Sensitive periods are times of development during which the effects of experience are unusually strong and long lasting. The peripubertal period has emerged as one such sensitive period, and a single administration of lipopolysaccharide (LPS) during this time reduces hormone-induced sexual behavior in adult female mice. During periods of high synaptic turnover, maturation, and elimination, as occurs during this sensitive period, microglia are particularly active. Estradiol also regulates microglial numbers, morphology, and activation. In addition, a good deal of evidence suggests that estradiol may confer this vulnerability to the effects of a stressor during the peripubertal period. Therefore, we investigated the effects of estradiol on microglial morphology, cytokine levels, and the sickness response to LPS. Estradiol levels were manipulated by implanting an estradiol-filled SILASTIC capsule (or oil-filled control) in ovariectomized mice or by administering the aromatase inhibitor, formestane (or oil control), to ovary-intact mice. We found that (1) estradiol elevates basal microglial Iba1 immunoreactivity in the ventromedial nucleus of the hypothalamus (VMH), (2) LPS induces higher levels of proinflammatory cytokines in the presence of estradiol, and (3) LPS causes hypothermia in the presence of estradiol. Taken together, these data suggest that estradiol enhances the effect of LPS during the pubertal sensitive period. The experience of trauma or extreme stressors, including immunostressors, during the peripubertal period can lead to marked and pervasive alterations of behavior. The underlying mechanisms contributing to this vulnerability during the peripubertal period are not understood. Microglia have emerged as a candidate through which stress might impact neurodevelopment and subsequently behaviors. Few studies have investigated how the developmental changes in levels of estrogens during the peripubertal period influence the microglia. Here, we find that exposure to estradiol during the peripubertal period enhances the microglial response to an immune challenge. These findings suggest a mechanism through which estradiol may confer vulnerability to an immunostressor during the sensitive, peripubertal period.Sensitive periods are developmental windows during which the effects of experience can lead to enduring effects . StressfDuring sensitive periods of development, there are marked changes in the architecture of brain regions via cell genesis, cell death, and synaptic remodeling . MoreoveOne of the hallmarks of the peripubertal period is the maturation of the reproductive axis, resulting in the secretion of gonadal steroid hormones. As these gonadal hormones impact neurobiological processes regulating reproduction at the molecular, cellular, physiological, and behavioral levels, it is likely that estradiol may shape these neuronal circuits during pubertal development. For example, full expression of female sexual receptivity in adulthood depends on peripubertal secretion of estradiol . AromataOne way in which estradiol can shape neuronal circuits is by regulating microglia. Microglia express estrogen receptors ER; , suggestad libitum access to food  and water in glass bottles. All procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and in accordance with the University of Massachusetts, Amherst Institutional Animal Care and Use Committee’ regulation. Experimenters were blinded to all experimental treatments.Female C57Bl/6 mice were shipped from Charles River Laboratories on P21 and housed in an all-female colony room. The room was under controlled temperature (24 ± 2°C) and reversed 14/10 h light/dark cycle . Mice were housed in groups of four in clear polycarbonate cages with On P28, mice were anesthetized with isoflurane , and a single incision was made to the dorsal skin and the muscle layer to locate the uterine horns. The uterine horns were clamped, the ovaries were removed, and the tips of the uterine horns were cauterized with an Acu-Cautery (Acuderm Inc). The muscle layer was sutured with an absorbable suture (Ethicon), and the incision was closed using surgical wound clips (Becton Dickinson). For capsule implantation, a small incision was made to the skin layer of the neck, and a 2.5-cm-long SILASTIC capsule  filled with estradiol or sesame oil was implanted . The incDuring the ovariectomy procedure, mice received a G2 E-mitter device (E-mitter Respironics Mini-mitter) to record core body temperature and activity levels. The E-mitter was sutured to the abdominal muscle wall to keep its placement stable. Before implantation, the E-mitter was washed with Tergazyme, a detergent with protease enzyme activity. Following