Improvements in oocyte cryopreservation techniques and establishment of cryopreserved donor oocyte banks have led to improved access to and lower cost of donor oocytes, upending the traditional practice of fresh oocyte donation. The objective of this study was to examine national trends in utilization and live birth rates with fresh versus cryopreserved donor oocytes.A retrospective analysis of 2013 through 2015 aggregate U.S. national data reported by the Society for Assisted Reproductive Technology which included 30,160 IVF cycles with either fresh or cryopreserved donor oocytes was performed.During the study period utilization of fresh oocyte donations rapidly declined by 32.9%, while cryopreserved oocyte donation increased by 44.4%. Fresh donor oocytes produced significantly higher live birth rates per recipient cycle start than cryopreserved donor oocytes (51.1% vs. 39.7%). Over the three-year study period fresh donor oocytes produced stable live birth rates per recipient cycle start while those with cryopreserved oocytes significantly declined year-by-year.Despite rising popularity of cryopreserved donor oocytes, prospective patients should be counselled that fresh donor oocytes still represent standard of care due to higher live birth rates. Improved survival of human oocytes following cryopreservation via the vitrification technique prompted a change in the position of the American Society for Reproductive Medicine (ASRM) on the experimental nature of oocyte cryopreservation in 2013 [We compared utilization and live birth rates of fresh versus cryopreserved oocyte donation cycles in 2013–2015 publicly available reports of the Society for Assisted Reproductive Technology (SART) . These rP-value of <0.05 was considered statistically significant. All statistical analyses were performed using SAS version 9.4 statistical software.Live birth rates were compared using the two-tailed Fisher’s exact test and Wilson confidence interval for binomial proportions. A Poisson regression model was used to analyze live birth rates overall trends. A P < 0.0001), while cryopreserved oocyte donation rapidly increased by 44.4%; (P < 0.0001). Concurrently, cycle cancellation rates with fresh oocytes declined from 11.7 to 9.1% (P < 0.0001), while those with use of cryopreserved oocytes increased from 8.5 to 15.0% (P < 0.0001). The table also shows that the number of embryos transferred over the study period was similar in both study groups.Table P < 0.0001) and per embryo transfer . Figure P = 0.2925), while those with cryopreserved oocytes declined year-by-year (P = 0.0094).Despite similar numbers of transferred embryos, live birth rates over the whole study period were higher with fresh than cryopreserved oocytes per recipient cycle start . Absolute and relative differences in live birth rates were, thus, smaller with eSET. While percentages of pregnancies resulting in term births were similar for eSET cycles with fresh and cryopreserved donor oocytes (84.6% vs. 83.1%).Analyzing outcomes only for cycles with elective single embryo transfer (eSET), live birth rates per embryo transfer were 53.7% with fresh vs. 46.5% with cryopreserved oocytes (Despite lower and, seemingly, over time further declining live birth rates with cryopreserved oocytes, here presented data demonstrate that oocyte donation in the U.S. is undergoing a rapid transition from fresh to cryopreserved oocytes. This trend is likely driven by improved access to, and lower cost of cryopreserved oocytes, and facilitated by the rapid establishment of cryopreserved donor oocyte banks .This analysis of aggregate outcome data did not allow adjustments for confounding patient characteristics which would require access to patient level data. Based on 2013 patient level national data, a recent report by investigators from the Centers for Disease Control and Prevention, where such adjustments were performed, also reported decreased likelihood of pregnancy and live birth per started cycle with cryopreserved donor oocytes but no difference when cycles were restricted to those proceeding to embryo transfer . We did Considering rising cancellation as well as declining live birth rates over the study period in cryopreserved oocyte donation cycles, here presented data not only confirm but actually strengthen our prior reports that suggested significant outcome differences in favor of fresh oocytes , 6.With increasing use of cryopreserved oocytes, live birth rates did not, as had been suggested , improveReasons for these increasing differences in live birth rates have yet to be determined. There are many potential explanations to consider: For example, it is possible that cryopreservation and subsequent thawing, negatively affects an oocyte’s and resulting embryo’s pregnancy potential. Though oocyte cryopreservation, with switch from slow-freezing to vitrification over the last decade, has greatly improved , 9, the The SART data set did not permit analysis of cumulative live birth rates, which include the initial IVF cycle plus any subsequent transfers of surplus cryopreserved embryos. In our clinical experience these rates are even more in favor of fresh oocyte donation since patients utilizing cryopreserved donor oocytes typically have fewer surplus embryos available following the initial cycle. Moreover, performance of surplus cryopreserved embryos derived from previously cryopreserved oocytes has not been well studied, though initial reports appear reassuring .Use of cryopreserved donated oocytes offers advantages over fresh oocytes, including simplified logistics of coordinating donor and recipient cycles, access to a broader pool of donors particularly for ethnic minorities, the ability to ship frozen oocytes over long distances, thereby reducing the need for reproductive tourism and, potentially, lower costs per treatment cycle . HoweverFresh oocyte donation must, therefore, still be considered the ‘gold standard’ in oocyte donation, and patients should be accordingly counselled. In this context it is also important to note that, according to ASRM recommendations, routine donor oocyte banking should not be recommended unless clinical data on safety and equivalent efficacy of oocyte cryopreservation become available .


The aim of the present study was to investigate whether a magnitude of deception of 5% in power output would lead to a greater reduction in the amount of time taken for participants to complete a 4000 m cycling TT than a magnitude of deception of 2% in power output, which we have previously shown can lead to a small change in 4000 m cycling TT performance.Ten trained male cyclists completed four, 4000 m cycling TTs. The first served as a habituation and the second as a baseline for future trials. During trials three and four participants raced against a pacer which was set, in a randomized order, at a mean power output equal to 2% (+2% TT) or 5% (+5% TT) higher than their baseline performance. However participants were misled into believing that the power output of the pacer was an accurate representation of their baseline performance on both occasions. Cardiorespiratory responses were recorded throughout each TT, and used to estimate energy contribution from aerobic and anaerobic metabolism.p = 0.01), with the difference in performance likely attributable to a greater anaerobic contribution to total power output (p = 0.06). There was no difference in performance between the +5% TT and +2% TT or baseline trials.Participants were able to finish the +2% TT in a significantly shorter duration than at baseline (Results suggest that a performance reserve is conserved, involving anaerobic energy contribution, which can be utilised given a belief that the exercise will be sustainable however there is an upper limit to how much deception can be tolerated. These findings have implications for performance enhancement in athletes and for our understanding of the nature of fatigue during high-intensity exercise. There is general agreement that the pacing strategy that athletes adopt during a closed loop, self-paced cycling time-trial (TT) is regulated in an anticipatory manner . The ratManipulating an athlete’s feedback could therefore distort the amount of effort they are able to attribute to an exercise challenge, and ultimately affect performance. We have previously shown that it was indeed possible to reduce the amount of time taken to complete a 4000 m cycling TT by misleading participants into believing they were exercising at the same intensity as in a previous trial, when in fact the power output was 2% greater . SeveralIt is difficult to compare the findings of these previous studies due to differences in experimental design. For example, the duration of the exercise bout varies and therefore the proportion and distribution of the anaerobic and aerobic energy contribution will be different. In addition, whilst two studies investigated the effects of deception on self-paced TT performance , 7, anotFor self-paced cycling TTs, deception has been shown to result in improvements in performance  and no cThe aim of the present study was therefore to investigate whether a magnitude of deception of 5% in power output would lead to a greater reduction in the amount of time taken for participants to complete a 4000 m cycling TT than a magnitude of deception of 2% in power output, which we have previously shown can lead to a small change in 4000 m cycling TT performance , 11.Ten male cyclists  and After the completion of the submaximal exercise test, participants were given 5 min to rest before the commencement of the maximal exercise test. Subsequently, participants were asked to select their preferred cadence, and then the resistance was adjusted to 200 W and increased by 5 W every 15 s. The test was terminated when cadence reduced by more than 15% or when participants felt that they could no longer sustain the required exercise intensity. The highest Participants completed a practice 4000 m TT, and three experimental 4000 m TTs on an electromagnetically braked cycle ergometer  interfaced with Velotron 3D software. The 3D software supports the instantaneous generation of an on-screen avatar that illustrates the cyclist’s progress as they undertake a TT on a track of a known distance. Comprehensive data from the performance can be stored and the avatar can be replayed, serving as an opponent for the cyclist to race against in future trials. Performance from participants first experimental 4000 m TT was set as their baseline (BL). During the subsequent experimental trials participants were deceived into thinking they were to race an avatar representing their BL performance, but the actual avatar was racing at a power output that was either +2% or +5% higher than BL. The view seen by the cyclist was from behind the slower of the two avatars, meaning that they were able to monitor the performance of both, and could estimate the distance separating their current and previous