Step kinematic variability has been characterized during gait using spatial and temporal kinematic characteristics. However, people can adopt different trajectory paths both between individuals and even within individuals at different speeds. Single point measures such as minimum toe clearance (MTC) and step length (SL) do not necessarily account for the multiple paths that the foot may take during the swing phase to reach the same foot fall endpoint. The purpose of this study was to test a step-by-step foot trajectory area (SBS-FTA) variability measure that is able to characterize sagittal plane foot trajectories of varying areas, and compare this measure against MTC and SL variability at different speeds. We hypothesize that the SBS-FTA variability would demonstrate increased variability with speed. Second, we hypothesize that SBS-FTA would have a stronger curvilinear fit compared with the CV and SD of SL and MTC. Third, we hypothesize SBS-FTA would be more responsive to change in the foot trajectory at a given speed compared to SL and MTC. Fourth, SBS-FTA variability would not strongly co-vary with SL and MTC variability measures since it represents a different construct related to foot trajectory area variability.We studied 15 nonimpaired individuals during walking at progressively faster speeds. We calculated SL, MTC, and SBS-FTA area.SBS-FTA variability increased with speed, had a stronger curvilinear fit compared with the CV and SD of SL and MTC, was more responsive at a given speed, and did not strongly co-vary with SL and MTC variability measures.SBS foot trajectory area variability was sensitive to change with faster speeds, captured a relationship that the majority of the other measures did not demonstrate, and did not co-vary strongly with other measures that are also components of the trajectory. Step kinematic variability has been characterized during gait using spatial and temporal kinematic characteristics such as step length, step height, step time, and cadence –4. PartiPast studies have demonstrated that foot trajectory variability, between individuals that walk more slowly compared to individuals that do not walk as slowly, can be an important marker for reduced CWS and increased fall risk. For example, greater variability of step kinematics is associated with decreased gait stability and increased risk of falls in fall risk populations , 13–16. T). Further, to capture differences in initial swing phase versus terminal swing, we divided that area measurement into two regions from foot off to midswing (H1), and midswing to initial foot contact (H2). The foot trajectory sub-phases between individuals are relatively consistent for healthy adults. However, we divided by these sub-phases because part of this current study is to establish a metric using healthy controls for individuals where these sub-phases are less consistent (chronic poststroke hemiparesis).We sought to capture the bi-dimensionality of foot trajectory in the sagittal plane by a curvilinear approach into an area measurement on a step-by-step basis. Therefore, the purpose of this study was to test a step-by-step foot trajectory area (SBS-FTA) variability measure that is able to characterize foot trajectories of varying areas, and compare this measure against MTC and SL variability at different speeds. The SBS-FTA variable was quantified by measuring step-by-step changes in the area under the toe marker during swing phase (HWith this paper we assessed which measure of variability was most responsive to change across a range of speeds. The types of variability responsiveness tested were SL variability and MTC variability using standard deviation (SD) and CV, and SBS-FTA variability. We hypothesize that the SBS-FTA variability would demonstrate increased variability with speed. Second, we hypothesize that SBS-FTA would have a stronger curvilinear fit compared with the CV and SD of SL and MTC. Third, we hypothesize SBS-FTA would be more responsive to change in the foot trajectory at a given speed compared to SL and MTC. Fourth, that SBS-FTA variability would not strongly co-vary with SL and MTC variability measures since it represents a different construct related to foot trajectory area variability. This can provide more information about how this endpoint control is modified by some scaling factor on a step-by-step basis, like walking speed, and may provide information that can be beneficial to study impaired populations whose endpoint control is affected.We recruited fifteen non-impaired individuals . Inclusion criteria were: older than 19 years of age, ability to walk independently, medically stable, and able to provide written informed consent. Exclusion criteria were: history of serious medical conditions, uncontrolled respiratory or metabolic disorders, uncontrolled blood pressure , presence of cerebellar and brainstem deficits, severe cognitive disorder, inability to follow simple commands, and major or acute musculoskeletal problems. This study was performed at the University of Alabama at Birmingham (UAB) and written informed consent was obtained from all participants. This study was approved by UAB’s Institutional Review Board under protocol number: F141212010. The study was conducted in accordance with the principles of the latest version of the Declaration of Helsinki. Recruitment and experimental procedures were completed by the investigators.Passive reflective markers were placed bi-laterally on the feet on the second and fifth metatarsal heads, lateral ankle malleoli, and the calcaneus and were collected by an 8 camera Qualisys motion capture system sampling at 100Hz. Marker positions were tracked using QTM 2.2 offline. Marker position data was processed via Visual3D software .This current study is a sub-analysis of data from a larger dataset that also involved participants that have chronic post-stroke hemiparesis. The larger study design involved the use of a robotic safety system to allow people, at risk for falls, to experience walking at very fast speeds without risk of harm if they were to lose their balance. The device has a fall harness applied at the waist. The KineAssist (KA) Gait and Balance Robotic SystemTM  was coupled to a Bertec treadmill (Bertec Corporation) to have similar conditions to the participants with chronic post-stroke hemiparesis. The pelvic harness of the KineAssist allowed 6-degrees of freedom at the pelvis and was used as a safety mechanism as the device would detect a loss of balance as a drop in the height of their pelvis, catching the individual in the harness system and simultaneously ceased from moving forward. The biomechanics of nonimpaired individuals walking in the KA has been described in detail in a previous study .After providing consent, the participant completed an over ground 10-meter walk test for their CWS. Then, participants were prepped for the kinematic collection and placed in the KA harness. Participants underwent a short familiarization process of walking in the KA coupled to the treadmill at comfortable speeds controlled by one of the investigators. We asked participants to decide when they felt comfortable walking in the KA and then concluded the acclimation process.We then asked people to walk at least thirty strides at a range of eleven different speeds . Order of presentation of speeds was randomized to reduce any order bias and to fulfill assumptions underlying tests for statistical inference. Rest breaks were provided as needed and, if not requested, were enforced every 5 trials.nd metatarsal head. Step kinematics of ‘foot on’ and ‘foot off’ were identified using Visual3D’s gait events algorithm using the 5th metatarsal head and calcaneus’s trajectory to isolate individual steps for analysis, and the data were visually inspected to ensure accurate representation of gait events. SL was calculated by dividing the belt speed by the step frequency on a step-by-step basis. The MTC was calculated by locating the local minima of each step during the swing phase. The coefficient of variation and standard deviation (SD) were calculated for SL and MTC for each trial as below  for any of the speeds. Trials where the individual adopted a gait other than walking were identified, and removed from analysis.1 and H2 SBS-FTA variability was calculated by bisecting the swing phase with the weighted centroid position. The weighted centroid position for each step was calculated to divide the anterior-posterior trajectory at mid-swing. The weighted centroid position was calculated by (1) locating data points from foot off to foot on, (2) the median A/P coordinate was identified and used to create the centroid position (Hposition .1) and the second half of swing (H2) were calculated. They were calculated for each half using Matlab  code for polyarea that returns the area of the polygon specified by the vertices of each data point in the fore-aft and vertical direction as seen in below  and 2) the second half of swing (H2), 3) SD of SL and 4) MTC, and 5) CV of SL and 6) MTC variability for each participant) across speed as the independent variable. The best-fit curve was determined by first fitting a linear model and then a quadratic model. If the quadratic model added a significant increase in R2 (significant F change between models), compared to the lower order model, then we chose the higher order relationship. We then compared H1 and H2 SBS-FTA, MTC, and SL changes on a step-by-step basis using paired t-tests to compare the slowest speed tested to the fastest speed (without a flight phase tested) to determine which measures had the greatest magnitude of change from a slow speed to a fast speed.Cadence and step length data were plotted against speed to determine the extent to which participants increased speed by linearly increasing step length and/or cadence and to ensure the data looked normal. Mean descriptive statistics for all measures at all speeds were calculated. We conducted simple linear and quadratic function regressions for each dependent variable , H1 SBS-FTA variability, H2 SBS-FTA variability, step length variability, and minimum toe clearance variability across the range of speeds to establish to what extent the variables were associated with each other. We used an alpha level of .05 for all statistical tests. Corrections were used for multiple comparisons where applicable. All statistical analyses were performed using SPSS v.24.We conducted Kendall Tau-b correlation analyses on total SBS-FTA variability (H2 = 0.95) and cadence and speed (R2 = 0.95).We characterized the way that each participant increased speed on the treadmill by calculating step length and cadence. Every participant exhibited strong positive linear relationships between step length and speed , CV SL had a negative linear fit, however, the quadratic model was a significantly better fit (CV SL sig. F change <0.01). SD SL did not have a significant fit for linear nor quadratic models. MTC increased monotonically but did not have a significant fit for any variability measure  measures using CV. HT, H1 and H2 had more variability relative to its mean compared to SL & MTC , we expressed relative variability magnitude for SL, MTC, HSL & MTC . H1 and 1 area magnitude was 11.9% (IQR = 8.34–14.1%), H2 area magnitude was 15.9% (IQR = 11.0–22.8%), HT area magnitude was 12.4% (IQR = 9.8–15.0%), while SL magnitude was 2.0% (IQR = 1.9–2.9%), and MTC magnitude was 4.4% (IQR = 3.1–5.8%)  .τ was to 1, the stronger the correlation [1 and H2 were strongly and positively correlated with each other (rτ >0.6), and weakly (rτ <0.4) and positively correlated with SD of SL and MTC, and CV of MTC  What does it mean that SBS-FTA increases with speed and why is it so responsive to speed? 2) Why does FTA capture the relationship with speed stronger than MTC and SL? 3) What are the implications that SBS-FTA do not strongly co-vary with SL and MTC if SL and MTC are points in the trajectory?SL variability values observed with this experiment were like those reported in the literature. For SL SD, Sekiya, Nagasaki  demonstrMTC variability values, observed with this experiment, were also similar to those reported in the literature. For MTC SD does not significantly change with increasing speed according to the model, but the mean increases monotonically with speed , 25. Sim1, foot off to midswing, the initiation of swing is important since the resulting swing trajectory is thought to be directed by initial momentum of the swing [2, midswing to foot on, the foot starts to travel with maximum horizontal velocity [What does it mean that SBS-FTA increases linearly with speed and why is it so responsive to speed? Increasing variability with speed could be viewed as walking performance becoming unstable or needing to transition to a new gait state , 27. Conhe swing . Initiathe swing ,30. For velocity  while movelocity , 33. In Why does SBS-FTA capture more variability relative to its mean compared to SL & MTC at a given speed? SBS-FTA captures the changing area under the toe marker, while SL and MTC are singular spatially tied data points that act independent of each other. SBS-FTA has more possible changes in area solely based on the number of trajectories that the foot can travel through on a step-by-step basis allowing us to observe more variability relative to a mean value. This may be indicative of observing more variability in the foot trajectory that the other measures may not capture.1 and H2 only weakly positively correlated with SD of SL and MTC and with CV of MTC indicating that the H variables appear to capture a unique construct of foot trajectory area variability. The combination of the measures not strongly covarying with H1 and H2, and H1 and H2 SBS variability increasing with speed, indicates that the numerous trajectories possible are not completely dependent on MTC nor SL.Is SBS-FTA a different construct? We hypothesized that SBS variability would not strongly co-vary with SL and MTC variability measures. We observed that SBS H1 and H2 SBS-FTA variability may be a better measure to use when examining changes in variability at faster speeds when compared to SL and MTC variability. MTC and SL provide information about points along that trajectory, but they do not provide information on the behavior of the continuous trajectory of the foot on a step-by-step basis. SBS-FTA may be a tool to acquire more sensitive information about endpoint control of the foot. Additionally, while the MTC and step length variability may be appropriate for non-impaired individuals, for individuals poststroke MTC is often not systematically identifiable and the different areas that the trajectory may generate for individuals poststroke are not accounted for [Is there a case where one of the variability measures would be better to use than the others? Hnted for , 35. Tho1 and H2 SBS foot trajectory area variability measure we used is a relatively new measure, which captured the change in area under the trajectory curve of the toe marker during the swing phase. We developed this measure in order to combine the variability of foot trajectory in both the vertical and fore-aft directions. Breaking the trajectory into halves allowed us to observe the combined fore-aft and vertical trajectory during the initial and terminal swing phases. Additionally, the data that was used for this analysis was only in the sagittal plane and did not account for medial-lateral movement. Due to the primary interest of comparing H1 and H2 SBS foot trajectory area variability to SL and MTC, we decided that it was more appropriate to utilize only sagittal plane data.There are a few study limitations that should be noted. The HSBS-FTA variability was sensitive to change with faster speeds, captured a relationship that the majority of the other measures did not demonstrate, and did not co-vary strongly with other measures that are also components of the foot trajectory. SBS-FTA foot trajectory area variability was sensitive to change with faster speeds, captured a relationship that the majority of the other measures did not demonstrate, and did not co-vary strongly with other measures that are also components of the foot trajectory. Future studies may be performed to determine if these measures may be useful in identifying people at risk for loss of control at faster speeds .


