N = 390), we examined the role of psychopathy, achievement goals, and self-efficacy as predictors of academic dishonesty. We found that the disinhibition aspect of psychopathy and mastery-goal orientation predicted the frequency of students’ academic dishonesty and mastery-goal orientation mediated the relationship between the disinhibition and meanness aspects of psychopathy and dishonesty. Furthermore, general self-efficacy moderated the indirect effect of disinhibition on academic dishonesty through mastery-goal orientation. The practical implications of the study include the identification of risk factors and potential mechanisms leading to students’ dishonest behavior that can be used to plan personalized interventions to prevent or deal with academic dishonesty.Academic dishonesty is a common problem at universities around the world, leading to undesirable consequences for both students and the education system. To effectively address this problem, it is necessary to identify specific predispositions that promote cheating. In Polish undergraduate students ( Academic dishonesty refers to behaviors aimed at giving or receiving information from others, using unauthorized materials, and circumventing the sanctioned assessment process in an academic context . The freExisting academic dishonesty prevention systems include using punishments and supervision , informiIt is also important to remember that implementing the aforementioned methods of prevention will not lead to a reduction in academic dishonesty if faculty members do not follow and apply the established rules . Facultyr = .27)—predictor of academic dishonesty according to a recent meta-analysis of 89 effects and 50 studies ) z = -1.85, p = .03) in men  than in women . The correlation between mastery-goal orientation and meanness was stronger  in men  than in women . And the correlation between disinhibition and academic dishonesty was stronger  in women  than in men . If we adjust for error inflation for multiple comparisons (p < .007) for these moderation tests, none of the Fisher’s z tests were significant. Therefore, we conclude the correlations were generally similar in the sexes. Men scored higher than women on meanness and disinhibition.Descriptive statistics, sex differences tests (see Bottom Panel), and correlations (see Top Panel) for all measured variables are presented in F = 18.60, p < .001]. The residuals for boldness , disinhibition , and a mastery-goal orientation  were correlated with academic dishonesty. Additional regression analysis revealed that both mastery-goal orientation and disinhibition strengthened the association between boldness and academic dishonesty, which on its own was not a predictor of the frequency of cheating–suppressor effect  = 4.40, p = .04]).To test the contribution of personality and motivation variables in predicting academic dishonesty, we conducted a standard multiple regression where the model explained 23% of the variance in academic dishonesty [To examine whether achievement goals mediated the associations between psychopathy and academic dishonesty we conducted a series of mediation analyses.Step 1 = .32, p < .001; 𝛽Step 2 = .24, p < .001), and the relationship between meanness and academic dishonesty . Initial non-significant negative relation between boldness and academic dishonesty  stayed unrelated after adding mastery-goal orientation to the model, but the value for the relation coefficient was higher and positive  suggesting a nonsignificant suppression effect.As shown in -0.03; 95% CI: -0.70, -0.003), however, the same analyses ran separately for men  and women  revealed moderated mediation only in women . Estimates for that model are presented in To test if the level of general self-efficacy moderated the aforementioned relationships between psychopathy, achievement goals, and academic dishonesty we ran a series of moderated mediations. Index for moderated mediation was significant only for the model with disinhibition and mastery-goal orientation  declared higher levels of academic dishonesty . An interaction between disinhibition and general self-efficacy  with the significant, negative index for moderated mediation means that the indirect effect of disinhibition on academic dishonesty through mastery-goal orientation is negatively moderated by general self-efficacy. The higher the level of the moderator, the weaker the effect of mediation, and for moderator values above one standard deviation from mean mediation become non-significant . In sum, the mastery-goal orientation partially mediated the associations that disinhibition had with academic dishonesty, however, this effect was absent for people with high levels of general self-efficacy.Psychopathy is an important predictor of engaging in unethical behaviors , includiIn the case of meanness, this relationship may indicate a tendency for dishonesty resulting from a lack of fear and, consequently, a diminished impact of the perceived risk of being caught cheating, sensation-seeking that involves engaging in destructive behavior regardless of possible negative consequences of such actions, and a propensity to exploit other student’s work or knowledge to pass classes , 54. TheBeyond these basic associations, we also examined the role of achievement goals as mediators for the relationships between psychopathy facets and academic dishonesty. Mastery-goal orientation mediated the relationships between two psychopathy facets and academic dishonesty. Both meanness and disinhibition led to low levels of students’ mastery-goal orientation which, in turn, contributed to cheating in the academic context. Low mastery-goal orientation might result from the fact that those who are characterized by meanness may have a propensity to be rebellious  and those who are characterized by disinhibition may have a propensity for impulsivity . Without motivation to acquire knowledge, students may cheat to achieve academic goals with no regard to the fairness  or the consequences  of their actions –33. In tLastly, we tested if general self-efficacy acts as a moderator of these mediation models and found evidence that it moderated the indirect effect of disinhibition on academic dishonesty through mastery-goal orientation. This means that disinhibited students who have a high sense of perceived ability to control their chances for success or failure, might be able to overcome the tendency to cheat resulting from their personality , and motivational  predispositions. However, that effect was found only for women, limiting any insights that can be drawn about men. Previous research showed that an increase in general self-efficacy reduced the risk of suicide among women . MoreoveIn the present study, we aimed to combine personality and motivation variables to describe the possible process leading to academic dishonesty assessed with a behavioral measure. Because Polish students do not constitute a typical W.E.I.R.D. sample , presented results can be used to generalize conclusions from research on academic dishonesty beyond typical W.E.I.R.D cultures. However, our study is not without limitations. First, the measurement of academic dishonesty was based on self-report, which, even after maximizing anonymity of the measurement, might have attenuated our results concerning the frequency of cheating. Thus, future studies should focus on measuring actual dishonest academic behavior. Second, we examined academic dishonesty as an overall frequency of committing different acts of cheating, which reflects the general propensity to cheat. It could be useful to further investigate the predictive power of described models in experiments, focused on the specific type of dishonest behavior. Third, the obtained range of academic dishonesty scores might result from sampling bias, which would require using different sampling procedure in future studies, or from non-normal distribution of academic dishonesty, which would be consistent with the results of the previous studies –4. FourtPreventing academic dishonesty is often made difficult by the lack of centralized and formalized university policies concerning cheating, faculty reluctance to take formal action against dishonest students, and limited attention paid to students’ personal characteristics associated with a tendency to cheat . Based oIn sum, we presented evidence that disinhibition and meanness are associated with the frequency of committing academic dishonesty. We described the possible underlying mechanism of those relations involving mediation effects of the mastery-goal orientation and, in the case of disinhibition, also a moderation effect of the general self-efficacy. Our research can be used by teachers to better identify factors conducive to dishonesty and to modulate their responses to fraud based on the personality and motivational predispositions of students.S1 Table(DOCX)Click here for additional data file.


Thermobifida fusca was a dominant actinobacterium in high-temperature corn stalk composts, but it was unable to grow alone in corn stalk solid medium. Interestingly, T. fusca showed good growth and secreted enzymes when cocultured with Thermomyces lanuginosus. T. lanuginosus grew firstly during the initial stage, whereas T. fusca dominated the system subsequently during cocultivation. The secretome indicated that T. lanuginosus mainly degraded xylan by expressing a GH11 xylanase . T. fusca was induced by xylan mainly to secrete a xylanase from GH11 family  which could rapidly degrade xylan to xylo-oligosaccharide (XOS) and xylose within 2 min, while high concentrations  of XOS or xylose suppressed the growth of T. fusca; which may be the reason why T. fusca unable to grow alone in corn stalk solid medium. However, T. lanuginosus could utilize the XOS and xylose produced by xylanases secreted by T. fusca. During the synergistic degradation of lignocellulose by T. lanuginosus and T. fusca, xylan was rapidly consumed by T. lanuginosus, the residual cellulose could specifically induced T. fusca to express a GH10 xylanase with a CBM2 domain  and 6 cellulases (2 exocellulases and 4 endocellulases). Moreover, T. lanuginosus increased the secretion of cellulases from T. fusca by 19–25%. The order of T. lanuginosus and T. fusca was consistent with the multilayered structures of lignocellulose and could be regulated by different concentrations of XOS and xylose. The novel synergism of T. lanuginosus and T. fusca gave a new sight for revealing more synergetic relationships in natural environments and exploring efficient microbial inoculants and enzyme cocktails for lignocellulose degradation.Effective artificial microbial consortia containing microorganisms with desired biological functions have the potential to optimize the lignocellulose-based bioindustry.  Plant biomass is the major sink for photosynthetically fixed carbon on earth, and efficient lignocellulose degradation has proven to be crucial for the maintenance of the global carbon cycle and bioprocess development . LignoceTrichoderma reesei and Penicillium oxalicum  were purchased from Sangon Biotech Co., Ltd.  