Acute myeloid leukemia (AML) is a heterogeneous disease, and communication between leukemic cells and their neighboring leukemia-supporting normal cells is involved in leukemogenesis. The bone marrow cytokine network is therefore important, and the mediator release profile seems more important than single mediators. It is not known whether the characterization of primary AML cell proteomes reflects the heterogeneity of the broad and dynamic constitutive mediator release profile by these cells. To address this, we compared the intracellular levels of 41 proteins in 19 AML patients with the constitutive extracellular release during in vitro culture, including chemokines, growth factors, proteases, and protease regulators. The constitutive release of most mediators showed a wide variation (up to 2000-fold differences) between patients. Detectable intracellular levels were seen for 10 of 41 mediators, but for most of these 10 mediators we could not detect significant correlations between the constitutive release during in vitro culture and their intracellular levels. Intracellular protein levels in primary human AML cells do not reflect the dynamics, capacity, and variation between patients in constitutive mediator release profiles. Measurements of these profiles thus add complementary information to proteomic detection/quantification regarding the heterogeneity of the AML cell contributions to the bone marrow cytokine network. Acute myeloid leukemia (AML) is an aggressive malignancy characterized by the infiltration of immature leukemia blasts in the bone marrow . SeveralAML is a very heterogeneous disease , and pro9/L) and/or percentage (>80%) of circulating AML blasts, and a highly enriched blast population could thereby be prepared by density gradient separation alone [The study was approved by the local Ethics Committee , and samples were collected after written informed consent. AML blasts were derived from 19 unselected patients admitted to our hospital for treatment of acute myeloid leukemia . The present study included all patients from a defined geographical area and during a defined time period who were below 70 years of age, had circulating AML blasts in the peripheral blood, and were regarded to be fit for intensive  antileukemic treatment. The patient characteristics are summarized in on alone ,15,16. TPrimary AML cells from all 19 patients were thawed and immediately processed for proteomic analysis/quantification according to the sample preparation protocol previously described in detail ,11. Briem/z 375–1500) were acquired in profile mode with a resolution of R = 120,000 at 200 m/z, an automatic gain control (AGC) target value of 3 × 106 and maximum injection time (IT) of 100 ms. The top 12 precursors (above the intensity threshold of 5 × 104 counts) were sequentially isolated for MS/MS. The scans were acquired in centroid mode with a resolution of R = 30,000, an AGC target value of 1 × 105 and a maximum IT of 110 ms. Normalized collision energy was set to 28%, the isolation window was 1.6 m/z (offset 0.3 m/z) and the dynamic exclusion lasted for 25 s. Furthermore, for spray and ion-source parameters, the ion spray voltage was at 1800 V, no sheath and auxiliary gas flow, and the capillary temperature was at 260 °C.The QExactive HF was operated in data-dependent acquisition (DDA) mode, thus switching between survey full scan MS and MS/MS acquisition. The full scans (from The raw MS files were searched in MaxQuant (v.1.5.6.0)  using de6 cells per mL; Nunclon, Roskilde, Denmark). The levels of 41 mediators were analyzed either by Luminex assays or ELISA analyses, as described previously [Our in vitro model for investigation of constitutive mediator release has been previously described in detail ,13,20. Beviously ,13,21. TSpearman’s test was used to assess the correlation between the intracellular and extracellular levels of the mediators .AML is a bone marrow disease, and the constitutive release of soluble mediators by leukemic cells is important for the communication between these cells and their neighboring AML-supporting cells in the common bone marrow microenvironment ,22,23,24r-values >0.49 and p-values < 0.04). For 13 of these 19 mediators the r-values were >0.60. We investigated the constitutive release of 41 soluble mediators by in vitro cultured primary human AML cells derived from 19 patients; for 38 of them, detectable release was seen for a majority of patients, while ADAM12 had undetectable levels in all patients. The overall results are summarized in We identified 5790 protein groups in our proteomic data after removing reverse hits and proteins only identified by site. The number of quantified proteins per patient varied from 3823 to 5102. Of these, 5777 proteins had a quantitative value, and only 10 of the 41 mediators of interest were identified/quantified , indicatProteomic analysis of the AML cells allows identification and quantification of the proteins and their relative abundance in a particular compartment at a specific time. This analysis therefore represents a static snapshot of ongoing biological processes. By exploring the correlation of intracellular protein abundances with the concentration released into the media after 48 h in vitro culturing, we intended to investigate the dynamics of mediator release in the 19 AML patients . p = 0.0078, r = 0.65) whereas only borderline significance was reached for CCL5  and CFD . For the seven other mediators, the p-values did not obtain statistical significance, and r-values were generally <0.25  correlation of intracellular and extracellular levels, indicating a steady relationship between protein synthesis and mediator release; (2) high intracellular levels and low constitutive release of soluble mediators, indicating mainly intracellular protein functions; or (3) low intracellular levels and high constitutive release of soluble mediators, indicating rapid mediator release for extracellular functions. A correlation between the in vitro culture and ex vivo proteomic levels was only seen for CCL5, CFD, and cystatin C/CST3; a highly significant correlation was seen for cystatin C/CST3 (ly <0.25 . Interesly <0.25 . By contWe hypothesize that the lack of correlation between intracellular and released extracellular levels for most mediators indicates that the intracellular levels, at least for certain mediators, reflect additional intracellular functions rather than the capacity of extracellular release. This may also be the explanation for the similar lack of correlation between intracellular and extracellular levels of osteopontin . At the To decrease the risk of inducing biological alterations in the leukemic cells by more extensive cell separation procedures ,16, we oIt has been suggested by others that cryopreserved AML cells should be cultured in vitro for a short period before proteomic analysis to reduce the effects of the cryopreservation-induced stress on the leukemic cells . HoweverWe have previously shown that cryopreservation and thawing of primary human AML cells will alter the proteome and phosphoproteome ,39, inclOur previous studies suggest that the capacity of constitutive mediator release is associated with a favorable prognosis , and it Communication between malignant and nonmalignant neighboring cells through the cytokine network is important in AML. AML cells show constitutive release of a wide range of soluble mediators, and the overall release profile or combined effects of several mediators may then be more important than the effects of single mediators. Our study shows that for most mediators, the ability of constitutive extracellular release by primary human AML is best evaluated by in vitro culture, and not by analyzing intracellular levels as a static representation of the potential secretome. However, several soluble mediators demonstrate a wide variation between patients both intracellularly and when constitutively released, thus reflecting the large heterogeneity in AML. A combination of different methodological strategies may be essential to capture this heterogeneity and for a deeper understanding of leukemogenesis and AML cells’ communication with the microenvironment.


This study investigated the incidence of transient hypothyroxinaemia of prematurity (THOP) associated with survival without composite morbidities and the predictability of THOP severity in extremely low birth weight infants (ELBWIs). We retrospectively reviewed the medical records of 546 ELBWIs who underwent initial thyroid function tests within 14 postnatal days, with 156 ELBWIs from 2000 to 2005 (period I) and 390 from 2006 to 2013 (period II). The infants were stratified into 23–24, 25–26 and 27–28 weeks’ gestation subgroups within each period; the initial thyroxine (T4) level, mortality, clinical characteristics and composite morbidities, including bronchopulmonary dysplasia, intraventricular haemorrhage, necrotizing enterocolitis, and retinopathy of prematurity were analysed. The predictive value of the initial T4 level, Apgar score at 5 min, and clinical risk index for babies II (CRIB II) score for estimating mortality and survival with or without composite morbidities was assessed. Comparing period II and period I, the incidence of THOP was significantly decreased along with significantly increased survival without composite morbidities in ELBWIs at 25–28 weeks’ gestation. The severity of THOP showed significant associations with mortality and composite morbidities. The initial T4 level was most effective for predicting outcome compared with Apgar and CRIB II scores. The low T4 levels reach a nadir at 7–10 days postnatal and could remain low for the first 3–6 weeks of the postnatal period, depending on the extent of prematurity4. Although the aetiologies of THOP are multifactorial, including the loss of maternal placental transfer of T4 after birth, immaturity of the hypothalamus-pituitary-thyroid axis, limited thyroid capacity to increase synthesis, and metabolism and drugs affecting thyroid function5, recent studies have demonstrated that THOP is associated with increases in perinatal mortality and morbidity6; prolonged mechanical ventilation7; and non-thyroidal illnesses such as respiratory distress syndrome (RDS)8, intraventricular haemorrhage (IVH)10, and cerebral white matter damage11. Furthermore, in concordance with the adult data on non-thyroidal illness, the illness severity in premature infants seemed to be an important determinant of low serum T4 levels13. Collectively, these findings suggest that THOP might just be the epiphenomenon reflecting the severity of the non-thyroidal illness in ELBWIs.Transient hypothyroxinaemia of prematurity (THOP), characterised by low thyroxine (T4) levels with normal or low thyroid stimulating hormone levels, occurs in more than half of the extremely low birth weight infants (ELBWIs) born at <30 weeks16. However, the temporal alterations in the incidence of THOP associated with the recently improved survival rates without composite morbidities of ELBWIs have not been studied yet. Furthermore, although prediction of mortality and morbidities in ELBWIs prior to discharge is of the utmost clinical importance, the predictability of THOP severity status for mortality, morbidities and survival without composite morbidities has not been tested yet. Therefore, in the present study, we