performances. The only other information that could be seen on the projector screen was distance travelled. All other feedback was blinded from the participants. Participants were instructed to complete the distance as fast as possible, and were able to modulate power output through changes in cadence and use of an electronics gearing system. Throughout each trial expired air was analyzed breath-by-breath.On completion of each trial, participants were asked to provide a global rating of perceived exertion using a 15-point scale  and to rmax. Breath by breath max using the equation GME = PTOT/PMET; where PMET is the aerobic metabolic power, which can be calculated as:Prior to each 4000 m TT, participants performed a standardized warm-up consisting of cycling for 5 min at 150 W and then a further 5 min at 70% of PIt was assumed that an RER greater than 1.0 was attributable to buffering, therefore in the calculation of metabolic work any RER values in excess of 1.0 were treated as if they equaled 1.0.AER) and anaerobic (PAN) contributions to total power output (PTOT) were calculated from PTOT, TOT data. These data were then averaged into ten, 400 m ‘bins’, in order to facilitate between-trial analyses. To calculate PAER, aerobic metabolic power was multiplied by GME for each 400 m bin. Given that PTOT is the sum of PAER and PAN, PAN was calculated as:Aerobic  repeated-measures ANOVA. Significant main effects were followed by Bonferroni post hoc tests. Statistical significance was accepted at p<0.05.Statistical analyses were conducted using SPSS 16.0 . Data are presented as mean ± SD. Differences in RPE, affect, peak 2, 18 = 6.665, p = 0.01) with the +2% TT finished more quickly (1.03%) than BL . There were no differences in completion time between either the +5% TT and +2% TT or the +5% TT and BL trials (p>0.05). Individual completion times for each trial are shown in 2, 18 = 5.560, p = 0.01). Pairwise comparisons revealed that power output was higher during the +2% TT compared with BL  however there were no differences in power output between the +5% TT and +2% TT or +5% TT and BL (p>0.05).Mean performance, cardiorespiratory and perceptual responses to 4000 m time trials are shown in TOT, PAN and PAER are shown in TOT at 2000 m  and 3600 m . Pairwise comparisons revealed that PTOT was significantly greater in the +5% TT than BL at 2000 m  and in the +2% TT than BL at 3600 m . Pairwise comparisons revealed a trend towards higher PAN during +2% TT compared with BL . There were no significant temporal variations in PAER between trials, but the between-trial difference in PAN at 3600 m approached significance  with a trend towards higher PAN during +2% TT compared with BL .The serial patterns of Pp > 0.05).Despite the small differences in power output, there were no discernible differences in the serial or mean trial respiratory responses between trials , affect  or Peak B[La] There were no between-trial differences in RPE  or moderate (+5%) increase in power output. When the difference between anticipated and actual exercise intensity of the avatar was small, participants were able to reduce the amount of time taken to complete the TT. However, when the magnitude of the deception was equal to a moderate difference in 4000 m TT performance, participants did not demonstrate an improvement in performance compared to BL, although the performance was also not significantly different to +2% TT. These observations support the assertion that deception can elicit improvements in simulated TT performance and adds the novel finding that the ergogenic effect of deception is magnitude dependent. What magnitude of deception is most efficacious for performance and how it varies between different durations, intensities and modes of exercise remains unclear; however in this study, which manipulated 4,000 m cycling time trials, an increase in mean power output of 2% elicited an improved performance compared to baseline while an increase of 5% was equivocal.The finding that participants were able to complete the +2% TT in a shorter amount of time is consistent with a previous study in our laboratory  and a moAnother reason for the discrepancy in RPE scores between studies might be due to the timing of the perceptual measurements. In the current study, a single post-exercise RPE score representing the whole trial was taken, whereas in the Shei et al. study  RPE was The present study used trained participants so it remains to be elucidated if elite-level riders would be able to detect a change in power output of this magnitude. Interoceptive sensitivity and physiological awareness are known to be developed through experience , 30 and The fact that a change in performance was found to be equivocal during the +5% TT might indicate this level of deception approximated to an upper limit regarding how much deception can be tolerated. It is likely that during +5% TT, the extent of peripheral disturbance associated with severe exercise such as depletion of phosphocreatine, and proton and myoplasmic phosphate accumulation, ultimately limited performance despite high levels of motivation during the task. Notably, 9 out of 10 participants  appear tPost-exercise RPE and affect responses were not significantly different in +5% TT compared to BL despite an increased PTOT being detected at 2000 m. However PTOT remained similar thereafter between the +5% TT and BL trials and so the post-trial perceptual measurements are perhaps reflective of this rather than the increased PTOT detected at 2,000 m. This would suggest that a decision was made to reduce exercise intensity during the +5% TT which served to ameliorate RPE and affect responses. This intuitive decision was likely based on participants’ levels of motivation, afferent feedback, progress of the avatars relative to one another and knowledge of the remaining trial distance. Renfree et al.  have pos-1) and PAN compared to BL. It has been suggested that lactate is an afferent signaling agent involved in complex systems control of self-paced exercise [The increased power output and speed during +2% TT was associated with a non-significant increase in B[La] . With trained participants, it is possible that there is a metabolic reserve that can be utilized through an increased central motor drive, to produce a 2% increase in power output, when participants are deceived during a competitive, highly motivating task. However if the increase in power output is greater (5% or more) then overall performance might not be enhanced, potentially due to afferent feedback reflecting excessive changes in metabolic substrate utilization, such as the finite anaerobic capacity being utilized too soon (or rapidly) and a rapid build-up of metabolic waste products. This interoceptive feedback integrated with a sense of the distance still remaining and the competitor avatar moving away rapidly, influenced a conscious or subconscious decision to reduce power output part way through the exercise bout. These findings provide support for the notion that variations in pace are determined in an anticipatory manner by the product of how an athlete feels momentarily and how much of the event remains. As the endpoint approaches, the risk of an excessive disturbance to homeostasis progressively decreases. Therefore, participants can access latent anaerobic energy resources during the final stages to improve their performance, but not necessarily during the early or mid-portion of an event. The use of deception has implications for performance enhancement in athletes, and for our understanding of the mechanisms underpinning variations in pace during severe exercise.S1 Dataset(XLSX)Click here for additional data file.


Clinically, increased cerebrospinal fluid (CSF) adenosine deaminase (ADA) level is an important diagnostic clue of tuberculous meningitis. However, increased CSF ADA level can be caused by other neurological diseases.We report a case of a 67-year-old woman with cryptococcal meningitis presented with increased ADA level of the CSF. In parallel with her recovery, the ADA level of CSF decreased steadily. This is the first case described the chronological change in CSF ADA level of the patient with cryptococcal meningitis in detail.Clinically, increased CSF ADA level is an important diagnostic clue of tuberculous meningitis. However, previously, it was reported that increased CSF ADA level can be caused by other neurological diseases. In this case, the patient was diagnosed with cryptococcal meningitis, and the possibility of coinfection with tuberculous meningitis has been discarded by the negative PCR, negative cultures and the clinical course. In addition, the chronological change in CSF ADA level was useful for follow-up assessment.Cryptococcal meningitis should be considered for the differential diagnosis for diseases presented increased CSF ADA. Clinically, increased cerebrospinal fluid (CSF) adenosine deaminase (ADA) level is an important diagnostic clue of tuberculous meningitis (TBM). However, increased CSF ADA level can be caused by other neurological diseases. We report of an older patient with cryptococcal meningitis associated with increased CSF ADA level, in which information on the chronological change in ADA level was useful for follow-up assessment.Cryptococcus neoformans. The mycobacterium cultures and polymerase chain reactions (PCR) of CSF, sputum, urine, and gastric juice repeatedly yielded negative results. Brain magnetic resonance imaging revealed hydrocephalus and an abnormal enhancement effect at the brain base  and meningeal irritation, hyperreflexia of all four-extremities, and positive Babinski response. Blood examination revealed normal findings, including inflammatory reactions, and negative results for human immunodeficiency virus antibody. In the CSF examination, the cell count was 46/µL ; the protein level, 142 mg/dL (reference range 10–40 mg/dL); glucose level, 31 mg/dL (reference range 50–75 mg/dL); and ADA level, increased to 12.7 U/L (reference range <9.0 U/L)  (Tuon et al. ADA is an enzyme that catalyzes the deamination of adenosine, forming inosine in the process (Erel et al. Previously, it was reported that increased CSF ADA level can be caused by other diseases such as sarcoid meningitis, meningeal involvement with leukemia or lymphoma, cerebral toxoplasmosis, cerebral infarction, neurosyphilis, listeria meningitis, and other aseptic meningitis (Nishida et al. Only two cases of cryptococcal meningitis associated with increased CSF ADA level have been described in the literature (Izumoto et al. In conclusion, we report an older patient with cryptococcal meningitis associated with increased CSF ADA level, in which information on the chronological change in CSF ADA level was useful for follow-up assessment. Although the determination of CSF ADA level can be useful for early differential diagnosis of TBM, other neurological diseases such as cryptococcal meningitis should be considered.