Lemurs, the diverse, endemic primates of Madagascar, are thought to represent a classic example of adaptive radiation. Based on the most complete phylogeny of living and extinct lemurs yet assembled, I tested predictions of adaptive radiation theory by estimating rates of speciation, extinction and adaptive phenotypic evolution. As predicted, lemur speciation rate exceeded that of their sister clade by nearly twofold, indicating the diversification dynamics of lemurs and mainland relatives may have been decoupled. Lemur diversification rates did not decline over time, however, as predicted by adaptive radiation theory. Optimal body masses diverged among dietary and activity pattern niches as lineages diversified into unique multidimensional ecospace. Based on these results, lemurs only partially fulfil the predictions of adaptive radiation theory, with phenotypic evolution corresponding to an ‘early burst’ of adaptive differentiation. The results must be interpreted with caution, however, because over the long evolutionary history of lemurs (approx. 50 million years), the ‘early burst’ signal of adaptive radiation may have been eroded by extinction. As diveIn this paper, I define an adaptive radiation as having the following properties. Organisms exploiting new ecological opportunities are predicted to undergo rapid lineage and phenotypic diversification as they fill open adaptive zones –5. The rAnolis lizards in the Caribbean [Microcebus) to around 10 kg, while some recently extinct lemurs were as large as the largest living primate (up to 160 kg). Species vary in their dietary preferences and activity patterns, and 10–14 species live in sympatry with discrete niches [Famous examples of adaptive radiations include aribbean , Darwin'aribbean  and Lakearibbean . These raribbean , and manaribbean . The endaribbean ,16. Lemuaribbean  in additaribbean . Living e niches . Lemurs e niches . These pe niches . This hye niches , howevere niches –24  If lemurs evolved via an adaptive radiation, then I predict the lineage speciation rates would have increased rapidly following the colonization of Madagascar. Subsequently, as species diversity ‘carrying capacity’  was approached, I predict that the speciation rate should have slowed to near zero. Alternatively, if environmental change or biotic interactions have affected diversification more recently, then I would instead expect that lemurs might show an increasing speciation rate towards the present, rather than the aforementioned pattern of ‘early burst’ followed by a slowdown in rate. Lastly, a mass extinction event may have perturbed diversification dynamics, with a spike in extinction eroding the signal of an ‘early burst’. For example, global cooling and drying during the end of the Eocene and beginning of the Oligocene epochs  have bee(2) If the diversification of lemurs is an adaptive radiation, then I predict speciation rates would have been higher in lemurs than in their sister clade, the lorisiforms. By contrast, if lemurs and lorisiforms had similar ecological opportunity, then I predict speciation rates in lemurs would not have been higher than those in close relatives.(3) If the diversification of lemurs is an adaptive radiation, then I predict that the rate of phenotypic evolution was initially high but would have declined through time towards the present. By contrast, if environmental or developmental constraints prevented phenotypic diversification, I predict a constant or gradual rate of phenotypic evolution.(4) If lemurs evolved via adaptive radiation, then I predict their phenotypes diverged in relation to shifts in niches of diet and activity pattern (adaptive zones). I predict optimal phenotypes, rates of evolution and evolutionary constraints differ among niches. If lemurs exhibited adaptive phenotypic and niche divergence, then I predict lemurs should occupy a wider breadth of multidimensional ‘ecospace’ than lorisiforms. If lemur phenotypic evolution was constrained by other forces, e.g. genetic constraints, then I predict phenotypes would not differ markedly among adaptive zones and the ecospace occupied by lemurs should be no different from that of lorisiforms.Based on the ecological theory of adaptive radiation ,5 and th2.2.1.n = 101) and lorisiforms (n = 19) with multiple primate outgroups [rBayes [Megaladapis following [To test the predictions of adaptive radiation theory, I used a phylogenetic comparative approach . I leverutgroups . The phyutgroups  implemen [rBayes , with 20ollowing , the MCCollowing , and theollowing  . BAMM also uses the same search algorithm to test for shifts in the evolutionary rate of phenotypic traits, assuming a Brownian motion model of trait evolution. I used this phenotypic algorithm to estimate the evolutionary dynamics of body mass (see below for body mass details). The models implemented in BAMM assess multiple possible diversification rate shift scenarios, but assumes there are no diversification rate shifts on lineages that go extinct. This assumption causes error in parameter estimates in trees simulated to have rate shifts on extinct lineages, but not for trees simulated to have constant rates [I estimated the speciation and extinction rate dynamics on the tree using several recently developed methods. First, I tested for shifts in rates across the tree and through time using Bayesian analysis of macroevolutionary mixtures (BAMM ). Brieflnt rates . The effnt rates . Anothernt rates . The resgeiger package [In addition to the speciation and extinction analysis in BAMM, I estimated diversification dynamics using an alternative approach: modeling evolutionary diversification using stepwise Akaike information criterion, MEDUSA  implemen package  for the  package . MEDUSA  package . To accoTESS, [Neither of the above methods tests the possibility of a dramatic shift in extinction, as would be predicted if a mass extinction event perturbed diversification dynamics. Such an extinction event may erode the signal of past diversification history, making it unlikely to detect the signature of an adaptive radiation. I tested the hypothesis that extinction rate increased markedly at the Eocene–Oligocene boundary (33.9 Ma) by comparing the probability of an extinction rate shift at that time period to the probability of no rate shift in a Bayesian framework  a single time-constant diversification rate, (ii) two time-constant rates, and (iii) a single time-varying speciation rate and constant extinction rate. For each tree, I calculated summary tree statistics and compared the values from the simulated trees to the empirical tree to determine if the macro-evolutionary model parameters could generate trees similar to the empirical tree .2.3.nlme package [ape [To determine if phenotypic evolution was adaptive, I compiled a database on strepsirrhine body mass, diet and activity pattern . I tested if variation in body mass could be explained by a combined diet and activity pattern niche variable using phylogenetic generalized least-squares (PGLS) regression in the  package  with funage [ape  for R. Fage [ape . I assesage [ape   Estimates of diversification rate variation through time did not support the prediction of high lineage diversification rates early followed by a decline in rates towards the present. Instead, initial diversification rates were low and increased towards the present, with no evidence of a slowdown in rates (figure 2Cenozoic  or depen−1) was double the rate estimated for crown lorisiforms . These results suggest that diversification dynamics in lemurs and lorisiforms may have been decoupled. Analyses of the whole strepsirrhine tree using automated detection of diversification shifts did not support a significant shift between lemurs and lorisiforms, however, suggesting that the support for a shift in rates is equivocal . Instead, a significant rate shift was inferred for the lemur genus Microcebus, which exhibited higher speciation rates than expected compared with the background speciation rate of the tree .(2) Supporting the prediction from adaptive radiation theory, the speciation rate estimated for the crown lemurs (approx. 0.15 lineages Myg RPANDA ). Modelsb; electronic supplementary material, table S7 and Results). The ‘early burst’ pattern has been difficult to detect with real datasets [(3) Concordant with predictions of adaptive radiation theory, the rate of lemur body mass evolution was highest after the colonization of Madagascar and declined towards the present (figure 2datasets . There w(4) Testing if phenotypes diverged adaptively, phylogenetic regressions support the prediction that body mass differed among adaptive zones ; nocturnal folivores and nocturnal omnivores had lower body masses than the mean, and the best-fitting phenotypic evolution models were Blomberg's g (early burst) and OU . These results support the early burst prediction that lineages co-evolved body mass with diet and activity niches, and that the rate of body mass evolution decelerated over time. Assessment of model adequacy, however, suggested there was rate heterogeneity across the tree .The major findings are presented in line with the predictions in the Introduction.To account for this heterogeneity in rates, I used generalized state-dependent OU models  to test As expected for an adaptive radiation, lemurs diversified to fill a wider breadth of multidimensional ecospace than lorisiforms . Two fam4.4.1.This study tested the long-held hypothesis that lemurs are an adaptive radiation and sheds light on the dynamics of lineage and phenotypic diversification. I found little evidence that lemur diversification dynamics fit the predictions of adaptive radiation theory. There was partial support for the prediction that lemur lineage diversification was higher than lorisiform rates—lemur speciation rates were twice that of lorisiforms, though a significant shift in diversification dynamics was not detected across the whole strepsirrhine tree. Contrary to predictions, there was no support for declining speciation or diversification rates, as would be expected if an equilibrium had been reached. Supporting the adaptive radiation hypothesis, lemur body mass evolution exhibited the ‘early burst’ pattern of high rates of change after colonizing Madagascar and declining rates towards the present, despite the fact that the ‘early burst’ pattern of phenotypic evolution is rarely observed in empirical datasets . LorisifFor an evolutionary radiation to be considered adaptive, the phenotypic evolutionary dynamics must reflect adaptations to rapidly diverging niches ,4. Indee4.2.Anolis lizards [The ecological limits hypothesis predicts that diversification rates decrease with increasing species richness as speciation and extinction rates reach a diversity-dependent equilibrium . I obser lizards ) may havLess attention has been paid to mechanisms causing increasing diversification rates through time, possibly because increasing rates are much less commonly observed . EvidencAnother factor that could explain prolonged positive diversification rates include biogeographic evolution. Geographical isolation and vicariance events lead to speciation , and vicEulemur [Microcebus [Eulemur [Microcebus [Microcebus species were between 1.15 and 3.84 Ma (interquartile range), with only two species pairs with divergence times less than 1 Ma. The relatively deep divergences, genetic and phenotypic differentiation, and biogeographic separation of species support higher taxonomic richness than was recognized in the past, but the true number of lemurs is still debated.One methodological artefact that may drive the observed increasing diversification rates in lemurs is the taxonomic richness recognized. The latest taxonomic assessments suggest there are approximately 100 species , while 5Eulemur , Microcecrocebus ). Diverscrocebus  suggeste[Eulemur  and Micrcrocebus  species.crocebus , the mea5.Lemurs are a relatively old primate group, characterized by their endemism on Madagascar, their species richness, and the unique ecological niches they occupy. This study tested predictions from the ecological theory of adaptive radiation. Lemur speciation rates were higher than those of their sister group, but there was no evidence of a slowdown in diversification rates. Therefore, based only on diversification dynamics, lemurs may not be considered an adaptive radiation. During the long lemur evolutionary history, extinction may have dampened the signal of past diversification dynamics, and without fossils it is impossible to corroborate the results from phylogenies with hard evidence. Lemur body mass evolution exhibited the early burst pattern, with phenotypic differentiation among lineages that occupy different niches early followed by declining body mass evolutionary rates. From these results, I argue that lemurs fit the phenotypic predictions of an adaptive radiation, but diversification dynamics based on the phylogeny of extant and recently extinct species do not support the predictions of a coincident exceptional burst of speciation.


Vincent and colleagues discussed some of the key issues related to antibiotic management in the critically ill, including problems associated with timing, duration, and dosing of antibiotics . In partVincent et al.’s review covered all infections in the critically ill but, with regard to the duration of antibiotic treatment, the authors referred to a guideline for the management of sepsis and septic shock only . DichotoAmong others, the consciousness state of the critically ill seems important for the initiation or discontinuation of antibiotic treatment. Ventilator-associated pneumonia is the most frequent intensive care unit-related infection in patients requiring mechanical ventilation, and comatose patients present a high risk of early-onset ventilator-associated pneumonia . For comDoctors in Western countries may say that it takes seven days to cure a common cold with medication but it will take a week to get better without it, but this advice does not apply to developing countries like China. Subsequent bacterial infections may prolong a common cold to as long as one month. The use of antibiotics varies substantially, even among developed countries. This discrepancy is particularly important for pediatric patients and the elderly with poor health. In conclusion, for antibiotic medication, general recommendations should be tempered by awareness of many local and specific factors in order to get the best effect from medicines. We thank Drs Jia and Jia for their interest in our recent review on antibiotic therapy . Indeed,Drs Jia and Jia also mention that antibiotic “prophylaxis” may reduce the occurrence of ventilator-associated pneumonia in brain-injured patients . NeverthRegarding the last statement on the use of antibiotics in patients with a common cold in developing countries, we respectfully disagree. Recommendations to rationalize the use of antibiotics in patients with acute respiratory infections have the common objective of minimizing unnecessary antibiotics because “antibiotic pressure” is one of the factors triggering bacterial resistance. Antibiotics are widely misused in medicine, especially for viral infections. Should subsequent bacterial infection develop, appropriate targeted antibiotics should then be started.


Microalgae have the potential to rapidly accumulate lipids of high interest for the food, cosmetics, pharmaceutical and energy (e.g. biodiesel) industries. However, current lipid extraction methods show efficiency limitation and until now, extraction protocols have not been fully optimized for specific lipid compounds. The present study thus presents a novel lipid extraction method, consisting in the addition of a water treatment of biomass between the two-stage solvent extraction steps of current extraction methods. The resulting modified method not only enhances lipid extraction efficiency, but also yields a higher triacylglycerols (TAG) ratio, which is highly desirable for biodiesel production.2O (Chl/Met/H2O) and dichloromethane/methanol (Dic/Met) showed respective lipid extraction yield enhancement of 72.3, 35.8, 60.3 and 60.9%. The modified acetone method resulted in the highest extraction yield, with 68.9 ± 0.2% DW total lipids. Extraction of TAG was particularly improved with the water treatment, especially for the Chl/Met/H2O and Dic/Met methods. The acetone method with the water treatment led to the highest extraction level of TAG with 73.7 ± 7.3 µg/mg DW, which is 130.8 ± 10.6% higher than the maximum value obtained for the four classical methods (31.9 ± 4.6 µg/mg DW). Interestingly, the water treatment preferentially improved the extraction of intracellular fractions, i.e. TAG, sterols, and free fatty acids, compared to the lipid fractions of the cell membranes, which are constituted of phospholipids (PL), acetone mobile polar lipids and hydrocarbons. Finally, from the 32 fatty acids analyzed for both neutral lipids (NL) and polar lipids (PL) fractions, it is clear that the water treatment greatly improves NL-to-PL ratio for the four standard methods assessed.Modification of four existing methods using acetone, chloroform/methanol (Chl/Met), chloroform/methanol/HWater treatment of biomass after the first solvent extraction step helps the subsequent release of intracellular lipids in the second extraction step, thus improving the global lipids extraction yield. In addition, the water treatment positively modifies the intracellular lipid class ratios of the final extract, in which TAG ratio is significantly increased without changes in the fatty acids composition. The novel method thus provides an efficient way to improve lipid extraction yield of existing methods, as well as selectively favoring TAG, a lipid of the upmost interest for biodiesel production.The online version of this article (doi:10.1186/s12934-017-0633-9) contains supplementary material, which is available to authorized users. Microalgae is an attractive platform for lipid production , 2. Micr2O), for lipid extraction from a wide range of biological materials [A short series of solvent-based methods have been largely used to perform lipid extraction from various biological materials. The Folch method  consistsaterials . More reaterials  as a subaterials . These mChlorella vulgaris, Araujo et al. [2O) [C. vulgaris, and showed that extraction efficiency level was higher using chloroform/methanol 1:1, then for chloroform/methanol 2:1, followed by dichloromethane/ethanol 1:1, hexane/isopropanol 3:2, acetone, diethyl ether, and methyl-tert-butyl ether/methanol 10:3. For the marine microalgae Tetraselmis sp., Li et al. [2O [2 [Isochrysis galbana, Grima et al. [2O 1:2:0.8, followed by hexane/ethanol 1:2.5, hexane/ethanol 1:0.9, butanol, ethanol, ethanol/H2O 1:1, and hexane/isopropanol 1:1.5. As it can be seen, lipid extraction efficiency differs with biomass type as well as with the solvent mixture.Comparative studies have been done with different microalgae species using different extraction systems. For the microalga o et al.  revealedal. [2O) , 12 is mal. [2O) , followeal. [2O) , while lal. [2O)  and soxhal. [2O) . Ryckeboal. [2O)  exploredi et al.  revealedi et al.  was the i et al. , Chl/Met al. [2O , 12, sup. [2O [2  and fina. [2O [2 . For Isoa et al.  have als2O [In this work, we thus test the hypothesis that a water treatment step added to current extraction protocols, between the two organic solvent extraction steps, increases cell material disruptions with an enhancement of lipid release from the cell. The four different extraction methods largely used for algal lipid extraction  were thChlorella protothecoides was cultivated under heterotrophic condition for biomass and lipid accumulation [g for 10 min, and were vacuumed (remove extra water) and freeze-dried  to determine the dry weight. Then the freeze-dried biomass was ground into a fine powder for subsequent extractions.mulation . The modmulation  was usedA mass of 35 mg of dried microalgae was used in each experiment. The four non-modified original extraction methods were applied in four control groups as detailed below.g at 4 °C for 5 min. Supernatants were transferred to a new test tube for lipid analysis, and the remaining cell pellets were re-extracted repeating the procedure.35 mg of dry samples were extracted with 5 mL of acetone under ultrasound in ice water for 30 min, and centrifuged at 4000g at 4 °C for 5 min. Cell pellets were kept for a re-extraction step and supernatants were transferred to a new test tube with 1.875 mL of H2O and shaken vigorously following a centrifugation at 4000g at 4 °C for 5 min. Then the lower layer of 5 mL chloroform with extracted lipids were pipetted