investigated whether survival without composite morbidities was associated with the altered incidence of THOP, and if applicable, the potential of THOP severity to predict outcome for ELBWIs. Specifically, we compared the predictive power of the initial T4 levels for estimating mortality, composite morbidities, and survival without major morbidities in ELBWIs with that of the Apgar score at 5 min and clinical risk index for babies II (CRIB II) score. The Apgar and CRIB II scores were found to be good predictors of mortality in our previous study17.Recently, we reported improved survival rate and increased survival rates without composite morbidities such as bronchopulmonary dysplasia (BPD), severe IVH, necrotizing enterocolitis (NEC), and retinopathy of prematurity (ROP) with better perinatal and neonatal intensive care for the ELBWIsTable Table Table The clinical outcomes of the infants were dependent on the initial T4 levels, with the highest mortality and composite morbidities rates and lowest survival without morbidities rates observed when initial T4 levels of <2.5 ng/dl and/or f T4 < 0.5 ng/dl, and the lowest mortality and composite morbidities rates and highest survival without morbidities rates observed when the initial T4 levels were ≥4.5 ng/dl and/or fT4 ≥ 0.9 ng/dl  infants with better perinatal and neonatal intensive care16. In this study, while the total GA and birth weight were significantly lower, the Apgar scores at 1 and 5 min were significantly higher, and the incidence of normal TFTs in ELBWIs at 25–26 and 27–28 weeks’ gestation significantly increased from 22% and 40% during period I to 37% and 61% during period II respectively. This increase was in strict concordance with the significantly increased survival without composite morbidities from 21% and 38% during period I to 38% and 58% during period II, respectively. To the best of our knowledge, this is the first study that studied temporal changes in the incidence of THOP. The change may have been linked to the continuing improvement in perinatal and neonatal intensive care resulting in a better outcome for EPT infants. Collectively, these findings suggest that THOP might be an epiphenomenon of non-thyroidal illness in ELBWIs, and its incidence could thus be reduced by attenuating non-thyroidal illness with better clinical management in ELBWIs.THOP has been known to be associated with increases in perinatal mortality and morbidities22. Overall, these findings suggest that the initial T4 levels reflect the severity of the non-thyroidal illness and thus could predict the outcome in ELBWIs.In the present study, the mortality, the prevalence of morbidities, composite morbidities and survival without morbidities was dependent on the initial T4 level, with the highest mortality and lowest survival without morbidities observed with the lowest T4 levels of less than 2.5 ng/dl. In concordance with our data, several studies have noted that the severity of the non-thyroidal illness is an important determinant of low serum T4 levels in premature infants23. Therefore, despite its transient nature and the epiphenomenon of severe illness, hypothyroxinaemia in ELBWIs could be a risk factor by itself for both acute mortality and morbidities10 and later neurodevelopmental deficits in ELBWIs26. However, a recent study reported that GA, but not THOP, played a key role in neuromotor dysfunction in preterm babies27. Moreover, thyroid hormone treatment did not attenuate the severity of RDS29, reduce mortality up to the point of discharge29, or improve long-term neurodevelopmental outcome29. Overall, these findings suggest that other factors associated with acute non-thyroidal illness in premature infants, rather than hypothyroxinaemia itself, might play a pivotal role in negatively impacting brain development and subsequent neurodevelopmental outcome. Further studies strictly limiting the study cohort to ELBWIs with THOP would be necessary to determine the potential beneficial effects of T4 supplementation10.Thyroid hormone has been known to play a critical role in perinatal neural development including brain myelination and production and maturation of oligodendrocytes30. In our previous study, the Apgar score at 5 min and the lowest serum albumin level were the best predictors of mortality during ≤7th and >7th postnatal days, respectively in ELBWIs17. In this study using the same data set, the initial T4 level showed the best predictability of mortality, composite morbidities, and survival without major morbidities compared with Apgar score at 5 min and the CRIB II score which was shown to be a good predictor for ELBWI mortality in our previous study17 As the initial T4 level reflects the severity of non-thyroidal illness, and is associated with the ensuing mortality and morbidities in ELBWIs in the present and other studies22, these findings suggest that an increased initial T4 level might best reflect recent improvements in clinical management and the resultant better outcome for ELBWIs, and thus could be most effective for predicting their prognosis compared with the currently used CRIB II and Apgar scores. Accordingly, further studies will be necessary to develop new and effective models for better prediction of prognosis in ELBWIs.Predicting the outcome of ELBWIs at the highest risk of mortality and morbidities would be very helpful for the difficult decision making regarding treatmentThe present study had several limitations including its retrospective and uncontrolled observational study design. We determined mortality and composite morbidities only at hospital discharge. Therefore, it was not apparent whether there was any causal relationship between the initial T4 levels and mortality and/or morbidities in these infants. Another limitation was that as our data were obtained from a single institution, the results obtained in this study might not be generalizable to other institutions. Nonetheless, we used a relatively large sample size of 546 ELBWIs born and admitted to a single centre with similar baseline clinical characteristics. Furthermore, although the consistency of clinical management was not measured, our data showing more antenatal steroid use and significantly higher Apgar scores at 1 and 5 min despite significantly lower GA and birth weight suggest that better perinatal and neonatal intensive care was provided during period II than during period I. Therefore, these findings imply that this single-centre study appropriately determined that the temporal alterations in the incidence and severity of THOP were associated with better clinical treatment and the ensuing improved outcomes in ELBWIs. These factors might also compensate for the limitations of the study.In summary, the incidence of THOP was significantly reduced in ELBWIs at 25–28 weeks’ gestation during period II compared to period I, in parallel with their significantly higher Apgar scores, decreased morbidities and increased survival without composite morbidities. In ELBWIs, the initial T4 level was the best predictor of mortality, composite morbidities, and survival without composite morbidities in comparison to CRIB II and Apgar scores.The data collection procedure was approved by the Institutional Review Board of Samsung Medical Center (2018-09-024), and the Institutional Review Board waived the requirement for informed consent in this retrospective chart review.17, and stratified them into the 23–24, 25–26 and 27–28 weeks’ gestation subgroups. We compared maternal and neonatal variables, survival and morbidity rates and incidence of thyroid dysfunction between the infants belonging to the 2 time periods.Medical records of 546 ELBWIs  with gestational ages (GA) between 23 and 28 weeks who were born at and admitted to the Samsung Medical Center neonatal intensive care unit and had undergone initial thyroid function tests (TFTs) within the first 2 postnatal weeks, between January 2000 and July 2013 were retrospectively reviewed. Among them, 156 were born between January 1, 2000 and December 31, 2005 (period I), while 390 were born between January 1, 2006 and July 31, 2013 (period II). We arbitrarily divided the study period according to the survival rate of these ELBWIs as estimated in our previous studiesth percentile), pregnancy-induced hypertension, gestational diabetes mellitus and antenatal steroid use, were analysed. GA was determined based on the maternal last menstrual period and the modified Ballard test. We analysed outcome measures, including death before discharge, BPD (≥moderate)31, IVH (≥grade 3)32, NEC (≥Bell’s stage 3b)33 and ROP requiring laser treatment.Clinical characteristics, including GA, birth weight, Apgar scores at 1 and 5 min, sex, delivery mode, small for GA (SGA)  level of <0.9 ng/dl with a TSH level of <20.0 µIU/ml and considered severe THOP to be having an initial T4 level of <2.5 ng/dl and/or fT4 level of <0.5 ng/dl. TFTs were followed up every 2–6 weeks until hospital discharge.We calculated the CRIB II score for each infant using the following variables: sex, GA, birth weight, and base excess.t-test or Mann-Whitney U test. Categorical variables were presented as percentages and frequencies and compared using the chi-square or Fisher’s exact test. We carried out binary logistic regression analyses with adjustment of gestational age, birth weight, sex and antenatal steroid use to obtain odds ratios of clinical outcomes according to the initial thyroxine level. The analyses of specificity and sensitivity of the Apgar score at 5 min, CRIB II score and initial T4 level were performed through receiver operating characteristic (ROC) curves, and the area under the curve (AUC) was conducted in order to define the differences with statistical significance in predicting mortality rate with selection of the most suitable cut-off point. SPSS version 19.09  was used for all statistical analyses, and a P < 0.05 was considered statistically significant.Continuous variables were presented as means ± standard deviation and compared using the Student’s