Saussurea involucrata plant, has anti-tumor and anti-inflammatory properties that ameliorate airway hyperresponsiveness in asthmatic mice. This study investigated whether acacetin has anti-adipogenic effects in 3T3-L1 adipocytes and whether it regulates the inflammatory response in adipocytes and macrophages. It also investigated whether acacetin ameliorates lipid accumulation in high-fat diet- (HFD) induced obese mice. Differentiated 3T3-L1 cells were treated with acacetin. The glycerol levels in the culture medium were measured, and the expression of proteins and genes involved in adipogenesis and lipolysis were assayed by Western blot and real-time PCR, respectively. Inflammatory cytokine signaling pathway activity was assessed in macrophages that were treated with acacetin and cultured with differentiated medium from 3T3-L1 cells. Intraperitoneal injections of acacetin were administered to HFD-induced obese mice twice a week for 10 weeks. Acacetin significantly increased the levels of glycerol in the culture medium and significantly inhibited lipid accumulation in adipocytes. Acacetin reduced the expression of adipogenesis-related transcription factors, including the expression of the CCAAT/enhancer-binding protein; it also increased sirtuin 1 expression and AMPK phosphorylation in adipocytes. In macrophages cultured with differentiated media from 3T3-L1 adipocytes, acacetin reduced the levels of inflammatory mediators and the activity of the mitogen-activated protein kinase and NF-κB pathways. In obese mice, acacetin reduced both body weight and visceral adipose tissue weight. These results demonstrate that acacetin inhibited adipogenesis in adipocytes and in obese mice. Acacetin also reduced the inflammatory response of macrophages that were stimulated with differentiated media from 3T3-L1 cells.Acacetin, a flavone that can be isolated from the  Obesity is a global public health problem that is associated with an increasing prevalence of chronic diseases such as metabolic syndrome, type 2 diabetes, cardiovascular disease, and cancer . White aA previous study found that in obese individuals, adipose tissue can secrete excessive chemokine ligand 5 (CCL5) and monocyte chemotactic protein-1 (MCP-1) protein to recruit macrophages that infiltrate the adipose tissue . AdipokiSaussurea involucrata Kar. et Kir. mainly grows in the high mountains of Xinjiang Province of China  induced obese mice. We also investigated whether acacetin reduced the inflammatory response of macrophages to adipogenic differentiation medium (DM).S. tridactyla, S. involucrata or other Asteraceae family (Acacetin (≥97% purity by HPLC), can be isolated from e family  was purcFor histological analysis, the mice were sacrificed, and the adipose tissue was fixed in formalin and embedded in paraffin, as described previously . AdipocyThe 3T3-L1 murine pre-adipocyte cell line and RAW264.7 murine macrophage cell line were purchased from the Bioresource Collection and Research Center .TM, Paisley, Scotland) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen-GibcoTM). The RAW 264.7 cells were routinely cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum . All cells were incubated at 37°C in a 5% CO2 atmosphere and subcultured twice a week.3T3-L1 cells were cultured in DMEM  for 24 h.Differentiated 3T3-L1 adipocytes were cultured with DMEM medium for 24 h, then the cultured medium (DM) was collected as described previously . RAW 264RAW 264.7 cells were cultured in DM plus acacetin for 1 h to collect medium for detected NO production. The nitrite measured as an indicator of NO in culture medium by Griess reagentas previously described . The levGlycerol was measured using the Glycerol Assay Kit (Sigma) according to the manufacturer’s protocol. Differentiated adipocytes were treated with various concentrations of acacetin for 24 h. Culture medium was collected and treated with the assay reagent. The levels of glycerol were determined by absorbance at 570 nm using a microplate reader .Differentiated adipocytes were treated with acacetin for 24 h and fixed with formalin as described previously . The celCells were treated with acacetin and lysed using protein lysis buffer (Sigma). Equal amounts of protein were separated on 8–10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes . The PVDF membranes were blocked, incubated with primary antibodies overnight at 4°C, incubated with secondary antibodies, and the signal was detected with Luminol/Enhancer solution (Millipore). Protein bands were quantitated using the BioSpectrum 600 system . The primary antibodies included antibodies that recognized the following proteins: phosphorylated-AMPKα, AMPK, IκBα, phosphorylated-IκBα, p65, and lamin B1 ; ERK1/2, phosphorylated-ERK1/2 (pERK1/2), JNK, phosphorylated-JNK (pJNK), p38, phosphorylated-p38 (pp38), fatty acid synthase (FAS), fatty acid binding protein (aP2), sirtuin 1 (Sirt1), SREBP-1c, and lipoprotein lipase (LPL) ; acetyl CoA carboxylase-1 (ACC-1), phosphorylated-ACC-1 (pACC-1), adipose triglyceride lipase (ATGL), C/EBPα, C/EBPβ, PPAR-α, PPAR-γ, hormone-sensitive lipase (HSL), phosphorylated-HSL (pHSL) ; and β-actin (Sigma).Table 1.RNA extracted from 3T3-L1 adipocytes using TRIzol reagent , and cDNA synthesized using a cDNA synthesis kit (Life Technologies). The specific DNA was amplified using real-time PCR with the SYBR Green Master kit  and a spectrofluorometric thermal cycler . The specific primers that were used for real-time PCR are shown in ELISAs were performed as described previously . 3T3-L1 post hoc test. The results are reported as means ± standard deviation (SD). P-values less than 0.05 were considered statistically significant.Statistical analyses were performed using one-way ANOVA with Dunnett’s The MTT assay was used to evaluate the cytotoxicity of acacetin in 3T3-L1 adipocytes.in vitro experiments.Acacetin at concentrations ≤ 100 μM showed no significant cytotoxicity in 3T3-L1 cells (data not shown). Therefore, 3–100 μM acacetin was used in the subsequent Figure 1A). Lipid accumulation in adipocytes was measured by treating the cells with isopropanol to release the oil droplets into the solution. Acacetin significantly reduced lipid accumulation compared to control cells . Triglycerides in adipocytes are broken down to produce free fatty acid and glycerol  .3T3-L1 adipocytes were treated with 0, 3, 10, 30, or 100 μM acacetin , and lipid accumulation was assessed by oil red O staining. Microscopy observations showed that acacetin treatment reduced lipid droplets in differentiated 3T3-L1 cells . However, acacetin significantly decreased the protein expression of C/EBPα, C/EBPβ, and SREBP-1c. Real-time PCR analysis showed that differentiated adipocytes treated with acacetin had lower mRNA levels of C/EBPα, C/EBPβ, and SREBP-1c but not lower mRNA levels of PPAR-α and PPAR-γ than the control cells .Western blot analysis showed that differentiated adipocytes treated with acacetin did not express significantly lower levels of the PPAR-α and PPAR-γ proteins than the control cells . Acacetin also significantly increased ATGL and pHSL protein expression and enhanced the expression of the ATGL and HSL genes compared to control cells . Aquaporin-7 (AQP7) is a channel protein on the adipocyte plasma membrane that helps regulate the release of glycerol from adipocytes into serum .Acacetin suppressed FAS, LPL, and aP2 protein and gene expression in 3T3-L1 cells in a concentration-dependent manner . The effects of acacetin on 3T3-L1 cells were similar to the effects of resveratrol (an AMPK activator) in that both agents enhanced Sirt1 expression and both increased the pAMPKα and pACC-1 levels . Furthermore, the levels of pAMPK, pACC-1, and Sirt1 were restored in 3T3-L1 cells co-treated with compound C (an AMPK inhibitor) and acacetin compared to the levels in cells treated only with compound C .Compared to control cells, acacetin significantly increased Sirt1 expression in 3T3-L1 cells and also increased the phosphorylation of AMPKα and ACC-1 in a concentration-dependent manner .Previous studies showed that TNF-α induces 3T3-L1 cells to become insulin-resistant adipocytes that release the chemokines MCP-1 and CCL5 and increase macrophage infiltration into adipocyte tissue . Here weFigure 5B). Acacetin also decreased COX-2 expression compared to the DM only group .Next we investigated whether acacetin could suppress the inflammatory response of macrophages to the fatty acids or adipokines released by adipocytes. RAW 264.7 macrophages were treated with acacetin or left untreated and then cultured in DM from differentiated 3T3-L1 cells for 24 h. We found that the RAW 264.7 cells cultured in DM medium showed significantly increased levels of nitrite, TNF-α, IL-6 and MCP-1, but acacetin treatment significantly reduced the levels of these inflammatory mediators . Acacetin also significantly inhibited the phosphorylation of ERK1/2 and JNK but did not reduce p38 phosphorylation .RAW 264.7 macrophage cells were treated with acacetin or left untreated and then cultured in DM from differentiated 3T3-L1 cells for 24 h. Acacetin reduced the levels of pIκB-α and attenuated the transport of the NF-κB subunit p65 into the nucleus . Histological analysis of the adipocyte tissue showed that acacetin decreased lipid accumulation and adipocyte size compared to untreated HFD-induced obese mice .We investigated the effects of acacetin on HFD-induced obese mice and found that acacetin treatment significantly reduced the body weight and visceral adipocyte tissue weight of obese mice .Real-time PCR analysis showed that acacetin did not significantly increase or decrease PPARα and PPARγ gene expression in mouse visceral adipocyte tissue compared with the HFD group. Interestingly, acacetin significantly decreased the gene expression of C/EBPα, C/EBPβ, SREBP-1c, and FAS, and it also significantly increased the gene expression of ATGL and HSL compared with the HFD group (Obesity is an important causal factor in many chronic diseases, including cardiovascular diseases, hypertension, hyperlipidemia, diabetes, and cancer  Morriso. OvernutMany studies found that some natural products may improve cardiovascular diseases and diabetes in obesity. Curcumin could modulate hypolipidemic effects, inhibit liver inflammation and decrease lipid accumulation of liver and adipose tissue in HFD-induced obese mice . ResveraQuercetin could regulate glucose uptake in skeletal muscle cell and hepatocytes for improved type 2 diabetes . In the Adipogenesis was the process that the differentiation of preadipocytes changed into mature adipocytes. Previous studies found that 3T3-L1 preadipocytes require the sequential action of adipogenesis-related transcription factors to induce and regulate their differentiation into mature adipocytes . PPARs . PPARα aHence, we also investigated the possible involvement of other transcription factors in adipocyte differentiation. C/EBP and SREBP-1c are two transcription factors that are involved in lipogenesis in the adipocyte differentiation process .Early stage of adipocyte differentiation, dexamethasone stimulated C/EBPβ expression for maintained the early stage of gene expression . Next, Cin vitro and in the adipocyte tissue of obese mice in vivo. Our results thus suggested that acacetin attenuated lipogenesis and had an anti-obesity effect in obese mice mainly by blocking the action of C/EBPs and SREBP-1c. Notably, SREBP-1c and C/EBPs bind to the promoter of FAS to promote lipogenesis and to increase aP2 and LPL expression in adipocytes (Acacetin significantly decreased C/EBPα, C/EBPβ, and SREBP-1c expression in mature adipocytes ipocytes . aP2 is ipocytes . FAS is ipocytes . LPL hydipocytes . Acacetiipocytes . We specReducing lipid accumulation and increasing lipid catabolism in adipocyte tissue has an anti-obesity effect in obese mice . In the AMPK is an energy sensor that is involved in the regulation of lipid and glucose metabolism , while SThe present study found that acacetin could break down triglycerides into glycerol and free fatty acids, and the free fatty acids were released into the culture medium. A previous study showed that mature adipocytes release fatty acids or adipokines to induce an inflammatory response in macrophages via toll-like receptor 4 and via activated MAPK and NF-κB signaling pathways . AdipokiExcessive lipid accumulation induces insulin resistance in adipocytes, hepatocytes, and skeletal muscle cells . InsulinIn vitro, acacetin suppressed the expression of transcription factors involved in lipid synthesis in mature 3T3-L1 adipocytes and increased lipolysis by increasing ATGL and HSL expression. Moreover, acacetin reduced macrophage inflammation when macrophages were cultured with DM from mature 3T3-L1 adipocytes. These findings provide evidences that acacetin inhibit adipogenesis in adipocytes and in obese mice, and acacetin is a natural product that has potential anti-obesity effects.Acacetin reduced both the body weight and visceral adipose tissue weight of HFD-induced obese mice and suppressed the expression of genes involved in lipogenesis in murine visceral adipose tissue. Designed the experiments: C-JL, S-JW, and W-CH; Performed the experiments: C-JL, S-JW, and C-YC; Analysis and interpretation of data: L-CC and K-WY; Drafting the manuscript: C-JL, L-CC, and W-CH.The 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.