Tergazyme, E-mitters were incubated in 3% glutaraldehyde to disinfect and sterilize the E-mitter and rinsed with sterile saline at least three times. Radio signals for locomotion and temperature were recorded by a receiver board (ER-4000 energizer receiver) underneath the cage housing each animal and stored via Vital View Data Acquisition System  in the laboratory computer. Mice were allowed to recover from E-mitter implant surgery for at least one week before onset of the experiments. Data were collected in 2-min bins, starting 24 h before the injection until decapitation (at either 6 or 24 h following LPS or vehicle injection).2 was obtained from Sigma-Aldrich. Estradiol was dissolved in sesame oil (vehicle) to a concentration of 50 μg/25 μl. Mice were randomly assigned to treatment groups and received a SILASTIC capsule (Dow Corning) filled with estradiol (50 μg E2/25 μl sesame oil) or sesame oil.United States Pharmacopeia (USP) grade 17β-estradiol Ein vivo in neonatal rats  to a concentration of 0.1 mg/ml. Mice were randomly assigned to treatment groups and received a single intraperitoneal injection of LPS (1.5 mg/kg body weight) or an equivalent volume of sterile saline on P42. Injections were administered within 1 h before the onset of the dark phase of the light/dark cycle. Animals were returned to their home cage immediately following injection. Mice were weighed immediately before injection, as well as 6 and 24 h following treatment to assess the effect of LPS on body weight.LPS from m, pH 7.4) at 4°C for 24 h, followed by incubation in 30% sucrose in 0.1 m PBS for at least 72 h. Brains were sectioned coronally in 30-μm-thick sections using a cryostat. Brain sections were stored in cryoprotectant at −20°C until they were immunostained.Mice were deeply anesthetized with an intraperitoneal injection of pentobarbital (100 mg/kg) and decapitated. Brains were removed from the skull and fixed in 4% paraformaldehyde in PBS  and also rinsed before each subsequent step, except between the blocking and primary antibody steps. Brain sections were incubated in a blocking buffer containing 20% normal goat serum, 3.5% hydrogen peroxide, and 1% bovine serum albumin in gel TBS for 1 h. Brain slices were incubated for 48 h at 4°C in a solution containing Iba1 primary antibody  at a 1:10,000 dilution. The primary Iba1 antibody was diluted in 2% normal goat serum and 0.5% Triton X-100 in gel TBS. After 48 h, sections were incubated for 90 min at room temperature in a solution of biotinylated secondary antibody in gel TBS. The avidin-biotin complex (ABC) was used to bind a complex of streptavidin-biotin peroxidase to the secondary antibody for 90 min. The reaction was then developed with diaminobenzidine  for 20 min to produce a colorimetric stain. Brain sections were rinsed in TBS and mounted on gelatin-coated slides, dehydrated, and coverslipped with Polymount.Free-floating brain sections were rinsed for 3 × 5 min in 0.05 Mouse Brain Atlas in Stereotaxic Coordinates, using the shape of the third ventricle as a landmark  and decapitated. Trunk blood was collected into an EDTA-treated tube (BD Vacutainer) and placed on ice. Blood was centrifuged at 4°C in a microcentrifuge (Eppendorf). Following centrifugation, plasma was aliquoted and stored at −80°C until assayed. Estradiol levels were determined using the mouse estradiol ELISA kit . The level of detection is 3 pg/ml, which is consistent with the results of Cytokine protein levels in plasma were measured 6 and 24 h after LPS administration. The 6-h time point was selected because, in a time course profile of cytokine levels 6 h post-LPS, this was the optimal time point to measure the peak levels of cytokines of interest. It is also the time point at which a single peripheral LPS injection causes significant breakdown of the blood brain barrier . The 24-2 for the ANOVAs  and hormonal milieu (estradiol or oil capsule in ovariectomized mice or formestane and oil controls in ovary-intact mice) on Iba1 immunoreactivity, cytokine protein levels, body weight, and estradiol levels using a two-way ANOVA. We corrected for multiple comparisons using Bonferroni corrections. All data in experimental 1 were normally distributed except for the VMH mean cell body area, the VMH total outgrowth, the VMH total branches, and the IL-1α 24 h. All data in experiment 2 were normally distributed except for VMH number of microglia, the total cell body area, the mean cell body area, IL-10 6 h, IL-1α 24 h, IL-1β 24 h, IL-10 24 h, TNF-α 24 h, the changes in body weight at 6 and 24 h, and the change in temperature. The statistical tests and observed powers were conducted using GraphPad Prism. Effect sizes estimates were calculated using