out for lipid analysis. The remaining cell pellets were re-extracted repeating the procedure.35 mg of dry microalgae samples were extracted with 7.5 mL of a mixture chloroform/methanol  under ultrasound in ice water for 30 min. The mixture was centrifuged at 4000g at 4 °C for 5 min. Then the lower layer of 5 mL chloroform with extracted lipids were pipetted out for lipid analysis. The remaining cell pellets were re-extracted repeating the procedure.35 mg of dry microalgae samples were mixed and homogenized with 5 mL of methanol, 2.5 mL of chloroform and 5 mL of water. The mixture was treated under ultrasound in ice water for 20 min. Another 2.5 mL of chloroform was added to the mixture and sonicated for 10 min. Then the mixture was centrifuged at 40002O. Lipids were then within the 5 mL dichloromethane phase. The remaining cell pellets were re-extracted repeating the procedure.This method was the same as the Folch et al. method. However, all extractions used dichloromethane/methanol  instead of chloroform/methanol. In order to layering the extracted mixture, 1.625 mL KCL solution (0.88%) was used instead of 1.875 mL H2O (deionized) and vortexed for 30 s at room temperature, and then centrifuged at 4000g for 5 min at room temperature; the treatment was done only once. After centrifugation, the aqueous phase extractions were also kept for total lipids quantification, but the concentration levels were all around or below the detection limit, thus confirming that no detectable amounts of lipids were released in the water phase. Solvent phases obtained from the first and second extractions are defined as stage 1 and stage 2 respectively in both control and test groups.Lipid extraction in the four test groups was carried out according to the four control groups (see above) with the following modifications. The 35 mg of dry microalgae samples were extracted two times as in the above-mentioned methods, but prior to the second solvent extractions, the pre-extracted fresh cell pellets were re-suspended in 5 mL dH2. Then each sample was re-suspended in 0.1 mL of acetone, and 0.9 mL of 1.5% sulfosalicylic solution was added. Each sample was shaken vigorously followed by a 30 min standing. The sample absorbance is read at 440 nm by UV–VIS determination  [C. protothecoides cells harvested at growth steady state [0.1 mL of extracted solvents were pipetted out from each solvent phase and evaporated under a stream of N UV/VIS) , 43, and2 and each sample was re-suspended in 500 µL dichloromethane to analyze lipid classes. Lipid classes were identified by TLC–FID according to Parrish’s method [All remaining solvent phases (~4.9 mL) collected in each group were evaporated under a stream of Ns method .Lipids were separated into polar  and neutral fractions  by column chromatography on silica gel micro-columns (30 × 5 mm I.D. Kieselgel 70–230 mesh Merck) as described in Marty’s method . The neuThree replicates were carried out for each experiment samples, and the variation within the replicates were assessed by calculating the standard deviation of the means. Evaluation of differences between the different extraction systems were carried out by analyses of variance (ANOVA) .2O (39.3 ± 13.5% DW in test and 13.6 ± 1.1% DW in control) and 3.0-fold using Dic/Met (38.2 ± 0.6% DW in test and 12.6 ± 0.5% DW in control). Lipid extraction efficiency thus improved by 72.3, 35.8, 60.3 and 60.9% respectively for acetone, Chl/Met, Chl/Met/H2O and Dic/Met by adding a water treatment between the two solvent extraction stages, which usually performed successively.In the present study, we evaluated a modification to current extraction methods for lipids in microalgae, adding a water treatment between two successive solvent extraction stages. The first solvent extraction stage was performed under the same condition in both control and test groups for the four different methods, with total lipids of 26.7 ± 1.1% DW in control and 26.5 ± 2.6% DW in test for method A; 17.4 ± 0.6% DW in control and 16.7 ± 7.9% DW in test for method B; 28.8 ± 0.1% DW in control and 28.7 ± 0.6% DW in test for method C; 26.1 ± 3.9% DW in control and 24.1 ± 4.0% DW in test for method D , 59.2 ± 15.5% (Chl/Met), 57.7 ± 0.4% (Chl/Met/H2O) and 61.3 ± 3.6% (Dic/Met) of the final lipids yield.Attempts have been done to enhance lipid extraction yield by adding more solvent to wash the post-extracted biomass, or washing the post-extracted biomass with the extracted mixture (solvent and lipids mixture), but without any improvement . Our resC. protothecoides biomass may rely on culture condition but it was reported reaching between 14.6 and 57.8  [ = 7.89, P < 0.05) and Chl/Met/H2O  = 249.93, P < 0.0001), which is in agreement with literature [ = 639.15, P < 0.0001), but not statistically different to that from Chl/Met/H2O and Dic/Met method  = 1.08, P = 0.397). We then moved further characterizing the effect of the water treatment on extracted lipids composition.Our data show that the total lipid extraction yield differs among the four original extraction methods. Lipid content in , w/wDW) , a range2O) and 34.0 ± 2.4% (Dic/Met). Moreover, HC was higher in Chl/Met/H2O (36.2 ± 1.0%) and Dic/Met (26.4 ± 0.2%), while PL was higher in Chl/Met (34.1 ± 0.2%) and Dic/Met (20.0 ± 0.1%). The water treatment affected differently the resulting lipid class distribution profile in the second solvent extraction phase depending on the method, but shows generally increased extraction yields. The second extraction stage led to significantly increased levels of HC in Chl/Met for both control (2.1 ± 0.1 µg/mg in stage 1 and 17.3 ± 0.3 µg/mg in stage 2) and test group (2.0 ± 0.2 µg/mg in stage 1 and 21.6 ± 0.5 µg/mg in stage 2). Using Chl/Met/H2O and Dic/Met also showed a high extraction efficiency for HC at the second stage with no significant effect of the water treatment, while acetone seems less efficient for HC extraction. Meanwhile, extraction of FFA, ST and AMPL was higher (or comparable) in the second stage for both control and test groups in all four methods. However, comparing control and test groups, HC extraction was only slightly improved in Chl/Met by water treatment (17.3 ± 0.3 µg/mg in control and 21.6 ± 0.5 µg/mg in test respectively), not significantly improved in acetone (2.2 ± 0.1 µg/mg in control and 2.4 ± 0.3 µg/mg in test respectively) and Dic/Met (11.8 ± 3.9 µg/mg in control and 12.1 ± 0.7 µg/mg in test respectively), while it was similar for Chl/Met/H2O (15.4 ± 1.1 µg/mg in control and 15.4 ± 0.9 µg/mg in test respectively). However, TAG, ST and PL revealed a high sensitivity to water treatment as showed by the significant extraction improvement in test groups compared to control groups in all four methods  and 11.5 ± 1.9-fold (Dic/Met) for the control groups in stage 2. Our results thus clearly show that the water treatment specifically favored the extraction of intracellular fractions of TAG, ST, and FFA compared to the membrane fractions of AMPL and HC (Table The major lipid classes identified include HC (hydrocarbons), TAG (triacylglycerols), FFA (free fatty acids), ST (sterols), AMPL (acetone mobile polar lipids) and PL (phospholipids) Fig. . HC are O 36.2 ± .0% and DO 36.2 ± .0% and D2O and Dic/Met method respectively) compared to control , with reduction of HC-to-total lipids ratio of 3.8, 6.6, 16.2 and 13.3% for acetone, Chl/Met, Chl/Met/H2O and Dic/Met method respectively . Chl/Met method led to the highest extraction levels of PL and HC . However, AMPL extraction level was similar in the four methods  composition and structure, such as carbon chain length and unsaturated degree, greatly affect the properties of resulting biodiesel , 52, theThe same result has also been reported in , where t2O and Dic/Met methods (6.9 ± 1.5 and 4.9 ± 0.5-fold of that in control respectively), followed by acetone (2.4 ± 0.2-fold) and to a lesser extent in Chl/Met method (1.3 ± 0.2-fold). However, C18:1n9 reached a similar final extraction yield of 66.9 ± 1.9 µg/mg in all methods after water treatment. Indeed, C18:2n6, C18:3n3, C16:0 and C18:0 all showed similar trends with a significant improvement using Chl/Met/H2O and Dic/Met, than acetone and Chl/Met. However, fatty acids in PL fraction differ from that in NL fraction, with C18:2n6 the predominant component in all methods. With a water treatment, extraction efficiency of all five components were improved in acetone, Chl/Met/H2O and Dic/Met methods at different extents. However, Chl/Met method resulted in a slightly but significant lower extraction efficiency than the control group  but more PL extracted . Acetone method thus shows the highest selectivity level for neutral lipids, with extraction yield ranked as acetone method > Chl/Met method > Dic/Met method > Chl/Met/H2O method. However, the PL extraction yield in stage one was ranked as Dic/Met method > Chl/Met/H2O method > Chl/Met method > acetone method  were increased compared to that in control groups . Indeed, a water treatment led to 2.1, 1.3, 5.1 and 3.8-fold that in control groups for acetone, Chl/Met, Chl/Met/H2O and Dic/Met method respectively. However, PL extraction in test groups was only improved in acetone, Chl/Met/H2O and Dic/Met methods , and resulted in lower yields than control in Chl/Met method (0.8 ± 0.0 of that in control). Therefore, the NL-to-PL ratio is greatly improved with a water treatment  compared with control . Of interest, the neutral lipids fraction is mainly stored in the cell while polar lipids fraction is mainly within the cell membrane, suggesting H2O treatment favors the release of intracellular storage lipids.We then compared extraction methods analyzing the partition of extracted fatty acids in neutral lipids fraction (FA-NL) and in polar lipids fraction (FA-PL) Fig. . Before C. protothecoides, lipid extraction yields efficiency is ranked as acetone-based method > Chl/Met/H2O method > Dic/Met method > Chl/Met method. This ranking agrees with the polarity degree of the extraction solvents; acetone and Chl/Met/H2O polarity being higher than Chl/Met and Dic/Met. Indeed, our results suggest that the water treatment increases solvent mixtures polarity and thus explains the resulting enhanced extraction yields. Moreover, since the cell membrane mainly contains polar lipids, the use of polar solvents could increase lipids diffusion phenomenon, as suggested by Araujo