Peripheral blood stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) from healthy donors are commonly used for allogeneic stem cell transplantation. The effect of G-CSF administration on global serum metabolite profiles has not been investigated before.This study aims to examine the systemic metabolomic profiles prior to and following administration of G-CSF in healthy adults.Blood samples were collected from 15 healthy stem cell donors prior to and after administration of G-CSF 10 µg/kg/day for 4 days. Using a non-targeted metabolomics approach, metabolite levels in serum were determined using ultrahigh performance liquid chromatography-tandem mass spectrometry and gas chromatography/mass spectrometry.Comparison of the metabolite profiles of donors before and after G-CSF treatment revealed 239 metabolites that were significantly altered. The major changes of the metabolite profiles following G-CSF administration included alteration of several fatty acids, including increased levels of several medium and long-chain fatty acids, as well as polyunsaturated fatty acids; while there were lower levels of other lipid metabolites such as phospholipids, lysolipids, sphingolipids. Furthermore, there were significantly lower levels of several amino acids and/or their metabolites, including several amino acids with known immunoregulatory functions . Lastly, the levels of several nucleotides and nucleotide metabolites  were also decreased after G-CSF administration, while methylated products were increased. Some of these altered products/metabolites may potentially have angioregulatory effects whereas others may suggest altered intracellular epigenetic regulation.Our results show that G-CSF treatment alters biochemical serum profiles, in particular amino acid, lipid and nucleotide metabolism. Additional studies are needed to further evaluate the relevance of these changes in healthy donors.The online version of this article (doi:10.1007/s11306-016-1139-x) contains supplementary material, which is available to authorized users. Peripheral blood stem cell (PBSC) grafts are commonly used for allogeneic hematopoietic stem cell transplantation for a wide range of hematologic malignancies. These grafts are generally prepared by administration of granulocyte colony-stimulating factor (G-CSF) to healthy donors which mobilizes hematopoietic stem cells (HSCs) into the blood before cells are harvested by leukapheresis. The clinical advantage of using PBSCs compared to bone marrow grafts includes accelerated neutrophil engraftment, which increases the likelihood to engraft, and in addition PBSC allografts contain a much larger number of T-cells compared to bone marrow grafts, which has been correlated with better outcome , mobilize sufficient stem cells for preparation of allografts and their age is also representative for donors used in routine clinical practice.The study was approved by the local ethics committee  and all samples were collected after written informed consent. Blood samples were collected from 15 consecutive healthy HLA-matched related allogeneic stem cell donors , with a mean age of 47 years (range 25–64 years)  and stored frozen at −80°C until analysed.Venous blood samples were collected into Vacuette Z Serum Clot Activator tubes with Gel Separator  from donors at two time points, (i) prior to administration of G-CSF and (ii) following G-CSF administration just before apheresis on day 4. All samples were collected at 9 am and were allowed to coagulate for 30 min at room temperature in upright position before being centrifuged at 1310×Levels of human G-CSF were measured using a Luminex assay , and the minimal detectable level was 20 pg/mL.® system . Briefly, samples were extracted using Metabolon`s standard solvent extraction method  to remove the organic solvent. Then samples were either stored overnight under nitrogen for LC or dried under vacuum overnight for GC, before preparation for analysis. Experimental samples were randomized across the platform and run with appropriate quality control samples spaced evenly among the injections. Compounds were identified by comparison to library entries based upon retention time/index, mass to charge ratio (m/z) and chromatographic data , and peaks were quantified using area-under-the curve.All mass spectrometry data were collected at Metabolon Inc . Each serum sample was accessioned into the Metabolon LIMS system and was assigned a unique identifier by this system which was used to track all sample handling and results. All samples were prepared using the automated MicroLab STARhttp://cran.r-project.org/).Two types of statistical analysis were performed: (1) significance tests (t-tests) and (2) classification analysis. Random Forest analysis is a supervised classification technique that provides an unbiased estimate of how well individuals can be classified into each group in a new data set. Statistical analyses were performed with the program R  prior to G-CSF administration and (ii) on day 4 after G-CSF administration. In total, 641 metabolites were identified (for a complete list see Supplementary Table 1), where levels of 239 metabolites were significantly changed (p ≤ 0.05); 149 metabolites had increased levels (62%) and 90 metabolites had decreased levels (38%) after G-CSF administration  which showed that the samples before and after G-CSF administration were generally distinguishable from each other Fig. ; one G-CRandom forest classification was used for further statistical analyses. Even though there was an overlap between samples collected before and during G-CSF therapy in the PCA plot Fig. , the ranLipid metabolism Among the main metabolites that distinguished between pre-and post-G-CSF administration groups, there was a consistent alteration of the amount of fatty acids indicating altered lipid metabolism   Fig. a. The poAmino acid metabolism was also altered; within the post-G-CSF samples there was a reduced level of dipeptides and amino acids including the aromatic amino acids tryptophan, phenylalanine and tyrosine and their metabolites, as well as branched-chain amino acids valine, isoleucine and leucine  levels of a wide range of metabolites reflecting an alteration of different metabolic pathways during this treatment in healthy individuals:We performed a metabolomic pathway enrichment analysis based on significantly altered metabolites (p < 0.001), to identify pathways that contribute to the major differences when comparing samples taken before and after G-CSF administration Fig. . This anMusculoskeletal pain and flu-like symptoms are common during G-CSF therapy  after G-CSF administration, which is in concordance with another study that measured S1P levels in donors undergoing G-CSF-induced mobilization   using whole genome expression profiling. Changes in global gene expression profiles were then described both at early time points after G-CSF administration (up to 5 days) . Furthermore, the increased levels of immature hematopoietic cells in the peripheral blood probably represent a minor part of the overall number of nucleated bone marrow cells, and one should also emphasize that the cells have not yet been harvested at the time of sampling for metabolite analysis. However, we cannot exclude the possibility that the metabolic changes are secondary to an increased proliferation of bone marrow cells, but if so this is in our opinion most likely caused by direct G-CSF stimulated proliferation during the whole treatment period rather than being a compensatory mechanism to altered compartmentalization during the last hours before stem cell harvesting.A possible explanation for our findings could be that they are secondary to an increased proliferation of immature hematopoietic cells to replace the cells that are lost from hematopoietic niches to the circulation. However, the CD34Several metabolites classified as xenobiotics were altered after G-CSF therapy. Many stem cell donors experience side effects during treatment  increased levels of lipids indicating altered fatty acid metabolism, (ii) increased levels of methylated nucleosides, (iii) changes associated with energy metabolism, and (iv) altered levels of amino acids, including reduced peptide levels indicating decreased proteolysis and altered levels of acetylated peptides. Long-term follow up studies have concluded that the use of G-CSF to mobilize stem cells appears to be safe Supplementary material 2 (DOCX 19 kb)Below is the link to the electronic supplementary material.


M. aurantiaca males across a PVC grid on the forest floor or enclosure floor to a speaker. For each playback, the following parameters were recorded to define the accuracy of phonotaxis: (1) number of jumps; (2) jump angles; (3) jump distances; (4) path straightness. During this experiment we observed that wild frogs had a similar behavioural (phonotaxis) response to calls independent of their source while frogs from Chester Zoo had a significantly stronger response to calls of other conspecifics held separately at Chester Zoo. The lack of appropriate phonotaxis response by captive bred frogs to the calls of wild conspecifics could have serious negative conservation implications, if the captive bred individuals were released back to the wild.Acoustic communication is highly influential in the expression of social behavior by anuran amphibians, transmitting information about the individual’s physical condition and motivation. We studied the phonotactic (approach movements) responses of wild and captive male golden mantella frogs to conspecific wild and captive playback calls to determine the impact of captivity on social behaviour mediated by vocalisations. Calls were recorded from one wild and two captive populations. Phonotaxis experiments were then conducted by attracting  Communication is the foundation upon which all social relationships between animals are built . AcoustiIn anurans significant information about the individual’s fitness is transmitted by acoustic signals ,4. AmongPhonotaxis is defined as any kind of movement or orientation towards specific acoustic signals . PositivMantella aurantiaca) to conspecific wild and captive playback calls.It is believed that the captive environment can significantly affect the vocalisations of animals to a point where their calls are no longer recognised by wild conspecifics . This woMantella aurantiaca) is a critically endangered species [ex-situ assistance is vital for the long-term survival of the golden mantella frog [The golden mantella frog  and Chester Zoo (UK). The phonotaxis experiments were performed with wild frogs in Madagascar and from captive frogs kept at Chester Zoo.Mangabe also known as the “blue forest” is a site of international biodiversity importance, divided into two administrative districts, Moramanga in the north and Anosibe An'ala to the south. Data sampling for this study was done in the Moramanga region. Most breeding ponds for the golden mantellas frogs are found in this area according to recent studies concerning conservation priority sites for mantella frogs.ex situ groups of M. aurantiaca, one is on public display at the zoo’s Tropical Realm exhibit from which calls were recorded and a second group is kept off show in a biosecurity container specifically designed for conservation-related research, where the playback experiment was conducted with these frogs. The biosecurity container is kept under temperature and humidity regimes to give the frogs a similar environment as they would experience in the wild. Enclosures are annually modified to keep animals under rainy and dry periods as per their natural environments.The zoo currently maintains two visually and acoustically isolated This community-run conservation organisation operates around the village of Andasibe in east-central Madagascar and it holds the first Malagasy biosecure facility to protect endangered amphibians. Fifteen local species including a genetically viable population of the golden mantella frog taken from the wild  collected at the Ambatovy area, and their F1 offspring are currently being kept at Mitsinjo. Only calls from the F1 frogs were recorded and used (no playback experiments were done here).All the research reported in this study was approved by the Chester Zoo’s Ethics Committee, UK and it conforms to all regulations and laws in all relevant countries in relation to care of experimental animal subjects. Furthermore we can confirm, from our post-experimental monitoring, that no animals suffered any injuries, became ill or had their survivorship negatively affected as a result of this study. Furthermore we followed the