ηe ANOVAs . Each coA total of 128 C57Bl/6 female mice were shipped on P21. Mice were ovariectomized and received a SILASTIC capsule implant on P35. Mice received an intraperitoneal injection of LPS or sterile saline vehicle on P42 A. ThirtyA total of 128 C57Bl/6 female mice were shipped on P21. Mice received a daily injection of formestane or sesame oil for seven consecutive days from P35 to P42 B. Mice ra1 in mice treated with saline control. This increase in microglial Iba1 immunoreactivity is not due to differences in the total number of microgliaa2 or differences in microglial cell body sizea3,a4  and a concomitant increase in macrophage-like microglia  and a concomitant increase in macrophage-like microglia  into the VMH causes a febrile response in rabbits . This region specificity may arise if the VMH is able to integrate signals from immune activity with the estradiol activity. For example, microinjections of prostaglandin E rabbits . In addiale pups . As LPS The notion that estradiol increases microglial Iba1 immunoreactivity, which can be associated with an enhanced response to immune activation, during pubertal development is supported by the cytokine, thermoregulatory, and metabolic changes observed in mice. LPS induces higher levels of TNF-α, hypothermia, and greater weight loss only in the presence of estradiol. Fever is a typical response to infection, but hypothermia is only induced in severe cases of inflammation . OverallThere are a couple of important caveats regarding the contributions of estradiol and other ovarian hormones to keep in mind when interpreting these data. First, these data indicate that presence of estradiol is required for the increased vulnerability to LPS during the peripubertal period; however, estradiol may not be the sole driver. In fact, one reason for the differences in microglial branching and cytokine expression between experiments 1 and 2 may be due to these other factors. Formestane, as an aromatase inhibitor, blocks the synthesis of estrogens, whereas the removal of the ovaries would also impact the estrogens and other ovarian hormones such as progestins. Therefore, adding back estradiol after removal of the ovaries is not the opposite of blocking synthesis of estrogens all over the body. The second relates to the estradiol levels. Although the route of administration of estradiol used in this study rescues the effects of ovariectomy on several markers of pubertal maturation , it is uAnother caveat to keep in mind when interpreting these microglia data are that they reflect the interaction between the hormone environment and an immune challenge at a single time point (24 h postinjection). Our behavioral, physiological, cytokine, and metabolic data showed dynamic changes in the immune response within the first 24 h after LPS administration. It is possible that the effect of estradiol on microglial morphology is also temporally regulated. In addition, the cytokine analysis was performed using plasma samples, so the cytokine levels in the hypothalamus are unknown. Therefore, future studies should be conducted to determine the cytokine levels in hypothalamic homogenates. Lastly, it is important to investigate whether these manipulations that impact the microglia also prevent the altered hormone-responsive behaviors in adulthood.While both, pubertal males and females, are sensitive to shipping from the breeder facility as indicated by reduction in the sexual behaviors in adulthood, females show a more robust response . It is uThe goal of the present work was to examine the potential cellular underpinnings of the vulnerability of female mice to LPS administered in the peripubertal period. The present work suggests that estradiol may be a key factor in conferring vulnerability to LPS during the peripubertal sensitive period. While not tested directly, the increase in estradiol-driven microglial Iba1 immunoreactivity may suggest that microglia in the VMH are more active during the peripubertal period. Regardless, estradiol seems to amplify the LPS-induced responses in female mice during pubertal development at the microglial, cytokine, and thermoregulatory levels. It is possible that there is an enhanced interaction between the endocrine and immune systems as part of typical pubertal development. However, exposure to an immune stressor like LPS intensifies the immune signaling pathways to a threshold that may have detrimental effects. Although the present experiments do not directly address the issue of the effects of peripubertal immune challenge or shipping stress on later behavioral response to estradiol, they suggest that estradiol enhances the effect of LPS during the pubertal sensitive period.