Xylan, 7-amino-1,3-naphthalenedisulfonic acid monopotassium salt monohydrate, trichloroacetic acid, dithiothreitol (DTT), iodoacetamide, trypsin and protease inhibitor cocktail were purchased from Sigma Chemical Co. . The corn stalk used in this study was collected from Dezhou, China. All other chemicals used were of analytical grade.Microcrystalline cellulose (MCC), cellobiose, glucose, xylo-oligosaccharide (XOS), xylose, and sodium cyanoborohydride  with the number CGMCC 3.15828 . A volume of 100 μL of fresh T. lanuginosus conidia (7.0 × 107/mL) were inoculated into 100 mL of potato dextrose agar medium at 55°C for 2 days as the fungal inoculant. T. fusca was firstly maintained following the supplier’s protocol, and then 0.1 g of fresh mycelia were inoculated into 100 mL of Czapek’s medium at 55°C for 2 days as the bacterial inoculant.The filamentous fungus  3.15828 . The actT. lanuginosus and T. fusca were obtained from previous research and the UniProt KB database1, respectively . A total of 50 g of corn stalk medium was added to each 300-mL Erlenmeyer flask, and they were sterilized at 115°C for 30 min. A 500 μL of fungal or bacterial inoculant was inoculated into the medium for individual culture and 500 μL of fungal and 500 μL of bacterial inoculants were inoculated into the medium for cocultivation, then they were cultivated at 55°C for 10 days. The samples were collected every 2 days. After collection, 50 mL of distilled water was immediately added to the medium, and the Erlenmeyer flasks were shaken at 200 × g for 1 h at 4°C. The substrates and mycelium were filtered with eight layers of gauze and centrifuged at 8000 × g for 20 min to obtain filtrates which were used as crude enzyme samples for further experiments. In order to inhibit the degradation of secreted proteins during the extraction process, we added 5 μL of protease inhibitor cocktail purchased from Sigma Chemical, Co.  to 1 mL crude enzyme samples. And the samples were stored at 4°C and used for further experiments as soon as possible. Three independent replicates were conducted for each carbon source.Corn stalk was washed three times with deionized water followed by drying at 60°C until constant weight. The cleaned corn stalk was crushed to powder by a knife mill and subsequently sieved in a sieve (Mesh 10). Corn stalk powder was mixed with the Czapek’s medium (without sucrose) with a ratio of 1:4 (w/v) to individually culture T. lanuginosus or 1.5 mL of T. fusca inoculant was inoculated into 300 mL of liquid-state medium. And for coculture, 1.5 mL of T. lanuginosus and 1.5 mL of T. fusca inoculants were inoculated into 300 mL of liquid-state medium. The strains were cultivated at 55°C and 200 × g for 5 days. Each day, 10 ml of medium was sampled. Substrates and mycelium biomass were removed by centrifugation at 10,000 × g at 4°C for 5 min. The supernatant was further filtered through a 0.22 μm membrane . The protease inhibitor cocktail was added to the supernatant as above. The supernatant was stored at 4°C and used as the crude enzymes in further experiments. Three independent replicates were conducted for each carbon source.Czapek’s medium (without sucrose) with different carbon sources as listed in T. lanuginosus and T. fusca on xylanase native zymograms were extracted by the Quantity One software. The mean of the gray values between the three replicates was used for further analysis using the Matlab software.The native zymogram method modified by T. lanuginosus and T. fusca determined by quantitative PCR (qPCR), respectively. DNA from the T. lanuginosus and T. fusca cocultured medium was extracted using the Soil DNA Kit  following the manufacturer’s instructions. Triplicate extractions were performed and then pooled into a single aliquot used as a representative DNA sample. The number of 16S rDNA gene representing relative content of T. fusca was amplified using the primer pair 16S-F (5′-GTGSTGCAYGGYTGTCGTCA-3′) and 16S-R (5′-ACGTCRTCCMCACCTTCCTC-3′) with amplicon size of 146 bp (T. lanuginosus relative content was performed by the primer pair ITS 1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS 4 (5′-TCCTCCGCTTATTGATATGC-3′) with amplicon size of 750 bp  and 16S rDNA genes are commonly used to detect fungal and bacterial species. The relative content of ITS and 16S rDNA genes are commonly used to represent the relative abundance of fungi and bacteria, respectively . The relf 146 bp , and thef 750 bp . The 20 T method  was usedT. lanuginosus and T. fusca are aggregated filamentous cells and they were mixed with cellulose solid, the biomass of T. lanuginosus and T. fusca were determined by measuring cytoplasmic protein content as described previously with slight modifications  containing 0.1 M DTT and 50% glycerol, which was then pulse-sonicated at 70% strength for 30 min. After centrifuging at 10,000 × g for 5 min, protein content in the supernatant was measured by the Bradford protein assay  sucrose solution which was used as loading buffer. Seven microliters of labeled sample was loaded in each well, and electrophoresis was performed at 7 mA for every plate. After electrophoresis, the gels were scanned using a ChemiDocTM MP System (Bio-Rad). Cellodextrins were used as the glucose marker  method . A volume marker . And 0.1T. lanuginosus and T. fusca were analyzed by LC-MS/MS as previously described . Then, proteins were precipitated by 10% (w/v) trichloroacetic acid, and dried at 50°C  at 37°C. A C18 Ziptip  was applied for desalination, and finally, the peptide samples were dissolved in 0.1% (v/v) trifluoroacetic acid. A Prominence nano LC system  coupled with an LTQ-Orbitrap Velos Pro ETD mass spectrometer  was used for analyzing eluted peptides. Three replicates were performed for each sample.The secretomes of escribed . Extrace at 50°C . Subsequq ≤ 0.05) were selected for further analysis. Identification of proteins required at least two peptides (q < 0.05) with false discovery rate at 1%. The relative abundance of proteins was determined based on the relative percentage of peptide spectrum matches (PSMs). A linear correlation between PSMs and protein abundance has been demonstrated in previous studies  and the SEQUEST search engine. Protein search parameters were set as follows: firstly, trypsin was used to digest proteins; secondly, mass tolerance was determined by setting a precursor mass tolerance to 10 ppm and a fragment mass tolerance to 0.8 Da; thirdly, the oxidation of methionine was selected as the dynamic modification; finally, carbamidomethyl of cysteine residues was chosen as the fixed modification. Peptides with at least six amino acid residues with 95% certainty ( studies . The meaT. lanuginosus (Accession No. ANHP00000000) and T. fusca (Accession No. CP000088) were analyzed. The distribution of the number and types of cellulolytic genes in the genomes of T. lanuginosus and T. fusca complemented each other  corn stalk liquid medium in their respective monoculture, indicating the growth of them in liquid medium  measurements showed similar regularity with native zymograms during cocultivation of T. lanuginosus and T. fusca (T. lanuginosus rapidly grew to be the dominant microorganism in the early phase (before day 4), accounting for 70% of gene content on day 4. Nevertheless, T. fusca dominated the system from day 5  and 1% XOS (w/v) suppressed the growth of T. fusca , even in the mixtures with MCC, indicating that increased in concentrations of XOS promoted the growth of T. lanuginosus  during 5-day cultivation, while the reducing sugar content hardly decreased during 5-day culture of T. fusca when XOS concentration over 0.5% (w/v)  . This re pathway , this acT. lanuginosus (10.88 ± 0.06 IU/mL) and T. fusca (10.75 ± 0.54 IU/mL)  , which ie of MCC . The celuginosus , which iuginosus . Althouguginosus , this fuuginosus , which wuginosus . The hign on MCC . The higT. fusca .T. fusca even in the presence of the MCC  XOS could also suppress the growth and enzymic secretion of  the MCC . T. fuscT. lanuginosus and T. fusca monoculture on different concentration of XOS, fluorescence-assisted carbohydrate electrophoresis (FACE) was performed  detected in the T. lanuginosus secretome  under the induction of xylan, in line with the xylanase activity detection (Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to further determine the secretome of ecretome , showingetection . The incetection .T. fusca, a GH11 family xylanase  was the most abundant protein when T. fusca was grown on xylan, while T. fusca secreted more GH10 xylanase Q47KR6  when MCC was used as the substrate  and four beta-1,4-endocellulases  for the effective degradation of crystalline cellulose  were simultaneously expressed by a in MCC . LinkingT. lanuginosus  XOS   of XOS supported the growth and xylanase secretion of uginosus , 5. In cw/v) XOS . AccordiT. fusca . At all ed first . The tim1% (w/v) . In addiT. fusca . These rPlant biomass composts are effective microbial lignocellulose-degrading systems that contain many microbial community members . The expThermomyces lanuginosus and Thermobifida fusca, was found to be the most dominant fungus and bacterium in many thermophilic lignocellulose composts , suggesting that it lacked the ability to efficiently utilize XOS and xylose. This result indicated that T. fusca has evolved a XOS and xylose inhibition mechanism to adapt to coexistence with T. lanuginosus. T. fusca utilized cellulose exclusively by secreting abundant cellulases.composts . T. lanud xylose . Previoud xylose . T. lanuly stage . As for  pathway . HoweverT. lanuginosus and T. fusca occupied different niches. T. lanuginosus absorbed oligosaccharides and oligopeptides in the saprophytic habitat and secreted a GH11 xylanase to degrade the external xylan, thus growing firstly. Meanwhile, T. fusca mainly secreted a GH11 xylanase  that degrading xylan into XOS and xylose, which were quickly utilized by T. lanuginosus  was detected in the genome of T. fusca, implying that they could not efficiently remove lignin; so that pretreatment lignocellulose was appropriate for the coculture of T. lanuginosus and T. fusca.aterials . In addiindustry . Neverthd xylose . Surprisellulose . A similtic acid . The cocindustry . HoweverT. lanuginosus and Lactobacillus  and a series of cellulases  of microorganisms from the environment are uncultivated microbes, but this does not mean that they cannot be cultured in the laboratory . One of uginosus . This reT. lanuginosus and T. fusca regulated by the accessibility and concentration of substrates. T. fusca possesses an efficient xylan-degrading system, but is inhibited by high concentration  of XOS or xylose. During cocultivation, T. lanuginosus was primarily responsible for xylan degradation and XOS removal. Exposed cellulose induced the production of a series of cellulases for external cellulose degradation by T. fusca, and as well as a GH10 xylanase with CBM2  for more efficient xylan residues removal. The inhibitory xylan-degrading products, XOS and xylose, were quickly consumed by T. lanuginosus, maintaining suitable condition for T. fusca’s growth. The cocultivation of T. lanuginosus and T. fusca has given new insight in synergetic relationships in natural environments, especially among microbes dominating natural habitats while could not be cultured in laboratory conditions. In future, continued researches on exploring the synergistic relationships of dominant lignocellulose-degrading microorganisms will contribute to further exploration of more efficient microbial inoculants and enzyme cocktails for the plant-based bioindustry.In summary, this study provided insight into a novel synergetic lignocellulose-degrading mechanism between Thermomyces lanuginosus could be obtained from DDBJ/EMBL/GenBank under the accession number ANHP00000000. The whole genome data of Thermobifida fusca could be obtained from GenBank under the accession number CP000088. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.This whole genome data of ZS designed the experiments, performed the experiments, and drafted the manuscript. CH, XZ, and LT revised the manuscript. LW conceived the idea and proofed the manuscript. All the authors read and approved the final 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.