Association for the Study of Animal Behaviour’s Guidelines for the care of animals .Frog calls were recorded using a digital audio recorder  with an omnidirectional microphone. Before recording calls, a pilot study was undertaken at the University of Manchester with their captive colony of golden mantella frogs to ensure the microphone and recorder had the appropriated sensitivity . Recordings were analysed for call characteristics using Raven software . The chaCall duration (s): Duration from the beginning of a call to its end.Call period (s): Duration from the beginning of a call to the beginning of the next call.Pulse rate: The number of individual components of each call.Interpulse interval (s): Time between the pulses of a call.Dominant frequency (Hz): The frequency with maximum intensity.M. aurantiaca from each population. In addition, to minimize intraspecific variance, we used mean values of the call parameters within and between individuals.We analysed three call sequences of 20 different males Prior to any experimentation, measurements of sound pressure (noise) levels that animals are already exposed to during routine husbandry at Chester Zoo were taken using a sound pressure meter  to avoid exposing animals to any extreme acoustic stimuli. Playback recordings were used with similar amplitude (i.e. volume) to what the animals were already exposed to in captive or natural environments. Calls were previously recorded from the three different populations using a digital audio recorder  with an omnidirectional microphone. Calls were edited for length and background noise using Audacity®  recordinM. aurantiaca males across a 100 x 60 cm PVC mat on the forest floor or enclosure floor to a Bluetooth speaker  broadcasting calls, similar to the method described by Mayer and colleagues (2014). During the experiment, 21 males from Chester zoo and 39 individuals from Mangabe had their phonotaxis response tested. Frogs were placed 10 cm away from the mat . Each animal was tested only once. Phonotaxis playback experiments were than conducted by attracting  mat see . Trials Allobates femoralis [Ranitomeya imitator [Previous playback studies with emoralis  and Raniimitator  revealedEach jump of an approaching male was plotted by manually digitizing the recorded videos in a stop-motion view with software called BORIS . The griposthoc test . In general, captive frogs took longer and used a lengthier and less accurate path to reach the speaker than wild frogs. All trials with Chester Zoo’s frogs resulted in a phonotaxis response, however, five trials  from Mangabe’s animals, had no phonotaxis response (i.e. no movement) and were, therefore, not analysed. All successful trials were scored for number of jumps, jump distances, jump angles, path straightness and duration .Generalised linear mixed models (GLMM) were used to compare the golden mantella frogs’ phonotactic movement in response to different playback treatments see . Calls wChester Zoo’s frogs had significant differences (p<0.05) in the number of jumps and duration to the speaker when their own call was presented, jump angles for Mangabe and zoo calls, and path straightness between all calls ; howeverWhen the responses of both populations were compared using a t-test  all the The analysis of different call parameters showed that calls from Chester Zoo’s frogs were statistically different from wild frogs’ vocalisations in all analysed characteristics. Whereas the call analyses from the colony held at Mitsinjo breeding centre showed greater similarities with the wild conspecifics. The implication of the observed differences could be negative in terms of reproduction if captive frogs were to be released to the wild. The breeding behaviour of golden mantella frogs involves males calling to court the females with multiple males vocalising simultaneously . Males wVocalisations are moulded by the acoustic environment in which the species is found ,25. A zoDuring the phonotaxis experiment we observed that wild frogs had a similar behavioural (phonotaxis) response to calls of conspecifics independent of their source (i.e. wild versus captive) while frogs from Chester Zoo had a significantly stronger response to their own calls. Wild frogs had more accurate response, reaching the speaker using a shorter path and in less time while captive frogs were using a longer path and more time, even though they had longer jumps. It is important to notice that wild frogs would recognize and react in a similar way to captive frogs despite the changes found in their calls. Captive frogs had a weak response to wild calls and, if captive frogs are not able to recognize wild calls or respond appropriately, this could, potentially have negative consequences, 27, sucThe golden mantella frogs breeding behaviour is characterized by groups of males competitively calling to attract females; in this scenario it is usual to observe males showing aggressive behaviour toward other males as a sign of competition for females. This aggressive behaviour have been describe in the wild and observed in captive populations . The phoSpecies recognition is a fundamental problem for animals in social contexts  for a reAnimals in captivity are in a confined space in close proximity to other males , which cFrogs characteristically avoid moving unless totally necessary, since it is both energetically costly and increases predation risk . The recLeontopithecus rosalia) [Animals may adjust the characteristics of their vocalizations in response to temporary changes in the background noise ,1. Such rosalia) .Communication can be crucial for breeding success in golden mantella frogs if individuals are being bred for conservation; it is of critical importance to make sure that captive animals, if released, will have the same chances of breeding as their wild counterparts. Captive breeding is growing as an indispensable tool in conservation tool for many species , especia


Nosocomial CNS infection (NI-CNS) is a common and serious complication in neurocritical care patients. Timely, accurate diagnosis of NI-CNS is crucial, yet current infection markers lack specificity and/or sensitivity. Presepsin (PSP) is a novel biomarker of macrophage activation. Its utility in NI-CNS has not been explored. We first determined the normal range of cerebrospinal fluid (CSF) PSP in a control group without brain injury before collecting data on CSF PSP levels in neurocritical care patients. Samples were analyzed in four groups defined by systemic and neurological infection status.CSF PSP levels in 15 control patients without neurological injury were 50–100 pg/ml. Ninety-seven CSF samples were collected from 21 neurocritical care patients. In patients without NI-CNS or systemic infection, CSF PSP was 340.4 ± 201.1 pg/ml. Isolated NI-CNS was associated with CSF PSP levels of 640.8 ± 235.5 pg/ml, while levels in systemic infection without NI-CNS were 580.1 ± 329.7 pg/ml. Patients with both NI-CNS and systemic infection had CSF PSP levels of 1,047.7 ± 166.2 pg/ml. In neurocritical care patients without systemic infection, a cut-off value of 321 pg/ml gives sensitivity and specificity for NI-CNS of 100 and 58.3%, respectively.CSF PSP may prove useful in diagnosing NI-CNS, but its current utility is as an additional marker only. Nosocomial infection of the central nervous system (NI-CNS) is a serious complication in neurocritical care patients, which leads to clinical deterioration, worsening of outcomes, and increased treatment costs –3. Risk The timing of antibiotic administration in neurocritical care patients with suspected NI-CNS is of pivotal importance. Early antibiotic therapy improves outcomes, reduces intensive care unit (ICU) stay, and decreases treatment costs –11. EquaIdeal antibiotic strategy can therefore only be achieved with accurate and rapid diagnosis of NI-CNS and pathogen verification. In many clinical situations, traditional diagnostic criteria are not specific. Alterations in consciousness and fever are common in neurocritical care patients and NI-CNS is one of a plethora of possible diagnoses. Meningism is typical of NI-CNS, but is also provoked by subarachnoid blood , 7, 14. Consequently, the search for new, reliable markers of NI-CNS is currently a focus of intensive research efforts. Recently, a new inflammation biomarker—presepsin —has been introduced into clinical practice –27. PSP Presepsin can also be detected in the CSF  and may This prospective observational study, carried out in accordance with Good Clinical Practice and the Declaration of Helsinki, was performed in a tertiary hospital setting. The study was approved by the local ethics committee and participants or their surrogates gave informed, written consent. The study was formed of two parts. The objective of the first component was to establish normal ranges of CSF PSP, while the second was to determine cut-off values predictive of NI-CNS and to provide data on the sensitivity and specificity of CSF PSP for NI-CNS. We also aimed to determine whether the presence of blood in the CSF affects PSP levels.In the first study component, a control group of adult patients undergoing elective surgery for urological pathology requiring spinal anesthesia were recruited. Exclusion criteria included individuals under 18 years of age, the presence of concomitant neurological or neurosurgical pathology, or refusal to participate in the study. In each patient, a CSF sample was obtained at induction of spinal anesthesia. Contemporaneous blood samples were not taken and only PSP was measured in the CSF.In the second component, neurocritical care patients aged 18 and older with suspected NI-CNS were recruited. In our institution, patients are considered for neurocritical care if they meet the following criteria: altered consciousness with a Richmond Agitation-Sedation Scale of +3 or +4 or −3 through −5, including those in coma or requiring prolonged sedation; respiratory insufficiency requiring mechanical ventilation; hemodynamic instability requiring inotropic or vasopressor support; or severe electrolyte disturbances  with high risk of complications.Patients were excluded if they had any contraindication to lumbar puncture (unless they had a ventricular drain). Other exclusion criteria comprised brain death and the refusal by the patient or their surrogates to participate. Patients were treated in accordance with international guidelines . In patiFor this study, NI-CNS was diagnosed when the following criteria were met with or without positive bacterial CSF culture: clinical suspicion , CSF ceU-test or the Wilcoxon test as appropriate), with statistical significance set at the p ≤ 0.05 level. Specificity and sensitivity of CSF PSP in the different patient groups were assessed using the receiver operating characteristic (ROC) toolbox in SPSS.Statistical analysis was performed using SPSS version 23.0 . The Shapiro–Wilk method was used to test for distribution normality. All comparisons between groups were carried out using non-parametric tests  59.3 ± 14.1 years and all were male. None required postoperative ICU admission, and outcomes were favorable in all cases. CSF PSP was 75.32 ± 25.32 pg/ml. Values were normally distributed, allowing us to conclude that the normal range of CSF PSP in patients with systemic infection, brain injury, or NI-CNS is 50–100 pg/ml.Twenty-one neurocritical care patients with suspected or confirmed NI-CNS were included in the study. Mean age was 50.7 ± 15.0 years. The pathologies leading to ICU admission are listed in Table Proteus mirabilis (1), Streptococcus pneumonia (1), and Enterococcus faecium (1). Risk factors for NI-CNS and outcomes are presented in Table Sixteen patients required mechanical ventilation during their stay (mean duration 11.3 ± 10.0 days), with fifteen undergoing a tracheostomy. The non-intubated patients were admitted either for vasopressor support in the context of hemodynamic instability (4 patients) or for treatment of hyponatremia . Mean length of stay in ICU was 16.5 ± 9.2 days, with total hospital stay 37.6 ± 37.0 days. NI-CNS was diagnosed in 10 patients. Onset of NI-CNS was 5.9 ± 3.5 days post-operation/ictus. Causative microorganisms were identified in three patients: In total, 97 pairs of CSF and blood samples were obtained. All pairs were divided into four groups defined by the presence or absence of NI-CNS and/or systemic infection Table . The disIn cases with neither NI-CNS nor systemic infection , CSF PSP was 340.4 ± 201.1 pg/ml. This level of CSF PSP, obtained from neurocritical care patients, is significantly higher than that found in the CSF of the control