The presence of either of these two risk factors can better reflect the condition of the donor than previous classification. However, on their last follow-up creatinine and estimated glomerular filtration rate values in those with or without these risk factors were not significantly different. For an ECD with a B2MG level of <7.18 and no history of RRT, kidney transplantation can be undertaken without considering the possibility of kidney discard.Kidney transplantations using expanded criteria donors (ECD) are being increasingly adopted, but no consensus tools are available to evaluate donor kidney status. Beta-2 microglobulin (B2MG) is a marker of kidney function, and herein, we evaluate the usefulness of assessing B2MG to evaluate donor kidney status. Fifty-seven kidney transplantations were performed from March 2017 to April 2019. Medical records were retrospectively reviewed, and relationships between clinical and laboratory variables and transplant outcomes were investigated. Thirty-eight patients received a standard criteria donor kidney and 19 patients an ECD kidney. Ten patients experienced delayed graft function (DGF), but no patient experienced primary nonfunction. Of the parameters studied, only donor renal replacement therapy (RRT) [odds ratio (OR) 24.162;  Renal replacement therapies such as dialysis or kidney transplantation (KT) are considered proper management options for patients with severe CKD or end-stage renal disease (ESRD), and KT is considered a better choice than dialysis in many respects3. Furthermore, as demand for KT increases, so does interest in the prognosis of patients with transplanted kidneys in terms of quality of life and cost-effectiveness.Chronic kidney disease (CKD) is becoming a major global health issue because of its prevalence and economic cost. Societal aging and the associated increases in the prevalence of hypertension and diabetes inevitably mean that the number of CKD patients will continue to increase5, but this has also inevitably increased discard rates6. Even patients fortunate enough to have undergone KT may experience primary nonfunction, delayed graft function (DGF), or rejection, and frequently, these conditions eventually result in poor graft outcomes or early graft failure. Furthermore, these conditions have been associated with prolonged hospitalization, higher costs, and mortality8.It is practically impossible to provide KT to all indicated patients due to donor shortages. To overcome this situation, kidneys from expanded criteria donors (ECDs) or donors after cardiac death (DCDs) have been used worldwideMany clinical and laboratory risk factors have been evaluated in attempts to predict the status of donor kidneys, but discard and DGF rates have not substantially changed. Accordingly, we considered the possibility of using novel predictors of kidney status and focused on assessing donor beta2-microglobulin (B2MG) serum level. It has been reported that the ability of serum B2MG to predict renal failure in the general population and CKD patients is unaffected by sex, age, or race. Also, the estimated glomerular filtration rate (eGFR) and calculated serum B2MG level have been reported to reflect renal function well. However, donor serum B2MG has not been evaluated in the context of the association between transplant outcomes and ECD and DCD kidneys.p = 0.523). The numbers of donors that received renal replacement therapy (RRT) in the SCD and ECD groups were 6 (15.8%) and 1 (5.3%), respectively (p = 0.405). Initial creatinine (Cr), final Cr, highest Cr values, urine and serum cystatin C, and urine and serum B2MG levels were not significantly different between the two groups. The baseline characteristics of donors in the two groups are displayed in Table Of the 57 recipients, 38 (66.7%) received a standard criteria donor (SCD) kidney and 19 (33.3%) an ECD kidney. Gender ratios and body mass indices (BMIs) were similar in the SCD and ECD groups. The frequency of acute kidney injury (AKI) > grade 2 was 11 (28.9%) in the SCD group and 4 (21.1%) in the ECD group (p = 0.469). Rates of biopsy-proven rejections were also similar. However, last follow-up median Cr (1.09 mg/dL) and eGFR (68.5 mL/min) were better in the SCD group than in the ECD group  . Baseline characteristics of recipients of SCD and ECD kidneys are shown in Table The demographic characteristics of the recipients, including age, gender, BMI, primary kidney disease, RRT type and duration before KT, were similar in the SCD and ECD groups, and the HLA mismatch and immunologic risk numbers, kidney weights, and cold ischemic times (CIT) were also similar. Ten recipients experienced DGF; 8 21.1%) in the SCD group and 2 (10.5%) in the ECD group  = 1.374 - 119.954] and donor B2MG level of ≥ 7.18 mg/L  were significant independent risk factors of DGF. Logistic regression analysis results are provided in Table Receiver operating characteristic (ROC) curve analysis showed donor final eGFR, highest Cr, serum cystatin C, serum B2MG level, and cold ischemic time significantly predicted DGF. Areas under curves (AUCs) of parameters are displayed in Table vs.7, 50%; p = 0.001). All living donor kidney recipients belonged to the group without a risk factor. Laboratory parameters including initial eGFR, highest Cr, final Cr, and final eGFR were much better in the group without risk factors than in the group with risk factors, and the prevalence of DGF and biopsy-proven rejection were lower in recipients without a risk factor. However, recipient last follow-up Cr and eGFR values were similar in the two groups. A