for acetone [Tetraselmis sp., a result that the authors explained by the lower polarity of hexane over the hexane & ethanol mixture. Rychecosch et al. [C. protothecoides and Scenedesmus dimorphus. Structural and composition differences of algal species may explain differences in extraction protocols efficiencies.The key step in the extraction and recovery of lipids from microalgae relies on their release from intracellular compartment, where stands the major lipid pool . Moreove acetone . It has  acetone . This re acetone  observedh et al.  and Lewih et al.  also demh et al.  showed t2O is a polar solvent having a high activity level that is thought to contribute perturbing cell membrane permeability, which is already highly weakened from the use of solvents in stage one. In addition, a hypotonic environment generated adding pure water results in the increase of cell volume  more available to the less-polar solvent mixture Chl/Met. However, two times successive solvent extraction stages shown leading to a similar effect, as shown in Chl/Met and Chl/Met/H2O with the release of HC from the membrane, no matter whether water treatment is applied or not.It is thus clear from this work Fig.  as well 2O treatment could lead to different lipid class compositions and significantly improve the sum of fatty acids extracted, the effect on the FA composition was less important. The most abundant FAs in the lipid extracts include C16:0, C18:1n9, C18:2n6 and C18:3n3. The FAs composition was not affected by the water treatment, with final FAs composition in each method being similar in control and test groups. For instance, acetone method led to 12.7% of C16:0 in control group and 12.4% in test group, 46.4% of C18:1n9 in control group and 49.1% in test group, 21.0% of C18:2n6 in control group and 20.2% in test group, 16.9% of C18:3n3 in control group and 15.4% in test group, and ~ 3.0% of other fatty acids in both control and test groups. Moreover, FAs composition was also found similar in the four methods, modified or not, compared stage by stage, which suggests that different extraction methods studied have limited impact on FAs composition selectivity, as proposed by Li [Finally, although Hed by Li . The mosed by Li .In the present work, we have clearly demonstrated that the classical extraction methods can be significantly improved from the addition of a water treatment between the two solvent extraction steps. However, all these methods were historically based on the use of dry microalgae biomass, while recent developments in the field propose the use of fresh biomass. Avoiding the drying process allows reducing process energy and costs, as well as it enables a positive energy balance between the process energy and that extracted from the microalgae biomass (e.g. biodiesel) . TherefoFinally, recent approaches propose replacing the use of ultrasounds to perform microalgae cells disruption , 57 with2O treatment between two-stage solvent extraction processes thus allows increasing the extraction efficiency, most probably through further perturbing cell membrane porosity and integrity. Furthermore, re-suspending the cells in pure H2O before extraction stage 2 increases the polarity of solvent mixture in the cellular vicinity thus enhancing the second stage extraction efficiency. The selection of the proper solvent system is crucial to the extraction process, because it may affect solvent penetration of the cell membrane and therefore lipids extraction. Finally, we conclude inviting to re-visit current productivity levels of microalgae bioprocesses by modifying extraction protocols adding a water treatment.Through the modification of four classical lipids extraction methods this study clearly demonstrated that water treatment of biomass after the first solvent extraction phase favors the release of intracellular lipids in the second solvent extraction step. Total lipid extraction yield as well as intracellular lipid class ratios in the final extract were thus significantly increased by the water treatment. The neutral-to-polar lipid ratio is also greatly improved after the water treatment, and the preferable lipid component TAG showed being increased up to 130.8% compared to the original extraction methods. H


YLFSVHWPPLKA) specifically targets c-Met receptor. Here, we evaluated 18F-labeled Met-pep1 for PET imaging of c-Met positive tumor in human head and neck squamous cell carcinoma (HNSCC) xenografted mice.c-Met is a receptor tyrosine kinase shown inappropriate expression and actively involved in progression and metastasis in most types of human cancer. Development of c-Met-targeted imaging and therapeutic agents would be extremely useful. Previous studies reported that c-Met-binding peptide  ([18F]FP-Met-pep1). The cell uptake, internalization and efflux of [18F]FP-Met-pep1 were assessed in UM-SCC-22B cells. In vivo pharmacokinetics, blocking and biodistribution of the radiotracers were investigated in tumor-bearing nude mice by microPET imaging.c-Met-binding peptide, Met-pep1, was synthesized and labeled with 4-nitrophenyl [18F]FP-Met-pep1 was over 55% with 97% purity. [18F]FP-Met-pep1 showed high tumor uptake in UM-SCC-22B tumor-bearing mice with clear visualization. The specificity of the imaging tracer was confirmed by significantly decreased tumor uptake after co-administration of unlabeled Met-pep1 peptides. Prominent uptake and rapid excretion of [18F]FP-Met-pep1 was also observed in the kidney, suggesting this tracer is mainly excreted through the renal-urinary routes. Ex vivo biodistribution showed similar results that were consistent with microPET imaging data.The radiolabeling yield for [18F-labeled c-Met peptide may potentially be used for imaging c-Met positive HNSCC cancer in vivo and for c-Met-targeted cancer therapy.These results suggest that  Mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase for hepatocyte growth factor (HGF), and activation of c-Met can lead to tumor progression, metastasis and angiogenesis –3. Studiet al. identified a c-Met binding peptide  targets explicitly c-Met receptor from a phage display of a combinatorial peptide library  is one of the most widely used positron emission isotopes for PET imaging because it has good imaging characteristics and ideally suited half-life (about 110 min) for the small-molecular-weight peptides -NPFP and further evaluated the in vitro cell uptake, internalization, and efflux studies on UM-SCC-22B cells and in vivo distribution pattern and c-Met-targeting efficacy using microPET imaging in HNSCC xenograft-bearing mice.Several attempts are being made to improve image quality and quantification using various radioisotopes. Fluorine-18 + = 1457.6571 (m/z), calc: 1456.7867.The c-Met binding peptide  with 20 mg of bis-4-nitropenyl carbonate (BNPC) in 0.3 ml of acetonitrile. The final product was purified by HPLC on a semi-prep Phenomenex Luna C18 column. The desired product was collected (Rt = 29 min) and trapped on a Waters Sep-Pak Plus C18 cartridge and eluted with 1 ml CH2Cl2 to a plastic tube.The 4-nitrophenyl [ications . Briefly2Cl2 in the tube containing [18F]NPFP was removed with argon flow at room temperature and 1.0 mg of Met-pep1 in 0.1 ml of DMSO containing 20 μl of DIPEA was added to the tube and heated at 80°C for 10 min. The reaction mixture was cooled and diluted with 0.7 ml of water containing 25 μl of acetic acid and injected onto a semi-prep HPLC column (Vydac C18) running a linear gradient starting from 5% A (0.1% TFA in acetonitrile) and 95% B (0.1% in water) for 2 min and increasing A to 65% at 32 min at 5 ml/min. The radioactive peak at retention time of 23.1 min was collected and added 10 ml of water and trapped on a Varian Bond Elut C18 column (100 mg). The radioactivity trapped on the C18 column was eluted with 0.3 ml of 1 mM HCl ethanol solution and the solvent was evaporated with argon flow and the product was re-dissolved in normal saline for further use.The CHnu/nu mice (Harlan Laboratories). Typically, 5 × 106 cells suspended in 50 μl of PBS were injected in the right or left front shoulder. Tumor growth was followed by caliper measurements of perpendicular diameters of the tumor. The tumor volume was estimated by the formula: tumor volume = a×b2 /2, where a and b were the tumor length and width, respectively, in millimeters. Small animal PET studies were performed when the tumor volume reached 100 to 200 mm3.The human head and neck squamous carcinoma (HNSCC) cell line UM-SCC-22B (obtained from the University of Michigan) was used in this study. The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 100 unit/ml penicillin, and 100 mg/ml streptomycin . Subcutaneous UM-SCC-22B tumor model was established in 4 to 6 weeks old male athymic 5 cells per well and incubated with 18.5 kBq (0.5 μCi)/well of [18F]FP-Met-pep1 at 37°C for 15, 30 and 60 min. Tumor cells were then washed twice with cold PBS and harvested by adding 250 μl of 0.1 N NaOH. Internalization assay was preformed similarly to the procedure described above. After 15, 30 and 60 min incubation of UM-SCC-22B cells with [18F]FP-Met-pep1 at 37°C, the cells were washed twice with cold PBS and then incubated for 1 min with acid-washing-buffer  to remove surface bound radioactive ligand. Thereafter, the cells were washed twice with cold PBS and harvested by adding 250 μl of 0.1 N NaOH. The blocking assay was preformed similarly to the procedure described above except that the unlabeled c-Met binding peptide was added into the wells (500 μg/well) and incubated for 15 min at 37°C before adding [18F]FP-Met-pep1. For efflux experiment, all UM-SCC-22B cells in a 24-well plate were initially incubated with 18.5 kBq (0.5 μCi)/well of [18F]FP-Met-pep1 at 37°C for 1 hour. The cells were then washed twice with cold PBS, and incubated with serum free DMEM medium for 15, 30 and 60 min. At each time point, the cells were washed twice with PBS, and harvested by adding 250 μl of 0.1 N NaOH. The cell suspensions were collected and measured in a gamma counter and the cell uptake, internalization and efflux were expressed as the percentage of the added dose (%AD) after decay correction to EOB. Each data point is an average of triplicate wells.For cell uptake measurement, UM-SCC-22B cells were seeded into a 24-well plate at a density of 1×102 (1.5–2% v/v) and intravenously injected with 3.7 MBq (100 