Podisus nigrispinus (Hemiptera) occurs via a paracellular or transcellular route. Light and transmission electron microscopies showed that short cell–cell contacts with well-developed occluding septate junctions were present in follicular cells with patency. Immunofluorescence microscopy revealed the presence of vitellogenin receptors in the plasma membrane and of vitellogenin in the cytoplasm of follicular cells. Data suggest that cell–cell contacts serve as a barrier to large vitellogenin molecules and that this protein is transported via a transcellular route of receptor-mediated endocytosis.Vitellogenin is the main yolk precursor protein in insect oocytes. It is synthesized in the fat body and released into the hemolymph. To reach the oocyte surface, vitellogenin must cross a single layer of follicular epithelium cells. The transport of vitellogenin across the follicular epithelium has been suggested to occur through the enlarged intercellular spaces (patency) by a paracellular route or by endocytosis by follicular cells and release onto oocyte surface in a transcelluar route. In this study, we investigated whether vitellogenin transport in the meroistic telotrophic ovary of  Each ovary has many functional units, the ovarioles, in which oocytes grow surrounded by a follicular epithelium4 and each ovariole is divided into terminal filament, tropharium, vitellarium, and pedicel5. The distal portion of the ovariole is composed of the terminal filament, formed by connective tissue6; a tropharium with oogonia (which differentiate into oocytes) and nurse cells, which produce cytoplasm compounds that are transferred to oocytes during development7.In Heteroptera (Hemiptera), which includes bugs, ovaries are of the meroistic telotrophic type8. Vitellogenesis is crucial for insect reproduction and comprises the synthesis of vitellogenin and yolk accumulation in the ooplasm9. Synthesis of the glycolipophosphoprotein vitellogenin occurs in the fat body under juvenile hormone control10. Vitellogenin is released into the hemolymph, taken up by growing oocytes, and stored as vitellin, the major yolk protein12.Oocyte maturation and vitellogenesis occur in the vitellarium15. LDLRs shares some structural features such as ligand-binding domain (LBD), epidermal growth factor precursor (EGFP) homology domain, O-linked sugar domain, a single transmembrane domain and a cytoplasmic domain16. Insects have two LBDs with different numbers of class A cysteine-rich ligand binding repeats (LBRs)20. These VgRs have been reported to combine with some different ligands in vertebrates21, and their mRNA are also detected in the hypopharingeal glands22 and fat body23 in insects, suggesting that VgR may recognize other ligands, but in insect ovary vitellogenin is the unique ligand of VgR endocytosed by oocytes24. However, to reach the oocyte surface, vitellogenin must be transported through the follicular epithelium25, which surrounds the oocyte6 and regulates the flow of vitellogenin to the oocyte7.Vitellogenin uptake into oocytes occurs via endocytosis, mediated by membrane vitellogenin receptors (VgRs) belonging to the low-density lipoprotein receptor (LDLR) superfamilyHyalophora cecropia (Lepidoptera)28 and Bemisia tabaci (Hemiptera)29, dynamics of the vital dyes trypan blue in Aedes aegypti (Diptera)30 and Evan’s blue in Rhodnius prolixus (Hemiptera)31 suggest the occurrence of paracellular transport of proteins trough the ovarian follicular epithelium. Conversely, in social Hymenoptera with meroistic polytrophic ovaries, ultrastructural studies indicate patency in the follicular epithelium25, but immunocytochemistry of vitellogenin and VgR show that both are localized in the plasma membrane and cytoplasm of follicular cells, suggesting a transcellular route in which vitellogenin initially diffuses through intercellular spaces to the follicular cells surface, bind to VgR, following clathrin-mediated endocytosis and transport to another plasma membrane domain (transcytosis) closely associated with oocyte surface32. This recent finding stimulated us to verify whether in other insects with meroistic telotrophic ovaries, patency is already an evidence of paracellular route for the vitellogenin transport to the oocyte surface for further endocytosis.A model proposed over fifty years ago to explain the vitellogenin transport through the follicular epithelium remains almost intact. Extracellular canals formed by enlargement of the intercellular spaces between the follicular cells (patency) facilitate diffusion of soluble proteins from the hemolymph until the oocyte surface via a paracellular route. Transmission electron microscopy and immunohistochemical studies in Podisus nigrispinus (Hemiptera: Pentatomidae) is an important generalist predator used in biological control of agricultural insect pests33. The development34, histology, cytology37, predator–prey interaction38, and biochemical processes39 of this predator have been studied. However, to the best of our knowledge, data on the morphology and reproductive physiology of P. nigrispinus females are scarce40 and the route for vitellogenin transport from the hemolymph to the oocyte surface has yet to be determined.P. nigrispinus and thus contribute to the comprehension of this insect’s reproduction cycle.The objective of this study was to investigate whether vitellogenin follows a paracellular or transcellular route through the follicular epithelium in Podisus nigrispinus oocytes in the vitellarium were enveloped by a single layer of binucleate follicular cells  increased in size, eggshell (chorion) synthesis occurred, and intercellular spaces of follicular cells narrowed Fig. .2. Therefore, we used transmission electron microscopy to evaluate intercellular contacts between follicular cells in previtellogenic and vitellogenic follicles. In the former, follicular cells showed narrow intercellular spaces along almost all their extension  at the onset of vitellogenesis, when cells of the follicular epithelium at the anterior and posterior poles are morphologically distinct from cells in the lateral portion41. The intercellular spaces between follicular cells of the epithelium lateral to the oocyte enlarge progressively with oocyte growth, whereas polar follicular cells continue to possess narrow intercellular spaces and might act as a barrier for the passage of yolk proteins43, but the physiological significance of this difference remains unknown.Our findings show that the follicular epithelium in vitellogenic follicles of P. nigrispinus, has been reported in H. cecropia27, R. prolixus41 and B. tabaci29. Those studies show accumulation of hemolymph proteins in the intercellular spaces of follicular cells, which has been interpreted as an evidence that these proteins, including vitellogenin, are transported to the oocyte surface via a paracellular route. However, in bees, ants, and wasps, although patency occurs, vitellogenin is transported to the oocyte surface by receptor-mediated endocytosis, that is, via a transcellular route32.Enlargement of intercellular spaces in the follicular epithelium (patency) during vitellogenesis, as observed in the present study in P. nigrispinus vitellogenic follicles but also revealed that follicular cells display septate junctions in small cell–cell contact regions. Septate junctions play an important role in maintaining the mechanical and physiological integrity of the epithelium44. In addition, septate junctions are important permeability barriers that control the transit of molecules through invertebrate epithelia48. Some chemical species such as lanthanum ions (La3+), which have high charge density and occur in a hydrated state, can cross septate junctions50; however, the passage of vitellogenin, which is a large protein with a molecular mass of 200–700 kDa51, seems unlikely, as septate junctions in different epithelia of insects are effective barriers to some ions and molecules larger than 7 kDa54.Light and transmission electron microscopies showed that patency occurs in the follicular epithelium of 32. However, the question remains as to why patency occurs in follicular cells of insects during vitellogenesis. A possible explanation would be that the almost complete separation of adjacent follicular cells allows the exposure of a larger area of the plasma membrane to the hemolymph and thus exposes many VgR molecules for vitellogenin uptake, resulting in a high transport rate of reserve proteins to the oocyte.Together, ultrastructural and immunofluorescence data show that intercellular spaces are blocked by septate junctions and that the plasma membrane of follicular cells contains VgR and vitellogenin, suggesting a transcellular route of vitellogenin transport to the perioocytic space, as has been demonstrated in HymenopteraThe present study provides insight into the transcellular route of vitellogenin transport through the follicular epithelium in meroistic telotrophic ovaries, which seems to be a conserved route in insects, as it also occurs in insects with meroistic polytrophic ovaries. Further investigation is needed to elucidate the routes of vitellogenin transport in insects with panoistic ovaries.P. nigrispinus females were obtained from the Biological Control Laboratory of the Federal University of Viçosa (UFV), Viçosa, Minas Gerais, Brazil.Mated Insects were cryoanesthetized at −4 °C for 90 s, and their ovaries were dissected in 125 mM NaCl. Five pairs of ovaries were transferred to 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.2. Then, samples were washed in the same buffer, dehydrated in a graded ethanol series , and embedded in historesin (Leica). Sections of 2 μm, obtained using a rotary microtome with a glass knife, were stained with hematoxylin and eosin and analyzed under a light microscope.Five females were cryoanesthetized at −4 °C for 90 s and dissected. The ovaries were transferred to 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, for 12 h. After being washed in the same buffer, samples were post-fixed in 1% osmium tetroxide for 2 h, dehydrated in a graded ethanol series , and embedded in LR White resin. Ultrathin sections, obtained using an ultramicrotome equipped with a glass knife, were stained with 2% aqueous uranyl acetate and lead citrate and analyzed using a transmission electron microscope (Zeiss LIBRA 120).Podisus nigrispinus females were dissected, and the ovaries were homogenized in 50 mM Tris-HCl buffer, pH 7.5, containing 10% protease inhibitor cocktail  using a cordless motor. Procedures were performed in triplicate. Then, samples were centrifuged at 10,000 × g for 15 min, and the supernatant containing soluble proteins was collected. The pellet was resuspended in Tris-HCl buffer, pH 7.5, 1% Triton X-100, and 10% protease inhibitor, homogenized, placed in an ultrasonic bath for extraction of membrane proteins, and centrifuged at 10,000 × g for 15 min. Supernatants containing soluble membrane proteins were submitted to SDS-PAGE55. The gel was incubated in transfer buffer  and transferred to a nitrocellulose membrane by electroblotting at 190 A and 4 °C for 3 h . Subsequently, the nitrocellulose membrane was incubated in Tris-buffered saline (TBS)  with 1% Tween 20 (TBST) and 5% skimmed milk powder for 16 h. The nitrocellulose membrane was rapidly washed with TBST, washed twice with TBST for 15 min, and incubated for 2 h with primary antibodies against vitellogenin receptor (anti-VgR)32 (1:500). After washing with TBST, the nitrocellulose membrane was incubated with peroxidase-conjugated anti-mouse IgG secondary antibody (1:100) for 2 h. Then, samples were revealed with a diaminobenzidine solution .32 (1:100 dilution in PBST) for 16 h. Another set of ovarioles were incubated in anti-vitellogenin primary antibody10 (1:100 dilution in PBST) for 16 h. Then, samples were incubated in a 1:400 dilution of FITC-conjugated anti-mouse IgG in PBST for 2 h in the dark. Samples were transferred to 50 μL of TO-PRO-3 iodide (Thermo Fisher Scientific) in PBS (1:1000), incubated for 30 min in the dark, mounted on slides with 50% sucrose., and analyzed on a Zeiss 510 Meta confocal fluorescence microscope.After dissection, the ovarioles had the peritoneal sheath removed with tweezers and were immersed in Zamboni fixative  for 2 h. Samples were transferred to PBS with 0.05% Triton X-100 (PBST) for 2 h and incubated in anti-VgR primary antibodyNegative controls were subjected to the same procedures, except that the primary antibodies were replaced with normal mouse (for VgR) or rabbit (for vitellogenin) serum.This article does not contain any studies with animals and humans participants performed by any of the authors.


