eliminated, with no person taking prophylactic antibiotic combination.P < 0.001).Overall, there were 483 (95.1%) patients in intervention group with rational prophylactic antibiotic selection, while there were 144 (42.1%) patients in baseline group. The difference between the 2 groups was very significant (P < 0.001), with 33 (6.5%) patients replacing prophylactic antibiotics unnecessarily.Furthermore, antibiotic selection was discordant between the first dose and the postoperative maintenance in both intervention and baseline groups. Then, we found that there were unnecessary changes of prophylactic antibiotics. In baseline group, 60 (17.5%) patients replaced antibiotics unnecessarily. Compared with the baseline group, it was significantly decreased in intervention group (P = 0.02) in intervention group (3.5%) than in the baseline group (1.2%).To evaluate clinical effect of pharmacist intervention, we estimated the influences on SSI rates in the baseline and intervention groups Table . There wP < 0.001) of length of stay in intervention phase (20.9 days), compared with the baseline group (23.3 days).Meanwhile, to evaluate economic effect, we assessed the influences of pharmacist intervention on length of stay in the baseline and intervention groups Table . There wP < 0.001) between the 2 groups, with the net acquisition cost reduced for antibiotic prophylaxis as $167.4. During intervention phase, the increase in total costs resulted from the time cost of dedicated pharmacist Zhou L. The benefit-to-cost ratio, calculated by dividing mean prophylactic antibiotics cost reduction by mean incremental cost, was 11.3:1 , acinetobacter baumannii (21.4%), enterobacter cloacae (12.7%), and staphylococcus epidermidis (8.7%) among positive cultures. Then, we isolated microorganisms resistant to antibiotic agents. We found the rate of klebsiella pneumonia resistant to cefuroxime, cefazolin, cefotaxime, ceftriaxone, cefepime, cefoperazone, piperacillin, and trimethoprim sulfamethoxazole was 67.9%, 62.5%, 71.4%, 62.5%, 16.1%, 58.9%, 60.7%, and 66.1%, respectively. The rate of enterobacter cloacae resistant to cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefoperazone, piperacillin, and trimethoprim sulfamethoxazole was 68.8%, 59.4%, 53.1%, 34.3%, 56.3%, 53.1%, and 53.1%, whereas the rate of staphylococcus aureus resistant to trimethoprim sulfamethoxazole was 60.0%. During intervention phase, the leading pathogen positively cultured was klebsiella pneumonia (35.7%), followed by acinetobacter baumannii (21.4%), aeruginosus bacillus (12.1%), and enterobacter cloacae (8.4%). It was noteworthy that cefuroxime, cefazolin, cefotaxime, ceftriaxone, cefepime, cefoperazone, piperacillin, and trimethoprim sulfamethoxazole-resistant klebsiella pneumonia and cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefoperazone, piperacillin and trimethoprim sulfamethoxazole-resistant enterobacter cloacae were recovered during intervention phase, with the significant differences between the baseline and intervention groups (P < 0.05), as shown in Table staphylococcus aureus, the rate of trimethoprim sulfamethoxazole resistance was significantly recovered in 14.8% (P = 0.02), and the rate of clindamycin resistance was decreased from 60.0% to 25.9% with pharmacist interventions, but the difference between the intervention and baseline groups was not significant (P > 0.05).During baseline phase, the pathogens most commonly yielded were annii 21.%, enteroP > 0.05).After the interventions were finished, we further estimated prophylactic antibiotic utilization in cardiothoracic surgery for half a year (from January 2015 through June 2015) to assess the sustainable effect of pharmacist intervention. During this period, all active pharmacist interventions were discontinued and we collected the data of 156 patients admitted to the cardiothoracic department with average age of 56.8 years, including 97(62.2%) males (Table P > 0.05). With regard to economic aspects, no significant differences were found in the length of stay and mean prophylactic antibiotic cost between the 2 groups (P > 0.05). As for clinical outcomes, SSI rates were 1.2% and 1.4% in intervention and postintervention groups, respectively, and the difference was not significant (P > 0.05).In postintervention group, 138 (88.5%) patients received prophylactic antibiotics. There was no difference between the intervention and postintervention groups in the proportion of patient who received antibiotic prophylaxis  patients in the postintervention group were treated preoperatively (0.5–2 hours) with the first dose of prophylactic antibiotics, while the proportion in the intervention group was 97.6%. Meanwhile, the difference of antibiotic combination between the 2 groups was negligible.The analysis on the duration of antibiotic prophylactic courses yielded a similar result. A total of 36.0% and 36.2% of patients received antibiotic prophylactics for less than 48 hours in the intervention and postintervention groups, respectively.P > 0.05). Moreover, the proportion of rational antibiotic selection was compared between the 2 groups and the difference was not significant (P > 0.05).Then we tested the rationality of antibiotic selection in the postintervention group. For the first dose, the leading antibiotics was cefuroxime (78.3%), followed by cefonicid (10.1%), clindamycin (8.7%), which were all the suitable selection. For the postoperative maintenance, the antibiotics were cefuroxime (80.6%), cefonicid (8.1%), and clindamycin (8.1%). No significant differences were found in the above antibiotic selection between the intervention and postintervention groups  in prophylactic antibiotic usage, clinical and economic outcomes between intervention and postintervention phases.In addition, we evaluated the sustained effect of pharmacist intervention after intervention phase, as enlightened by a literature about interventions of antibiotic utilization in intra-abdominal infections.20 found that prophylaxis in 2 hours before surgery also reduced the risk of wound infections.The first dose of antibiotic prophylaxis was given during a period up to 2 hours before the first surgical incision which was indicated in internal policy. Besides, Finkelstein et al38 However, we did not identify any other interventions during the study periods that could potentially have influenced antimicrobial use. During the whole study period, there were no new guidelines or official documents and the medical stewardship in the cardiovascular ward was the same. Even more, we also utilized identical time periods to eliminate any potential seasonal influence. In a future study, we will implement a more intrinsically rigorous design to evaluate pharmacist interventions and will take further measures to shorten antibiotic prophylactic duration.Unfortunately, the study had several limitations. Although the proportion of patients receiving antibiotic prophylaxis not more than 48 hours was increased during intervention phase, it (36.0%) was not satisfied. Another limitation of this study was the pre-to-post quasiexperimental design. This retro-prospective study was less convincing than a randomized controlled trial.In conclusion, our study demonstrates that real-time pharmacist intervention in cardiothoracic surgery results in a profound culture change in prophylactic antibiotic prescribing practice and a high adherence to evidence-based treatment guidelines. With the improved rationality of antibiotic utilization, favorable clinical and economic outcomes are attained and bacterial resistance is decreased. When the intervention is discontinued, the effects are still sustained. The results from this study raise the atmosphere of irrational drug utilization and emphasize the role of pharmacists in clinical treatment. In the future, more efforts are needed to expand pharmacist's practice scope to manage medication activity to address clinical inertia in prescribing habits.


Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1), an important regulator of actin polymerization in cells.in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA) phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE) analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK), an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS), GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1.We performed in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with phosphorylation of a certain pool of intracellular Pfn1 on the T89 residue. In summary, we propose that T89 phosphorylation could have major functional consequences on Pfn1. This study paves the way for further investigation of the potential role of Pfn1 phosphorylation in PKA-mediated regulation of actin-dependent biological processes.We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89), a novel site. Consistent with PKA’s ability to phosphorylate Pfn1  These factors harbor poly-L-proline (PLP) domains through which they interact with profilins (Pfn), a family of actin-monomer binding proteins that strongly inhibits spontaneous nucleation and elongation at the pointed ends of actin filaments but promotes barbed-end elongation through the addition of ATP-bound monomeric actin . HEK-293 cells were maintained on culture dishes  coated with type I collagen  for all experiments. MDA-MB-231 cells  were cultured in EMEM  growth medium . For activation of cAMP/PKA pathway, cells were serum-starved for 6 hours prior to treatment with 50μM FSK  for 5–10 min. In some experiments, cells were pretreated with 10μM H89 (Sigma) or DMSO (vehicle control) for 15 min prior to changing to FSK-containing media in the continued presence of H89 (or DMSO).Mus musculus Pfn1 were introduced into either a bacterial expression vector encoding GST-Pfn1  or a bicistronic mammalian expression vector  encoding myc-Pfn1  using the primers summarized in Phosphomimetic  and phosphodead (T89A) mutations of His-tagged Pfn1  was added to kinase buffer  and incubated with or without 0.5U/μL catalytic subunit of bovine PKA (Sigma) at 30°C for 1 hour with gentle mixing every 15 minutes. The reaction was stopped by boiling the products for 5 minutes in the presence of 2-mercaptoethanol. Reaction products were run on SDS-PAGE and visualized by silver staining.In gel trypsin digestion was carried out as previously described . PKA trehttps://chorusproject.org/pages/index.html) under the project “Phosphorylation of Pfn1”.Proteolytic peptides from in gel trypsin digestion were analyzed by a nanoflow reverse-phased liquid chromatography tandem mass spectrometry (LC-MS/MS). Tryptic peptides were loaded onto a C18 column  using a Dionex HPLC system  operated with a double-split system  to provide an in-column nano-flow rate (~300 nl/min). Mobile phases used were 0.1% formic acid for A and 0.1% formic acid in acetonitrile for B. Peptides were eluted off the column using a 52 minute gradient  and injected into a linear ion trap MS  through electrospray. The LTQ XL was operated in a date-dependent MS/MS mode in which each full MS spectrum  was followed by MS/MS scans of the 5 most abundant molecular ions determined from full MS scan . Dynamic exclusion was enabled to minimize redundant selection of peptides previously selected for CID. The MS raw files have been deposited to Chorus  against a UniProt human proteome database (January 2013 release) from the European Bioinformatics Institute (http://www.ebi.ac.uk/integr8). The following modifications were used: static modification of cysteine , variable modification of methionine  and variable modification of serine/threonine/tyrosine . The mass tolerance was set at 1.4Da for the precursor ions and 0.5Da for the fragment ions. Peptide identifications were filtered using PeptideProphet™ and ProteinProphet® algorithms with a protein threshold cutoff of 99% and peptide threshold cutoff of 95% implemented in Scaffold™ .MS/MS spectra were searched using the SEQUEST search engine implemented in Proteome DiscovererCells were scraped in the presence of 2D lysis buffer  and collected into a bead-beater tube  containing 50mg Glass Beads (Sigma). Isoelectric focusing was performed using the Zoom IPG Runner System (Life Technologies) according to manufacturer’s instructions with modification. Briefly, lysates were reconstituted in rehydration buffer . IPG strips underwent isoelectric focusing using the following program: 175V for 45 minutes; linear ramp 175-2000V over 45 minutes; 2000V for 45 minutes. IPG strips were briefly washed with running buffer  and sealed on Tris-HCl polyacrylamide gels with running buffer containing 0.5% agarose (Life Technologies) and 0.005% (w/v) bromophenol blue before performing electrophoresis in the second dimension followed by immunoblotting.kip1 .Total lysates were prepared by extracting cells with modified RIPA buffer (25mM Tris—HCl (pH 7.5), 150mM NaCl, 1% (v/v) NP-40, 5% (v/v) glycerol, 1mM EDTA, 50mM NaF, 1mM sodium pervanadate, and protease inhibitors). In some experiments, SDS and/or urea were added to RIPA buffer at final concentrations of 2% and 6M, respectively. Immunoblotting conditions for the various antibodies were: monoclonal GFP , monoclonal Pfn1 , monoclonal actin , monoclonal myc , monoclonal α-tubulin , monoclonal VASP  and monoclonal p27Phalloidin staining—Cells were washed with DPBS, fixed with 3.7% formaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 5 min and then blocked with 10% goat-serum for 1 h at room temperature. Cells were incubated with rhodamine-phalloidin (Life Technologies) for an hour at room temperature. Stained cells were washed two times with PBS containing 0.02% tween, two times with PBS, and then once with distilled water before mounting on slides for imaging using a 60X oil-immersion objective on an Olympus IX71 inverted microscope.Bacteria carrying various forms of GST-Pfn1 were grown at 37°C under constant agitation in the presence of antibiotics until the optical density at 600 nm was between 0.6 and 0.9. Cells were induced with 0.1mM isopropyl-β-D- thiogalactopyranoside (IPTG) for 3 hours, and