μCi) of [18F]FP-Met-pep1 in a volume of 100 μl saline. For blocking experiments, [18F]FP-Met-pep1 was co-injected with 500 μg of unlabeled Met-pep1 peptide. Each group contained four mice. The images were reconstructed by a three-dimensional ordered subsets expectation maximization (3D-OSEM) algorithm with maximum a posteriori (MAP) algorithm and the frame rates were 10 × 30s, 5 × 60s, 5 × 120s and 10 × 240s, and no correction was applied for attenuation or scatter. Static images at 1 h time point were also acquired as 10-min static images for region of interest (ROI) quantification. Image analysis was done using ASI Pro VMTM software. PET/CT images were co-registered and fused automatically with ASI software. For each microPET scan, three-dimensional ROIs were drawn over the tumor, liver, kidney, and muscle on decay-corrected whole-body coronal images. The average radioactivity concentration within a tumor or an organ was obtained from mean pixel values within the ROI volume, which were converted to counts per milliliter by using a pre-determined conversion factor.To perform a microCT scan by a high-resolution Inveon MicroCT scanner  before microPET scan, an anesthetized nude mouse bearing UM-SCC-22B tumor was mounted on a turntable bed that could be moved automatically in the axial direction. Whole body CT acquisition parameters were as follows: voltage 70 kVp, current 400 μA, angular sampling 1° per projection for a full 360° scan, and effective pixel size 58 μm. Scan time was approximately 12 min. The image was reconstructed in real-time by a modified Feldkamp cone bean algorithm with a Shepp-Logan filter and appropriate center offset determined prior to scanning. PET scans were performed using an Inveon microPET scanner . Tumor-bearing mice were anesthetized using isoflurane/OAfter microPET imaging, the blood of mice bearing UM-SCC-22B tumor xenografts was drawn from the orbital sinus, then mice were sacrificed by cervical dislocation and selected organs were excised. Blood, tumor, and major organs tissues were collected and weighed (wet weight). The radioactivity in each tissue was measured using a gamma-counter (Packard Instrument). Distribution of activity was calculated as percentage of injected dose per gram of organ (%ID/g). For each mouse, the radioactivity of the tissue samples was calibrated against a known aliquot of the injected dose and normalized to a body mass of 30 g. Values were expressed as mean ± SD for a group of four animals.Quantitative data are expressed as mean ± SD. The One-way ANOVA and Two-tailed Unpaired t test, confidence interval of 95% (95% CI), were used to analyze group differences. Results of statistical analyses were considered significant if P values of ≤ 0.05. was first prepared on a peptide synthesizer with 98% purity. Radiosynthesis of [18F]FP-Met-pep1 is shown in Fig 1B. Total of 8 runs were performed for [18F]NPFP and specific activity were about 55.5 GBq/nmol (1500 mCi/nmol) at the end of bombardment (EOB). The radiochemical yield for [18F]FP-Met-pep1 was over 55% with specific activity of 7.4 GBq/nmol (200 mCi/nmol). The radiochemical purity of [18F]FP-Met-pep1 was over 97% by analytical HPLC analysis.The c-Met binding peptide, Met-pep1  and decreasing at 60 min . Similarly, [18F]FP-Met-pep1 also showed a high level of internalization after 15, 30 and 60 min incubation, respectively. Zhao et al study have proved that Met-pep1 internalizes cells via receptor specific binding followed by endocytosis. Approximately 2/3 of the observed uptake was due to the internalization of [18F]FP-Met-pep1 into the cells in the early phase, which was decreased to about 1/3 at 60 min. In contrast, dramatic inhibition of cell uptake was observed in the presence of 500 μg of unlabeled Met-pep1 peptide , indicating specific cellular binding and prominent uptake of [18F]FP-Met-pep1 in c-Met positive tumor cells. When the labeled cells were incubated in serum-free medium devoid of radioactivity, the [18F]FP-Met-pep1 showed dissociation and efflux from the cells during the time . During 15 min, approximately 47% of [18F]FP-Met-pep1 had effluxed out of the UM-SCC-22B cells. The efflux rate of [18F]FP-Met-pep1 became slower after 30 min. At the end of incubation, about 36% of the radiotracer remained bound with the cells.The cell uptake, internalization and efflux of FP-Met-pep1 are shown in Fig 3A and S1 Fig. The UM-SCC-22B tumors were clearly visible in relation to the contralateral background with uptakes of 4.72 ± 0.67, 3.83 ± 0.55 and 3.11 ± 0.25%ID/g at 30, 60 and 120 min post injection, respectively . Both kidney and liver showed higher uptake at 30 min post-injection with %ID/g of 6.15 ± 0.71 and 11.5 ± 0.78 respectively, but the tracer was washed out during time . There was rapid clearance and excretion of radioactivity, primarily through the renal pathway, with about 65% excreted within 2 h after injection , suggesting that it is mainly excreted through the renal-urinary routes. In contrast, muscle had very low uptake during the all timepoints.To assess tumor-targeting efficiency, microPET imaging study was performed Fig 4A). As shown in Fig 4B, in the absence or presence of 500 μg of unlabeled Met-pep1 peptide, the tumor uptake of [18F]FP-Met-pep1 was significantly lower  than that of unblocked tumors . Note that reduced uptakes were also observed in other organs and tissues in the presence of blocking dose of c-Met binding peptide, but the differences in normal organs were less marked than that in the tumor.The receptor specificity of the tracer accumulation was confirmed by a blocking assay, in which 500 μg of unlabeled Met-pep1 peptide was injected 30 min before the tracer injection . The circulation time of [18F]FP-Met-pep1 was longer since blood tracer concentration increased from 1.55 ± 0.48 to 2.85 ± 0.76%ID/g after blocking. Interestingly, the uptake in the pancreas was also increased after unlabeled peptide blocking. Similarly, the tracer accumulations in the kidney and liver were much higher  and even after the peptide blocking. There was no significant change of uptake in other organs, including heart, lung, spleen, bone marrow, stomach and intestine.To further confirm the microPET imaging quantification, the biodistribution of -2-fluoropropionate ([18F]-NPFP) was chosen as the labeling agent due to its small size that will have minimal disturbance on the affinity of the peptide. The peptide also has an α-amine on tyrosine residue which could also be labeled, however, the reactivity is much lower than the amine on lysine and the majority of the label is attached on the lysine. After labeling with [18F]-NPFP, a similar time-dependent cellular uptake and internalization were seen for [18F]FP-Met-pep1 in c-Met positive UM-SCC-22B cells, and about 54% of the radioactivity uptake was internalized within 30 min by UM-SCC-22B cells. Despite tumor uptake dropped rapidly at 60 min, 45% of radioactivity uptake  was still retained with the cells . Similar patterns were observed in vivo imaging study, except for two mice at 120 min post-injection in which the tumors became much less visible . We speculate that the signal decrease was primarily due to rapid clearance from the bloodstream and excretion via the renal-urinary pathway. We also confirmed that tumor uptake is specific since the rapid cellular uptake of the tracer could be effectively blocked by the extra amount of unlabeled peptides. In PET imaging study, the tumors are clearly visualized at early time points with high contrast after intravenous injection of [18F]FP-Met-pep1 and the image quality is much better than that those reported in the literature obtained by planar nuclear scintigraphy. The main route of the tracer clearance was through kidney and it was verified by the time-activity curves which showed fast uptake and moderate clearance of [18F]FP-Met-pep1 through renal-urinary routes leading to good target to background radio. The clearance may be caused peptide degradation inside cells and subsequent efflux of radiolabeled fragments. In our 18F-labeled c-Met binding peptide, it is not a major concern since the radiolabeled fragments can be cleared through the kidneys, but this could be a major issue for 125I-labeled peptide due to the release of radioactive iodide. In agreement with the PET imaging study, the ex vivo biodistribution results further validated higher tumor uptake and lower background.In this study, we achieved site-specific labeling of c-Met peptide with 18F-tracer, which binds to human c-Met with high affinity, has a favorable kinetic profile, exhibits specific uptake in c-Met positive H&N tumor mouse model, and rapid renal clearance. However, certain limitations are inevitable in our study: 1) we only reported the 18F-c-Met peptide imaging results in male mice not in females, gender-specific differences in effective dose and biodistribution might occur; 2) we haven’t tested whether it can differentiate between high and low c-Met-expressing HNSCC; 3) we haven’t tested the usage in other types of tumor models. These limitations point to the need for further research and that will be considered in future investigations.In this study, we chose a novel, peptide-based molecular imaging agent with 18F-labeled c-Met peptide specifically binds to c-Met-expressing tumor cells in vitro makes it possible to visualize the abnormal alteration of c-Met expression in vivo and in real time. Targeting c-Met in conjunction with these pathways may lead to more effective therapeutic strategies and stronger treatment modalities in the near future. On further optimization and development, [18F]FP-Met-pep1 may be translated into the clinic for more applications such as patient screening for c-Met-targeted therapeutics, monitoring therapeutic effects, evaluating prognosis, and analysis for resistance mechanism.It is worth pointing out that c-Met also plays a major role in compensating for inhibition of RTK pathways that drive proliferation and metastasis in many tumors including HNSCC tumor. Our S1 Fig18F]FP-Met-pep1 in UM-SCC-22B tumor–bearing mice. (B) Time-activity curves of [18F]FP-Met-pep1 in UM-SCC-22B tumor, blood, liver, kidney and muscle.(A) Dynamic decay-corrected whole-body coronal microPET images after injection of 3.7 MBq (100 μCi) of FP-Met-pep1. Tumors are indicated by arrows.(TIFF)Click here for additional data file.