This study was designed to investigate its therapeutic effects on acute lung injury (ALI) and explore the underlying mechanisms of action. Rats from treatment groups were subject to oral administration of MAN for 3 consecutive days beforehand, and then ALI was induced in all the rats except for normal controls via an intraperitoneal injection with lipopolysaccharide. The severity of disease was evaluated by histological examination and hematological analysis. Protein expressions in tissues and cells were examined with immunohistochemical and immunoblotting methods, respectively. The levels of cytokines and nicotinamide adenine dinucleotide (NAD) were determined using ELISA and colorimetric kits, respectively. It was found that MAN treatment significantly improved histological conditions, reduced leucocytes counts, relieved oxidative stress, and declined TNF-α levels in ALI rats. Meanwhile, MAN treatment decreased expressions of nicotinamide phosphoribosyltransferase (NAMPT) and Sirt1 both in vivo and in vitro, which was accompanied with a synchronized decline of NAD and TNF-α. Immunoblotting assay further showed that MAN downregulated HMGB1, TLR4, and p-p65 in RAW 264.7 cells. MAN induced declines of both HMGB1/TLR4/p-p65 and TNF-α were substantially reversed by cotreatment with nicotinamide mononucleotide or NAD. These results suggest that downregulation of NAMPT/NAD by MAN treatments contributes to the alleviation of TLR4/NF-κB-mediated inflammations in macrophage, which is essential for amelioration of ALI in rats. Xanthone is a naturally occurring polyphenol with the hybrid structure of flavone and anthraquinone. Similarly, xanthones also possess various notable bioactivities and have drawn intense attentions worldwide . Increas in vivo and in vitro, the underlying mechanisms are still not well understood. It is believed that, as a typical polyphenol, the beneficial effects of xanthone on inflammations result from its antioxidative activities, and downregulation of certain oxidative stress sensitive pathways such as COX-2, NF-κB, and MAPKs is responsible for reduced inflammatory reactions [κB in rodent arthritis models, which subsequently alleviated the arthritis severity [Although numerous studies have solidly confirmed the anti-inflammatory properties of xanthones botheactions –8. Indeeseverity –11. Howeα-mangostin  on experimental arthritis and found that MAN compromised the biosynthesis of nicotinamide adenine dinucleotide (NAD) in fibroblast-like synoviocyte (FLS) via downregulation of nicotinamide phosphoribosyltransferase  and subsequently protected the joints from destruction (unpublished). We believe that the decrease in energy metabolism during this process is critical for the clinical outcome. However, the effects of MAN in FLS could have little to do with the alleviated systemic symptoms, because most of the circulating extracellular NAMPT (eNAMPT) and NAD are contributed by leucocytes [κB mediated inflammations.Recently, we carried out a metabolomics study to elucidate therapeutic mechanisms of ucocytes , 13. Fro E. coli 055:B5), phosphate buffered saline (PBS), BCA protein quantitative kit, NAD, nicotinamide mononucleotide (NMN), HRP conjugated streptavidin, and HRP/biotin conjugated secondary antibodies were purchased from KeyGen Biotech . Fetal bovine serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM) were from TianHang Biotechnology . Enhanced chemiluminescence (ECL) detection kit, defatted milk powder, and bovine serum albumin (BSA) were from Thermo Scientific . Primary antibodies used in both western blot and immunohistochemical assays were obtained from Affinity Biosciences . Ultrapure water was prepared by using a Milli-Q purification system .MAN with the purity of 98%  was brought from SanHerb Bioscience Inc. . Lipopolysaccharide  according to the manufacturer's instructions. The lung was promptly dissected, washed by cold PBS, and fixed in buffered formalin for further examinations.Male SD rats  were used in this study. All the animal protocols described below were approved by the Ethical Committee of Yijishan Hospital, Wannan Medical College (No. YJS 2018-5-011), and were strictly in accordance with the Guide for the Care and Use of Laboratory Animals . Before experimental procedures, all the animals were kept for 7 days to get accommodated. Afterwards, the rats were assigned into 4 groups randomly. Two MAN treated groups received MAN treatments by gavage (suspended in CMC-Na solution) for 3 consecutive days and 15 and 45 mg/kg/day served as the low and high doses, respectively. The other two groups were used as normal healthy and ALI model controls and treated with 0.5% CMC-Na instead. One hour after the last administration, lung injury was induced in all rats except for the normal controls via an intraperitoneal injection of LPS at the dose of 10 mg/kg. The animals were then intensely observed and sacrificed under anaesthesia with chloral hydrate 12 h later. The whole blood was collected through abdominal aorta into anticoagulation/promoting coagulation tubes. Anticoagulated blood was used for complete blood count (CBC) analysis on an automated hematology system , and the coagulated blood was used to separate serum for the serological analyses. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were measured using colorimetric kits , and the concentrations of TNF-μm thickness. After the staining of hematoxylin and eosin, the lung injury was evaluated based on the following histological changes: (a) thickness of the alveolar walls, (b) infiltration or aggregation of inflammatory cells, and (c) alveolar hemorrhage [The fixed lung was embedded in paraffin and then sectioned at 4 morrhage . Each pa in vivo. The cells were grown in DMEM supplemented with 10% FBS and penicillin-streptomycin (100 U/ml) at 37°C under the humid atmosphere containing 5% CO2. Cells were passed every 2 days.Because macrophage is deeply implicated in sepsis and relevant complications, we used RAW 264.7 cells  in the following experiments to mimic effects2 culture flask and treated with MAN with a series of concentrations or in combination with NAD/NMN after an overnight incubation for attachment. After 24 h treatment, the cells were collected and counted. NAD was extracted from cells using the reagent provided in the NAD determination kit with the aid of heating in a boiling water bath , and supernatant of the extraction was obtained after a high speed centrifugation  at 4°C. The concentrations of NAD in the processed samples were determined according to the manufacturer's instructions and then normalized to the number of cells to calculate the relative NAD levels in cells.To evaluate the effects of MAN on production of NAD, cells were seeded in a 75 cm5 cells/well. According to the experimental arrangement, some cells were pretreated with LPS (1 μg/ml) for 1 h after attachment. Afterwards, the cells were treated with MAN or in combination of NAD/NMN for 24 h based on a predetermined schedule. By the end of treatments, supernatants of the culture medium were collected for TNF-α and eNAMPT quantitative analyses using commercial available ELISA kits , and the harvested cells were lysed on ice using RIPA buffer supplemented with 1% PMSF. Supernatants from the lysates were obtained after centrifugation at 12,000 RPM under 4°C. Samples containing the equal amount of proteins  were subject to SDS-PAGE, and the separated proteins were then transferred onto nitrocellulose filter membranes, which were then blocked with 5% defatted milk for 2 h at room temperature and incubated with primary antibodies at 4°C overnight. The target proteins on the membranes were probed by HRP conjugated secondary antibodies (2 h at room temperature), and specific signals of protein complexes were finally developed and detected using an ECL detection kit on a Tanon 5200 system  [Cells at exponential growth stage were seeded into 6-well plates at the density of 1×10, China) .The 3D conformation construction and energy minimization of MAN were achieved using the ChemBio3D Ultra 14.0 software . Crystal structures of NAMPT in complex with small molecular ligands were chosen and downloaded from RCSB Protein Data Bank. The docking simulation procedures were mainly performed on the 5LX3 conformation, while 3DKJ and some other cocrystallized structures were used to test results. To prepare the protein for simulation procedures, atoms of water and ligands were removed from the molecule, and polar hydrogens were added by the aid of AutoDockTools 1.5, the graphical interface of AutoDock software. MAN and NAMPT structural data were then converted into the PDBQT format and fed to AutoDock 4.2 for docking simulation. A grid box with the size of 40×40×40 was generated to completely encompass the binding site cleft with the coordinate of PHE 193 as the center reference. Lamarckian genetic algorithm (GA) was adopted. Interactions between MAN and the protein were analyzed and visualized using PyMol graphic system 2.1  . in vivo. Specifically, neutrophil and monocyte were significantly increased. Individual rat's responses varied substantially to MAN regimen, especially in the low dose group; however, the tendency of dose-dependent decrease of these indicators was quite obvious . Given the newly developed conception about feedback between energy metabolism and inflammation [π-π interaction with the aromatic ring of MAN, and the planar structure of MAN was especially favored for the sandwich-like insertion [Previously, we found that MAN treatment efficiently reduced production of NADammation , manipulammation . Similarnsertion . Additionsertion , the occα in culture medium  to restore the decreased intracellular levels of NAD under MAN treatments. Cotreatment with both NAD and NMN significantly reversed the MAN induced inhibition of NF-κB activation and TNF-α release (κB pathway and alleviated inflammation.To further validate the connection between NAD production and TLR4/NF- release , which s release . All the in vivo. The current study firmly confirmed this hypothesis and further revealed its relevance to the anti-inflammatory properties.Due to its high abundance in nature and versatile bioactivities, MAN is one of the most investigated naturally occurring xanthones. Its potentials in therapies of immune and inflammatory disorders are especially meaningful, because MAN rich mangosteen pericarp has been used as a traditional medicine to cure infections and inflammations for centuries in Southern Asia . In prev in vivo, because the lipid peroxidation is usually connected to accumulation of fatty acids in blood [Most researches on anti-inflammatory activities of xanthones mainly emphasized their antioxidative capability –7. We alin blood . in vivo and in vitro. Given its well recognized anti-inflammatory role [Available evidences show that Sirtuins regulate key aspects of lipid metabolism, and Sirt1 promotes lipid mobilization and oxidation of fatty acids . We founory role , the redory role . From thAccumulating evidences support that, apart from its decisive role in NAD biosynthesis, extracellular NAMPT also acts as an adipokine cooperating in glucose and lipid metabolism. By exerting an insulin-mimetic effect, NAMPT regulates carbohydrate metabolism and promotes fat deposition. Hence, overexpression of NAMPT is usually linked to development of obesity . Taken tκB activation in macrophage. Despite we still do not know why the decrease in NAD leads to reduced expression of TLR4, there are some available clues to clarify this phenomenon from other perspectives. Camp et al. have shown that eNAMPT is a unique endogenous agonist of TLR4. It can potently activate the TLR4/NF-κB pathway alone without the need of partner molecules [κB activation under MAN treatments. Considering their roles in lipid mobilization and fat deposition, decreases in Sirt1 and eNAMPT under MAN treatments could cut down free saturated fatty acids in blood, which will further curb the activation of TLR4/NF-κB, because they can bind to TLR4 and directly elicit inflammation reactions [Increased leucocyte counts and neutrophil/macrophage recruitment in lung are important hallmarks of sepsis induced ALI , which wolecules . Logicaleactions . Due to eactions , which weactions . These mκB mediated inflammation reactions by manipulation of NAMPT/NAD, and the regulation of fat metabolism could be an effective therapeutic strategy in therapies of inflammation related disorders.As a well known naturally occurring bioactive compound, MAN possesses a notable clinical potential in treatments of many diseases. This study provides further evidences to support its anti-inflammatory properties and partially elucidates the underlying mechanisms from a unique perspective. The results of this study suggested that MAN suppressed TLR4/NF-