However, the temporally variant nature of hypoxia can complicate interpretation of imaging findings. We explored the relationship between hypoxia and vascular function in breast tumours through combined 18F-FMISO-PET/MR examination. Tumour hypoxia was assessed using influx rate constant Ki and hypoxic fractions (%HF), while parameters of vascular function  and cellularity (ADC) were derived from dynamic contrast-enhanced (DCE) and diffusion-weighted (DW)-MRI, respectively. Additional correlates included histological subtype, grade and size. Relationships between imaging variables were assessed using Pearson correlation (r).Women with histologically confirmed, primary breast cancer underwent a simultaneous Ktrans correlated negatively with Ki  and %HF , though parametric maps exhibited intratumoural heterogeneity with hypoxic regions colocalising with both hypo- and hyperperfused areas. No correlation was observed between ADC and DCE-MRI or PET parameters. %HF correlated positively with lesion size .Twenty-nine women with 32 lesions were assessed. Hypoxic fractions > 1% were observed in 6/32 (19%) cancers, while 18/32 (56%) tumours showed a %HF of zero. The presence of hypoxia in lesions was independent of histological subtype or grade. Mean tumour 18F-FMISO-PET correlated negatively with Ktrans from DCE-MRI, supporting the hypothesis of perfusion-driven hypoxia in breast cancer. Intratumoural hypoxia-perfusion relationships were heterogeneous, suggesting that combined assessment may be needed for disease characterisation, which could be achieved using simultaneous multimodality imaging.Hypoxia measured by At the tumour level, hypoxia measured by 18F-FMISO-PET was negatively correlated with perfusion measured by DCE-MRI, which supports the hypothesis of perfusion-driven hypoxia in breast cancer.• No associations were observed between 18F-FMISO-PET parameters and tumour histology or grade, but tumour hypoxic fractions increased with lesion size.• Intratumoural hypoxia-perfusion relationships were heterogeneous, suggesting that the combined hypoxia-perfusion status of tumours may need to be considered for disease characterisation, which can be achieved via simultaneous multimodality imaging as reported here.• The online version of this article (10.1007/s00330-020-07067-2) contains supplementary material, which is available to authorized users. In breaeristics . In vitreristics  and downeristics . Severaleristics –8.Although tumour hypoxia can be broadly categorised as diffusion or perfusion limited, it is generally accepted that the tumour microenvironment is a highly dynamic entity, exhibiting temporally varying perfusion patterns and heterogeneous oxygen-tension gradients . Experim18F-labelled nitroimidazoles can provide specific measures of intracellular hypoxia [18F-fluoromisonidazole (18F-FMISO) has been used for the evaluation of response to anti-angiogenic and HER2-targetted treatment [18F-FMISO uptake at baseline has been associated with shorter disease-free survival [Imaging methods, including positron emission tomography (PET) and magnetic resonance imaging (MRI), have been used for the non-invasive assessment of the tumour microenvironment. Dynamic contrast-enhanced (DCE) MRI has shown utility in characterising tumour perfusion and vascular permeability in clinical studies , while d hypoxia . In breareatment , 16 and reatment , 18. Addsurvival .Despite the intrinsic link between tumour hypoxia and perfusion, multimodal imaging approaches to characterise this aspect of cancer pathophysiology have been limited in the clinical setting , 19–24. 18F-FMISO-PET/MRI. To our knowledge, this is the first such study in breast cancer.The primary objective of this study was to examine the association between hypoxia and vascular function in patients with treatment-naïve breast cancer using simultaneous Women aged > 18 years with histologically confirmed primary breast cancer and a tumour diameter > 10 mm on mammography and/or ultrasound were eligible for the study (February 2017 to November 2018). Pregnancy, lactation, previous surgery or radiotherapy for cancer or benign breast disease, inadequate renal function and contraindications to MRI were exclusion criteria for the study. The research was approved by a National Research Ethics Committee (14/EE/0145). All study participants provided written informed consent before PET/MRI examination.18F-FMISO. The uptake period post injection (p.i.) was used to enhance hypoxic-to-normoxic tissue contrast and allow the free 18F-FMISO concentrations in tissue and blood to reach equilibrium [Ki) determination by Patlak analysis [Participants underwent a 60-min simultaneous PET/MR scan of the breasts in the prone position on a SIGNA PET/MR scanner , using a 16-channel bilateral breast array  120 min  after injection of 306 ± 14 MBq ilibrium , 26, a r.5 min) aPET: Emission data from 120 to 180 min p.i. (12 × 5-min frames) were reconstructed using time-of-flight ordered-subsets expectation-maximisation (TOF-OSEM) with 4 iterations and 28 subsets  derived from existing data, permitting calculation of Ki [on of Ki –31).MRI: The MRI protocol involved a 2-point Dixon sequence for PET attenuation correction, T1- and T2-weighted images, DWI, and a DCE series. Sequences were also acquired to measure B1+ transmission-field non-uniformity, using a Bloch-Siegert method, and baseline T1 (T10) as required for the pharmacokinetic analysis of DCE-MRI data [MRI data . DCE-MRITumour regions were manually delineated in OsiriX, version 8.0.2 (Pixmeo SARL), by three radiologists in consensus . Regions were drawn on the peak-enhancing volumes of the DCE-MRI series on all contiguous axial sections encompassing the invasive part of the tumour and including multifocal/multicentric disease , using the extended Tofts’ model [Ktrans; efflux-rate constant, kep; extravascular-extracellular volume fraction, ve; and plasma volume fraction, vp  maps was performed in OsiriX, using b values of 0 and 900 s/mm2. Mean lesion ADC was calculated by manually outlining whole tumour regions on the b = 900 s/mm2 image  and maximum tumour-to-plasma (Tmax/P) and tumour-to-muscle (Tmax/M) ratios within the regions defined on the DCE-MRI. The influx rate of 18F-FMISO into the trapped (hypoxic) tissue compartment (Ki) was determined by Patlak-plot analysis, utilising all frames in the registered 18F-FMISO series and the scaled population-based AIF. Hypoxic fractions (%HF) in tumour regions were calculated as the percentage of voxels with Ki values > 2 × standard deviations (SD) of the mean Ki of normoxic muscle , progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) status was obtained from core biopsies or surgical tumour specimens. Cancers with positive ER or PR expression were classified as hormone-receptor (HR) positive.r). t tests were used for comparison between means of two groups, and ANOVA when more than two groups were compared. Where data were not normally distributed, or normality could not be assessed, Mann-Whitney U and Mood’s median or Kruskal-Wallis H tests were employed for comparisons between two or more groups, respectively. p values < 0.05 were considered statistically significant.Statistical analysis was performed in IBM SPSS Statistics for MacOS, v25.0 (IBM Corp.) or Matlab 2016b. Continuous data were assessed for normality using the Anderson-Darling test. Correlations between continuous variables were assessed using the Pearson correlation coefficient , who successfully completed the DWI examination.Two thirds of the lesions 21/32; 66%) were invasive ductal cancers (IDC). The majority of cancers  were either grade 2 or 3. HR-positive expression was noted for 31/32 (97%) lesions, with 24/32 (77%) cancers being HER2-negative. Tumour characteristics are summarised in Table /32; 66% Ki or %HF are illustrated in Fig. Ki and Ktrans, ve and vp , but not for Ki vs. ve  or vp . Associations between %HF and DCE-MRI parameters followed similar trends, also indicating a decrease in hypoxia with increasing Ktrans, ve and vp  and ve . No correlation was observed between kep and either Ki  or %HF .Scatter plots indicating the relationships between DCE-MRI parameters and Ki and Ktrans parametric maps of four tumours of different histological subtypes, indicating heterogeneous spatial relationships between hypoxia and perfusion; other DCE-MRI parametric images are given in Supplemental Fig. Figure Ki, %HF and 18F-FMISO uptake parameters showed no significant difference between different histological subtypes or grades . Furthermore, analysis of hypoxia and Ktrans values in the most vascularised area of the tumour (hotspot on DCE-MRI) yielded no significant differences among different subtypes or grades  cancers with an additional 8/32 (25%) lesions displaying hypoxic fractions greater than zero but less than 1%; the remaining 18/32 (56%) tumours had a %HF of zero. Dot plots of %HF vs. tumour histological subtype and grade are presented in Fig.  0.13–0.3 vs. 0.3018F-FMISO-PET parameters correlated positively with size; %HF significantly correlated with pathological size , while 18F-FMISO-PET uptake metrics displayed associations of moderate strength with longest diameter on MRI.Table Ktrans: r = 0.24, p = 0.34; ve: r = 0.29, p = 0.25; vp: r = 0.20, p = 0.43), except for kep which correlated negatively with ADC  and DCE-MRI metrics. In contrast, in human head-and-neck cancer, where hypoxia is often marked, 18F-FMISO SUV measurements were negatively correlated with both Ktrans and kep [18F-FMISO trapping rather than non-specific tracer accumulation. Due to the higher contribution of non-specific 18F-FMISO accumulation at low hypoxia levels [No or weak positive correlations were found between static  and kep . A plausa levels , the useKi were noted in both grade 2 and 3 tumours, and less so in grade 1 cancers. These findings are concordant with previously reported small differences in hypoxia between low- and high-grade breast malignancy [No significant correlation was observed between PET hypoxia parameters and tumour grade or subtype. Our sample size of non-IDC cases was small for evaluating the impact of histology on tumour hypoxic status, but the presence of non-zero hypoxic fractions was observed in all histological subtypes studied. Hypoxic fractions and higher 18F-FMISO-PET hypoxia parameters and size. The size-related hypoxia changes could be ascribed to diffusion-limited hypoxia, concomitant perfusion decreases or increased interstitial fluid pressure [Correlations between DCE-MRI functional parameters and pathological size or MR tumour diameter yielded moderate negative relationships and conversely positive associations between pressure .ADC has been shown to inversely correlate with cellular density , and theKi. Despite the higher variability associated with kinetic parameter estimates, our choice was based on two considerations. First, several authors have reported lack of correlation between 18F-FMISO uptake ratios and pO2 measurements casting doubt on the accuracy of thresholds derived from static PET imaging for hypoxic quantification [Ki, have provided superior correlations with physiological measures of hypoxia from pO2 histography and immunohistochemistry [We calculated hypoxic fractions based on a specific parameter for hypoxia namely influx rate constant fication , 55. Kinhemistry , 55. SecThe main limitations of our study are the small sample size and that the majority of cancers were HR-positive ductal carcinomas. Though our findings cannot be generalised to the full spectrum of histological/molecular subtypes encountered in breast cancer, our study indicates the presence of hypoxia in all histological subtypes studied independent of nuclear grade. While the majority of lesions 56%) examined were found to be non-hypoxic, it should be noted that breast tumours are generally less hypoxic than cancers of the head and neck, cervix or lung and show greater variability in hypoxia among molecular subtypes, with basal-like subtypes being the most hypoxic [% examineOur demonstration of in vivo simultaneous measurement of perfusion and hypoxia is clinically important for three reasons. First, previous reports have indicated that tumours with a high hypoxia-perfusion ratio (i.e. hypoxia due to low perfusion) have a poorer prognosis and suboptimal treatment response , 58. In 18F-FMISO-PET, and markers of perfusion and vascular function from DCE-MRI, endorsing the hypothesis of perfusion-driven hypoxia in breast cancer. No associations were observed between 18F-FMISO-PET parameters and tumour histology or grade, but hypoxic fractions increased with lesion size. The intratumoural heterogeneity observed in hypoxia and perfusion images is consistent with the known complex relationship between perfusion and the hypoxic tumour micromilieu. The combined hypoxia-perfusion status of tumours may need to be considered in determining treatment efficacy or informing therapy selection in breast cancer, which could be achieved using simultaneous multimodality imaging as reported here.In conclusion, we found a negative relationship between tumour hypoxia, measured by ESM 1(DOCX 1303 kb)ESM 2(DOCX 21 kb)