summary of the results is provided in Table Contrary to the traditional SCD/ECD classification, DGF developed more frequently in those recipients with a risk factor  in all nucleated cells, and under the above conditions, a large number of cells may be generated and shed B2MG25. Furthermore, brain death induces a profound inflammation reaction, which influences multiple transplantable organs and causes kidney tubular damage by generating cascades of activated cytokines27. Ischemic-reperfusion injury can induce similar events. B2MG is an indicator of systemic inflammation, but not of kidney status, and the outcomes and functions of transplantable organs are significantly influenced by inflammation reactions. Thus, B2MG status, which is strongly correlated with GFR and inflammation, may be a meaningful marker of transplantable organ function, including kidney function. Most B2MG is reabsorbed in proximal tubules, and thus, a high level of urinary B2MG indicates tubule damage25. However, Cooper et al.28 reported that acute tubular injury did not affect transplant outcome. In the present study, urinary B2MG was not predictive of DGF, and the urinary B2MG level was not different between the SCD and ECD groups. In a previous study, the effectiveness of serum B2MG level as a biomarker of adverse outcomes in CKD was investigated. In patients with ESRD or chronic allograft damage after renal transplantation, a previous study reported that compared to urinary B2MG level, serum B2MG level predicted kidney status more accurately25. However, there are no reports evaluating donor kidney status or predicting the incidence of DGF by assessing serum B2MG levels. Even though our study was conducted at a single institution and on a small number of patients, serum B2MG showed good predictive power for DGF with RRT, and it reflected the condition of donors more accurately than those derived by using the SCD and ECD classification approaches.In this study, we reviewed several risk factors believed to result in DGF. Of these, only donor RRT and B2MG were observed to be related to the development of DGF. Donor RRT means that the donor has a kidney with an AKI > grade 3 according to the Kidney Disease Improving Global Outcomes (KDIGO) definition. Even though many studies have reported that KT using an ECD with AKI have outcomes comparable to those using an ECD without AKI or SCD, subgroups that needed RRT or showed Cr level elevation showed poor outcomes29. If identification of DGF risk factors before transplantation were possible, recipient outcomes would be improved, and donors would have an additional incentive to prevent kidney discard. The therapeutic interventions used to prevent DGF have been widely investigated and reviewed. A recent Cochrane review showed that machine perfusion provides a better means of preserving donor kidneys than cold storage in terms of preventing DGF and improving outcome30. Modifications of immunosuppressant agents have been investigated, and the selection of induction agents is a well-debated issue. Rabbit anti-thymocyte globulin (rATG) produced better results than interleukin-2 receptor blockers in some studies32, but other studies produced contradictory results33. In the present study, rATG was used to a greater extent in the recipients with risk factors group, and its use was not associated with reductions in DGF and rejection rates. However, final creatinine and eGFR levels in the recipients with risk factors group were similar to those in the recipients without risk factor group despite the presence of DGF and rejection. Even though machine perfusion can produce better results, many institutions cannot use machine perfusion due to resource limitations. Thus, modulation of immunosuppressant agents may be a more realistic option in many situations.DGF is not an uncommon complication during the period immediately following transplantation and is associated with prolonged hospitalization, increased cost, and the possibility of graft failureIn conclusion, we increased the use of ECDs and DCDs to address donor shortage, which means we now attach greater importance to detecting patients at risk to prevent DGF and improve long-term outcomes. In this regard, it is important to find kidney function indicators that can be quickly and repeatedly assessed. Although our study was conducted on a small cohort at a single institution, the results show that if serum B2MG is <7.18 mg/L and therapeutic, and there is no history of donor RRT, KT from an ECD with AKI can be performed without the need to consider kidney discard.From March 2012 to July 2019, 135 patients underwent KT at Wonju Severance Christian Hospital, Wonju, South Korea, with all transplants performed by a single surgeon. B2MG samples were prospectively obtained beginning in March 2017, and the present retrospective study was performed using the data of 57 patients that registered from March 2017 to April 2019. This study was approved beforehand by the Institutional Review Board of Yonsei University Wonju Severance Hospital (CR318083). The requirement for informed consent was waived because of the retrospective nature of the study. All studies were conducted according to the principles of the Declaration of Helsinki. All living and deceased donors were evaluated for infections of hepatitis B or C virus  or human immunodeficiency virus (HIV) before transplantation. Transplantation was conducted in cases when donors did not show evidence of HBV, HCV, and HIV infections. All procurements and transplantations were performed by surgeons of our