Mycobacterium avium subsp. paratuberculosis (MAP), affecting virtually all ruminants as well as other animals. MAP is also suspected to be involved in the etiology of some human diseases, like Crohn’s disease and others. In surveillance studies, different analytical methodologies were employed to detect MAP, showing different results and incidence in dairy products. The aim of this study was to evaluate the performance characteristics of three analytical methods  for MAP detection in raw, heat-treated and powdered milk. The methods were evaluated according to performance characteristics defined for qualitative methods in ISO 16140-2:2016. To estimate sensitivity (including trueness) and LOD, 720, and 900 test portions, respectively, were blind tested by two laboratories. Considering all matrices, different sensitivities, expressed as the percentage of positives from the total of true positive test portions, were obtained for IS900 qPCR (94%), f57 qPCR (76%), culture (83%), and PMS-phage (40%). Trueness, expressed as results correctly assigned (including positive and negative) to the reference value, was 93% for the IS900 qPCR method, 89% for culture and 49% for the PMS-phage. The LODs obtained in this study were similar to the LODs previously published for cultural and qPCR methods. However, for the PMS-phage method, the obtained results showed higher LOD values compared to the limited data available in the scientific literature. Our results highlight that while the PMS-phage assay is workable in pure liquid culture for estimation of MAP counts, its usage for surveillance of dairy matrices should be treated with a lot of caution as performance characteristics obtained were lower than for the two other methods tested. qPCR and culture are the most appropriate methods to detect MAP in milk-based matrices according to ISO 16140 methodology. Cultural techniques are considered the gold standard for detection of viable MAP, but qPCR, which is widely used in analytical and surveillance studies, can be considered a suitable and recommendable alternative to cultural methods for screening, if confirmation of MAP’s viability is not requested.Paratuberculosis is a chronic enteric infection, caused by  Mycobacterium avium subsp. paratuberculosis (MAP), a member of the M. avium complex group. MAP is also suspected to be involved in the etiology of some human diseases, like Crohn’s disease and others. More precisely, a zoonotic role of MAP has not been definitively proven, but, on the other hand, it has never been ruled out  aimed at detecting MAP DNA in various food and clinical samples have been regularly used . Among tMore recently phage-based assays, coupled or not with peptide-mediated magnetic separation (PMS) for MAP cell capture, have been developed for the detection of MAP in milk . This meThe aim of this study was to evaluate the performance characteristics of three methods for MAP detection in raw milk, heat-treated milk and milk powder. The methods assessed were: culture , IS900 qFour reference strains of MAP , were used to spike the test portions. Each freeze-dried MAP strain was grown as described by TM kit  according to manufacturers’ instructions. Milk test portions were spiked with declumped MAP cells by performing different 10-fold dilutions according to the estimated level of inoculum required.Prior to enumeration and inoculation, MAP cultures were declumped by filtration according to Three different milk matrices were used in the study: heat-treated milk (four sources), milk powder (five sources), and raw milk (four sources). For heat-treated milk, commercial whole pasteurized, UHT whole, semi-skimmed and skimmed milk samples were purchased from Swiss, French, and Spanish supermarkets. For skimmed milk powder, three items typically used as constituent of culture media for microbiological methods  and two commercial items obtained from Swiss supermarkets were used. Skimmed milk powder samples were reconstituted in sterile water at a ratio 1:10 (w:v). Raw bovine milk was collected from four Friesian farms from the Basque Country (Northern Spain) participating in an experimental paratuberculosis control program since 2006 . The proTest portions (50 ml each) were distributed in centrifuge tubes, spiked (when required), randomly coded, frozen at −20°C and delivered to the participating laboratories. All test portions were confirmed frozen at arrival and stored frozen until analysis. All laboratories analyzed the test portions in blind within 4 weeks of receipt using cultural, qPCR (IS900 and f57) and PMS-phage methods.The concentration of MAP cells in raw milk, heat-treated milk and reconstituted milk powder test portions after spiking was estimated by using the Most Probable Number (MPN) enumeration technique described in ISO 7218:2007 . For eacHomogeneity and stability studies to assess MAP detectability among replicates and during storage time were performed for every batch of spiked test portions at levels in which positive results were expected. For homogeneity, at least 10 test portions from each batch were analyzed at time 0 by cultural and IS900 qPCR methods as described in the analytical methods section. Stability was performed on test portions during a storage period of 4 weeks at −20°C. Every week, at least three test portions were defrosted at 4°C and analyzed by the cultural method.Three non-spiked test portions of each batch were analyzed by cultural and qPCR methods to check for the absence of MAP.0), 20% inoculated at low level, between 2 and 4 Log10/50 ml (L1) and the remaining 40% inoculated at high level (L2), between 3 and 5 Log10/50 ml  of each milk type  and comprising four milk items per milk type described previously. For each food item, 40% of the test portions were not inoculated  according to the following table and equations to determine the performance characteristics :Sensitivity (SE), which was estimated as:Trueness (T), which was estimated as:Acceptability Limits (AL), which was estimated as the AL = ND-PDAcceptability limits are the maximum acceptable difference between the reference value of a test portion and an individual result obtained when applying the analytical method . The AL For an unpaired study (results from each method are obtained from different test portions from the same sample), maximum limits are AL ≤3 for a single food type and AL ≤5 when three food types are tested  .0), 20 test portions were spiked with a different MAP strain at low level (L1); 20 test portions at intermediate level (L2); and five test portions at high level (L3). Fractional results were expected at least for the L2 or L3  of each milk type , with one item per milk type in this LOD study. For each food item, laboratory and method, five test portions were not spiked (LL2 or L3 .50) or 95% (LOD95) allows the estimation of MAP concentration, obtained with each method, for which the probability of detection is 50 or 95%, respectively.Limit of detection 50%   and decontaminated at room temperature for 5 h. Heat-treated milk and reconstituted milk powder pellets were not decontaminated because significant background microflora was not expected. After decontamination, raw milk test portions were centrifuged again under the same conditions and the supernatants were discarded. The pellets obtained from heat-treated milk, milk powder, and decontaminated raw milk test portions were resuspended in 1 ml of PBS supplemented with 0.05% Tween 20 .The cultural method applied was similar to previously used methods , 2013. TThe resuspended pellet was distributed in two slants of homemade HEYM medium supplemented with Chloramphenicol (30 mg/l) and Amphotericin B (50 mg/l) (HEYM/CAF) and in two slants of homemade or commercial  HEYM supplemented with Amphotericin B (50 mg/l), Nalidixic acid (50 mg/l), Vancomycin (50 mg/l), and sodium pyruvate (4 g/l) (HEYM/ANV). The slants were incubated up to 18 weeks at 37 ± 1°C.g. Pellets were resuspended in 10 ml of sterile distilled water and MAP DNA was extracted using AdiapureTM kit  according to manufacturers’ instructions.Fifty ml of each milk test portion were centrifuged for 15 min at 2,500 ® Exogenous Internal Positive Control mix  and 0.5 μl TaqMan® Exogenous Internal Positive Control DNA . Amplification reactions were performed for each test portion using Applied Biosystems 7500, 7500 Fast Real-Time PCR, or StepOne Plus System (Life Technologies), with the following thermal cycling conditions: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. For each qPCR run, positive and negative controls were also analyzed.DNA was amplified using qPCR targeting IS900 and f57 sequences . BrieflyTM Tosyl-activated magnetic beads  and 250 μl of MyOneTM Tosylactivated Dynabeads®  were coated with 0.25 mg/ml of each peptide following the instructions of Bioclone and with 1 mg of each peptide previously resuspended in 60 μl of molecular grade water according to The procedure for coating magnetic beads with biotinylated-aMp3 (NYVIHDVPRHPA) or biotinylated-aMptD (GKNHHHQHHRPQ) peptides was based on Following the PMS, the test portions were subjected to the phage amplification assay according to TM Gel DNA recovery Kit  according to manufacturer’s instructions, followed by qPCR targeting IS900 as described above.After the PMS-phage assay, plaques were counted and one to 10 plaques per plate were harvested immediately after the overnight incubation and pooled to extract the MAP DNA using Zymoclean1 and 46 ND out of 96 spiked test portions from L2. No potential relationship  were found to explain this high number of ND.Different sensitivities, expressed as the percentage of positives from the total of true positive test portions (spiked samples), were obtained for IS900 qPCR (94%), f57 qPCR (76%), culture (83%), and PMS-phage (40%), when comparing all matrices . The lowTrueness, expressed as results (including positive and negative) correctly assigned considering the reference value, was highest for the IS900 qPCR method (93%) and lowest for the PMS-phage (49%) . The tru10 CFU/50 ml and 3.06 Log10 CFU/50 ml) with three different strains. For the PMS-phage method, results beyond the AL  were observed in both laboratories and all three dairy categories and were associated to a high number of ND for all inoculum levels, food items and strains analyzed. Only laboratory A obtained acceptable results with raw milk but still with a high number of ND (10) and a relative high number of PD (7).Considering AL for all milk categories , AL abovMycobacterium avium subsp. paratuberculosis was detected in all test portions tested for homogeneity. Only one out of 12 results from the raw milk test portions was not available due to overgrowth of background flora. The stability results were positive for all test portions of heat-treated milk and raw milk stored up to 4–6 weeks. For milk powder, one test portion (Skimmed milk powder spiked with strain reference DSM 44135) was positive after 3 weeks of storage but negative after 4 weeks. Despite that, this set of test portions showed positive results for the sensitivity study by both laboratories, which processed the test portions within three weeks after reception.Prior to test portion preparation for LOD study, the amount of MAP pure culture needed for spiking the test portions to reach the desired level for each milk type was determined by several rapid methods as shown in After spiking, the reference values were determined by MPN to calculate LOD .In general, the values are aligned but differences were observed between MPN and calculated values from pure culture enumerations because of the matrix effect and the analytical method used.50 and LOD95 were estimated considering combined results from both laboratories when possible  showing better sensitivities than those obtained in this study. In this regards, it should be pointed out how our results were probably highly influenced by the presence of background microflora, which was already known for its potential to inhibit the growth of MAP  for IS900 (10 CFU/50 ml) for f57  for a PMS phage-based method in raw milk but it is important to highlight that these results were obtained with reference values estimated in PFU rather than using a common standard reference   ; however50’s for heat-treated milk and milk powder were higher for the PMS-phage method compared to all other qPCR and cultural methods. From 126 non-spiked milk test portions, analyzed using the PMS-phage method by the two laboratories, 80 test portions generated phage plaques and 29 test portions were confirmed positive by qPCR. Some of these non-spiked milk test portions showed a high number of plaques (>300), which were not confirmed by qPCR, questioning the efficacy of the FAS treatment to inactivate exogenous phages.Variants of the PMS phage-based method have been reported and applied by a limited number of groups . In ordeIn contrast, no PD for culture and f57 qPCR and only a low number of them for IS900 qPCR (eight PD detected in one laboratory) were observed among all these 126 non-spiked test portions.While the PMS-phage assay is workable in pure liquid culture for estimation of MAP counts, its usage for surveillance of dairy matrices (raw and processed) should be treated with a lot of caution as performance characteristics obtained were inferior than for the two other methods tested.Our study highlights that qPCR and culture are the most appropriate methods to detect MAP in milk-based matrices according to ISO 16140 methodology. Cultural techniques are considered the gold standard for detection of viable (and cultivable) MAP . DespiteAll datasets generated for this study are included in the manuscript and/or the supplementary files.DT and SB contributed to the design of the study, prepared the samples, and wrote the manuscript. MR and IS contributed to the design of the study, tested the samples, and wrote the manuscript. LM prepared the samples and helped to write the manuscript. SR, EM, and MT tested the samples and helped to write the manuscript. NA and JG supervised the study and helped to write the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pregnancy complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR) promotes endothelial cell (EC) dysfunction. Our in vitro study aimed to evaluate the endothelial cell morphology after acute and chronic exposition to medium supplemented with serum taken from healthy pregnant women and women with IUGR and IUGR with PE. In the same condition, ECs viability, proliferation, reactive oxygen species (ROS) production, and serum concentration of vascular endothelial growth factor (VEGF) were also measured. Pregnant women with IUGR and IUGR with PE-delivered babies with reduced body mass and were characterized in elevated blood pressure, urine protein loss, and reduced level of VEGF. The 24 hours of exposition did not exert any morphological changes in ECs, except the reduction in cell viability, but prolonged exposition resulted in significant morphological changes concerning mostly the swelling of mitochondria with accompanying ROS production, cell autophagy, reduced cell viability, and proliferation only in complicated pregnancies. In conclusion, the sera taken from