participants investigated during the first part of the study  are seen in the context of simultaneous NI-CNS and systemic infection. Finally, the presence of blood in the CSF does not appear to significantly influence CSF PSP levels.The diagnosis of NI-CNS in contemporary neurocritical care is frequently presumptive due to the low sensitivity and specificity of traditional markers, a fact well demonstrated in our cohort Table . New, reAlmost nothing is known regarding PSP behavior in the CSF of adult neurocritical care patients, with or without NI-CNS. It was therefore necessary to establish a normal range using an appropriate control population without brain injury. In our cohort of urological patients undergoing routine spinal anesthesia, the normal range of CSF PSP was 50–100 pg/ml. Certain caveats pertain to this finding; this small control group consisted only of older males prior to undergoing surgery for urological tumors. These factors  may influence CSF PSP and care should be exercised in extrapolating these values to other groups. For comparison, 123 control participants had a median serum PSP of 123 pg/ml (IQR: 89–155 pg/ml) , and thuNeurocritical care patients invariably have some degree of brain injury . Thus, iSystemic infections have considerable effects on neurological function . DeliriuInfection of the CNS stimulates an immune response and microglia activation. It may be expected that this response would be more pronounced in NI-CNS than in brain injury without infection or in neurocritical care patients with systemic infection only. These data indicate that CSF PSP levels in such cases (640 ± 235.5 pg/ml) are indeed higher than the other two groups, but not significantly so.The highest level of CSF PSP was observed in patients with neurological injury with both NI-CNS and systemic infection, with levels frequently over 1,000 pg/ml.Receiver operating characteristic analysis of these preliminary data indicates that a CSF PSP cut-off value of 321 pg/ml and above has a sensitivity of 100% and a specificity of 58.3% for NI-CNS in neurocritical care patients without systemic infection. The relatively low specificity was in keeping with the high SD observed in these two groups of patients, with the optimal cut-off value falling below the mean of the group without NI-CNS (340 ng/ml; Figure Taken collectively, these data provide grounds to use CSF PSP in the diagnosis of NI-CNS only as an additional marker. Indeed, CSF PSP discriminated particularly poorly between our patients with systemic infection and those with isolated nosocomial CNS infection. However, there is reason to be optimistic that it may be possible to define sensitive and specific cut-off values for CSF PSP for particular clinical situations such as isolated brain injury, intraventricular hemorrhage, CNS infection, and so on, as is the case with PCT , 50. ComBlood in the CSF complicates the diagnosis of NI-CNS for several reasons. First, cell counts are always raised in subarachnoid hemorrhage and postoperative patients and, while centrifugation of the CSF sample may aid in discrimination between a postictal state and CNS infection, this is not always reliable . Second,This preliminary, prospective analysis is subject to the limitations inherent to such pilot studies. The number of patients and samples were small and from a single institution and, while all data were normally distributed and patients were representative of the target group in which PSP may be used, the cut-off value indicated by the ROC analysis should be interpreted with caution.Cerebrospinal fluid PSP holds potential as a diagnostic marker for NI-CNS. The normal level of PSP in the CSF is 50–100 pg/ml, whereas a CSF PSP of more than 321 pg/ml in neurocritical care patients without systemic infection is associated with 100% sensitivity and 58.3% specificity for NI-CNS. Currently, the role of CSF PSP in diagnosing NI-CNS is limited to application as an additional marker, aiding the interpretation of the clinical picture, including other routine infection parameters.All procedures performed in studies involving human participants were in accordance with the ethical standards of the 1964 Helsinki declaration and its later amendments. The study was approved by the research ethics committee of the Burnazian State Research Center. Informed consent was obtained from all individual participants included in the study.NK, KB, IL, MZ, AS and EC designed and carried out the study, analyzed and interpreted data and reviewed the manuscript. SA designed and carried out the study, analyzed and interpreted data and wrote the manuscript. KK and TK performed statistical analysis, analyzed and interpreted data and reviewed wrote the manuscript. OP and YU analyzed and interpreted data and reviewed the manuscript. KP conceived and acted as sponsor and guarantor of the study, designed and carried out the study, analyzed and interpreted data and wrote the manuscript.The 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.


This paper reports the profile of the Australian chiropractic workforce and characteristics of chiropractic care from a large nationally-representative sample of practitioners.n = 4,684) in Australia in 2015 via both online and hard copy mail out.A 21-item questionnaire examining practitioner, practice and clinical management characteristics was distributed to all registered chiropractors , and the sample is largely representative of the national chiropractic workforce on a number of key indicators. The average age of the chiropractors was 42.1 years, nearly two-thirds are male, and the vast majority hold a bachelor degree or higher qualification. Australian chiropractors are focused upon treating people across a wide age range who mainly present with musculoskeletal conditions. Australian chiropractors have referral relationships with a range of conventional, allied health and complementary medicine (CAM) providers.The survey attracted a response rate of 43% (The chiropractic profession represents a substantial component of the contemporary Australian health care system with chiropractors managing an estimated 21.3 million patient visits per year. While the Australian chiropractic workforce is well educated, research engagement and research capacity remains sub-optimal and there is much room for further capacity building to help chiropractic reach full potential as a key integrated profession within an evidence-based health care system. Further rich, in-depth research is warranted to improve our understanding of the role of chiropractic within the Australian health care system. Chiropractic constitutes a substantial component of health care seeking in Australia and worldwide , 2, withAs for the Australian workforce characteristics of the chiropractic profession, previous surveys show that the majority of chiropractors are male (64–67%) and aged between 25 and 54 years 80.3%); with 16% of practitioners aged 55 years or older %; with 1.Yet, in-depth, national Australian chiropractic workforce data is vital for helping understanding the existing workforce, supporting future workforce planning, and helping determine if chiropractic training, education and research adequately reflect the practice characteristics and techniques/methods used in grass-roots daily routine care. Moreover, such workforce data also helps appreciate the current and potential interface of chiropractors with other health providers and the role of chiropractors within the wider Australian health system.In direct response to this significant research gap, this paper provides an in-depth examination of the profile of the Australian chiropractic workforce and characteristics of chiropractic care from a large nationally-representative sample of practitioners.This paper reports analyses from a questionnaire distributed as part of the recruitment for the Australian Chiropractic Research Network (ACORN) project — a national practice-based research network (PBRN) independently designed and conducted by senior researchers at the Australian Research Centre in Complementary and Integrative Medicine (ARCCIM), Faculty of Health, University of Technology Sydney. As part of the recruitment for the ACORN PBRN a 21-item practitioner questionnaire was distributed to all registered chiropractors across Australia . While cRecruitment for the ACORN PBRN including the practitioner questionnaire reported in this paper was conducted between March and June 2015 and consisted of an invitation pack distributed to all registered chiropractors via both professional organisations and a profession-wide mail out based on publically available information (including non-members). The invitation pack was also distributed via a number of regional chiropractic-related conferences and events and was also made available online through the ACORN website during the recruitment period. All participants were offered opportunity to complete the practitioner questionnaire via either online access (SurveyGizmo™) or hard copy. A reminder invitation pack was distributed four weeks following initial invitation pack distribution via the same channels and 4 reminders were sent to potential participants via email where possible. Further details regarding the ACORN PBRN recruitment and promotion strategies can be found elsewhere .p = 0.634) and age (p = 0.065). While the workforce sample is also generally representative of the wider chiropractic population regarding practice location, we found slight differences in terms of the distribution of State of residency with the workforce sample slightly over-represented by chiropractors from South Australia, the Australian Capital Territory, Tasmania and the Northern Territory (p < 0.01) [At the time of recruitment there were 4,684 registered chiropractors in Australia and 2,005 chiropractors completed the questionnaire providing a response rate of 43%. Of those 1,119 (55.8%) returned a hard copy questionnaire and 886 chiropractors (44.2%) participated in the online survey. In total, 92.8% of respondents were members of one or both of the two major organisations (CAA and COCA) and 2.7% of respondents were not members of either organisation. Compared to the entirety of registered chiropractors as registered by AHPRA in March 2015 [The instrument used for this study consisted of a 21-item questionnaire including examination of practitioner characteristics, practice characteristics, and clinical management. The questionnaire was developed by an interdisciplinary ACORN Project Steering Committee and subsequently pilot tested amongst a sample of chiropractors to ensure the final format, content and wording were practitioner-sensitive, readily understood and as timely to complete as reasonably possible.The chiropractic practitioner characteristics examined included age, gender, number of years in private chiropractic practice, highest level of chiropractic professional qualification attained, professional organisation membership, and roles as a chiropractor in the previous 12 months. The practice characteristics included average patient care hours and patient visits per week, number of practice locations, area of practice location , State/Territory of practice, other health professionals working in their practice location, professional referral relationships , use of diagnostic imaging, and use of electronic records. Clinical management measures included the frequency of discussion with patients regarding lifestyle aspects of care/management plans, frequency with which chiropractors treat people across a range of conditions and broader patient subgroups, and the frequency with which chiropractors employ a range of techniques/methods and musculoskeletal interventions within their patient management.All data were imported into the statistical software Stata 13.1. Data were checked for plausibility and cleaned for outliers. Data are presented in absolute and relative frequencies for dichotomous or categorical variables, as well as means and standard deviations for continuous variables.Of the 2,005 chiropractors who participated, 62.4% were male and the average age was 42.1 (SD = 12.1) years. Nearly all chiropractors (97.1%) had a bachelor degree or higher, with the majority of chiropractor’s highest professional qualification being a bachelor or double bachelor degree (34.6%), followed by a Master’s degree (32.7%), Doctor of Chiropractic (28.9%) or PhD (0.9%). Only a small number of chiropractor’s highest professional qualification was a diploma (2.1%) or advanced diploma (0.8%). The average number of years in practice was 15.8 (SD = 11.3) years.Other than private practice, the