After neoadjuvant chemotherapy of breast cancer pathologic complete response (pCR) indicates a favorable prognosis. Among non-selected patients, pCR is, however, achieved in only 10–30%. Early evaluation of tumour response to treatment would facilitate individualized therapy, with ineffective chemotherapy interrupted or changed. The methodology for this purpose is still limited. Tumour imaging and analysis of macromolecules, released from disrupted tumour cells, are principal alternatives.− 1) and tumour volume, can be used for early prediction of pathologic response.To investigate whether a metric of cell-loss, defined as the ratio between serum concentration of thymidine kinase1 , 48 h after cycle 2 (n = 104) and prior to cycle 2 (n = 57). The performance of the metric was evaluated by association with pathologic tumour response at surgery 4 months later.One hunred four women with localized breast cancer received neoadjuvant epirubicin/docetaxel in 6 cycles, supplemented with bevacizumab in cycles 3–6. The cell-loss metric was established at baseline (p = 0.01). In 80 patients with remaining tumour, tumour size was inversely related to the metric (p = 0.002). In 57 patients studied before treatment 2, positive and negative predictive values of the metric were 77.8 and 83.3%, compared to 40.5 and 88.7% 48 h after treatment 2.Treatment caused a rise in sTK1, a reduction in tumour volume and a marked increase in the cell-loss metric. Patients were subdivided into quartiles according to the baseline cell-loss metric. For these groups, baseline means were 0.0016, 0.0042, 0.0062, 0.0178 units. After subtraction of baselines, means for the quartiles 48 h after treatment 2 were 0.002, 0.011, 0.030 and 0.357 units. pCR was achieved in 24/104, their distribution in the quartiles  differed significantly .PROMIX (Clinical  Neoadjuvant chemotherapy (NACT) has become a treatment option for patients with early stage breast cancer (BC) –4. The aNACT provides valuable opportunities also in the perspective of clinical research. With pCR as endpoint, the effectiveness of new treatments may be established without several years of follow-up, as would be the case with disease-free or overall survival. For instance, pertuzumab for treatment of high-risk early stage BC received, therefore, an accelerated FDA-approval . LikewisThe growing insight into the diversity of BC has generated an increasing demand for methods that may facilitate, in the individual patient, early evaluation of the response to NACT. Identification of tumours with poor response would permit a switch in chemotherapy or motivate proceeding with immediate surgery - and suffering due to fruitless cytotoxicity could be avoided. Hence, individualized or response-guided therapy has become a prominent subject in present oncology. Nevertheless, a general obstacle is that tumour sensitivity to drugs can only be established in a minority of patients.-18F-fluorothymidine, response assessment criteria have still not been established [Several available methods have the potential of predicting pathologic tumour response during therapy: (i) measurement of changes in tumour size, (ii) estimation of tumour metabolism using radioactive tracer uptake, and (iii) measurements of the concentration of macromolecules released from disrupted tumour cells into the blood circulation. Most frequently used are anatomical measurements of tumour size, and criteria of response are defined in the Response Evaluation Criteria in Solid Tumors (RECIST) . For traablished . A generablished , 14. ForIn the present study the release into the blood circulation of thymidine kinase1 (TK1) during chemotherapy has been used to create a measure of cell loss. The cytoplasmatic TK1 is a key enzyme in DNA synthesis, catalysing thymidine into deoxythymidine monophosphate from extracellular sources via the salvage pathway. TK1 is cell cycle dependent: being undetectable in G0/G1, its concentration increases at the G1/S-phase border and reaches peak values during S-phase/G2. It is finally degraded in mitosis by ubiquitination , 16. In The aim of the present study was to investigate the usefulness of a measure of cell loss, defined as the ratio between sTK1 and tumour volume. We hypothesized that, whereas sTK1 is most likely dependent on tumour volume, the cell-loss metric would be more closely related to functional properties of the tumour, i.e. the occurrence of cell loss in undisturbed tumour growth or the enhanced cell loss during chemotherapy. To this end, in BC patients the cell-loss metric, established prior to NACT and in conjunction with the 2nd cycle of therapy, was related to pathologic response at surgery as objective end-point 4 months after initiation of chemotherapy. An association of the cell-loss metric with pathologic response would also confirm the tumour specificity of sTK1, thereby highlighting the issue of possible pathways for elimination of disrupted tumour cells during chemotherapy.Trials.govNCT000957125). The study was approved by the Ethics Committee at Karolinska University Hospital, 2007/1529–31/2, and informed written consent was obtained from all patients. The inclusion criteria and treatment protocol are fully described elsewhere [This study is part of the neoadjuvant, multicentre single-arm Phase II clinical trial, PROMIX  every 3 weeks, and in the absence of clinical complete response (cCR) after the 2nd cycle, for the addition of bevacizumab (15 mg/kg i.v.) on day 1 of cycles 3–6. Within 3 weeks after completing chemotherapy the patients underwent surgery and were eventually further treated in accordance with the Swedish national guidelines.The patients were scheduled for 6 cycles of epirubicin and docetaxel . For 57 of the patients, sTK1 and tumour volume had also been obtained prior to the 2nd cycle; these data were used for comparisons with the data 48 h after the 2nd cycle but were not included in the overall analysis.Serum thymidine kinase1 concentration: For collection of serum, venous blood was drawn in 5 ml plastic tubes. The tubes were inverted 10 times, the blood sample was allowed to clot for 30–60 min and centrifuged for 10 min at 1500 RCF = g at room temperature. After transfer of serum to a new tube, it was centrifuged at 3000 RCF = g for 10 min at room temperature, and transferred to new tubes in aliquots of 0.5 ml to be immediately frozen at -20 °C or -80 °C for storage at -80 °C until analysis.www.arocell.com). Samples were blinded with respect to patient identity, clinical data or tumour pathology.The concentration of TK1 protein in serum was measured at the Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden, with the new sandwich TK210 ELISA, produced by AroCell AB, Uppsala, Sweden. This test is based on two monoclonal antibodies against the C-terminal region of the TK1 protein and was performed in accordance with the manufacturer’s instruction (3) were calculated by assessment of the largest diameter from caliper examinations, mammography and/or ultrasound. Tumour volume was measured at baseline and after the 2nd cycle of therapy.The tumours were considered to be spherical and their volumes  was defined as absence of invasive cancer in the breast; residual non-invasive DCIS was allowed. Remaining cancers were classified according to size into pT1-pT3, and volume of the tumours was calculated from their largest diameter. Regional lymph node status was not taken in account for pCR because response to therapy could not be assessed during therapy.p-value below 0.05 was considered as indicating statistical significance. Concerning baseline characteristics and pathological outcome, analysis of variance was applied to examine the associations. Receiver operating characteristic (ROC) curves was used to assess the discriminating power for differentiating pCR from patients with incomplete response. All analyses were done using the statistical software Statistical Analysis Software, SAS, Cary, NC. USA.To obtain an estimate of the proportion of proliferating tumour cells being disrupted due to chemotherapy, the value of sTK1 48 h after the 2nd cycle was divided by the measure of tumour volume obtained between the 2nd and 3rd cycle. From this cell-loss metric, the baseline metric was subtracted. The cell-loss metric at baseline reflects the spontaneous disruption of proliferating tumour cells together with the background release from a minority of normal cells. Based on the cell-loss metric at baseline, the 104 patients were divided into quartiles. For each quartile the percentage of pCR was calculated. Additionally, for a subgroup of 57 patients the cell-loss metric, corrected for baseline, was also established before cycle 2. Possible differences in percentages between groups were examined with Fisher’s exact test and for absolute changes Wilcoxon test. A two-sided In the flow chart  the reason for missing information and excluding patients from the analyses are accounted for. Table For the 57-patient subgroup baseline demographic data are presented in the additional material (Table A1). The subgroup did not deviate in any respect from the main group.p < 0.001).A general observation was that treatment caused an increase in sTK1 while there was a reduction in tumour volume. Consequently, the cell-loss metric showed a marked increase 48 h after the 2nd treatment cycle (group mean 0.107) compared to baseline (group mean 0.007). Table p = 0.01).Table In a receiver operating analysis for distinguishing pCR from remaining tumour, 1-specifity and sensitivity were 0.31 and 0.71, respectively, at a cut-off value for the cell-loss metric of 0.026.The treatment aim to achieve a tumour free breast was reached in 24/104 23.1%) of the patients. 