women with IUGR and IUGR with PE show a detrimental effect on ECs, reducing their viability, proliferation, and generating oxidative stress due to dysfunctional mitochondria. This multidirectional effect might have an adverse impact on the cardiovascular system in women with IUGR and PE. Preeclampsia is a hypertensive disease that complicates 2–8% of pregnancies characterized by high blood pressure and signs of damage to another organ system, most often the liver and kidneys. It is postulated that, in preeclampsia, hypoxia/reperfusion, which generates reactive oxygen species (ROS) production and an apoptotic/necrotic cascade causes morphological changes in the syncytium of preeclamptic placentas. Roland et al. describe many morphological changes of placental syncytium and their implications for the pathogenesis of this complex disorder ,2. This In this present paper, based on extensive medical reports that show ECs dysfunction in IUGR and IUGR complicated with PE, we evaluated the morphological abnormalities in endothelium after being exposed to sera taken from pregnancies complicated with PE and PE with IUGR. The novelty of our data is to show the variety of changes in ECs ultrastructure, visualized by the electron microscope.This study was carried out in accordance with the Declaration of Helsinki of the World Medical Association and approved by the Ethical Committee of Poznan University of Medical Sciences (Decision number: 1212/16). All included study participants fulfilled the criteria and completed the study. They were fully informed about the research, and all of them gave written informed consent before their examination.In the years 2017–2018, a total of 215 women in the third trimester of pregnancy, aged 18–48, were admitted to the Department of Perinatology and Gynaecology at the Poznan University of Medical Sciences or were treated in outpatient clinics. All consecutive pregnant women underwent a careful interview and a clinical examination, and their history was evaluated on the basis of available hospital and outpatient data. Each time, specialist gynecologists, using well-known recognized criteria based on symptoms, laboratory results and imaging tests, qualified a woman into one of three research groups, i.e. (i) control group—healthy women with normal fetal weight, (ii) IUGR group—women with estimated fetal weight at or below the 10th percentile for gestational age but without preeclampsia (PE), (iii) IUGR + PE group—women who met the criteria for PE and IUGR.n = 20); deficiencies in medical records (n = 3); chronic disease (n = 26); multiple pregnancy (n = 27); taking medications during pregnancy that may affect the coagulation system, e.g., acetylsalicylic acid, heparin (n = 18); intrauterine growth retardation due to structural defects, fetal genetic abnormalities or intrauterine infection (n = 18); smoking and/or alcohol abuse in the past 3 years (n = 13) and withdrawal of consent to participate in the study (n = 6). In addition, haemolysis was revealed in 8 blood samples.The exclusion criteria from this study were: no written consent to be included in the study  according to the manufacturer’s instructions. C-reactive protein (CRP) was measured by using a high sensitivity assay from DRG Medtek . The sensitivity of the assays was: 12.2 pg/mL for VEGF, and 0.1 mg/mL for high-sensitivity C-reactive protein (hsCRP).v/v fetal calf serum . The cultures were maintained at 37 ºC in a humidified atmosphere of 95% air and 5% CO2.The tests were performed using HUVEC line EA.hy926  . The celAll the reagents were purchased from Sigma-Aldrich . Cell culture plastics came from Nunc .v/v fetal calf serum (FCS) and 10% human pooled serum for one and 10 days. Human pooled serum was composed of an equal volume of serum taken from 22–25 women from the control group , from pregnant women with IUGR (n = 25), and pregnancies with IUGR + PE group (n = 24). The created medium was then sterilized using siring filters . This process protects the endothelial cell culture against potential infection, especially during prolonged exposition (10 days). During a prolonged exposition medium was exchanged every three days. It would have been much better if endothelium had been exposed to each serum per se. However, due to the small amount of serum taken from pregnant women with complicated pregnancies, we decided to prepare one pooled serum to perform all experiments.HUVECs were exposed to the culture medium supplemented with 10% v/v fetal bovine serum, and it was the recommended milieu for HUVEC culture, as described at the beginning of the method section (Con = standard medium). The second control consisted of medium supplemented with 10% v/v human serum taken from non-pregnant women with matched age (HS). The third control consisted of medium supplemented with 10% v/v human serum taken from healthy pregnant women (HP). Controls second and third are not a physiological milieu for the HUVECs and were made on the purpose of the experiment, which aims to evaluate diverse human serum.During our experiments we used three types of controls. One control consisted of medium supplemented with 10% 3 cells/well and 2 × 104 cells/well respectively for 10 days and 24 h of exposition to tested sera. After one and 10 days of exposition to medium supplemented with pooled human sera , cell viability was measured using the MTT assay, which measures the metabolic conversion of the MTT salt -2,5-diphenyl-tetrazolinum bromide) by active dehydrogenases derived in this test from endoplasmic reticulum -thymidine incorporation. Briefly, cells in 48 well plates were plated at a density of 2 × 104 cells/well, allowed to attach for 24 hours, and then pulsed with [3H]-thymidine  for 24 hours. After the incubation, the cells were harvested, precipitated with 10% (w/v) trichloroacetic acid, and dissolved in 0.1 M NaOH. The radioactivity released was measured in a beta liquid scintillation counter . The data were expressed in two ways, as a percentage of (i) control HS (cells cultured in a medium supplemented with human serum taken from non-pregnant women) and (ii) control HP .After one and 10 days of exposition to human sera, HUVECs proliferation was assessed by [3 cells/well and 2 × 104 cells/well respectively for 10 days and 24 h of exposition to tested sera. Detection of ROS was assayed by 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) labeling. Briefly, cells (2 × 104) were incubated with 10 μM H2DCFDA  for 45 min at 37 ºC and then solubilized with the lysis buffer . The measure of ROS generation in cells was the degree of fluorescence of the H2DCFDA dye, which under intracellular ROS was oxidized to the fluorescent product Fluorescence emitted by cell lysate was measured in a spectrofluorometer , using wavelengths of 485 and 535 nm for excitation and emission, respectively. The positive control during the experiment consisted of 100 µM H2O2. The data were expressed in two ways, as a percentage of (i) control HS (cells cultured in a medium supplemented with human serum taken from non-pregnant women) and (ii) control HP .The experiments were performed in 96-well plates. Cells were seeded in following densities depending on the time of the experiment: at 4 × 104 cells/well and incubated with standard medium (Con), and medium supplemented with pooled human sera  for 10 days. The microphotographs were taken every 1–3 days using the Axio Observer D1 inverted microscope  up to the 10th day of exposition.ECs were seeded in 24-well plates at 4 × 105 cells/well and 105 cells/well respectively for one and 10 days of exposition to tested sera. After 1 and 10 days of exposition to standard medium (Con), and medium supplemented with pooled human sera , ECs were harvested using trypsin/EDTA, centrifuged, and subsequently fixed in 0.1 M phosphate-buffered saline (PBS) containing 2.5% glutaraldehyde for 1 hour at room temperature, post-fixed in 1% osmium tetraoxide for 30 minutes at room temperature, washed and dehydrated in graded series of ethanol solutions (40–100%). Then, cells were infiltrated and embedded in Epon . Ultrathin sections (40–60 nm) were mounted on mesh copper grids, contrasted with 2% aqueous uranyl acetate (30 minutes), and lead citrate (30 minutes) and analyzed with a Jeol transmission electron microscope at 80 kV .The experiments were performed in 6-well plates. Cells were seeded at 4 × 10p-value < 0.05 was considered significant. The data were expressed as means ± standard deviation (SD) or median (min–max).Statistical analysis was performed using GraphPad PrismTM 6.00 . The data were interpreted with repeated measures analysis of variance—one-way ANOVA—using a post hoc test for multiple comparisons (Dunn’s or Tukey’s tests). A Women with complicated pregnancies delivered babies with reduced newborn weight when compared to women without complications. The IUGR and IUGR + PE group was also characterized by higher systolic and diastolic blood pressure and higher urine protein loss. We detected about four times higher levels of VEGF in women with healthy non-complicated pregnancies in contrast to IUGR and IUGR with PE groups. For the characteristics of the study participants see The exposure of HUVECs to medium supplemented with human serum taken from healthy pregnant women, pregnant women with IUGR, and IUGR with PE for 24 hours reveals a significant decrease in ECs’ viability in the groups from pregnancies with complications A,C. SeruAny of the tested groups generate endothelial ROS production during acute exposition A,C. The We did not observe any morphological changes in ECs after one day of the exposition, using light microscopy. In all tested groups, ECs maintained the typical cobblestone appearance A–E. The To determine ultrastructural changes in HUVECs’ morphology, transmission electron microscope studies were performed. After one day of exposure to modified medium no significant differences between study groups in the cell ultrastructure were observed A–E. The Our study confirmed that patients with IUGR and IUGR with PE are characterized by the impaired function of vascular endothelial cells. During the chronic exposition of ECs in vitro, exposed to the medium supplemented with pooled serum taken from women with complicated pregnancies, we documented the intensification of oxidative stress and reduced cell viability. Additionally, we have observed reduced ECs proliferation which could be partially attributable to decrease pro-angiogenic VEGF found in the serum from complicated pregnancies. The pathological alteration in the placenta stimulates the release of many antiangiogenic factors but VEGF blockade seemed to be crucial for ECs’ proliferation and migration, which may have an adverse effect on the cardiovascular system. Soluble fms-like tyrosine kinase (sFlt)-1 prevents VEGF interaction with receptors located on vascular ECs blocking its stimulation, leading to maternal endothelial dysfunction in pregnancies complicated with IUGR and PE ,5, but tThe diversity of morphological changes in the ECs was confirmed in the observation of ultrastructure of these cells in an electron microscope. In all tested groups, among cells with typical morphology, distinct ultrastructural changes were observed, especially pronounced in IUGR and IUGR with PE. Observed abnormalities were generally concerned with the accumulation of (i) lipid droplets in the cell cytoplasm, (ii) changes in mitochondrial structure, and additionally in the IUGR with PE group, to (iii) presence of autophagic vacuoles. Among observed ultrastructural changes, abnormalities in mitochondrial structure and the presence of autophagosomes can serve as potent factors leading to cell death. Cell death can be defined as an irreversible loss of plasma membrane integrity. Simplifying, historically three basic forms of cell death have been distinguished in mammalian cells by morphological criteria: necrosis, apoptosis and autophagy . The perMalperfusion of the placenta is a potent inducer of oxidative stress which has been linked to the complication of pregnancy including PE and IUGR . OxidatiMitochondrial swelling is a hallmark of mitochondrial dysfunction and is one of the most important indicators of the opening of the mitochondrial permeability transition pore. It is a cell-specific ultrastructural change visualized by electron microscopy, which allows the detection of temporal dynamics of mitochondrial swelling in living specimens . DysfuncIn electron microscopy, we frequently observed lipid droplets (LDs), characteristic for metabolic dysfunction. Its presence could be partly explained by the disability of ER and mitochondria, especially in IUGR and PE groups. LDs store energy as triacylglycerols and sterol esters and are initiated in the ER. They dynamically interact with ER, mitochondria, lysosomes, and peroxisomes. They grow in size and number due to increased triacylglycerols synthesis when cells enter the stationary phase. Utilization of LDs is driven by the hydrolysis of triacylglycerols during growth resumption . The exiMaternal constitutional factors  are involved in defective placentation. Hypoperfusion of the placenta releases many factors seem to be involved in vascular endothelial dysfunction and clinical symptoms of PE, and often in IUGR. Endothelial dysfunction presented in complicated pregnancies as IUGR and PE has its bases in cell ultrastructural changes, mainly related to mitochondria but also to the accumulation of lipid droplets and autophagic vacuoles formation. These lesions could be responsible for the abnormal endothelial secretory phenotype, angiogenic properties, and its capability to produce ROS. Deterioration of endothelial functions leads to clinical symptoms characteristic for cardiovascular diseases as hypertension, proteinuria, activation of pro-coagulant activity, inflammation, and incorrect redistribution of fluids. Our results can help to understand the nature of the disorders studied in pregnant women and contribute to their more effective treatment.