departments who were registered into Korean Network for Organ Sharing (KONOS), under the permission of KONOS. There is no procurement from prisoners.34. SCD, ECD, DGF, and acute AKI were defined as we described in a previous article13.Samples of blood and urine to assess serum cystatin C, creatinine, B2MG, and urine cystatin C levels were collected and evaluated one day before procurement of a deceased donor or living donor kidney. B2MG level was measured by enzyme-linked fluorescent assay (ELFA) using a Vidas analyzer . The eGFR was calculated using the Modification of Diet in Renal Disease four4-variable equationRecipients at immunologic risk were defined as those that had undergone a second or ABO-incompatible KT, had a panel reactive antibody level > 20%, or had received a kidney from a deceased donor with an AKI grade > 3.13 with the exception that corticosteroid administration was started as 500 mg of methylprednisolone followed by dose tapering as previously described13. We checked cytomegalovirus (CMV) and BK virus infection status repeatedly. CMV infection status was checked postoperatively at 1 day, 1 week, 1 month, and 3 months. The BK virus status was checked postoperatively every three months for 1 year and then every 6 months. If quantitative CMV and BK virus test results were positive, we performed qualitative tests. As needed, oral valganciclovir was used to treat CMV infection, and a reduced level of immunosuppressive agents was applied for BK virus treatment.When neither donor nor recipient had any risk factor, we chose basiliximab as the induction agent. Briefly, 20 mg of basiliximab were injected on the day of the operation and again at four days after surgery. However, when a recipient was deemed high immunologic risk, rATG was the induction agent and was administered at 4.5 mg/kg/day in the first three days. We used a triple regimen of tacrolimus, corticosteroid, and mycophenolate mofetil as the maintenance immunosuppressant agents. Trough levels of tacrolimus and mycophenolate mofetil dosage were controlled as we previously describedThe baseline characteristics of SCDs and ECDs were compared and their effects on transplantation outcomes were investigated. Because there was no case of primary nonfunction, we performed analysis to identify risk factors for DGF. We also compared recipient baseline characteristics were compared and transplantation outcomes analyzed according to the absence or presence of risk factors.t-test, respectively. Cut off values of factors predictive of DGF were determined by performing receiver operating characteristic (ROC) analysis. Variables significantly associated with DGF were identified by univariate logistic regression analysis. Multivariate analyses were performed using the logistic regression analysis posterior Wald test.Statistical analyses were performed using SPSS Ver. 25.0 . Continuous variables are presented as medians and ranges, and categorical variables as numbers and percentages. Categorical and continuous variables were compared using Fisher’s exact t test and the


Parents influence their infants’ diets and are the providers of healthy foods such as fruit and vegetables. Parental motives can influence infant’s diets directly or through parental feeding practices.This study aimed to assess the associations between parental food choice motives and infants’ fruit and vegetable intakes and to examine whether parental feeding practices mediated these associations.A total of 298 parents participated in the Norwegian Food4toddlers study. Before the child’s first birthday (mean age = 10.9 months), the parents completed an online baseline questionnaire. Five parental food choice motives were assessed: health, convenience, sensory appeal, price, and familiarity. Infants’ fruit and vegetable intakes and three health-promoting feeding practices were also assessed. For each food choice motive and its relation to fruit or vegetable intake, three single mediation models were conducted. Mediation effects were examined using MacKinnon’s product of coefficients procedure, and bootstrap confidence intervals (CIs) were used for inferential testing.τ = 0.394, P < 0.001). No other significant associations were found between food choice motives and fruit or vegetable intake. The feeding practice of shaping a healthy environment mediated the relationships between health motive and both fruit  and vegetable  intakes. The feeding practice of encouraging balance and variety mediated the relationships between health motive and vegetable  intake and between sensory appeal motive and vegetable intake .Higher scores on the motive of health were positively associated with infants’ vegetable intake (High levels of parental health motive are associated with higher infant vegetable intake. Our study contributes to understand the structure of parental feeding behaviors that may have implication for nutrition interventions targeting parents. The parental food choice motive of health is associated with higher infant vegetable intake.Health-promoting feeding practices mediate the relationships between the parental food choice motives of health and sensory appeal and their infants’ fruit and vegetable intakes, and the feeding practices of shaping the environment and encouraging balance and variety are the strongest mediators on these associations.The findings contribute to the understanding of parental feeding behaviors.Food is fundamental for health, growth, and development and also plays a central part in the increasing childhood obesity rates . Eating Fruit and vegetables are valuable