respondents report being involved in a number of other roles as a chiropractor in the previous 12 months, including: university teaching (7.2%), research (6.0%), clinical supervision (10.5%), volunteer work (18.3%), and professional organisation activities (19.4%).The majority of chiropractors are based in (the State of) New South Wales (34.4%), followed by Victoria (24.3%), Queensland (14.5%), Western Australia (13.3%), South Australia (8.9%), Australian Capital Territory (2.3%), Tasmania (1.5%), and the Northern Territory (0.8%). Overall 7.3% of chiropractors routinely consulted patients in a language other than English, with 61.4% of those chiropractors consulting in European languages, and 36.6% in Asian languages (Multiple languages possible). It should also be mentioned that 1.4% of those chiropractors consulted in some sign languages.Most chiropractors are located in an urban area (73.6%); with 75.1% of chiropractors routinely consulting in one location only. Of those who practice in more than one location, 80.9% practice in two locations and 13.3% practice in 3 locations.The majority of chiropractors 78.1%) practice in a multi-practitioner location, with 46.0% of all chiropractors working with one other health practitioner, and 19.2% working with two other health practitioners (Table % practicThe respondents report spending an average of 27.3 (SD = 12.6) hours per week on patient care and providing an average of 87.3 (SD = 57.7) patient visits per week. Diagnostic imaging is utilised as part of practice by 47.7% of the chiropractors on an ‘often’ basis, and 39.6% of the chiropractors on a ‘sometimes’ basis. One in five (20.2%) of the chiropractors reported having imaging facilities or scanning tools available on site, including X-ray (14.9%), MRI (3.2%), surface electromyography (SEMG) (4.1%), thermography (4.5%), ultrasound (2.8%), and other (4.3%). Electronic records are used by 41.7% of all respondents, with 33.0% of all the chiropractors using electronic records for initial history, 39.4% for subsequent patient visits, and 34.1% for examination findings.The chiropractors report discussing a range of topics as part of their care/management plans Table . The mosThe chiropractors report employing a number of techniques and methods in their patient management Table . The mosThis is the first in-depth chiropractic workforce survey drawing upon a nationally representative sample of Australian chiropractors. This survey not only confirms basic findings from prior enquiries but also reveals a number of new and significant insights.Our study findings add further weight to evidence that the national chiropractic workforce is becoming increasingly female over time. This trend was highlighted in a previous study , and seeIt is noticeable however that only 0.9% of chiropractors in our study currently have a PhD. This level of PhD award represents only a small increase compared to the 0.7% identified in 2010  and whilIn the context of research capacity building, it is interesting to note that 6% of the chiropractors in our study did report a personal engagement in research activities and while the specific type of research engagement is not known (it may possibly range from conducting or collaborating on a research activity to possibly having been a targeted participant in a research project) this finding does suggest there may at least be a relatively substantial level of research familiarity amongst chiropractors that the profession may be able to build upon for future research capacity building. Further research is required to explore the research-practitioner interface amongst chiropractors including examination of the perspectives and experiences amongst the ranks of the Australian chiropractic profession towards empirical enquiry and an evidence-based platform for practice.Chiropractors in Australia also report an average of 27 patient contact hours per week across a wide patient age range. Previous reports have found chiropractors working an average of 33 h per week , 16, indAnother interesting finding from our analyses relates to the inter-professional relationship between chiropractors and other health care providers in Australia. While the vast majority of chiropractors in our study report working with another chiropractor or massage therapist in the same practice location, and only a minority of chiropractors report a GP or physiotherapist as working in their practice location, a large number of chiropractors are interconnected to GPs, physiotherapists and other health care professionals by sending and/or receiving referrals. Indeed, a substantial percentage of chiropractors in our study report having a referral relationship with GPs, physiotherapists and other health care professionals. This aligns with previous research that has found more than half of GPs regularly refer to chiropractors , 34 and The vast majority of chiropractors report being consulted by patients with musculoskeletal conditions such as back and neck pain, which is in line with the traditional chiropractic focus on spinal health , 37. It Our findings may be limited due to several factors. The survey data was collected via self-report and this may have introduced a possible recall bias. Furthermore, the depth to which our questionnaire examined some workforce issues was further limited in view of encouraging a reasonable response rate and some of these issues will require follow-up in future research. Due to space limitations for example no detailed information on education and trainings, ethnicity, and attitudes and beliefs of chiropractors were explored; those issues will be examined in further sub studies. Nevertheless, despite these limitations, previous workforce surveys of Australian chiropractic have reported substantially lower response rates , and no The chiropractic profession represents a substantial component of the contemporary Australian health care system. While the Australian chiropractic workforce is well educated, research engagement and capacity remains poor and there is much room for future initiatives to help chiropractic reach full potential as a key integrated profession within an evidence-based health care system. Further rich, in-depth research is required on a number of specific topics relating to the practice of chiropractors in Australia in order to ensure safe, effective and coordinated health care for all patients.


Familial and acquired thrombophilia are often etiologic for idiopathic hip and jaw osteonecrosis (ON), and testosterone therapy (TT) can interact with thrombophilia, worsening ON.Case 2: A 32-year-old hypogonadal Caucasian male had 10 years of unexplained tooth loss, progressing to primary jaw ON with cavitation 8 months after starting TT gel 50 mg/day. Coagulation studies revealed FVL heterozygosity, PAI 4G/4G homozygosity, and the lupus anticoagulant. TT was discontinued. Jaw pain was sharply reduced within 2 months.Case 1: A 62-year-old Caucasian male (previous deep venous thrombosis), on warfarin 1 year for atrial fibrillation (AF), had non-specific right hip-abdominal pain for 2 years. CT scan revealed bilateral femoral head ON without collapse. Coagulation studies revealed Factor V Leiden (FVL) heterozygosity, 4G/4G plasminogen activator inhibitor (PAI) homozygosity, high anti-cardiolipin (ACLA) IgM antibodies, and endothelial nitric oxide (NO) synthase (eNOS) T786C homozygosity . Apixaban 5 mg twice daily was substituted for warfarin; and L-arginine 9 g/day was started to increase NO. On Apixaban for 8 months, he became asymptomatic. Idiopathic ON, often caused by thrombophilia-hypofibrinolysis, is worsened by TT, and its progression may be slowed or stopped by discontinuation of TT and, thereafter, anticoagulation. Recognition of thrombophilia-hypofibrinolysis before joint collapse facilitates anticoagulation which may stop ON, preserving joints. Osteonecrosis (ON) is often secondary to high-dose, long-term steroids or alcoholism, with primary (idiopathic) ON defined when known secondary etiologies are ruled-out . The devStudies were carried out following a protocol approved by the Institutional Review Board with signed informed consent.To provide a frame of reference for the two cases in the current report, thrombophilia-hypofibrinolysis measures in 240 cases with idiopathic osteonecrosis sequentially referred to our center for coagulation studies are provided, along with comparisons to 110 previously reported healthy, normal controls [PCR measures of the Factor V Leiden, Prothrombin, methylenetetrahydrofolate reductase (MTHFR), and plasminogen activator inhibitor (PAI-1 gene) mutations were done along with serologic measures of Lp (a), homocysteine, factors VIII and XI, antigenic proteins C, total S, free S, and antithrombin III, lupus anticoagulant, and anticardiolipin antibodies (ACLA) IgG and IgM, using previously reported methods , 12. SerX2 tests were used.The data were processed by SAS 9.4. Comparison of measures of thrombophilia-hypofibrinolysis between cases and controls was done by Chi-Square analysis, excepting those comparisons where the expected cell size was <5, when Fisher’s exact A 62-year-old Caucasian male had a past history of deep venous thrombosis (DVT) in the right lower extremity , and superficial venous thrombosis of the right greater saphenous vein . He was receiving warfarin for 1 year for paroxysmal atrial fibrillation (AF) (status-post ablation and failed cardioversion). He was evaluated for abdominal-flank-hip pain progressing over a 2-year period. An abdominal CT scan revealed previously undiagnosed ON of both femoral heads without collapse (Ficat  stage I)A 32-year-old hypogonadal Caucasian male had a 10-year history of unexplained progressive tooth erosion and tooth loss. Tooth loss accelerated 8-months after starting clomiphene (50 mg/day) to raise serum total and free testosterone, and a CT scan revealed cavitating ON of both jaws. The patient was referred to our center by his dental surgeon for evaluation. There were no etiologic factors for secondary ON. There was no evidence for excessive stomach acid or trauma (including teeth-grinding) related to tooth erosion, and no alcohol abuse, high-dose/long-term corticosteroids, or other secondary causes of ON. He had never received bisphosphonates, associated with jaw ON . CoagulaTT was discontinued, with a sharp reduction in jaw pain within 2 months.n = 110), except in the comparison of eNOS [To provide a frame of reference to the two patients of the current report, Table  of eNOS  mutationThe two patients of our current report were heterozygous for the FVL mutation, one had elevated ACLA, and one was homozygous for the eNOS T786C mutation . Our 240Arthralgia of the hips  and kneeThere appear to be etiologic associations between factor V Leiden heterozygosity , 28, 32,In patients with early stage primary-idiopathic ON, before segmental collapse of hips or knees has occurred  primary ON of the knee . In 6 paIn the jaw, when left untreated, ON leads to progressive tooth loss with failure to heal, jaw bone cavitation, and chronic pain syndromes , 16. ParIn patients with thrombophilia-hypofibrinolysis and thrombotic events on TT, continuation of TT, even with adequate concurrent anticoagulation, leads to repetitive thrombotic events .When the thrombus-promoting TT therapy is stopped, and anticoagulation started, we speculate that venous outflow is restored, increased venous pressure in the bone is reduced, facilitating increased arterial flow, reducing osseous ischemia , 26. ThiHistorically, in adults with primary ON of weight bearing joints, the most common treatments include invasive forms of secondary and tertiary prevention, including core decompression  with or Primary ON is often caused by underlying familial and acquired thrombophilia and hypofibrinolysis, and can be worsened by TT or testosterone-elevating clomiphene. In patients with primary ON, it is important for diagnostic and therapeutic reasons to determine whether thrombophilia-hypofibrinolysis are present, and whether TT is being used. To stop progression of early primary ON at Ficat Stage I or II before joint collapse in patients with thrombophilia-hypofibrinolysis, discontinuing TT is essential and may slow or stop progression of the ON. Initiating anticoagulation may stop progression of ON in thrombophilic patients without joint collapse and without progression to jaw cavitation, thus, avoiding the usual natural history of untreated ON, which is total joint replacement within 2 years of initial diagnosis, and in the jaw, chronic pain syndrome with jaw cavitation and recurrent supra-infection.