extracted with lysis buffer (25mM Tris-base (pH 7.4), 150mM NaCl, 1% NP-40, 1mM EDTA, 5% glycerol) followed by sonication. Bacterial extracts were clarified at 18000g for 30 minutes before incubating with glutathione-agarose beads (Sigma) for 2 hours at 4°C with constant rotation. Beads were washed 5 times with lysis buffer and subjected to pull-down assay with HEK-293 lysate overnight at 4°C. After washing the beads 5 times with lysis buffer, protein was eluted by boiling beads for 5 minutes in the presence of 2-mercaptoethanol and subjected to SDS-PAGE and immunoblot analyses.Mus musculus Pfn1 was created from a crystal structure of bovine Pfn1 bound to actin (PDB 2BTF). The model was created by manually performing the following mutations using Pymol: N9S, N41S, I49V, I100V, and M122L. All mutations could be performed without introducing significant steric clashes. Additional Pfn1 models were created by mutating T89 to either alanine or aspartic acid to mimic phospho-dead and phosphorylated states as well as to phosphothreonine. All simulations were performed with GPU accelerated Amber version 14 with the ff14sb force field [A homology model of ce field . Parametce field . The homce field .We performed either Student’s t-test or one-way ANOVA (when more than two groups were involved) for statistical comparison of the means between the different groups and p<0.05 was considered to be statistically significant.in vitro phosphorylation assays using recombinant His-tagged Pfn1 and the catalytic subunit of PKA. We observed acidic shift of a significant population of His-Pfn1 (unmodified isoelectric point ~6.5) as evident from several new charged states demonstrating that PKA is capable of directly phosphorylating Pfn1 at multiple residues at least in vitro (data not shown). As the protein coverage was incomplete, we could not rule out the possibility of other PKA-phosphorylation sites of Pfn1.To predict potential phosphorylation sites of Pfn1and the likely kinases associated with these sites, we performed sequence analyses of both human and mouse Pfn1 using two different bioinformatics tools , the results of which are summarized in in vitro . To idenin vitro . Note thin vitro. As spot #2 accounted for >95% of total pool of myc-Pfn1, we used the relative intensities of spot #2 at a very low exposure  between control and FSK-treatment groups as a correction factor for loading control in these experiments to quantify the fold-changes in the intensity of spot #4 in response to FSK. Using this correction factor, we estimated that FSK treatment led to approximately 9-fold (data summarized from 4 independent experiments) increase in the intensity of spot #4  is much better resolvable than endogenous Pfn1  on IEF, likely owing to its acidic nature (it circumvents the problem of reducing agent DTT migrating away the protein). We found that even under serum-starved condition, myc-Pfn1 exists in multiple charged states as evident from three distinct spots (spots #1 through 3) with spot #2 representing the predominant pool of myc-Pfn1 and an extremely weak fourth spot (#4) representing the most negatively charged form of the protein . The mos spot #4 . In a se spot #4  further Phosphopeptides of Pfn1 involving S56, S57, T89, S91, T92 have been previously found in global proteomic screens of mitotic cells, cancer and immortalized T cell lines, and endothelial cells suggesting that Pfn1 can be phosphorylated on these residues at least in some cell types under certain conditions ,19,20. Hin silico prediction of extreme destabilization of the intramolecular T89:F98 interaction upon T89 phosphorylation. Therefore, T89 appears to be a structurally important residue of Pfn1 that can be modified by phosphorylation.We next performed GST-pull-down assays of bacterially expressed forms of either WT (control)- or various phosphomimetic Pfn1 using HEK-293 cell lysate to qualitatively assess the changes in Pfn1:actin binding conferred by T89D substitution. To our surprise, the pull-down assay failed for T89D-Pfn1 as this mutant protein was largely insoluble in non-denaturing lysis buffer and therefore could not be extracted from the bacteria . T89D-Pfin vitro kinase assay condition) made little to no difference in the overall effect of Pfn1 on the kinetics of actin polymerization (data not shown). This was not totally unexpected because if only a very small fraction of Pfn1 is modified on T89 by PKA, the resulting effect would be hard to detect in a bulk polymerization assay such as the pyrene-actin assay. It is possible that PKA-mediated phosphorylation on other residues may have a confounding effect masking the effect of T89 phosphorylation.Since a direct biochemical assessment of the effect of T89 phosphorylation on actin-binding of Pfn1 was not possible due to protein insolubility issues, we considered two indirect approaches. First, we performed the standard pyrene-actin polymerization assay to determine whether PKA-mediated phosphorylation has any effect on Pfn1’s ability to inhibit actin polymerization. In the absence of PKA, Pfn1 inhibited actin polymerization by ~30% when added to actin at a 1:1 molar ratio. Unfortunately, pre-incubation with PKA  in MDA-MB-231 (MDA-231) breast cancer cells. As in HEK-293 cells, T89D-Pfn1 a) expressed at a much lower level than either WT- or T89A-Pfn1 , and b) in vitro , an alteWe finally asked whether any subcellular pool of Pfn1 is post-translationally modified on T89 even under the basal condition. Unfortunately, we were unable to directly confirm T89 phosphorylation of Pfn1 by mass-spectrometry of immunoprecipitated myc-Pfn1 from HEK-293 cells. We speculated that this could be either due to a) a possibility of only a very negligible fraction of Pfn1 that is being modified at the T89 residue, and/or b) detergent insolubility of T89-phosphorylated Pfn1  thus precluding T89-phosphorylated Pfn1 from the immunoprecipitated sample. Therefore, we adopted an indirect approach where we expressed either WT- or non-phosphorylatable T89A forms of Pfn1 as myc-tagged proteins in HEK-293 cells and analyzed their post-translational