sources for a wide range of micronutrients, fiber, and antioxidants and important for growth and development , 8. FurtParents of infants play a key role in what their children eat. They provide food and shape the food and eating environment for their children –17. The coercive control practices  and how they affect children  and infants’ fruit and vegetable intakes. Further, we aimed to examine the potential mediating effects of three health-promoting feeding practices  on these associations.This study used baseline data from the Food4toddlers randomized controlled intervention study. Food4toddlers is a digital intervention aiming to promote healthy dietary habits among toddlers  months . The recParents of infants in Norway were recruited through tailored advertisement  on social media (Facebook). In Norway, 67% of the population uses Facebook daily . The FacApproximately 1–2 months before the infant’s first birthday, those who signed up for the study received an email with a link to the baseline questionnaire. Data were collected using SurveyXact, an online survey software tool. The protocol for the present study was approved by the Norwegian Centre for Research Data  and is in accordance with the Helsinki Declaration of 1975, as revised in 2008.We recruited 404 parents of infants through Facebook. One to two months before each infant turned 1 year old, a baseline questionnaire was sent to the parents. A total of 298 (response rate 73.8%) parents who originally signed up for the study answered more than half of the questions in the baseline questionnaire and were included in the present analyses.Most participants were mothers (98.0%), and the mean age was 31.7 years (standard deviation [SD] = 4.2). Most parents lived in two-adult households (99.0%), 86.7% of the parents were born in Norway and the majority of participants were from Eastern Norway (43.3%), which has the densest population. See Each participant reported their age, the age of the child, the number of persons in the household, the county of residence, and their own level of education. These items have previously been used and tested in Norway . The parThe Food Choice Questionnaire (FCQ) was used to assess parents’ motives underlying their selection of food. Developed by Steptoe et al. , the FCQThe FCQ comprises 36 items grouped into nine factors , and responses to the original FCQ were on a four-point scale . Fotopouα).In the questionnaire, participants were asked to rate their level of endorsement of statements such as ‘It’s important to me that the food I eat on a typical day […]’, rating each statement from 1 (extremely unimportant) to 7 (extremely important) . The relα values presented are for our sample): health , convenience , sensory appeal , price , and familiarity . These five factors were included in the baseline questionnaire for the Food4toddlers intervention because they were regarded as important precursors for the development of a healthy food and eating environment for toddlers. The Cronbach’s α values for this study were slightly lower than those reported by Pollard et al. (familiarity) and higher for three out of five items  compared with the study of Fotopoulos et al. , showed acceptable-to-excellent correlations for the factors used .We did not perform a full-scale reproducibility study; however, in October 2018, the items were tested for reproducibility through a test–retest study at two time points (2 weeks apart) with 29 participating parents who did not participate in the intervention recruited from several local kindergartens. The standardized measure, Pearson’s correlation coefficient . Similar recoding has previously been used by others , and there was also the additional item of ‘other fruits/vegetables’. The reported weekly consumption scores for these items were aggregated into sum scores and divided by seven. Results for fruits (11 items) and vegetables (13 items) showed the daily frequency of fruit and vegetable intakes.disagree to agree or from never to often. The CFPQ has been validated and tested for reliability for parents of children in different age groups  . The CFPe groups –38, incle groups .α values presented are for our sample): encouraging balance and variety , shaping a healthy environment , and healthy modeling .Of the 12 subscales, five can be considered healthpromoting feeding practices. We investigated three of these  and the exposure variables of interest (food choice motives and feeding practices) to assess the need to control for demographic variables in later analyses. No significant associations were found, so no covariates were included.n = 5,000)  –42.τ) between food choice motives (predictor variables) and fruit and vegetable intakes (outcome variables) were calculated by regressing the outcome variables on each food choice motive .The overall associations (path α). Second, the conceptual theory test estimates the association between each potential mediator  and the outcome variables, adjusted for the predictor variables (path β). The indirect effect was calculated by multiplying the α-coefficient by the β-coefficient. Bootstrap CIs  were used instead of the SOBEL test (which tests the significance of a mediation effect) to conduct the inferential tests of the indirect effects, as recommended by Hayes (αβ) by the overall association (τ-coefficient) (αβ and τ’ should have the same sign (τ) is not a necessary condition for mediation  . A samplficient) . In addiame sign . A signiediation , 44.All analyses were conducted using IBM SPSS Statistics , Version 25. Hayes’ Process 3.1 for SPSS  was usedMean daily servings of fruits and vegetables consumed by the infants were quite high at 2.85 (SD = 1.60) and 3.15 (SD = 1.59), respectively see . Food chThere were high scores on all measured motives except familiarity. In terms of the examined feeding practices, balance and variety  had the highest score.