3/24 cases of pCR were found in each of quartiles 1 and 2, 6/24 in quartile 3, and 12/24 in quartile 4 , and the pathological findings of quartile 2 from those of quartile 3 (p = 0.029). There was a borderline difference between quartile 3 and 4 (p = 0.08).pCR of quartile 1 and 2 differed from those of quartile 4 , had any significance for the cell-loss metric 48 h after the second cycle of therapy.In order to evaluate the significance of the baseline cell-loss metric for the cell-loss metric established 48 h after cycle 2, all data shown in Tables n = 104), before cycle 2 (n = 57), and 48 h after the 2nd cycle (n = 104). The results are shown in Table Finally, patients were subdivided according to pathologic outcome into pCR and non-pCR. For these two subgroups, tumour volume, and sTK1 per se, and the cell-loss metric were compared at three points in time, namely baseline (n = 104).Notably, in the two groups tumour volume showed a similar (58%) decrease between baseline and as obtained between the 2nd and 3rd cycle, but there was no association between these early measures of tumour volume and pathologic response. However, the cell-loss metric differed significantly between responders and non-responders already at baseline as well as prior to and 48 h after cycle 2. A further observation was the relatively high discriminating power of the cell-loss metric obtained before cycle 2, with positive and negative predictive values of 77.8 and 83.3%, respectively (n = 57). For the metric obtained 48 h after cycle 2, the positive and negative predictive values were 40.5 and 88.7%  but also by a multitude of less well-known factors. If the cell-loss metric thus reflects a sum effect of several mechanisms, including the rate of proliferation, then, adding Ki67/Mib1 would not contribute to the predictive value of the metric. In the practical perspective, the cell-loss metric might be considered causally closer to the outcome of treatment.The finding that a number of tumour properties did not differ between the quartile groups does not imply that they are clinically insignificant but that they are independent of the cell-loss metric. Therefore, it is logically possible that some of them would improve the prediction of pathologic response. This is the main theme of a following study (to be published), where it was found that combining the cell-loss metric with histopathologic markers, such as receptors for oestrogen and progesterone, improves the predictive power in terms of both sensitivity and specificity.The clinical value of tumour biomarkers is to guide therapy. A distinction is made between prognostic markers, supposed to provide information about long-term outcome, and predictive markers, which reveal a tumour’s response to treatment. Ideally, the adequate choice of therapy would be based on tumour or patient characteristics established before treatment. For a defined type of tumour there is, nevertheless, always an inter-patient variability in the response to treatment. Therefore, predictive markers for early detection of the effects of treatment would be a valuable complement to tumour characteristics established at diagnosis. Among the most well-established tissue markers in oncology are the receptors for oestrogen, progesterone and growth-factor 2 . These aNevertheless, there is a paucity of methods for the early evaluation of tumour response during treatment. Such methods would give a valuable contribution particularly in the management of patients for whom the statistically calculated benefit of a standard treatment is low and has to be balanced against unnecessary side effects. For instance, in low-grade, low-stage ER+/HER-2neu luminal-A tumours, pCR after cytotoxic treatment was achieved in less than 10% of patients and, in addition, pCR was not prognostic for long-term survival  and fromEGFR-DNA 14 days after initiating treatment with erlotinib (a tyrosine kinase inhibitor) was associated with tumour shrinkage 2 months later [A few studies deal with the release of macromolecules early during chemotherapy and how such early response markers are associated with pathologic outcome or long-term survival. In patients with lung cancer a high activity of TK1 in serum after the first and second cycles of cytotoxic treatment was associated with a significantly longer survival . Analogohs later . Likewishs later .In BC, no significant changes in nucleosome levels have been found during the first two treatment cycles of NACT . HoweverTo our knowledge there are no studies which address the clinical value of a measure that relates the levels of a macromolecule, released from disrupting tumour cells, to the volume of the tumour. The usefulness and predictive power of the TK1-based cell-loss metric have the potential of being improved in several ways. A limitation of the present study was that the patients were examined and treated in five different clinics. Methods for estimating tumour size included caliper measurement, mammography and ultrasonography, the accuracy of which ranges between 57 and 79% . MethodsReactions of lymph nodes on therapy could not be assessed, but release of TK1 from metastatic lymph nodes cannot be excluded. Another issue is the time point for establishing the cell-loss metric. The precise time course for treatment-induced changes in sTK1 remains to be clarified, and it may, in addition, be dependent on the type of treatment. As already noted, the predictive value of the cell-loss metric appears to be higher prior to the 2nd treatment than 48 h after treatment. Advantages of the present study were the prospective layout of the original clinical trial and the absence of patients with distant metastases, which would have constituted sources of TK1 with unknown volumes. Prospective studies should be performed to confirm the present findings, to establish the optimal time points for the cell-loss metric during different treatments, and to define cut-off values for discriminating between responders and non-responders.The present study introduces a measure of cell loss, obtained by combining the serum level of TK1, released from disrupted tumour cells, with tumour volume. Established early during chemotherapy, this metric showed a considerable inter-patient variability and a significant association with later pathologic response. Thus, it appears to reflect an inherent property of the tumour, of importance for tumour growth and response to treatment. In the practical perspective, monitoring treatment response by means of the cell-loss metric could be valuable in individualized therapy as well as in the development of new cytotoxic drugs or targeted therapies.Additional file 1. Flow chartAdditional file 2: Table A1. Baseline characteristics for the subgroup of 57 women.Additional file 3: Table A2. Pathologic findings in the breast and axillary lymph nodesAdditional file 4: Table A3. Cell-loss metric 48 h after the 2nd cycle of therapy without baseline subtractionAdditional file 5: Table A4. Pathologic outcome and cell-loss metric 48 h after the 2nd cycle of therapy without baseline subtractionAdditional file 6: Table A5. Baseline cell-loss metric and pathologic outcomeAdditional file 7: Table A6. Pathologic complete response in relation to baseline variables


The AMEPS was less sensitive to lubricants compared to the NEPS and Starch 1500®. Similarly, the dilution potential of the AMEPS was superior to the NEPS and S1500®. The AMEPS accommodated up to 50% of paracetamol while the NEPS and S1500® were able to hold only up to 30%. Pharmacopoeial specifications for disintegration and dissolution were met by the paracetamol tablets prepared by AMEPS. Thus, considering all the results obtained, spray-dried AMEPS could be a potential alternative directly compressible tablet excipient.Ethiopian potato is one of the tuber-bearing members of the family Lamiaceae. It is an indigenous crop in Ethiopia and important source of starch. Unprocessed native starches are structurally weak and functionally restricted for application in pharmaceutical technologies. Consequently, starch is usually modified either chemically or physically to make it convenient for industrial use. The aim of the study was to prepare and characterize acid-modified Ethiopian potato starch (AMEPS) and evaluate its functionality as a direct compressible excipient in tablet formulations. The extracted starch from Ethiopian potato tuber was modified using 6% HCl concentration for 8 days, then dried using oven and spray drying techniques, and subsequently evaluated and compared with the native Ethiopian potato starch (NEPS) and S1500® as a direct compressible excipient. Acid modification of the NEPS decreased the moisture content and swelling power while increased the percent solubility. The X-ray diffraction revealed that both the NEPS and AMEPS have B-type crystal patterns. The AMEPS showed improved flowability compared to the NEPS. This improvement was further enhanced by the spray drying process. The compactability study revealed that the tensile strength of spray-dried AMEPS (16.76 kg/cm Today's technologic desire is to simplify the manufacturing processes while at the same time getting cheap and quality products. Regarding tablet manufacturing, direct compression (DC) had become one approach that simplifies the tablet manufacturing process . HoweverStarch can generally be recognized as the most common pharmaceutical excipient , 5 whichEthiopian potato tubers have a starch yield value of about 80.4% on a dry weight basis . Thus, tFresh Ethiopian potato tubers were collected from Shashagalle kebele, Wolaita Zone, Southern Nations, Nationalities, and Peoples' Region, and authenticated by the National Herbarium of Ethiopia, Department of Plant Biology and Biodiversity Management, College of Natural Sciences and Computational Sciences, Addis Ababa University. Croscarmellose sodium , disodium hydrogen orthophosphate dihydrate , magnesium stearate , paracetamol powder , and potassium dihydrogen orthophosphate  were kindly donated by Addis Pharmaceutical Factory. Starch 1500®  was kindly donated by the School of Pharmacy, College of Health Sciences, Addis Ababa University. Hydrochloric acid , sodium hydroxide , sodium metabisulphite , and sodium chloride  were of analytical grade and used as received.w/v). Then, the suspension was passed through a fine muslin cloth and the resulting filtrate was allowed to sediment. After the suspension had sediment, the supernatant was decanted and the sediment starch was repeatedly washed with 0.075% (w/v) sodium metabisulphite solution until the supernatant became clear (translucent). Then, it was washed several times with distilled water, dried in open air, milled, sieved using a 250 μm mesh sieve, and stored in an airtight container for further use.Starch, from Ethiopian potato (EP) roots, was extracted following the method described by Assefa . First, w/v) HCl solution at room temperature for 0, 2, 4, 6, 8, 10, and 14 days. Similarly, the effect of HCl concentration on the recovery yield of starch was studied by suspending two grams of NEPS (dry basis) in 10 mL of 0, 2, 4, 6, 8, 10, and 12% (w/v) HCl solution at room temperature for 8 days. The resulting suspension was then neutralized with 10% (w/v) of sodium hydroxide solution to terminate the hydrolysis. The starch slurry was then washed five times with distilled water until the pH of the filtrate became 7. The resulting starch was dried for 24 hours at 40°C in a hot air oven . This dried starch was then powdered using mortar and pestle, passed through a 250 μm mesh sieve, and stored for further studies : in the range of 3-64° of the 2θ with a microprocessor equipped with a single-crystal graphite monochromator.The X-ray diffraction patterns of the NEPS and AMEPS samples were obtained using the method described by Nwokocha and Williams  in an X-ρB) and tapped density (ρT) were calculated from the bulk and tapped volumes using Equations , which provides 300 taps/minute, and operated for one minute. Finally, the resulting volume was recorded, and the bulk density (quations  and (5),Flow rate and angle of repose of the starch powders were determined by using a powder flowability tester . First, 50 g of powder, from each sample, was filled into the stainless steel funnel of the machine having a 15 mm outlet nozzle. The funnel was held at a height 10 cm above the base having a diameter of 10 cm. The stirring speed of the blade was held at 5 rpm. Then, the machine was allowed to operate, and the readings for the flow rate and angle of repose were recorded .Wi and Wf are the weights of the sample powder before and after drying, respectively.The moisture content of the powders was measured as per the method described by Olayemi et al. . The perWds) and the weight of the sediment remained after decantation (Ws) using Equations (The swelling power (SP) and water solubility index (WSI) of the NEPS, AMEPS, and S1500® were determined following the methods described by Odeku and Picker-Freyer . The staquations  and 8),,Wds) andWb) and after equilibration (Wa) usingThe moisture sorption properties of the NEPS, AMEPS, and S1500® were investigated based on the method described by Gebre-Mariam and Schmidt (1996). Accordingly, the percentage of moisture sorbed (MS) by the samples was calculated from the weight of the samples before equilibration (−1 at 25°C. The spectrum was obtained after the samples were scanned 20 times.Drug-excipient compatibility was studied using a Fourier transform infrared spectroscopy (FT-IR) . This study was conducted for pure paracetamol and the mixture of paracetamol and acid-modified starch. These samples were then placed in the FT-IR machine and scanned in the