We aimed to assess the positivity, distribution, quantitative degree of vessel inflammation, and clinical characteristics of IgG4-related aortitis/periarteritis and periarteritis (IgG4-aortitis), and to examine the difference in these characteristics between cases with and without IgG4-aortitis, using fluorodeoxyglucose positron-emission tomography/computed tomography (FDG-PET/CT) co-registered with contrast-enhanced CT (CECT).We retrospectively evaluated 37 patients with IgG4-related disease (IgG4-RD) who underwent both FDG-PET/CT and CECT. The arterial SUVmax and its value normalized to the background venous blood pool (BP)—the target-to-background ratio (TBR) in the entire aorta and the major first branches—were measured. Active vascular inflammation was considered in cases with a higher FDG uptake than BP and a thickened arterial wall (>2 mm).p < 0.0001). The IgG4-aortitis-positive group patients were older  and had a higher male predominance  than the negative group, although the differences were not significant .Fifteen (41%) patients exhibited IgG4-aortitis. Most patients (80%) showed multiple region involvement. The entire aorta, including the major first branches, were involved, typically showing a thickened wall and high FDG uptakes. The most common site was the iliac arteries (35%), followed by the infrarenal abdominal aorta (33%), thoracic aorta (8%), first branches of the thoracic aorta (8%), suprarenal abdominal aorta (6%), and the first branches of the abdominal aorta (5%). The IgG4-aortitis-positive vessel regions were thickened, with an average maximal wall thickness of 6.3 ± 2.9 mm. The SUVmax and TBR values were significantly higher in the IgG4-aortitis-positive regions  than in the IgG4-aortitis-negative regions (median 2.1 [1.2–3.7] and 1.3 [0.9–2.3], respectively; We investigated the image characteristics of IgG4-aortitis. The entire aorta and major branches can be involved with more than 2-fold higher FDG uptake than the venous background pool, and with wall thickening. The most common involved site is the iliac arteries, followed by the infrarenal abdominal aorta. Immunoglobulin G4-related disease (IgG4-RD) is a newly recognized disease entity characterized by the infiltration of IgG4-positive plasma cells and fibrosis in systemic organs along with the elevation of serum IgG4 levels . AutoimmVascular involvement has also been detected in IgG4-RD , 9. The In patients with IgG4-RD, whole-body organ involvement manifests as increased FDG uptake in multiple organs on fluorodeoxyglucose (FDG) positron-emission tomography (PET) and FDG-PET/computed tomography (CT) –26. AlthIn the present study, we aimed to assess the positivity, distribution, quantitative degree of vessel inflammation, and clinical characteristics of IgG4-aortitis, and to examine the differences in these characteristics between cases with and without IgG4-aortitis on FDG-PET/CT co-registered with CECT.This retrospective study was approved by the institutional review board. The requirement for obtaining informed consent was waived. Forty-three patients who were diagnosed with definite IgG4-RD according to the 2011 comprehensive diagnostic criteria for IgG4-RD proposed by Umehara et al.  and who The background of the patients is summarized in Table The serum IgG4 levels within 2 months from PET/CT were available in 28 patients.FDG-PET/CT was performed using a hybrid scanner , with lutetium oxyorthosilicate crystal detectors, covering an axial field of view of 21.6 cm, and a 64-slice multi-detector computed tomography scanner. The patients were instructed to fast for more than 6 h, and their blood glucose level was measured  before injecting the FDG. A mean total dose of 240 ± 35 MBq of the FDG was administered intravenously, which was computed as 4.2 ± 0.6 MBq/kg. Following the injection, the patient was allowed to rest comfortably for 60 min. Image acquisition was initiated with a non-contrast-enhanced CT scan for attenuation correction and co-registration. This was followed by three-dimensional PET acquisition, with a 3-min time-limit per position, from the top of the skull to the mid-thigh.Standard diagnostic CT scans were performed with a 64-multislice CT scanner . The CECT protocol in the present study included a single-phase scan performed from the neck to the pelvis, 70–180 s after the injection of contrast material (450–560 mg·I/kg), with a 5-mm section thickness.Vascular involvement was assessed at the following six regions: thoracic aorta, suprarenal abdominal aorta, infrarenal abdominal aorta, iliac arteries , first branches of the thoracic aorta , first branches of the abdominal aorta .Of the 222 vascular regions in 37 patients, 220 regions were evaluated; two regions could not be assessed due to suprarenal and infrarenal aortic replacement. Below the kidney level, abnormal vessel FDG uptake was differentiated from uptake in the urinary tract using co-registered CECT images Fig. .Fig. 1CEFDG-PET/CT and CECT images were simultaneously reviewed by two board-certified radiologists (one with 10 years of radiology experience and another with 20 years of experience and a certification in nuclear medicine) in consensus.The diagnostic criteria for CECT were based on the findings of a previous study , 31, 32.The diagnostic criteria of the FDG-PET/CT were based on visual evaluation of a higher abnormal vessel wall uptake than that in the blood pool. For qualitative evaluation, the vessel standardized uptake value (SUVmax) of each vascular region was defined as the maximal value by drawing several volumes of interest (VOIs) with a radius of 5 mm. For normalizing the SUVmax value, we also calculated the ratio of the arterial SUVmax to that of the venous blood pool, also known as the target-to-background ratio (TBR). Rudd et al. described the TBR as a reproductive and quantitative method for assessing the arterial wall . Besson When abnormal findings were simultaneously detected on both CECT and FDG-PET/CT images, the region was considered to have positive inflammation due to IgG4-aortitis (IgG4-aortitis-positive region). IgG4-aortitis was differentiated from atherosclerosis as follows: if there was an abnormal finding only with CECT or FDG-PET/CT, or if there was no abnormal finding on both CECT and FDG-PET/CT, the region was considered as IgG4-aortitis-negative region. This sorting was applied to six regions independently in each patient. If a patient had at least one positive vascular region, the patient was assigned to the IgG4-aortitis-positive group. Patients without vascular involvement were assigned to the IgG4-aortitis-negative group. The involvement of other organs, such as lymph node, salivary gland, pancreas, and chest  were simultaneously assessed. The diagnostic clue for lymph node involvement is abnormal swelling and diffuse enhancement. The salivary gland shows unilateral or bilateral diffuse or focal swelling and homogeneous enhancement. The pancreas shows diffuse or focal enlargement with irregular narrowing and loss of the pancreatic cleft and smooth rim (capsule-like rim). Pleural and paravertebral involvements are indicated by thickened or flattened soft tissue density areas along the pleura and vertebra , 32, 35.To evaluate the disease distribution, the positivity ratio was calculated for each of the six regions.t test was used to compare the continuous variables with normal distribution, including age and serum IgG4 levels, between the two groups.We compared the differences in the patient’s background  between the IgG4-aortitis-positive and IgG4-aortitis-negative groups. The categorical variables, including male-to-female ratio and diabetes mellitus, were compared using Fisher’s exact test. An unpaired Student For region-based comparisons, the vessel SUVmax and TBR values were compared between the IgG4-aortitis-positive and IgG4-aortitis-negative regions. Since the vessel SUVmax and TBR values were not normally distributed, they were compared using the nonparametric Wilcoxon rank-sum test.p value <0.05 was considered significantly different.Continuous variables are presented as mean ± standard deviation or as median (range), whereas categorical variables are presented as percentages. A All statistical analyses were performed using dedicated software JMP 12.2.0 .Fifteen of 37 patients 41%) had at least one positive IgG4-aortitis-positive region and were assigned to the IgG4-aoritis-positive group  of 37 patients showed aneurysmal aortic dilatations. Nine of 37 patients (24%) exhibited aneurysmal changes in the iliac arteries, and four patients (11%) exhibited abdominal aortic aneurysms, while three patients showed overlaps between these regional involvements. There was no case of aortic dissection. A representative case with abdominal aorta involvement is described in Fig. p = 0.17 and p = 0.06, respectively). There was no significant difference in the incidence of diabetes mellitus between the groups . The mean serum IgG4 levels (749 ± 392 mg/dL) in the IgG4-aortitis-positive group were higher than those in the IgG4-aortitis-negative group (545 ± 288 mg/dL), although the difference was not significant (p = 0.15).The IgG4-aortitis-positive group was older  and had a higher male predominance  than those in the negative group, although the differences were not significant  was significantly higher than that in the IgG4-aortitis-negative regions  was significantly higher than that in the IgG4-aortitis-negative regions  and wall thickening . IgG4-aortitis-positive regions exhibited more than 2-fold the FDG uptake of the background blood pool . The typical finding of IgG4-aortitis-positive group was multiple vascular region involvements (80%). The entire aorta and major branches can be involved with more than 2-fold the FDG uptake of the venous background pool. The most common site was the iliac arteries, followed by the infrarenal abdominal aorta.To assess IgG4-RD, modalities such as CT, magnetic resonance imaging, ultrasonography, Gallium-67 scintigraphy, endoscopic retrograde cholangiopancreatography, endoscopic ultrasonography, intraductal ultrasonography, and FDG-PET or FDG-PET/CT have been used individually or in combination for both morphological and functional evaluation , 36–39. Previous studies have reported the prevalence of vascular involvement as 14–23% , 32, 43.There have been two previous studies that reported the distribution of IgG4-aortitis. Inoue et al. reported that 13 (76%) of 17 patients had infrarenal abdominal aorta or iliac arteries involvement, while the thoracic aorta was involved only in four patients (24%) . This reSome form of aneurysmal change was observed in the aortas of 10 patients (27%) in our study, while Perugino et al. reported that 11 of 160 (7%) patients demonstrated thoracic or abdominal aortic aneurysms . These dIn our study, patients with IgG4-aortitis tended to be older and male as compared to those in the negative group, which was also consistent with the previous report .The present study has certain limitations. First, the study design was retrospective in nature, which may have led to possible selection bias and the restriction of evaluable regions. Moreover, we included only newly diagnosed patients, who had not been exposed to immunosuppressive treatments, and hence, not all the patients diagnosed with IgG4-RD were enrolled in this study. Second, the evaluable region may have been restricted due to the non-ECG-gated body CECT scanning protocol; hence, we could not evaluate the head and neck region, and particularly the carotid arteries. The coronary arteries were also not evaluated due to motion artefacts. Third, FDG-PET/CT and CECT images were simultaneously reviewed for division into IgG4-aortitis-positive and IgG4-aortitis-negative regions. Because both a thickened arterial wall on CECT and a higher uptake of FDG in the visual assessment were used for determining IgG4-aortitis-positivity, there was a potential bias that the positive region would show a higher SUVmax value. The IgG4-aortitis-positive region may include both active inflammation and atherosclerosis; however, our aim was not to differentiate active inflammation from atherosclerosis, but to examine the degree of FDG uptake in active regions. Finally, we did not perform biopsies for vascular involvement, as there is no pathological gold standard for diagnosis. Moreover, it is difficult, and may not be safe, to obtain vessel wall specimens. Hence, a non-invasive method is required for the diagnosis of IgG4-aortitis.We assessed the positivity and distribution of vascular involvement in IgG4-RD using FDG-PET/CT co-registered with CECT. The entire aorta and major branches may be involved, with more than twice the FDG uptake as compared to the venous blood pool. The infrarenal abdominal aorta and iliac arteries were the most commonly involved sites.