modification patterns by two-dimensional gel electrophoresis (2D-GE). We reasoned that if any cellular pool of Pfn1 is phosphorylated on T89, rendering it non-phosphorylatable should result in a basic charge shift of the isoelectric profile of Pfn1 in 2D GE. We observed multiple charged states of myc-Pfn1 with the second spot representing the predominant pool of myc-Pfn1. Rendering T89 non-phosphorylatable by alanine substitution resulted in basic shift of a small population of myc-Pfn1 . As spotin vivo events. By MDS, we established similarities between the intramolecular changes caused by actual T89 phosphorylation and T89D substitution suggesting T89D-Pfn1 reasonably mimics a T89-phosphorylated state of Pfn1. We further showed that T89D-Pfn1 exhibits hallmarks of proteins in an alternative three-dimensional conformation including protein aggregation when expressed in cells, insolubility and marking for proteolytic degradation. Therefore, T89 appears to be a structurally critical residue of Pfn1 that is a target for phosphorylation leading to significant biochemical consequence.It is well recognized that Pfn1 is a vital cog in the machinery that regulates actin polymerization in cells. Therefore, how Pfn1’s interactions with actin and other major controllers of actin polymerization are regulated is a fundamentally important biological question in the context of actin-driven cellular events. There is emerging evidence that phosphorylation plays an important role in tuning Pfn1’s ligand interactions. At present, only two phosphorylation events  have been directly linked to functional alterations of Pfn1. This study demonstrates that Pfn1 can be directly phosphorylated by PKA, and accordingly, post-translationally modified in a PKA-dependent manner in cells. We identified at least three PKA phosphorylation sites . As phosphopeptides of Pfn1 involving all of these residues were previously found in various proteomics screens ,19,20, tIn silico analyses predicted that phosphorylation of T89 could potentially result in an ‘actin-friendly’ conformation because the backbone oxygen of T89 is more accessible to Y166 of actin through destabilization of intramolecular T89:F98 interaction. T89D-Pfn1:actin co-clusters in cells even in the near absence of endogenous Pfn1 expression is consistent with a direct interaction of T89D-Pfn1 and actin, as predicted by the in silico analyses.Previous studies have mutated residues near and adjacent to T89, including R88 and K90, with no apparent impact on solubility ,23. Recein silico prediction of the increased strength of Pfn1:actin binding conferred by T89 phosphorylation consistent with reduced level of actin polymerization in cells upon T89D-Pfn1 overexpression? Pfn1 has dual effects on actin cytoskeleton. Pfn1 can either inhibit actin polymerization  or promote barbed-end directed actin polymerization. Even for barbed-end elongation, Pfn1 must also dissociate from G-actin following shuttling of G-actin to the barbed ends. If T89 phosphorylation dramatically enhances Pfn1’s affinity for actin to a point that dissociation of the Pfn1:actin complex is greatly inhibited, in an overexpression setting T89D-Pfn1 should not only competitively inhibit the endogenous Pfn1:actin interaction, but it could also act as a G-actin sequestering protein resulting in an overall inhibition of actin polymerization. There is, however, an alternative possibility that T89D-Pfn1 induces local actin polymerization at the sites of its aggregation and in turn make less G-actin available for actin polymerization elsewhere in the cell. We think this is unlikely since phalloidin staining did not reveal any F-actin clusters at the sites of T89D-Pfn1 aggregates. Although it remains to be seen whether activating the PKA pathway triggers T89 phosphorylation of Pfn1 in cells, if T89-phosphorylation of Pfn1 inhibits actin polymerization of Pfn1 as suggested by the F-actin phenotype of T89D-Pfn1 expressors in this study, it would be at least consistent with the recent finding of FSK-induced stellation of astrocytes in a Pfn-dependent manner [Is our t manner .Another interesting finding of this study was the faster protein turnover of Pfn1 conferred by the phosphomimetic mutation on the T89 residue. Although the structural basis for this observation is currently unclear, one simple explanation could be that T89D substitution affects protein folding of Pfn1, and that the misfolded protein becomes a target for degradation. Previous proteomic studies have found evidence of ubiquitination of the adjacent residue K90 ,26. ThusIn conclusion, we have identified T89 phosphorylation as a novel post-translational modification of Pfn1 which has important biochemical consequence on Pfn1. The actual biological significance of T89 phosphorylation still needs to be resolved in future studies. Although phosphorylation of other PKA sites identified herein  residues did not show any obvious change in Pfn1’s ability to co-precipitate actin and PLP ligand in a GST-pull down assay, without a quantitative binding assay, we cannot absolutely rule out subtle changes in ligand affinity of Pfn1 due to these mutations. Furthermore, whether phosphorylation of any these residues has any influence on other post-translational modifications or impacts other functionalities of Pfn1 (such as PPI binding), particularly if they occur simultaneously, need further investigation. Future studies should also examine whether PKA signaling influences actin dynamics and actin-dependent biological processes such as cell migration through phosphorylation of Pfn1.S1 FigCell lysates were immunoblotted with anti-myc antibody to demonstrate that myc-tagged T89D-Pfn1 is insoluble in non-denaturing lysis buffer.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 TableIn addition to predicted sites that are annotated by the plus sign, this table also includes a few kinase-specific experimentally validated phosphorylations, e.g. Src/Y128, PKC/S137 and ROCK/S137, even though these did not meet the cut-off or prediction accuracy criteria set in our analyses.(TIF)Click here for additional data file.