wavenumber range from 500 to 4000 cmAll tablet formulations in this study were prepared using the DC method compressed at a constant compression force (15 ± 2 kN), using a single punch tablet machine  fitted with a flat-faced punch and a die of 10 mm diameter. The prepared tablet formulations were evaluated and compared for compaction property, lubricant sensitivity, and dilution potential of the excipients used which are presented in the respective sections below.f(%)), and disintegration time (Dt). The lubricant sensitivity of the spray-dried NEPS, spray-dried AMEPS, and S1500® was evaluated as per the method described by Assen et al. [The compaction property of NEPS, AMEPS, and S1500® was evaluated following the method described by Okunlola and Akingbala . Accordin et al. . Fifty gTo evaluate the dilution potential of spray-dried NEPS and AMEPS, paracetamol was blended with each excipient at different ratios as indicated in D is the diameter and T is the thickness.The crushing strength (CS) of tablets was determined using a hardness tester . Ten tablets were randomly selected from each formulation, and the CS of each tablet was measured . The tabTen tablets from each batch were weighed and placed in a plastic chamber of a friability tester  and operated at a revolution speed of 25 rpm for 4 min. The tablets were then collected, dedusted, and weighed, and the percentage weight loss was calculated and displayed as percentage friability .The disintegration test was carried out using a USP disintegration tester  for each batch taking six tablets randomly. Each tablet was placed in a disintegration apparatus containing 900 mL of distilled water and maintained at 37 ± 2°C. The machine was then allowed to operate, and the time taken for complete disintegration and to pass through the mesh was recorded as the disintegration time.λmax of 243 nm. Each withdrawn sample was replaced with an equal volume of fresh dissolution medium which kept at the same temperature.The dissolution tests were performed according to the USP/NF  specificp values of ≤0.05 were considered statistically significant. All the data measured and reported are averages of a minimum of triplicate measurements, and the values are expressed as mean ± standard deviation.Statistical analysis was performed using analysis of variance (ANOVA) with SPSS statistical software package (IBM SPSS Statistics 20). Origin8® software  was used to plot graphs. At a 95% confidence interval, The acid recovery yield of EP starch at different hydrolysis times is presented in θ with other minor peaks . The presence of strong diffraction peaks at around 17° and small peaks at around 5.6°, 20°, 22°, and 24° 2θ angles indicated that the NEPSs have B-type crystals which are in line with other tuber starches [θ. This may indicate that the internal structure of the starch granules had changed and resulted in a more crystalline structure [The X-ray diffractograms of NEPS and AMEPS are presented in Figures starches . Similarstarches . Howevertructure , 19, 32.p < 0.05). This higher bulk density is advantageous in tableting due to a reduction in the fill volume of the die [The density and related properties of the NEPS, AMEPS, and S1500® are presented in  the die . The cha the die .p < 0.05) indicating poor flow property of the NEPS. The CI and HR of spray-dried AMEPS were found to be 11.48% and 1.13, respectively, indicating good powder flow properties. This result indicated that spray drying had significantly improved its flow properties (p < 0.05). This might be due to the presence of greater amounts of spherical-shaped particles produced during the spray drying process. Similarly, spray drying decreased the CI and HR of both the NEPS and AMEPS. On the other hand, S1500® showed excellent flow properties which might be due to its higher densities and particle size [The air- and spray-dried NEPS exhibited significantly higher CI and HR values compared to the AMEPSs (the oven- and spray-dried) p < 0.0 indicatip < 0.05). The flow rate of the air-dried NEPS and oven-dried AMEPS could not be determined for the same reason mentioned above in the angle of repose.The results of the angle of repose for the spray-dried NEPS indicated poor flow characteristics while the spray-dried AMEPS showed excellent powder flowability. The air-dried NEPS and oven-dried AMEPS, on the other hand, could not flow through an opening. This indicated that the spray drying had improved the flow properties of both the NEPS and AMEPS. The flow improvement, however, was significantly higher for the AMEPS compared to the NEPS (excellent versus poor flowability). The difference could be attributed to the acid modification of the NEPS prior to the spray drying. On the other hand, no significant difference was observed in the angle of repose of spray-dried AMEPS and S1500® indicating excellent powder flowability. The flow rate, as can be noticed in p < 0.05). This could be attributed to the difference in the relative crystallinity of the starches. However, no significant difference was observed in moisture content within the NEPSs and similarly between the AMEPSs (p > 0.05). This result indicated that the spray drying process did not bring about any significant change in the moisture content of the NEPS and AMEPS (p > 0.05) in contrast to the study done by Assen et al. [w/w) [The moisture contents of the NEPSs, AMEPSs, and S1500® are presented in n et al.  and Tessn et al. . On the n et al. . Generall. [w/w) .p < 0.05). The lower SP of the NEPS and AMEPS may be related to the strong bonding forces within the granules making the starches relatively resistant to swell [p < 0.05). This could be attributed to the reduction in chain length and hydrolysis of the amorphous parts of the starch granules as a result of the acid modification. Amylopectin chain length is one of the factors that affects SP [The SP of all the starch powders studied is depicted in to swell . In contto swell . On the to swell . It coulfects SP .p < 0.05) which might be due to the changes in the structure of starch granules and amylose leaching because of acid hydrolysis [p > 0.05). Thus, the drying methods had no significant effect in this case.The solubility of the NEPSs, AMEPSs, and S1500® increased with increasing temperature as can be noticed in drolysis . Due to p > 0.05). All the starches studied showed a slight increase in their moisture sorption between 20 and 60% RH. However, beyond 75.5% RH, the moisture sorption significantly increased. This sharp increase above 75.5% RH could be attributed to change in mechanism of sorption. This reinforces the necessity for moisture avoidance during storage [The moisture sorption profiles of the NEPSs, AMEPSs, and S1500® are depicted in  storage .−1 (O-H stretching), 3155.54 cm−1 (N-H stretching), and 1651.07 cm−1 (C=O (amide) stretching) functional groups, whereas amide II band stretching at 1562.34 cm−1, OH deformation -C-O stretching at 1253.73 cm−1, C-H stretching at 3000 cm−1 to 2800 cm−1, and aromatic ring stretching vibration band at 1608.63 cm−1 were also observed . The higher CS and TS of the AMEPS tablets indicated that it has a higher compactability which could be attributed to the acid modification. Acid hydrolysis cleaves the amorphous region and increases the relative crystallinity. These crystalline regions could be forced closer with increased intermolecular force during compression resulting in a higher tablet hardness [The tablet characteristics of the blank tablets prepared for the compaction study are presented in Figures hardness . The reshardness , 44, 45.p < 0.05). On the other hand, the disintegration time was ranked in the order of S1500®>spray-dried AMEPS>spray-dried NEPS (p < 0.05). The spray-dried AMEPS tablets showed prolonged disintegration time than the spray-dried NEPS which could be attributed to their higher hardness. Generally, all the blank tablets were able to disintegrate into their primary particles within 15 minutes.The resistance to friability and disintegration time of the blank tablets prepared from NEPS, AMEPS, or S1500® are in line with the values of their hardness. This is to mean that their values increased with increasing tablet hardness. Hence, the friability of the tablets in this study was ranked in descending order as spray-dried NEPS>S1500®>spray-dried AMEPS (p < 0.05) with an average CS of 66.73, 13.9, and 33.46 N, respectively, at 2% lubricant concentration. On the other hand, the spray-dried NEPS and S1500® produced soft tablets with an average CS of 13.9 and 33.46 N, respectively, at 2% magnesium stearate concentration. On the other hand, as depicted in The CS of all the prepared tablets for lubricant sensitivity studies generally decreased as the concentration of magnesium stearate increased . This cop > 0.05) at all levels of magnesium sterate concentration. However, the former possesses a shorter disintegration time even without considering its higher CS. The longest disintegration period observed in the tablets made of S1500® could be related to the formation of a gel-like layer, which is formed in combination with water preventing their disintegration. The shortest disintegration time was registered for tablets of spray-dried NEPS at all levels of magnesium stearate which could be attributed to their lower CS.The hardness of tablets could be expressed in terms of CS and TS which are related to the mechanical strength of tablets . Crushinp < 0.05). This could be related to the poor compressibility characteristic and high elastic recovery of paracetamol. The tensile strength of tablets from the AMEPS was significantly higher than both the NEPS and S1500® at all levels of paracetamol concentration (p < 0.05). This implies that the AMEPS underwent a higher plastic deformation that predominates the disruptive elastic recovery of paracetamol [The TS of the paracetamol tablets prepared from NEPS, AMEPS, and S1500® were given in acetamol . The resacetamol . The NEPp < 0.05). Tablets of the spray-dried AMEPS fulfilled the acceptance criteria for friability at all paracetamol contents while tablets of the spray-dried NEPS and S1500® fulfilled this specification only up to 30%. The higher friability values of spray-dried NEPS and S1500® at higher paracetamol concentration could be due to their low tablet mechanical strength as discussed above. There is an inverse relationship between tablet mechanical strength and friability in such a way that the latter declines as the former increases [The friability test results of all paracetamol tablet formulations are depicted in ncreases .p < 0.05)  . This cop > 0.05). This is in line with the disintegration time of the respective tablets. A similar trend was also observed for tablets containing 30% of paracetamol. In both concentrations of paracetamol, the prepared tablet formulations fulfilled the dissolution specification set by the USP 30/NF 25 [The dissolution profiles of all the selected paracetamol tablets of the spray-dried NEPSs, spray-dried AMEPSs, and S1500®s are presented in 30/NF 25 , which iThe main limitation of this study was only a single compression force was used in the powder compaction property study. The compaction property would have been better explained if compression force of 3-4 levels versus hardness profile was measured.The result of this study indicates that acid modification improves the flow properties of the native Ethiopian potato starch. This improvement was further enhanced by the spray drying process. In addition, the spray-dried AMEPS showed a higher compactability compared to both the spray-dried NEPS and S1500® by yielding tablets of higher mechanical strength and acceptable friability values. The spray-dried AMEPS was also able to incorporate up to 2% of magnesium stearate satisfying the tablet specifications while the spray-dried NEPS and S1500® incorporated only up to 0.5%. Similarly, it was noticed that only the spray-dried AMEPS was capable of incorporating up to 50% of paracetamol content with acceptable pharmacopoeial specifications. From the perspective of the above mentioned results, spray-dried AMEPS could be a potential alternative candidate as a directly compressible tablet excipient.