Introduction: There are increasing concerns world-wide with growing rates of antibiotic resistance necessitating urgent action. There have been a number of initiatives in the Republic of Srpska in recent years to address this and improve rational antibiotic prescribing and dispensing despite limited resources to fund multiple initiatives.Objective: Analyse antibiotic utilization patterns in the Republic of Srpska following these multiple initiatives as a basis for developing future programmes in the Republic if needed.Methods: Observational retrospective study of total outpatient antibiotic utilization from 2010 to 2015, based on data obtained from the Public Health Institute, alongside documentation of ongoing initiatives to influence utilization. The quality of antibiotic utilization principally assessed according to ESAC, ECDC, and WHO quality indicators and DU 90% (the drug utilization 90%) profile as well as vs. neighboring countries.Results: Following multiple initiatives, antibiotic utilization remained relatively stable in the Republic at 15.6 to 18.4 DIDs, with a decreasing trend in recent years, with rates comparable or lower than neighboring countries. Amoxicillin and the penicillins accounted for 29–40 and 50% of total utilization, respectively. Overall, limited utilization of co-amoxiclav (7–11%), cephalosporins, macrolides, and quinolones, as well as low use of third and fourth generation cephalosporins vs. first and second cephalosporins. However, increasing utilization of co-amoxiclav and azithromycin, as well as higher rates of quinolone utilization compared to some countries, was seen.Conclusions: Multiple interventions in the Republic of Srpska in recent years have resulted in one of the lowest utilization of antibiotics when compared with similar countries, acting as an exemplar to others. However, there are some concerns with current utilization of co-amoxiclav and azithromycin which are being addressed. This will be the subject of future research activities. The discovery of antibiotics is one of the greatest discoveries of modern medicine. However, since their discovery, it is acknowledged that bacteria have the ability to become resistant to antibiotics if they are not used rationally  of the Republic of Srpska MoHSW, .Antibiotics can only be dispensed on prescription by pharmacist. There are considerable fines if this is abused  responsible for monitoring and controlling of AMR and proposing measures to improve rational antibiotic prescribing and utilization.On the proposal of the National Committee, the MoHSW adopted the national Program to reduce AMR resistance in the Republic of Srpska from 2016 to 2020, harmonized with the WHO Global Strategy for Containment of Antimicrobial Resistance WHO,  and the Box 2Activities to enhance rational prescribingThe set of Clinical Guidelines (CG) with diagnostic and therapeutic principles have been developed for Primary Health Care physicians. These include guidelines for upper and lower acute respiratory infections in children , urinary tract infections in children, as well as urinary tract infections in adults (2004).The list of antibiotics funded by the Health Insurance Fund of the Republic of Srpska (HIF) are in accordance with the CG recommendations.HIF adopted a positive list of medicines with reference prices comprised of List A and List B. List A is a basic list of medicines for which HIF covers 90 or 100% of the reference price of the medicine depending on the category of the insured person. List B is a supplementary list, which includes more expensive medicines, for which HIF covers 50% of the costs for all insured patients. For medicines not included in the positive list, the patient must pay 100% of the price. In addition, if the price of drug is higher than the reference price adopted by HIF, the patient has to pay the price difference.The antibiotics included in List A are: doxycycline, amoxicillin, phenoxymethylpenicillin, benzathine-phenoxymethylpenicillin, cefalexin, sulfamethoxazole with trimethoprim, and erythromycin. Norfloxacin is included in List B.Activities to enhance rational dispensingThere is permanent education of pharmacists and pharmacy technicians on the appropriate management of infectious diseases to address concerns with knowledge of infectious diseases, especially viral infectious diseases, which has been a concern in other countries .Publishing articles on AMR and rational antibiotic use in professional journals.Discussions concerning current AMR surveillance in the Republic of Srpska among key stakeholder groups and agreement of specific activities to improve AMR reporting and monitoring.The MoHSW and the Public Health Institute (PHI) marking European Antibiotic Awareness Day and World Antibiotic Awareness Week in November each year starting from 2013 with support of European Centre for Disease Prevention and Control (ECDC).Activities to enhance public awareness on AMR and rational antibiotic useCampaigns are organized to raise awareness about AMR and rational antibiotic use among the general public, patient associations and health professionals.Promotional materials , recommended by ECDC, are provided to patients and health professionals.Consequently, the aim of this study was to analyse total antibiotic utilization in the Republic of Srpska in recent years and to assess the influence of these various initiatives to improve antibiotic prescribing and dispensing in the Republic. Subsequently, compare the findings with those of other European and neighboring countries to review potential additional measures that could be introduced in the Republic of Srpska if needed to further improve the rational use of antibiotics. As a result, seek to reduce future AMR rates.This was a retrospective observational study on outpatient antibiotic utilization in the Republic of Srpska from 2010 to 2015, based on drug utilization data obtained from the Public Health Institute (PHI). Data collection was based on quantitative and structural medicinal reports that pharmacies in the Republic send to the PHI each year for collation /DDD (Defined Daily Dose) methodology WHO, , which iAntibiotic utilization and associated quality indicators were used to compare the Republic of Srpska's utilization patterns with that from other European countries, as well as former Soviet Union Republics, to place this in perspective  as a % of total antibiotic use,Penicillins combination utilization such as amoxicillin with clavulanic acid (co-amoxiclav) as a percentage of total antibiotic use,Total utilization of cephalosporins expressed as DIDs,Percentage utilization of third- and fourth-generation of cephalosporins vs. all cephalosporins,Total utilization of J01F group  expressed as DIDsUtilization of short-acting macrolides, intermediate and long-acting macrolides  as a percentage of total antibiotic use,Utilization of quinolones (J01M) expressed in DIDs as well as percentage of fluoroquinolones (J01MA) of total antibiotic use.A number of these indicators are due to concerns with the over use of co-amoxiclav, macrolides, third and fourth generation cephalosporins as well as fluoroquinolones , lower than a number of neighboring countries  comprised 98% of total utilization of anti-infectives (J group). Utilization of the J01 group varied between 15.6 and 18.4 DIDs during the observed period  were the most consumed antibiotics comprising 50% of total antibiotic utilization on average, followed by other beta-lactam antibiotics (14%). The highest utilization of penicillins was observed in 2013 with 10.0 DIDs, while the lowest (7.9 DIDs) observed in 2014. The utilization of broad-spectrum penicillins (J01CA) was 6.04 DIDs on average (Table 4). The first-generation cephalosporins (J01DB) were the most used within J01D group (67.6%), of which cefalexin was the only one used. Its utilization decreased from 2.0 DIDs in 2010 to 1.5 DIDs in 2015, which represents a reduction of 25%. Overall, utilization of the second and third generation of cephalosporins (J01DC and J01DD) also declined during this period. Regarding the second-generation cephalosporins, cefuroxime had very stable utilization ranging from 0.45 to 0.56 DIDs during the study period, while utilization of cefaclor decreased from 0.15 in 2010 to 0.05 DIDs in 2015 (67%). Overall, third generation cephalosporins constituted 7.9% of total cephalosporins, or 1.2% on average of total antibiotics (Table 4). After declining from 2010 to 2014, utilization of third generation cephalosporins subsequently increased in 2015 (Table 4). The fourth generation cephalosporins, monobactams, and carbapenems, were not used at all.Utilization of cephalosporins J01D) decreased from 3.0 DIDs in 2010 to 2.3 DIDs in 2015 (Tables D decreasErythromycin was the most used macrolide (0.68 DIDs) followed by azithromycin (0.57 DIDs) and clarithromycin (0.32 DIDs). Overall, macrolides constituted 9.4% of total antibiotic utilization, which is about five times lower than the penicillins. The use of the macrolides was highest in 2011 (1.78 DIDs) and then dropped thereafter to 1.55 DIDs in 2014 (Table Doxycycline was the most used tetracycline J01A), with quinolone utilization (J01M) ranging from 1.5 DDDs in 2010 to 1.7 DIDs in 2015 , cephalexin, ciprofloxacin, doxycycline, and sulfamethoxazole with trimethoprim were the most used antibiotics over the observed period , macrolides and fluoroquinolones, expressed as percentage of total antibiotic use, was very stable over the observed period Table . However5 and Table Figures Total antibiotic utilization was relatively constant during the study period in the Republic of Srpska Table , with miSecondly, a number of initiatives and activities have been implemented in the Republic in order to improve knowledge, attitudes and practices of pharmacists to reduce antibiotic self-medication and improve appropriate management of patients with URTIs Boxes , 2, espeThese combined initiatives also appeared to result in high use of beta-lactam antibiotics and limited use of third and fourth generation cephalosporins and fluorquinolones in the Republic in recent years in accordance with agreed quality indicators Table , comparaAmoxicillin, which is included in the HIF positive A list, was the most utilized penicillin in the Republic of Srpska, with utilization of co-amoxiclav approximately 4 times lower than amoxicillin. The low utilization of co-amoxiclav in the Republic compares favorably with neighboring countries Figure . HoweverC. difficle (Dancer, Encouragingly, given concerns with the development and spread of resistant strains of  Dancer, , there w Dancer, , 4. In a Dancer, . There w Dancer, , 4. This Dancer, . However Dancer, , 4, resu Dancer, .Concerns with the recent increased utilization of co-amoxiclav and azithromycin, as well as higher use of fluoroquinolones than some neighboring countries Table , has alrAlongside this, pharmacists and family physicians will be encouraged to communicate more to their patients about rational antibiotic use and AMR, building on current initiatives. This will be combined with planned activities to further raise public awareness regarding the potential harmful effects of excessive, inappropriate, and unnecessary use of antibiotics. These programmes will also be the subject of future research projects.We believe this study clearly shows that even lower income countries such as the Republic of Srpska can appreciably improve rational medicine utilization by introducing multiple interventions and initiatives, acting as an exemplar to other European countries and wider.We are aware of a number of limitations of this study. These include no access to prescribing data to be able to assess the quality of antibiotic prescribing against standard treatment guidelines. However, we believe our methodology, including the use of total antibiotic utilization via the PHI datasets, as well as comparisons with neighboring countries, adds robustness to our findings and conclusions.In conclusion, without looking specifically at prescribing indications, we believe there appears to be rational antibiotic utilization in the Republic of Srpska in recent years compared to neighboring countries, with favorable use of penicillins combined with moderate or low use of co-amoxiclav, cephalosporins including third and fourth generation cephalosporins, macrolides, and quinolones. This again demonstrates that multiple interventions with key stakeholder groups can favorably improve medicine utilization patterns.LB, VM-P, MÐ, RŠ, JB, and BG: developed the concept; LB, AK, VM-P, RŠ, and BG: undertook the analysis and the first write-up of the manuscript. All authors critiqued the initial and subsequent drafts before submission.VM-P is employed by the Ministry of Health and Social Welfare in the Republic of Srpska and LB, MÐ, and JB are employed by the Public Health Institute. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.