Introduction. Antimicrobial resistance is a global health issue. Studies have shown that improved antibiotic prescription education among healthcare professionals reduces mistakes during the antibiotic prescription process. The aim of this study was to investigate novel educational approaches that through the use of Augmented Reality technology could make use of the real physical context and thereby enrich the educational process of antibiotics prescription. The objective is to investigate which type of information related to antibiotics could be used in an augmented reality application for antibiotics education.Methods. This study followed the Design-Based Research Methodology composed of the following main steps: problem analysis, investigation of information that should be visualized for the training session, and finally the involvement of the end users the development and evaluation processes of the prototype.Results. Two of the most important aspects in the antibiotic prescription process, to represent in an augmented reality application, are the antibiotic guidelines and the side effects. Moreover, this study showed how this information could be visualized from a mobile device using an Augmented Reality scanner and antibiotic drug boxes as markers.Discussion. In this study we investigated the usage of objects from a real physical context such as drug boxes and how they could be used as educational resources. The logical next steps are to examine how this approach of combining physical and virtual contexts through Augmented Reality applications could contribute to the improvement of competencies among healthcare professionals and its impact on the decrease of antibiotics resistance. The widespread inappropriate use of antibiotics provokes the manifestation of antibiotic resistance organisms. Antimicrobial resistance (AMR) is one of the biggest public health challenges . The effThere are efforts being made widely around the world between cultures and regions on multifaceted and multilevel interventions that defy local barriers and beliefs. Recently reviewed educational strategies indicate the essential role of education for health-care workers, laboratory staff and the public in appropriate antibiotic use and antimicrobial resistance . DefininAugmented Reality (AR) is a technology that provides the opportunity for computer-generated virtual imagery information to be overlaid onto a direct or indirect live real world environment in real time . AR bridHowever, AR applications are not yet being used on a large scale in the educational system . AlthougIn the 2010 and 2011 Horizon reports, augmented reality has been mentioned as a promising technology for education. According to A number of studies were found for augmented reality in medical education and more specifically AR training systems for medical learning tasks such as visualizing parts of human body and laparoscopy training session with augmented reality. These studies are attempting to explore the dynamics of augmented reality for complex learning domains in medical education .Another study from Antibiotics have been considered a determining factor for saving lives and minimizing the suffering of patients for more than sixty years. The widespread inappropriate use of antibiotics provokes the manifestation of antibiotic resistance organisms. Antimicrobial resistance (AMR) is a major public health challenge . PreviouTherefore the aim of this study is to investigate how the visualization of information in medication boxes from an AR scanner may support antibiotics prescription education.A design-based research approach was applied, and consisted of four connected phases :•Analysis•Development of solutions•Iterative cycles of testing•Production of design principlesThe approach of this study is based on the principles and the basic structure of building applications for educational purposes. This research approach is being used widely in education since it investigates the innovation with the usage of technology-based initiatives because according to According to The problem as it has been set in the introduction is antimicrobial resistance. Since this global health issue is very complex, combining several scientific and social areas, this project focused on the educational structures governing this area. Different explorative methods had been used for mapping the educational field of antibiotics. For this purpose a web survey consisting of 15 questions was used in this study. This web survey was not based on any specific standard of surveys since it was the first phase of approaching the researched area, and the goals were to explore information regarding the antibiotic educational process, the prescription process in hospitals, the private prescription process, possible training sessions for antibiotic education, and courses in medical schools which are important for the participants for antibiotic knowledge. Eight resident doctors and two registrar doctors participated in this study. All of them were working within the Stockholm area in public hospitals. The recruitment was based on an open announcement in Södertälje hospital in Stockholm, Sweden. With this survey, the practitioner’s experience regarding the educational experiences they had in antibiotics as well as the tools that they currently use when they prescribe antibiotics were explored. Since the design research approach focuses on the knowledge of the practitioners and seeks to use their insights on the research and design and thereby providing potential solutions to the educational process, the collection of these data was critical for this study.In parallel with the web survey, a literature review was conducted in order to explore information regarding the educational area of antibiotics, as well as to examine which technology could possibly be useful for supporting the educational process and enriching the educational experiences. This process also supported the study by providing information from other research in similar areas. Moreover, the literature review supported the exploration of research in technological educational tools. More specifically, it focused on researchers whose main academic interests are mobile educational tools and augmented reality in the field of education.For the literature review, different databases have been used according to different science fields. The Karolinska Institutet’s e-library databases  have been used for medical and healthcare material. More specifically, research in the field of antimicrobial resistance, antibiotics, antibiotic education, public health, educational tools in antibiotic education, antibiotic guidelines, antibiotic policies, virtual patients in antibiotic education, clinical pharmacology. For the technological material, Stockholm’s University e-library databases have been used . A significant number of papers were found in the fields of mobile, mobile educational tools, augmented reality, mixed reality, augmented reality and education, mixed reality and education, augmented reality integration, augmented reality and data collection, contextual learning and ubiquitous learning.Following the study of A “think aloud” session was organized and included eight final-year undergraduate medical students from the Karolinska Institutet. The recruitment of the students was based on an open announcement. The selection criteria of this group of students were based on the fact that the designed AR prototype will be used by medical students, and the fact that the testing prototype requires that the user have a basic knowledge of antibiotics in order to understand the prototype’s principles and dynamics .Thinking-aloud tests conformed to the traditional The prototype was tested in terms of functionality and viability. A functional test plan was conducted with the usage of test cases. The test cases can reveal flaws in the functional specs. The users were asked to use their phones and install an AR scanner. In parallel, we ran a test case based on a training session for antibiotic prescription.The prototype was built on the idea that the antibiotic medication box could be useful for the antibiotic educational process since it is used in hospitals following the completion of their studies. One antibiotic box was used for this prototype (Kåvepenin) visualizing additional information when the box was scanned by a mobile device. More specifically, the Kåvepenin box was chosen as a marker. This prototype was the backbone upon which we based the “thinking-aloud” session in order to facilitate the students in understanding how AR works and what its main features are. At the beginning of the session, a short presentation took place, during which videos and images were used to show students the AR technology in the medical field as well as other fields.Following this, we started discussing the potential usage of AR. The discussion started by setting the context in which the students assumed that this prototype could fit better. The potential of using real antibiotic products and the features that AR could provide in this design were examined. Moreover, we examined the contextual framework within which the AR technology could be incorporated and designed. Whenever a discussion was loaded from information a short presentation on the projector showcased how the prototype would look. This fact triggered the beginning of new ideas regarding the prototype’s User Interface and its different functions. Notes were kept from their observations and proposals, and the conversation moved forward in this manner. For analyzing the data of the web-based questionnaire, thematic context analysis was used. This technique structures the collective answers by dividing them into categories of identified themes.From the collected information, it was concluded that physicians in a real clinical setting follow a routine when they prescribe an antibiotic to a patient. However, the process is complex and in some cases different prescription methods are used. This study explores the most common habits in the prescription process; it does not investigate individual patients’ incidents.http://www.janusinfo.se/In-English/) for the same purpose  as a guideline consulting tool and six out of ten use the “wise list” (Kloka Listan) Matters regarding antibiotic use and containment of antibiotic resistance.