The role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms.2+ level [Ca2+i] were investigated with microfluorometry.Fluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists  on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca2+-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [Both norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α1-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of β-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor. Ca2+ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.Our results prove the direct role of α A predominant amount of the aqueous layer is produced by the lacrimal gland (LG).–,1 and β1-adrenergic agonists could result in protein secretory response in whole LG pieces of mouse and rat, the role of α1-adrenergic receptors is expected to be more relevant.,1D and not the most common α1A or α1B subtypes.,1D-adrenergic receptor activation are not clearly understood. Additionally, involvement of the NO/cGMP pathway was suggested in the phenylephrine-induced protein secretion of rat LG.Autonomic regulation of the ductal function is not fully explored. Parasympathetic pathways are the main regulatory system of the LG, whereas sympathetic effects have been supposed to play indirect role through blood flow regulation.Therefore, the aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion of isolated LG duct segments and to study the intracellular mechanisms underlying adrenergic stimulation.Parts of the results in this paper have been presented in abstracts in the Annual Meetings of the Association for Research in Vision and Ophthalmology : 2256; Tóth-Molnár E., et al. IOVS 2018; 59(9): 4923).Mouse exorbital LGs dissected from 12 to 16 week old wild type FVB/N mice  were used throughout the study. Animals were narcotized intraperitoneally with ketamine (80 mg/kg) and xylazine (10 mg/kg), and euthanized with pentobarbital overdose (100 mg/kg).All experiments were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Ethical Committee for the Protection of Animals in Research of the University of Szeged, Szeged, Hungary, and conformed to the Directive 2010/63/EU of the European Parliament.1D-adrenergic receptor inhibitor BMY-7378 were purchased from Sigma-Aldrich Corp. . Collagenase was purchased from Worthington Biochemical Corp. . FURA2-AM was purchased from Invitrogen . The compositions of solutions used in our experiments are summarized in the 3−/CO2− buffered solution was gassed with 95% O2/5% CO2 at 37°C.Media and its supplements for LG duct isolation and culture , phenylephrine, isoprenaline, propranolol, phentolamine, norepinephrine, carbachol (carbamylcholine chloride), endothelial nitric oxide synthase (eNOS) inhibitor L-NAME, guanylyl cyclase inhibitor ODQ, and α2.Mouse LG interlobular ducts were isolated as previously described by our laboratory.,Video-microscopic method was used for the measurement of ductal fluid secretion. The technique was originally described for the investigation of pancreatic ducts and was adapted by our laboratory for the measurement of ductal fluid secretion.2+-sensitive fluorescent dye FURA 2AM (5 µM) was used for the measurement of intracellular Ca2+ concentration [Ca2+]i as described earlier.2+]i were measured using an imaging system . Four to 5 small areas (region of interests [ROIs]) of 5 to 10 cells in each intact duct were excited with light at 340 nm and 380 nm, and the 380 / 340 fluorescence emission ratio were measured at 510 nm. One [Ca2+]i measurement was obtained per second.CaP value of < 0.05 was regarded as significant.For the analysis of ductal fluid secretion, effects of the stimulatory agents  were considered as “fixed effects.” The effect of the individual “duct” and the “duct and effects of phenylephrine/isoproterenol/ norepinephrine interaction”  were taken into account as random effects. For the investigation of the inhibitory effect of L-NAME, ODQ, and BMY-7378, data were expressed as the percent change of the LV above baseline LV (baseline LV was considered 1.0). A mixed ANOVA model was used for statistics, by using SigmaPlot version 12.5 , results were presented as means ± SEM. A 2; 10 µM: 189.6 ± 13.9 pl/min/mm2; and 20 µM: 181.5 ± 11.7 pl/min/mm2 in the first 10 minutes of stimulation). The most effective concentration of norepinephrine proved to be 10 µM  were used to determine the secretory response and dose-response relationship. To ensure the blockade of β-adrenergic receptors, phenylephrine was administered in the presence of β-adrenergic antagonist propranolol (1 µM). Phenylephrine stimulation caused a rapid fluid secretory response in the isolated duct segments . Therefore, concentration of 10 µM was used throughout the additional phenylephrine experiments. It is important to mention that no statistically significant difference was detected between the extent of the fluid secretory rates evoked by phenylephrine in the presence of propranolol versus norepinephrine (P = 0.42) and the kinetics of these stimulated secretions were also similar.Isolated mouse LG ducts were used for the investigation of the effect of various adrenergic agonist on ductal fluid secretion. In the first series of experiments, ducts were stimulated with various concentrations  of the natural adrenergic agonist norepinephrine  to determine the secretory response and dose-response relationship. Norepinephrine stimulates both α- and β-adrenergic receptors causing a complete adrenergic upset. Application of norepinephrine initiated a dose-dependent, rapid fluid secretory response . Effect of β-adrenergic stimulation on ductal fluid secretion was also investigated. β-adrenergic agonist isoproterenol was administered in the presence of α-adrenergic antagonist phentolamine (10 µM) to ensure the selective β-adrenergic stimulation. Isoproterenol failed to elicit any detectable secretory effect in all applied concentrations  for 30 minutes and then phenylephrine (10 µM) was added to the superfusate. BMY-7378 reduced phenylephrine-induced ductal fluid secretion in a dose-dependent manner . The difference between baseline LV and the LV measured following phenylephrine stimulation in the presence of 100 µM BMY-7378 was statistically not significant (P = 0.081). Therefore, administration of 100 µM BMY-7378 completely abolished phenylephrine-induced ductal fluid secretion proving the role of α1D-adrenergic receptors in the observed secretory response  for 30 minutes and then 10 µM of phenylephrine was added to the bath. Phenylephrine-evoked ductal fluid secretion was reduced by L-NAME in a dose-dependent manner . However, even at the maximal inhibition effect of L-NAME, a significant difference (P = 0.023) was found between baseline LV and LV measured following phenylephrine stimulation in the presence of L-NAME (Because the mechanisms underlying αf L-NAME . These rP = 0.0008) was proved between baseline LV and LV measured following phenylephrine stimulation in the presence of ODQ  for 30 minutes before administration of phenylephrine (10 µM). Inhibition of guanylyl cyclase with ODQ decreased phenylephrine-induced LV increase in a dose dependent manner . Maximal inhibition occurred at 10 µM ODQ concentration. Although the inhibitory effect of ODQ was visible, a significant difference . The extent of this increase was much smaller , no statistically significant difference could be demonstrated in the fluid secretion between BAPTA-AM-treated and non-treated ducts (P = 0.052).Phenylephrine-induced ductal fluid secretion was measured in BAPTA-AM pretreated ducts in order to investigate the role of Ca2+]i, the effect of L-NAME on phenylephrine-induced secretion was investigated in the presence of intracellular Ca2+-chelator BAPTA-AM. In these experiments, isolated ducts were pre-incubated with the most effective dose of L-NAME (100 µM) and BAPTA-AM (10 µM). Co-administration of L-NAME and BAPTA-AM completely blocked phenylephrine-induced ductal fluid secretion .In contrast to BMY-7378, eNOS inhibitor L-NAME considerably reduced but not completely abolished phenylephrine-induced ductal fluid secretion. To investigate the potential role of phenylephrine-evoked elevation of i and ductal fluid secretion was investigated.2+]i, no statistically significant difference could be demonstrated in the fluid secretion between the Ca2+-chelator BAPTA-AM-treated and nontreated ducts.Tear secretion is regulated by the autonomic nervous system. Besides the generally accepted decisive role of parasympathetic innervation, there is accumulating experimental evidence about the direct effect of sympathetic regulation of LG function.2+]i in the α-adrenergic stimulation-enhanced fluid secretion, further series of experiments were performed. In these experiments, Ca2+ signaling was excluded by co-administration of intracellular Ca2+-chelator BAPTA-AM either with L-NAME or ODQ. Under these circumstances, complete blockade of phenylephrine-induced ductal fluid secretion could be reached demonstrating the apparent role of NO/cGMP pathway-independent Ca2+ signaling mechanism.To specify the role of the observed increase in [Ca2+]i plays some role. Blockage of the cGMP pathway alone markedly reduced but not completely abolished fluid secretion, whereas in combination with depletion of [Ca2+]i resulted in complete stoppage. On the other hand, because the elevation of [Ca2+]i was small, distraction of Ca2+ itself did not result in significant reduction in fluid secretion, although some tendency of lower secretory rates could be noticed .Although the main intracellular event in the fluid secretion evoked by phenylephrine is the activation of the guanylyl-cyclase-cGMP pathway even though minor elevation of [Ca1D-adrenergic receptors in mouse LG ducts. Inhibition of phenylephrine-induced ductal fluid secretion by α1D-adrenergic receptor antagonist or by reduction of fluid secretion by either eNOS or guanylyl cyclase inhibitors suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor stimulation to increase fluid secretion, but involvement of a NO/cGMP pathway-independent Ca2+ signaling mechanism is also assumed.In conclusion, our data strongly suggest the direct role of α-adrenergic stimulation in LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response and the similar secretory effects of norepinephrine and phenylephrine suggest that the determining adrenergic pathway is via α