Coxiella burnetii infection and the contribution of Q fever to APOs.Q fever has been associated with perinatal complications. We conducted a prospective follow-up study to assess both the incidence of adverse pregnancy outcomes (APOs) associated with C. burnetii antibodies was defined using indirect immunofluorescence for a phase 2 IgG titre ≥1:64. Acute Q fever was defined for a high phase 2 IgG titre ≥1:256 (compatible with recent or active infection) or the detection of C. burnetii genome in miscarriage products and placentas. Incidence rate ratios (IRR) for Q fever related APOs  were assessed using Poisson regression models for dichotomous outcomes controlling major confounders.Between May 1 and October 31, 2013, within the regional perinatal health care centre of Saint Pierre, Reunion island, we investigated unexplained miscarriages, stillbirths, preterm births or small-for-gestational age children. Seropositivity for C. burnetii exposure and acute Q fever were independently associated with APOs .Over a 6-month period, 179 pregnant women suspected or diagnosed with an APO were investigated for Q fever, of whom 118 met the definition for an APO. Of these, 19 were seropositive and 10 presented a profile indicative of an acute infection. For three women with an acute Q fever, the chronology between the onset of infection, the APO  and the seroconversion suggested causality in the pathogenesis. The cumulative incidence of Q fever related APOs was estimated between 2.2‰ and 5.2‰, whether causality was required or not. Both In the endemic context of Reunion island, acute Q fever may lead to APOs. To limit the burden of Q fever on reproduction, pregnant women should be kept away from farms and avoid direct contact with ruminants. Coxiella burnetii, an obligate Gram negative intracellular bacterium maintained in wildlife through mammals, birds and arthropods , serving as reservoirs -2 or neonatal herpes simplex) pathogens ruled out.Biological plausibility was defined as a positive RT-PCR or the seroconversion of phase 2 IgG. The relationship between exposure to Q fever and APOs was deemed causal when temporality and biological plausibility criteria were met and TORCH  of each Q fever related APO were estimated using Poisson regression models for dichotomous outcomes with the robust variance option adjusted for hypertensive disorders, diabetes , and maternal addictions . Attributable risk percent  among the exposed and population attributable fractions were generated to estimate the contribution of C. burnetii exposure to APOs.Miscarriage, stillbirth, preterm birth, small-for-gestational age as well as a composite outcome of all these APOs were compared according to P value < 0.05 was considered significant.All these analyses were performed using Stata 14.2® . For all estimations, a Between May 1 and October 31, 2013, 2331 pregnant women gave birth or aborted within the level-3 maternity. Of these, 850 (36.4%) were suspected of an APO  and 10 presented a profile indicative of an acute infection . It was worth noting that Q fever genome was not recovered from all the 10 birth products sampled from early or late miscarriages. Except patient n°1, who exhibited fever and mild hepatitis, all parturient women were asymptomatic, and the diagnosis was made incidentally at the occasion of the APO event. No persistent infection was identified upon follow-up. The serological profiles and perinatal complications of the seropositive women are presented in Table n = 9) was the most common perinatal complication followed by SGA (n = 5), PTB (n = 3), stillbirth (n = 2), late miscarriage and oligohydramnios (n = 1). For three women with an acute Q fever , the chronology between the onset of the infection and the APO  with respect to the kinetics of antibodies  suggested causality in the pathogenesis of the complication.Of the 21 APOs observed within the 19 seropositive women, early miscarriage (The incidence of Q fever related APO in the reproductive population was estimated at between 2.4‰ and 5.4‰ (1.3 to 4.3‰ in the study sample), whether causality was requested or not, which was in the observed range for TORCH pathogens. Miscarriage featured almost half of this burden  and this follow-up study (under the rules of standard care research). We believe however this stringent methodological option (known as tip of iceberg epidemiology) makes the results from our local study that more interesting, the real burden being likely underestimated.In support to the eventuality of underreporting, there is a potential for Q fever to be associated with other pregnancy outcomes, such as oligohydramnios, hydramnios, or preterm premature rupture of membranes, as suggested by several case series , 23.Q fever is circulating on Reunion island, including among pregnant women. Given that specific strains have been found to be associated with different clinical manifestations and given the increased risk of miscarriage in our cohort study, it would now be interesting to identify whether the circulating strain is abortive, as that harbouring the QpDV plasmid .As for mitigation measures aimed at limiting the burden of Q fever on reproduction, pregnant women should be kept away from farms to rule out airborne transmission, avoid direct contact with ruminants, or to consume fresh farm products. In addition, whenever possible, at risk women of childbearing age should be screened before conception or early in pregnancy and be treated with antibiotics to prevent potential harm to the foetus. Our findings are of particular interest to public health stakeholders and policy makers based in countries where Q fever is endemic to protect communities, especially in rearing areas.Additional file 1. Methodological appendix; Table S1 Maternal and foetal characteristics in the South Réunion island reproductive population and the study population; Table S2 Adverse pregnancy outcomes (APO) in the eligible population and the study population according to maternal and foetal characteristics; Supplemental findings; Table S3 Adverse pregnancy outcomes associated with Q fever seropositivity in bivariate and multivariate analysis; Table S4 Cumulative seroincidence rates of adverse pregnancy outcomes (APOs) associated with positive Q fever serology (any cut-off) in prospective observational studies reported in the literature.