(2)http://en.strama.se/dyn//,85,3,78.html).Facilitates an interdisciplinary and locally approved working model, ensuring involvement by all relevant stakeholders including national and local authorities and non-profit organizations”  drug box the buttons appeared in the right side of the box as it is presented in the following figure. By clicking one of the buttons, the information appearing in the user’s display .The prototype was evaluated by the end users (students) in terms of functionality and viability. It was observed that the end users were not familiar with the augmented reality technology and its functions. The functionality test, though, shows a high level of acceptance of AR technology as a training tool. The users were satisfied with the combination of a real object (drug box) which they will use in the real context in the future, with the digital information. The process was also very fast without the need of the users to use special equipment, log in information or special training to use the application.On the other hand, some observations regarding the user interface and the functionality of the application from mobile devices should be mentioned. Some of the users found it difficult to use their mobile phones and interact with display information.Some of the student’s quotes considered for evaluating the drug box prototype are presented below:Participant 6:It seems useless to have a training session with the drug box…for me the interface is quite difficult to use it”“Participant 8The drug boxes are changing every six months approximately…probably we need to keep only the brand name as a tracker and not the whole drug box”“Participant 2:It would be useful to have a picture that refers to antibiotics and with the usage of AR it takes real life”“Participant 5:I want also in this prototype to have a video for informing the students about the causes of non-proper prescription and the antimicrobial resistance”“Participant 6:I would like to have pictures or 3D objects of current active antibiotics that the hospitals are using in Sweden and to visualize the basic guidelines of each agent”.“Antimicrobial resistance is currently one of the biggest global health threats in the world. This problem is complex and incorporates many different scientific fields. This study approached the problem from the educational perspective and attempted to answer the scientific questions that were set in order to examine the potentiality of modern technologies, such as Augmented Reality, to be applied as an educational tool in the education of antibiotics. The research question to identify the aspect that are important during a prescription process was answered. This information seeded the study in terms of prototype development.For the development of this prototype we used the Metaio Platform Beta Version. On the one hand, in this version the developer is flexible enough to develop an AR prototype in limited time. The platform also provides the opportunity to upload the prototype and test it in real time. On the other hand, in this version the development features are limited. The developer is confined to using the specific tools that the platform provides. Future studies might consider more flexible platforms such as Unity Vuforia for their prototype development.Since we know from our research what information of antibiotics can be used in antibiotic prescription training session, it is a matter of design how this information will be designed and set for AR visualization. For the drug box prototype we used 2D clickable buttons as visualized objects. We chose this structure in order to separate the different information categories.Approaching the antibiotics field from the training perspective, this study shows which information on antibiotics is important during a prescription process in a hospital. Based on this information, we developed an augmented reality prototype which aims to support the training of antibiotic prescription education. The results indicates that augmented reality technology had a high level of acceptance among the medical students who participated in this study, but the final prototype needs further improvement.This study investigated the design process of an AR prototype for supporting education about antibiotic prescription. The Design Research methodology was followed and the structure was based on its principles. This study didn’t complete more than one cycle of testing for the developed prototype. This fact should be taken under consideration. Going deeper in the methodology different methods could possibly be implemented, such as observation in real context and the examination of educational processes for antibiotics. Another possible approach would be to involve teachers in the development process and take their insights under consideration. As an alternative, the model methodology was examined: The basic principle of this methodology is “the purposeful abstraction of a real or a planned system with the objective of reducing it to a limited, but representative, set of components and interactions that allow the qualitative and quantitative description of its properties.” This alternative should be examined by the future researchers, especially by computer scientists, in terms of AR model development and AR model in medical education.http://www.janusinfo.se/In-English/) the dynamics of mobile technology and how it could be used for enriching the educational process in different areas by using real objects in real time without requiring physical presence in a study place. These dynamics need further exploration in order to identify ways to increase the interactivity, understandability and reduce complexity in medical education with mobile technology.Our results show how AR technology can be used to support a training session for the antibiotic prescription process and open the way for future research by using real objects as educational resources. It also illustrates (


Autoimmune diseases (AIDs) consist of a group of physiological disorders with highly diversified pathogenesis and clinical manifestations In this special issue, we aimed to give an overview on the most recent progress of biomarker studies on some of the important autoimmune-related diseases, such as systemic lupus erythematosus (SLE), psoriasis, systemic sclerosis (SSc), and primary Sjogren’s syndrome (pSS). Dr. Tianfu Wu’s group The prevalence of pSS is hard to be estimated due to the inconsistent classification criteria of the disease. It also varies largely among surveys in different age groups, ethnicities, and geographical regions with an obvious bias in female population SSc is a chronic multisystem AID with an estimated prevalence in the US to be around 240 cases/million adults SLE is the prototypic AID predominately affecting woman with the production of various autoantibodies as one of the major clinical manifestations. Proteomic microarray, as a powerful high throughput autoantibody screening approach, has been extensively utilized for identification of autoantibodies associated with pathogenesis of SLE. A review from Dr. Quan-Zhen Li’s group The review from Dr. Haitao Niu’s group The multiple omics technologies have greatly facilitated the discovery of biomarkers and improved our knowledge of the etiopathology of AIDs. In the meantime, the advances in high-throughput screening technologies offered excellent opportunities to identify new biomarkers potentially useful for early diagnosis, prognosis, and even prediction of therapeutic responses. Given the high complexities of the immune system and diversification of AIDs, the exploration of novel biomarkers and their underlying molecular mechanism will continue to be hot topics in the study of AIDs. Putting these articles together in this special issue might be helpful to reveal the intercommunication of the AIDs in the new bioinformatics and big data picture and may shed light on other less-studied AIDs or the discovery of universal biomarkers. In addition, novel biomarkers, especially the non-invasive biomarkers, are greatly demanded. Methodologically, application of the state-of-art novel technologies, such as NGS, as well as high-throughput proteomics and metabolomics approaches, in combination with various data analysis platforms, would greatly facilitate the discovery of multiple disease biomarkers and assessment of genes, proteins, metabolites, and network analysis of complex pathways implicated in the specific AID conditions. Comprehensive understanding of the relationship between the biomarkers and AIDs will not only provide valuable tools for disease prediction, early diagnosis, and prognosis, but also shed light on the identification of novel therapeutic target for treatment of the diseases.The authors have declared no competing interests.