Synovial fluid culture is the standard investigation for the preoperative diagnosis of periprosthetic joint infection (PJI). However, the culture has limited sensitivity and requires several days until result. We evaluated the value of isothermal microcalorimetry for real-time diagnosis of PJI based on heat produced by microbial growth in synovial fluid.Patients undergoing aspiration of prosthetic hip or knee joint before revision surgery were prospectively included between 2014 and 2015. The performance of microcalorimetry was compared to synovial fluid culture using McNemar’s chi-squared test. Pearson’s correlation coefficient was calculated for synovial fluid leukocyte count and microcalorimetric heat.r = 0.366) was found between leukocyte count and microcalorimetric heat in synovial fluid (p < 0.001). The median time to positivity of microcalorimetry was 9 h  vs. 3 days for cultures .Of 107 included patients (58 knee and 49 hip prosthesis), PJI was diagnosed in 46 patients (43%) and aseptic failure in 61 patients (57%) according to institutional criteria. In 26 PJI cases (56%) the pathogen grew in synovial fluid and intra-operative cultures. The sensitivity of synovial fluid culture and microcalorimetry was both 39% and the results were concordant in 98 patients (92%). In patients with PJI, microcalorimetry missed 4 pathogens which grew in synovial fluid culture, whereas culture missed 4 pathogens detected by microcalorimetry. A linear correlation (Microcalorimetry of synovial fluid allows thermogenic diagnosis of periprosthetic joint infection in synovial fluid. The diagnostic performance of synovial fluid microcalorimetry is comparable to culture and delivers results considerably faster.NCT02530229.This prospective study was registered on August 21, 2015 with the public clinical trial identification  An early and reliable diagnosis of periprosthetic joint infection (PJI) is critical for its successful management –3. JointIsothermal microcalorimetry is a novel method for real-time detection of growth-related heat production of reproducing microorganisms in biological fluid . This hiMicrocalorimetry of synovial fluid has not yet been evaluated for the diagnosis of PJI. The aim of this prospective study is to assess the diagnostic performance and speed of microcalorimetry in the diagnosis of PJI of the hip and knee and to compare it to current standard diagnostic methods employed in clinical routine, including synovial fluid leukocyte count and microbial culture. We hypothesized that microcalorimetry of synovial fluid could improve and accelerate the diagnosis of PJI.https://clinicaltrials.gov/ct2/show/NCT02530229).Our institutional review board issued its approval for this prospective study (EA1/306/14) and written informed consent was obtained for all patients. This study is compliant with the Declaration of Helsinki and was registered under the public clinical trial identification NCT02530229  or (ii) growth of at least two low virulent organisms  [Positivity of synovial fluid culture was considered for a confined microbial organism in solid media culture. For enrichment broth, positivity was contemplated only for high-virulent pathogens. Positivity for culture of periprosthetic tissue was defined for (i) growth of one or more highly virulent pathogens (robioma) . Growth robioma) .Asepsis was applied to the skin before joint aspiration. The corresponding joint was aspirated with a sterile 18-gauge spinal needle in consonance with aseptic directives. Synovial fluid was aspirated into a 10-ml syringe. The needle was relocated inside the joint without trespassing the skin if the initial joint aspiration was void.A pediatric blood culture bottle  with at least 1 ml of synovial fluid was employed for culture (at 37 °C) and monitored for 14 days (or until positivity). For gram stain an additional minimum of 1 ml was applied. Further explorations comprised Schaedler, chocolate and blood agar plates, and thioglycolate broth. Aerobic and anaerobic cultures at 37 °C were monitored daily for 7 and 14 days, respectively. Exploration for pyrophosphate and urate crystals and inflammatory cells was performed with polarization microscopy on an additional 1 ml of synovial fluid. An ethylenediaminetetraacetic acid (EDTA) vial was filled with at least 1 ml of synovial fluid to determine leukocyte count and differential. 10 μl of hyaluronidase  was applied on clotted samples at room temperature conditions for a 10 min-period.For microcalorimetric analysis, an aliquot of 1 ml synovial fluid was inoculated into a 4 ml microcalorimetry vial pre-filled with 2 ml of tryptic soy broth. The vial was inserted into an isothermal microcalorimeter with 48 measuring channels . According to the manufacturer, the sensitivity of the microcalorimeter is 0.225 μW. First, an equilibration process was performed for 15 min in order to reach 37 °C and another 45 min to get accurate measurement of heat flow. Then, heat production was continuously measured for a minimum of 5 days, expressed in μWatts over time. Calorimetry heat peak values were measured as the difference between the maximum heat value of the curve and its baseline value  and histopathologic examination (at least 1 sample). Similar to the aforementioned processing of synovial fluid, homogenization and culture of periprosthetic tissue samples was performed on aerobic and anaerobic agar plates and thioglycolate broth. Microbial pathogens were automatically determined according to standard procedure . Sonication of the extracted implant was performed following standard technique .p < 0.05 were considered statistically significant). For aseptic calorimetry curves, we explored the association between leukocyte count in synovial fluid and heat production with Pearson’s correlation coefficient r . The relationship between leucocyte count and peak heat production was explored with linear regression.The diagnostic methods were evaluated with McNemar’s Chi-squared test , 5 with aspiration within 6 weeks of surgery, 4 patients with periprosthetic fracture and 8 patients with a prosthesis dislocation. The demographic characteristics of included patients is shown in Table Of 46 (43%) patients diagnosed with PJI, the infecting microorganism was identified in 26 (56%) cases. The pathogen was isolated in 18 (39%) PJI cases by synovial fluid culture only. The pathogen was detected in an additional 8 cases with the addition of periprosthetic tissue and sonication fluid cultures. Of these 8 patients with negative synovial fluid cultures, 6 cases had positive periprosthetic tissue cultures, 4 cases had positive sonication cultures and 2 cases had both. Hence, 26 (56%) PJI cases had any positive culture.For 20 PJI cases (44%), no pathogen was identified as all  cultures remained negative.Streptococcus spp. in 8 (44%) cases, coagulase-negative staphylococci in 3 (17%) cases, Escherichia coli in 3 (17%) and Enterococcus spp. in 3 (17%) cases. No polymicrobial infections were detected by synovial fluid culture.Out of the 18 identified pathogens in synovial fluid culture, the most recurrent causative organisms detected were The results for the diagnostic methods are listed in Table E. faecalis (2 isolates), coagulase-negative staphylococci and E. coli (1 isolate each). In contrast, microcalorimetry was positive for 5 cases with negative synovial fluid culture (Table In 98 of 107 patients (92%), synovial fluid culture and microcalorimetry showed concordant results, including 38 PJI and 60 aseptic cases .Pearson’s correlation coefficient for log leucocyte count and log peak heat production showed a weak linear correlation coefficient of Non-microbiological tests such as synovial fluid leukocyte count and periprosthetic tissue histopathology demonstrated high sensitivity for diagnosing PJI. However, these methods are unable to identify the causing pathogen and its antimicrobial susceptibility. Moreover, periprosthetic tissue histopathology is not feasible in pre-operative diagnostics as representative samples of periprosthetic tissue are difficult to obtain by arthroscopy , 23.To our knowledge, this is the first report on microcalorimetry of synovial fluid for the diagnosis of PJI. Microcalorimetry allows real-time analysis of a pathogen’s heat production curve. The heat production profile can be used for rapid identification of the corresponding pathogen . Hence, Microcalorimetry was positive for 5 cases with negative synovial fluid culture. Given the clear clinical manifestations for cases 1, 2 and 3 Table , synoviaMicrocalorimetry was positive in one aseptic failure case with negative synovial fluid culture. The agar plating of this microcalorimetric probe was negative and the case was considered a false-positive. The misclassification of this case may be associated with a misinterpreted heat spike. The source of the heat spike could be related to increased synovial fluid leukocyte count, as a high leukocyte count initially produce a heat peak as has been demonstrated here Fig. .There are several limitations of our study. First, the sensitivity of microcalorimetry was lower 39%) than in previously reported results –4, 6, 239% than iMicrocalorimetry of synovial fluid allowed thermogenic diagnosis of periprosthetic joint infection in synovial fluid, showing a similar sensitivity and specificity than culture but providing faster results . However, given its limited sensitivity, microcalorimetry presents restrictions similar to culture for the diagnosis of PJI. Hence, with further improvement of its performance, microcalorimetry could complement conventional cultures and support rapid, real-time decisions in orthopedic-device related infections.