The adsorption of macromolecules on solid surfaces is of great importance in the field of nanotechnology, biomaterials, biotechnological, and food processes. In the field of oenology adsorption of wine macromolecules such as polyphenols, polysaccharides, and proteins is much less desirable on membrane materials because of fouling and reduced filtering performance. On the other hand, adsorption of these molecules on processing aids is very beneficial for achieving wine clarity and stability. In this article, the effect of surface chemical functionalities on the adsorption of white, rosé, and red wine constituents was evaluated. Allylamine, acrylic acid, and ethanol were selected as precursors for plasma polymerization in order to generate coatings rich in amine, carboxyl, and hydroxyl chemical groups, respectively. The surface chemical functionalities were characterized by X-ray photoelectron spectroscopy (XPS) and the ability of different surface chemical functionalities to adsorb wine constituents were characterized by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). The results demonstrated that the amine and carboxyl modified surfaces encourage adsorption of constituents from white wine. The hydroxyl modified surfaces have the ability to preferentially adsorb rosé wine constituents, whereas red wine adsorbed to the highest extent on acrylic acid surface. Adsorption of macromolecules on solid surfaces and the changes in surface properties caused by macromolecules adsorption are major concerns in a number of fields, such as biology, medicine, biotechnology, and food processing ,2,3. ForIn wine industry, many significant and unresolved problems such as selective removal of undesired constituents from wines—i.e., removal of smoke taint compounds, bitter molecules, haze forming proteins, or managing deposits from the surface of the manufacturing equipment for maintains or sanitary conditions —can be sOur previous work described the role of functional groups  in adsorption of red wine constituents before and after filtration . In the 2), carboxyl (–COOH), and hydroxyl (–OH) chemical groups, respectively.It is well known that different grape varieties and yeast strains make different types of wines. Since wines vary in their chemical composition our hypothesis is that different wine components from different wines will adhere on the various functionalized surfaces. For this purpose, allylamine, acrylic acid, and ethanol were selected as precursors for plasma polymerization in order to generate coatings rich in amine  was used for the assessment of particle size and concentration . Figure White wine sample had the lowest particle concentration (2.04 E8/mL), the d90 was 327 nm and the mean diameter was 190 nm. The data suggest that there is a significant difference in particle characteristics such as d90, total particle concentration, and mean particle diameter between all studied wine samples. Overall, the results of the study demonstrated that there was a decrease in d90 and mean particle diameter in the following order: red wine > white wine > rosé wine. On the other hand, there was a decrease in total particle concentration in the following order: red wine > rosé wine > white wine.Plasma polymerized films from acrylic acid, allylamine, and ethanol were produced and analyzed using XPS. The survey spectra were used to obtain the surface O/C and N/C content of the plasma polymers and the C1s core levels were fitted to obtain information about functional groups. The XPS survey spectra of ppAcrA revealed only carbon (76.17%) and oxygen 23.83%) in the deposits . The C1s.83% in tThe effect of the carboxyl functional group on wine adsorption was also investigated. Three wines were brought into contact with the QCM sensors coated with acrylic acid plasma polymer. After 40 min of adsorption, washing with MilliQ water was performed to remove any non-surface-bound wine material and later on, the XPS was performed on these samples. The high resolution C1s spectra for wine-bound surfaces are shown in The O/C and N/C atomic ratios before and after the adsorption of wines are presented in On the other hand, the N/C ratio substantially increased on ppAcrA and ppAA surface after white and rosé wine was adsorbed and almost did not change its value after red wine adsorption on ppAcrA and ppAA surfaces when comparing with wine samples directly dried on the silicon wafer. In the case of ppET surface, the N/C ratio decreased regardless of the wine.When the ratios are compared with bare plasma polymerized surfaces it can be seen that the O/C ratio almost did not change after white and rosé wine adsorption and increased on the ppAcrA red wine coated sample as compared to bare ppAcrA coating A. The N/The XPS survey scans were also made of plasma-polymerized surfaces prepared from allylamine. Those deposits contained carbon (75.07%), oxygen (8.95%), and nitrogen (15.99%). The XPS data were quantified and the results are shown in To verify wine molecules and wine colloids binding to ppAA surfaces the high resolution C1s spectra were obtained by XPS. The ppAA/wine C1s peak can be fitted with six components as shown in The XPS survey spectra of ppET revealed only carbon (81.63%) and oxygen (18.37%) in the deposits. The O/C ratio was measured and is shown in The static water contact angles on the surfaces of uncoated gold sensor and pp coatings are shown in The gold-coated quartz crystals were first treated with acrylic acid, allylamine, and ethanol plasma in a glow-discharge apparatus in order to deposit carboxyl, hydroxyl, and amino groups on their surfaces. Then the coated crystals were exposed to three different wines  in QCM-D experiments. Due to the large change to the dissipation energy loss observed during measurements , Voigt-b2 modified substrates the adsorbed amount of white and rosé wine are higher than red wine. The trend was similar on –COOH modified substrates (white > rosé > red wine) but adsorbed mass was much higher than on –NH2 modified substrates. Finally, on –OH modified substances the wine mass uptake was as follows: the highest for rosé wine, followed by red wine and white wine. The levels of mass uptake of wine molecules on three different surfaces are distinct. On –NHTopography of bare pp surfaces as well as adsorbed wine constituents on pp surfaces was assessed through tapping mode Atomic Force Microscopy . The AFM height and phase images of the bare QCM sensor  are depicted in The combination of the QCM-D and AFM allowed to get an estimation of water content in the adsorbed wine layers. The volume of adsorbed wine over the AFM image area can be estimated from the AFM images by multiplying the average layer thickness by the area fraction of the wine coverage . CalculaAs shown in 2 groups at pH 3, the surface potential at the ppAA surface is high and positive (210 mV) [2 modified surfaces the highest adsorbed amount was obtained for white wine (12 mg/m2) followed by rosé (9 mg/m2) and red wine (7 mg/m2). According to the wine analysis, white wine has the highest protein concentration (134 mg/L), does not contain tannin and has the lowest polysaccharide content (222 mg/L). Rosé wine has a little protein (only 5 mg/L), some tannin (57 mg/L), and more polysaccharides than white wine (315 mg/L). Finally, red wine has the highest polysaccharide (520 mg/L) and tannin content (909 mg/L) and only little protein . The amine group is protonated at low pH values thus leading to a more hydrophilic surface. As pH increases, deprotonation makes the surface neutral and its wettability decreases. As we measured previously by colloid probe AFM, due to the complete protonation of the NH(210 mV) . The XPS(210 mV)  and condThe above results indicate that there are at least two possible mechanisms of adsorption onto amine surface: (1) electrostatic adsorption between positively charged amine surface and negatively charged wine constituents (acidic polysaccharides and condensed tannins); and (2) covalent immobilization of wine proteins onto amine surface.2. As mentioned previously and presented in The zeta potential of carboxyl-rich surface is neutral at pH 3 and became negative with increasing pH, which is consistent with the weak acidity of –COOH groups . At wine2 among all investigated wine samples. The peak fit of rosé wine bound ppET surface revealed that 22.85% of the carbon was in alcohol/ether and imine environment, 8.75% of the carbon was in carbonyl and amide environment and 3.46% of the carbon was in a carboxylate-type environment (carboxylic acid or ester).The hydroxyl groups are neutral and hydrophilic. They are less attractive than amines and carboxyls as platforms for covalent immobilization of biomolecules in aqueous solution due to their lower nucleophilic character and thus lower chemical reactivity. The –OH groups can bind, for instance, a range of peptides without any crosslinking or coupling agent which appears to be a physisorption rather than, for example, covalent interfacial linkage. The QCM-D demonstrates that the ppET enhanced rosé wine adsorption with the highest adsorbed amount of 17.5 mg/mThree wines were selected for this study: 2012 AWRI sequential harvest trial Shiraz (Red), 2014 unfined SA Riesling (White) and 2014 Pinot Noir from Adelaide Hills (Rosé). 2. The methyl cellulose precipitable (MCP) tannin assay was used to measure the total wine tannin in red and rosé wine sample, whereas phenol-sulphuric acid assay was used to measure total polysaccharides concentration in wines. Proteins in wines were analyzed by HPLC method [2O (Solvent A) and 0.1% TFA/ACN (Solvent B), at 0.75 mL/min and 35 °C column temperature. The mobile phase gradient was: 0–1 min 10%–20% Solvent B, 1–4 min 20%–40% B, 4–6 min 40%–80% B, 6–7 min 80% B, 7.01 min 10% B, and 7–10 min 10% B. Detection of proteins was achieved by Diode Array monitoring at 210 nm. Identification of proteins was achieved by comparing the retention times of sample peaks with isolated standards and quantitation was achieved by comparing the peak areas with those of a standard curve of thaumatin . The concentration of phenolic substances of the wines was assessed using absorbance at 280 nm.Basic chemistry of the wines—such as alcohol, pH, titratable acidity, glucose/fructose, and volatile acidity—were analyzed by the AWRI Commercial Services using a Foss WineScan FT 120 and is provided in the C method  with somv/v Ajax Finechem, Thermo Fisher Scientific Australia Pty Ltd., Vic, Australia) were used as received.Allylamine (AA) , acrylic acid (AcrA) (99% Aldrich), and ethanol (ET) (absolute 99.5% 2), carboxyl (–COOH), and hydroxyl (–OH) chemical groups, respectively. Deposition was carried out at a precursors pressure of 0.2 mbar. Allylamine vapor was introduced at 10 sccm, and plasma was applied at 10 W for 4 min. Deposition of acrylic acid was carried out at a flow rate of 0.5 sccm, power of 10 W for 10 min. Whereas, conditions for ethanol deposition were as follows: 4 sccm, 40 W and 4 min. Thickness of the deposited plasma polymers is shown in QCM-D sensors and clean silicon wafers were polymerized in a plasma reactor operated with a 13.56 MHz radiofrequency generator as previously described ,38. AllyX-ray photoelectron spectroscopy was carried on a Kratos Axis Ultra DLD spectrometer  using a monochromatic Al radiation source . The surThe contact angle was measured by the sessile drop method using a custom-built contact angle goniometer. A droplet of water was deposited on ppAcrA, ppAA, and ppET surfaces. Images of the droplet were captured using a horizontal digital microscope. Contact angles were determined by drawing the tangent close to the edge of the droplet using a drop shape analysis software ImageJ with the DropSnake plugin . Five measurements were taken at different locations on the investigated substrates, and an average value was reported. Experiments were performed at room temperature in a clean room (class-100).The quartz crystal microbalance with dissipation (QCM-D) was used to study wine adsorption. The instrument consisted of an E4 QCM-D unit  coupled with a peristaltic pump  . During Atomic force microscopy analyses were acquired using an NT-MDT SPM  with a 100 μm scanner. Images were obtained using non-contact mode in air. Gold coated, silicon nitride non-contact tips with a resonance frequency between 65 and 100 kHz were used. The amplitude of oscillation was 10 nm, and the scan rate was 1 Hz. The AFM parameters were determined using 5 μm × 5 μm images. The AFM software  was used to assess the root-mean-squared (RMS) roughness and the peak-to-valley (PTV) distance for the imaged surfaces. The area fraction of wine coverage was determined using ImageJ.Nanoparticle tracking analysis was performed with a NanoSight NS300 , equipped with a sample chamber with a 635 nm laser and a syringe pump. The wine sample was injected in the sample chamber with a sterile 1 mL syringe. A video clip of the wine samples submitted to their natural Brownian motion was captured over 60 s and analysed by the analytical software NanoSight NS300 NTA 2.3 . Analysis was performed with a constant and controlled sample flow at room temperature. The samples were measured with manual shutter and gain adjustments. The ‘extended dynamic range mode’ was used as recommended for polydispersed samples. NTA post-acquisition settings were optimized and kept constant between samples, and each video was then analysed to give mean particle size together with an estimate of the concentration.2 and –COOH modified surfaces were shown to promote adsorption of constituents from white wine. Among all wine samples, rosé wine exhibited a strong preference for the –OH terminated plasma polymer layer. Red wine adsorbed to the highest extent on acrylic acid surfaces as compared to two other surfaces. These results demonstrate that wine constituents’ adsorption depends on the composition of the wine. It was also found that wine constituents can form poorly hydrated  or highly hydrated layers  with different characteristics depending on the underlying substrate.Three different precursors, such as allylamine, acrylic acid and ethanol were used in plasma polymerization process to produce thin films rich in amine, carboxyl, and hydroxyl functional groups. The use of QCM-D and AFM allowed quantification of the properties of wine constituents at homogenous surfaces with three different surface chemistries. The results demonstrate that the chemical surface composition generated by plasma polymerization can affect behavior of wine adsorption. The –NHMoreover, the overall results suggest that tuning the surface chemical functionalities is an effective way to modulate wine adsorption. These data may have implications in future design of wine processing technology, such as membranes, and promote the development of tailored sensing systems.