Brazil was the first middle-income country to provide free and universal access to AIDS treatment. Understanding the impact of this policy is key to promote ongoing improvement of current intervention strategies. The aim of this study was to compare mortality rates and survival in a cohort of AIDS patients before and after the introduction of antiretrovirals (ARV) and to investigate predictors of survival.A retrospective cohort study of AIDS patients aged 13 years or more living in the city of Sao Paulo was conducted. All patients were recruited from an STD/HIV outpatient clinic between 1988 and 2003 and followed up until 2005. We estimated AIDS mortality rates in person-years (py) and carried out a survival analysis using the Kaplan-Meier method. The Cox proportional hazards model was used to assess predictors of survival in AIDS patients.3 (HR 1.2) and less than 350 cells/mm3 at AIDS diagnosis (HR 1.3).The study cohort comprised 6,594 patients. The yearly mean mortality rates were 17.6, 23.2, and 7.8 per 1,000 py for the study periods 1988-1993, 1994-1996, and 1997-2003, respectively. Median survival time was 13.4 and 22.3 months for patients entering the study in the first and second study periods and survival time was 108 months or more in 72% of those entering the study during 1997-2003. Factors independently associated with shorter survival included: AIDS diagnosis during the 1994-1996 (HR 2.0) and 1988-1993 (HR 3.2) periods; 50 years of age or more (HR 2.0); exposure category of injection drug users (IDU) (HR 1.5); 8 years of schooling or less (HR 1.4); no schooling (HR 2.1); and CD4+ counts between 350 and 500 cells/mmThe study showed a strong impact following the introduction of HAART in 1996 with decreased AIDS mortality, increased survival rates, and benefits with early introduction of HAART. However, some groups of patients were less likely to benefit from the new drug regimens. Public policies promoting health equity create an enabling environment helping AIDS control programs in developing countries to achieve their goals as effectively as in developed countries.The online version of this article (doi:10.1186/s12879-014-0599-8) contains supplementary material, which is available to authorized users. The introduction of highly active antiretroviral therapy (HAART) has dramatically changed the course of the HIV/AIDS epidemic. It has decreased AIDS mortality rates and both increased survival and improved quality of life of people living with HIV/AIDS . UnderstBrazil was the first middle-income country to provide in 1996 free and universal access to HAART for AIDS treatment . This poThere is a need to better understanding the impact of the Brazilian policy of universal access to AIDS treatment as many other developing countries have implemented similar guidelines in recent years. However, most studies in Brazil have focused their analyses on the first years following the introduction of HAART ,7]..7].The aim of this study was to compare mortality rates and survival of AIDS patients before and after the introduction of antiretrovirals (ARV) and to investigate predictors of survival in a cohort of AIDS patients living in the city of Sao Paulo who were diagnosed between 1988 and 2003 and followed as of the end of 2005.A retrospective cohort study of people living with AIDS in the city of Sao Paulo, southeastern Brazil, was conducted. Brazil's largest metropolitan area with a population of around 11 million, the city of Sao Paulo has been hit hard by the HIV/AIDS epidemic accounting for about 15% of all cases reported in the country .st, 1988 and December 31, 2003 and followed up until December 31, 2005  outpatient clinic. The study cohort comprised AIDS patients aged 13 years old or more living in the city of Sao Paulo with AIDS diagnosis confirmed between January 13 regardless of other existing causes of immunodeficiency.The AIDS case definition was any HIV-infected individual with at least one AIDS-defining illness and/or CD4+ counts below 350 cells/mmThe AIDS case definition for adults changed over the study period following the Brazilian Ministry of Health recommendations: 1) CDC criteria; 2) Rio de Janeiro/Caracas criteria; 3) death exceptional criterion; and 4) adapted CDC criteria. Because there was insufficient information available to apply for all cases the latest AIDS case definition, we chose to use the case definition that was in effect in each period studied . The expWe considered the date of inclusion in the study as the date of confirmation of AIDS diagnosis. Only outpatients enrolled in the CRT-DST/AIDS clinic were included in the study. There were excluded: 1) individuals infected through mother-to-child transmission or blood transfusion; 2) those who died within thirty days after AIDS diagnosis; and 3) those attending a single visit only or followed up for less than 30 days.The study variables included sociodemographic and behavior characteristics; characteristics related to diagnosis (CD4+ count at AIDS diagnosis) and treatment regimen; and time from AIDS diagnosis to AIDS-related death.Data was obtained from the following sources of information: CRT-DST/AIDS database ; Integrated São Paulo AIDS Database (BIPAIDS) which is a linkage database created from the State Data System Foundation (SEADE) death database and the São Paulo AIDS Program (PEDST-AIDS) Notifiable Diseases Database (SINAN-AIDS) ; and thest, 1988 and December 31, 2003. This database was linked to BIPAIDS, SISCEL and SICLOM databases. Of these, there were excluded 4,398 (28.1%) patients with negative confirmatory HIV tests; 996 (6.3%) who were HIV-infected, but AIDS-free; 634 (4.0%) who were not diagnosed within the study period; 2,814 (18.0%) who attended a single visit only or were followed up for less than 30 days, 147 (0.9%) who were diagnosed with AIDS at death; and 80 (0.5%) who were infected through vertical transmission. The remaining 6,594/15,663 patients (42.1%) were eligible to participate in the study.The initial cohort included 15,663 individuals enrolled as CRT-DST/AIDS outpatients between January 1A descriptive analysis of the main patient characteristics was performed in the pre- (1988-1996) and post-HAART periods (1997-2003).We estimated AIDS mortality rates in person-years (py). The numerator was the number of deaths and the denominator was total person-years at risk of death (in months). The denominator (person-years) was calculated from the date of AIDS diagnosis to the date of AIDS-related death or censoring date.Data censoring occurred at three different situations: 1) at end of the study period on December 31, 2005 for living patients at follow-up (administrative censoring); 2) at the date of death for those who died from a non-AIDS-related cause; and 3) at the date of the last visit for those lost to follow-up for one year.We conducted an AIDS survival analysis using the Kaplan-Meier method. Peto and log-rank tests were used to compare time to the event (AIDS-related death) among the groups.AIDS survival time was estimated from the date of AIDS diagnosis to the date of AIDS-related death or censoring date (in months). Kaplan-Meier curves were generated for the three AIDS diagnosis periods studied: 1988-1993, 1994-1996, and 1997-2003. Combination ARV therapy and HAART became available in the last two study periods, respectively. HAART became available in 1996. These study periods were used as a proxy for different drug regimen exposures and measures of populational effectiveness of these interventions -14]..14].