As a systemic disease, atherosclerosis commonly affects intracranial and extracranial carotid arteries simultaneously which is defined as co-existing plaques. Previous studies demonstrated that co-existing atherosclerotic diseases are significantly associated with ischemic cerebrovascular events. The aim of this study was to investigate the characteristics of co-existing intracranial and extracranial carotid atherosclerotic plaques and their relationships with recurrent stroke by using 3D multi-contrast magnetic resonance (MR) vessel wall imaging.Patients with recent cerebrovascular symptoms in anterior circulation and at least one carotid plaque were recruited. All patients underwent cardiovascular magnetic resonance (CMR) for brain and intracranial and extracranial arteries. Presence/absence of atherosclerotic plaque at each arterial segment was identified. The maximum wall thickness (Max WT), length, stenosis of each plaque was measured. The presence/absence of calcification, lipid-rich necrotic core (LRNC), and intraplaque hemorrhage (IPH) was assessed. Cerebral old and acute infarcts in anterior circulation were evaluated.p = 0.030) and co-existing intracranial and extracranial carotid artery plaques  was significantly associated with recurrent stroke. After adjusting for traditional risk factors, the number of co-existing plaques was still significantly correlated with recurrent stroke . No correlations were found between recurrent stroke and Max WT, length, stenosis, and compositions of plaques.Fifty-eight patients  were recruited. Of the 58 patients, co-existing intracranial and extracranial carotid artery plaques were found in 45 patients (77.6%), of which 7 (15.6%) had first time acute stroke and 26 (57.8%) had recurrent stroke. For these 33 patients with stroke, the number of intracranial plaques (OR = 11.26; 95% CI, 1.27–100; Co-existing intracranial and extracranial carotid artery plaques are prevalent in symptomatic patients and the number of co-existing plaques is independently associated with the risk of recurrent stroke. Cerebrovascular atherosclerotic disease is the major cause of ischemic stroke or transient ischemia attack (TIA) , 2. As aCurrently, noninvasive angiography-based imaging techniques, such as computed tomography angiography (CTA) and cardiovascular magnetic resonance (CMR) angiography have been largely utilized for assessment of cerebrovascular atherosclerotic disease severity. However, increased evidences showed that only measuring luminal stenosis by angiography will underestimate the severity of atherosclerotic disease due to the arterial positive remodeling effect . InvestiVessel wall CMR has been demonstrated to be capable of determining atherosclerotic plaque vulnerability in extracranial carotid arteries . In contIn this study, we sought to investigate the characteristics of co-existing atherosclerotic plaques in intracranial and extracranial carotid arteries and their relationships with recurrent stroke risk by using 3D multicontrast vessel CMR.Patients with clinically confirmed recent ischemic stroke or TIA in the anterior circulation (<2 weeks) and atherosclerotic plaque in at least one carotid artery determined by ultrasound were consecutively recruited in this study. All subjects underwent MR imaging for brain and intracranial and extracranial carotid arteries. The exclusion criteria include: 1) cardiogenic stroke; 2) hemorrhagic stroke; 3) stroke in posterior circulation; 4) contraindications to CMR. The clinical characteristics including age, gender, body mass index (BMI), history of hypertension, diabetes mellitus, smoking, hyperlipidemia, stroke, TIA, and coronary heart disease and the levels for cholesterol and the blood pressure were collected from the clinical record. The study protocol was approved by institutional review board and the written consent forms were obtained from all the subjects prior to the initiation of this study.3 and isotropic spatial resolution of 0.8 × 0.8 × 0.8 mm3. Gadolinium contrast media was not applied to the vessel wall CMR. A clinically standard imaging protocol including T1-weighted (T1W), T2-weighted (T2W) and diffusion weighted imaging (DWI) was used to acquire the axial images of brain.CMR was performed on a 3 T MR scanner  with a custom-designed 36-channel neurovascular coil . For intFour radiologists with >5 year experience in neurological radiology independently interpreted the CMR images with consensus using MR workstation . The intracranial and extracranial carotid arteries were divided into the following segments: M1 segment of middle cerebral artery (MCA), C3-7 segment of internal carotid artery (ICA), C2 segment of ICA, C1 segment of ICA, bulb, and common carotid artery (CCA). The vascular CMR images were reviewed by two radiologists (Y.X. and X.Z.) blinded to brain images and clinical information. Presence or absence of atherosclerotic plaque which is defined as the eccentric thickening of vessel wall at each arterial segment was identified. The maximum wall thickness (Max WT), length, stenosis of each plaque was measured. The luminal stenosis was measured by using the NASCET  and WASITen subjects were randomly selected to determine the intra-observer and inter-observer reproducibility in identification of the presence of plaque and the plaque compositions, including calcification, LRNC, and IPH. The reproducibility for the measurements of Max WT, length, and stenosis of each plaque was also evaluated. A time interval of 3 months was set for testing the intra-observer reproducibility to minimize the bias of memory.p value of p <0.05 was considered as statistically significant. All statistical analyses were performed using SPSS 16.0 .The continuous variables were described as mean value and the corresponding standard deviation (SD) and the binary variables were presented as frequency. The prevalences of atherosclerotic plaque and plaque compositions in intracranial arteries, extracranial carotid arteries and co-existing plaques were determined. Logistic regression was utilized to determine the odds ratio (OR) and corresponding 95% confidence interval (CI) of plaque characteristics in discriminating presence of recurrent stroke in patients with co-existing intracranial and extracranial carotid artery plaques. Cohen’s kappa was calculated to determine the inter-observer and intra-observer agreement in identification of arterial plaque. The intra-class correlation coefficient (ICC) was analyzed to assess inter-observer and intra-observer agreements in quantitatively measuring the atherosclerotic plaque. A p = 0.009). No significant differences can be observed in other clinical characteristics between these two patient groups  who were included in the final analysis, 34 are males, 40 (69%) had hypertension, 21 (36.2%) had diabetes, 49 (84.5%) had hyperlipidemia, and 30 (51.7%) had history of smoking. The clinical characteristics of this study population are summarized in Table In total, 209 atherosclerotic plaques were detected in all 58 patients. Of 209 plaques detected, 114 (54.5%) were found in extracranial carotid arteries and 95 (45.5%) occurred in intracranial arteries. Co-existing intracranial and extracranial carotid artery plaques were found in 45 patients (77.6%). Figure p = 0.030) and the number of co-existing intracranial and extracranial carotid artery plaques  were found to be significantly associated with recurrent stroke. After adjusting for traditional risk factors, including age, gender, BMI, history of hypertension, smoking, diabetes, and hyperlipidemia, and the recurrent stroke was still significantly correlated with the number of co-existing intracranial and extracranial carotid artery plaques  but not with the number of intracranial artery plaques . No significant correlations were found between recurrent stroke and Max WT, length, and stenosis of plaques  had first time acute stroke, 26 (57.8%) had recurrent stroke in the territory of anterior circulation, 8 (17.8%) had only old infarcts, and no infarcts were evidenced on MR images in 4 (8.9%) patients. For the 33 patients with co-existing intracranial and extracranial carotid artery plaques and either first time acute stroke or recurrent stroke in anterior circulation, the number of intracranial plaques . For the intra-observer agreement in assessment of presence of plaque compositions, the kappa value was 0.85, 0.74, and 0.71 for calcification, LRNC, and IPH, respectively . The intra-observer ICC was 0.98 , 1.00 , and 0.96  for measuring Max WT, length, and stenosis, respectively.For intra-observer agreement in identification of presence of atherosclerotic plaque, the kappa value was 1, 0.89, 0.88, 1, 0.83 and 0.77 for CCA, bulb, C1, C2, C3-7, and M1 segment, respectively . For the inter-observer agreement in assessment of presence of plaque compositions, the kappa value was 0.66, 0.69, and 0.67 for calcification, LRNC, and IPH, respectively . The inter-observer ICC was 0.95 , 0.95 , and 0.91  for measuring Max WT, length, and stenosis, respectively.For inter-observer agreement in identification of presence of atherosclerotic plaque, the kappa value was 1, 0.89, 0.76, 1, 0.61 and 0.62 for CCA, bulb, C1, C2, C3-7, and M1 segment, respectively  [In the present study, the number of co-existing intracranial and extracranial plaques was found to be significantly associated with recurrent stroke. Our finding is in line with previous reports. Presence of co-existing plaques in intracranial and extracranial arteries may be an effective indicator for poor prognosis of ischemic stroke patients , 6, 24. = 0.002) . The und= 0.002) . Wong et= 0.002)  found th= 0.002)  reported= 0.002)  showed t= 0.002)  reportedIn the present study, co-existing intracranial and extracranial artery plaques were more frequently found in men. The male preponderance of co-existing plaques in multiple vessels has been demonstrated in coronary arteries in previous studies. Lansky et al.  reportedThis study has several limitations. First, this is a cross-sectional study. Prospective studies are warranted to investigate the predictive value of co-existing intracranial and extracranial carotid artery atherosclerosis for future events. Second, we recruited patients with existing carotid plaques. This study population didn’t include those with intracranial atherosclerosis but without extracranial carotid artery plaques. Third, this pilot study has smaller sample size. Future studies with larger sample size are warranted. Finally, 6 patients with poor image quality were excluded from the final analysis. This may introduce the patient selection bias.The co-existing intracranial and extracranial carotid artery plaques are prevalent in symptomatic patients and the number of co-existing plaques is independently associated with the risk of recurrent stroke.