(p < 0.25) and biologically plausible were included in the final Cox proportional hazards model adjusted for treatment regimen. We used the likelihood ratio test to evaluate model fit. Schoenfeld residuals were used to test the proportional risk assumption.The Cox proportional hazards model was used to assess factors associated with time from diagnosis of AIDS to AIDS-related death and to estimate crude and adjusted hazard ratios (HR) with 95% confidence intervals (95% CI). Variables that were statistically significant in the bivariate analysis This study was approved by the University of São Paulo School of Public Health research ethics committee (protocol number 1612) and the CRT-DST/AIDS research ethics committee (protocol number 211/06).The follow-up of the 6,594 patients with AIDS studied is shown schematically in Figure A comparison of patient characteristics in the pre- (1988-1996) and post-HAART periods (1997-2003) showed that 52% entered the study in the pre-HAART period.p < 0.001) and a greater proportion of heterosexuals , which can be explained by an increase in the number of non-IDU women infected, and patients with more than eight years of schooling .Compared with pre-HAART, there was in the post-HAART period a lower proportion of injecting drug users (IDU)  and the second period studied  (p = 0.283). Similarly, there was no significant difference in viral load at AIDS diagnosis between these two periods, ranging from 400 to 100,000 copies/mL in 75% of the patients (p = 0.054). However, it should be noted that only 546/3,464 (15.8%) patients had their viral load measured in the first period.Of the 6,594 patients studied, 2,936 (44.5%) died during follow-up. From 1988 to 1993, there were 1,552 deaths with a yearly mean mortality rate of 17.6 per 1,000 py. During 1994-1996, there were 731 deaths with a yearly mean mortality rate of 23.2 per 1,000 py, i.e., a 31.8% increase. From 1997 to 2003, there were 653 deaths with a yearly mean mortality rate of 7.8 per 1,000 py, a 66.4% decline in AIDS mortality rate compared to the previous period  was 10.6% and median survival time (MST) was 13.4 months (1.1 years) among patients diagnosed with AIDS from 1988 to 1993. Among those diagnosed from 1994 to 1996, the cumulative probability of survival was 24.4% and MST was 22.3 months (1.8 years). In the last period studied (1997-2003), the cumulative probability of survival over a maximum follow-up time of 108 months was 72.0%. Over 50% patients were alive at the end of the follow-up.p < 0.001).Figure Figure p < 0.001); greater survival rates in heterosexuals followed by MSM and IDU ; and increase in survival rates with increasing schooling (log-rank test for trend p < 0.001) and CD4+ count at AIDS diagnosis   and below 350 cells/mm3  : AIDS diagnosed from 1994 to 1996 , and from 1988 to 1993 ; male sex ; 50 years of age or more , white skin color ; MSM  and IDU ; 8 years of schooling or less ; no schooling ; and CD4+ count at AIDS diagnosis between 350 and 500 cells/mm3  and below 350 cells/mm3   and from 1988 to 1993 ; 30-49 years of age ; 50 years of age or more ; MSM  and IDU ; 8 years of schooling or less ; no schooling ; and CD4+ count at AIDS diagnosis between 350 and 500 cells/mmThis is one of the few studies examining survival of AIDS patients in Brazil in the pre- and post-HAART eras, most likely the latest available data -7],15],[15]7],7],. AlthougThe AIDS epidemic in Sao Paulo has taken a favorable course similar to that seen in developed countries ,18]. It. It18]. In addition to a sharp decline in AIDS mortality and a significant increase in survival time, a key finding of this study is the fact that not all patients benefited equally from new drug regimens and these benefits varied according to individual characteristics of AIDS patients. These differences, however, tended to be less remarkable after the introduction of HAART. The study found that shorter survival time was associated with being diagnosed with AIDS in the pre-HAART period (as discussed before), more advanced age, exposure category of IDU, low schooling and lower CD4+ counts at AIDS diagnosis (a proxy measure for late diagnosis).The inverse association between age and survival time found in this study is consistent with other studies that reported lower effectiveness of ARV in individuals over 50. These studies have attributed it to higher proportion of late diagnosis in this age group ,22], hi, hi22], Another important finding is that shorter survival time was independently associated with the exposure category of IDU, which is consistent with the literature. A possible explanation may be a higher prevalence of co-infection of HIV and hepatitis B and C in this group ,18],,.The independent association between low schooling and lower survival of AIDS patients may be explained by the fact that disease perception is affected by level of education; educated individuals are more likely to have an early diagnosis and adhere to treatment . If we tThe study results indicated an association between low CD4+ counts at AIDS diagnosis and lower survival, which corroborates the findings of several other studies. CD4+ count is a major biological marker of immune status -29] and and29] a3 ,,[Although factors affecting HAART effectiveness may vary in different populations and backgrounds, our results were obtained under conditions that are in effect comparable to those found in other low- and middle-income countries. These countries have greatly expanded access to ARV treatment in recent years , which sThis study emphasizes the impact of HAART introduction, lowering the mortality rates and increasing survival of AIDS patients. It indicated benefits with early initiation of HAART, and identified groups of patients that benefited less from new drug regimens. This finding should be taken into consideration to improve AIDS control programs. Our results also suggest that public policies promoting health equity create an enabling environment helping AIDS control programs in developing countries to achieve their goals as effectively as in developed countries.MVT and EAW designed and developed the study. MVT and EAW analyzed the data. MVT prepared the manuscript. MVT and EAW discussed data analysis and interpretation. MVT and EAW reviewed the manuscript and approved the final version.


Antibiotic prophylaxis has been proven to reduce surgical site infection (SSI) rates in adult cardiac surgical patients. However, current guidelines are vague and in this era of increasing antibiotic resistance it is essential that we inform and develop a consensus opinion.Our aim was to acquire an overview of antibiotic prophylaxis in adult cardiac surgical centres in the UK and compare this to national and international guidelines.We developed a 10 point questionnaire and obtained results via telephone interview of on-call cardiac surgery registrars in all UK units in April 2015.A total of 32/35 (91%) of on-call registrars responded, with the majority  being unaware of their local SSI rate. Only one cardiac centre used a surgeon-specific policy while the remaining 31 centres (97%) used a unit-specific policy. Although all units complied with guidelines, there was wide variation in practice. For indexed operations e.g. CABG and isolated valve procedures, the majority  of units used single antibiotic prophylaxis, most commonly cefuroxime . The remaining centres used combination antibiotic prophylaxis , most commonly flucloxacillin and gentamicin . Most units  prescribed prophylaxis for 24 hours post operatively, 3 units (9%) for 48 hours and 4 units (12.5%) administered only a single dose.Approximately half of centres used MRSA skin preparation bundles even when MRSA status was unknown and 7 centres (22%) routinely used anti-MRSA antibiotic agents.The results of this survey demonstrate that while all UK adult cardiac centres adhere to guidelines, there is significant divergence in policy between different centres.While it is clear that large scale multi-centre studies are required to develop more specific guidelines, particularly in terms of antibiotic selection and duration, the results of this national survey will help to inform ongoing debate and guide future policy development.