τ = 0.394, P < 0.001)  see . The effα = 0.136, P < 0.001), environment , and modeling . All relationships were in the expected positive direction, with greater values for health food choice motive associated with higher scores on these feeding practices. The food choice motive of sensory appeal was also positively associated with balance and variety  and modeling . The food choice motives of convenience, price, and familiarity were not significantly associated with any of the feeding practices.The results from the single mediation analysis are shown in β shows the associations between health-promoting feeding practices and fruit and vegetable intakes, adjusted for the food choice motives. When adjusted for the motive of health, the conceptual theory tests revealed that the feeding practice of environment was associated with fruit intake , whereas both environment  and balance and variety  were related to infants’ intake of vegetables.As When adjusted for the other food choice motives , the tests revealed that environment was associated with fruit intake, whereas all three practices were associated with vegetable intake. All statistically significant relations were positive, such that greater scores on these feeding practices were associated with a higher intake of fruits or vegetables among the infants.αβ = 0.085, CI: 0.030–0.150) and environment  mediated the relationship between health motive and vegetable intake  emerged as a mediator in this relationship. However, the effect size was small.Despite the lack of a direct significant association between health motive and fruit intake see , the feeαβ = 0.047, CI: 0.005–0.103) and modeling  mediated this relationship. The effect sizes were again small.No significant association was observed between sensory appeal motive and vegetable intake, but balance and variety  as a recruitment channel, participants from the entire country  could beAn important limitation of this study involves its cross-sectional design, which hindered causal interpretation of the findings . Some quRegarding the generalizability of our findings, participants were more highly educated, compared with national figures . In addiOur results confirm previous findings on the importance of health motives for infant and children’s vegetable consumption. We also see that the health-promoting feeding practices assessed mediate associations between some food choice motives  and fruit and vegetable consumption, but not to a large degree.Health-promoting feeding practices may mediate associations between parental characteristics other than food choice motives, such as knowledge, attitudes, and general parenting style, and infants’ fruit and vegetable intakes. Such associations should be examined in further studies to identify which feeding practices and potential predictors of these feeding practices should be targeted in interventions to enhance the intake of fruits and vegetables among infants and toddlers.Our results contribute to understand the underlying motives of parental feeding behaviors in this age group. The results may be different investigating older children because, for example, the family interaction and tastes changes by age. Continuing to study the interplay between infant’s food intake and parents’ motives and practices about healthy and unhealthy eating behaviors is an important endeavor.NCØ, ERH, and FNV conceived the study. MR, ERH, FNV, and NCØ initiated and designed the study and developed the intervention. MR performed the data collection, supervised by ERH, FNV, and NCØ. MR performed the analysis, supervised by WVL and NCØ. MR drafted the manuscript with substantial input from ERH, FNV, WVL, and NCØ. All authors contributed to read and approved the final version of this manuscript.The authors declare that they have no competing interests. This study is funded by the University of Agder. The financial contributor was not involved in designing the study, collection, analyses, and interpretation of data or in writing the manuscript.


Acanthamoeba are widely distributed in the environment and are known to cause blinding keratitis and brain infections with greater than 90% mortality rate. Currently, polymerase chain reaction (PCR) is a highly sensitive and promising technique in Acanthamoeba detection. Remarkably, the rate of heating–cooling and convective heat transfer of the PCR tube is limited by low thermal conductivity of the reagents mixture. The addition of nanoparticles to the reaction has been an interesting approach that could augment the thermal conductivity of the mixture and subsequently enhance heat transfer through the PCR tube. Here, we have developed hexagonal boron nitride (hBN) nanoparticle-based PCR assay for the rapid detection of Acanthamoeba to amplify DNA from low amoeba cell density. As low as 1 × 10−4 wt % was determined as the optimum concentration of hBN nanoparticles, which increased Acanthamoeba DNA yield up to ~16%. Further, it was able to reduce PCR temperature that led to a ~2.0-fold increase in Acanthamoeba DNA yield at an improved PCR specificity at 46.2 °C low annealing temperature. hBN nanoparticles further reduced standard PCR step time by 10 min and cycles by eight; thus, enhancing Acanthamoeba detection rapidly. Enhancement of Acanthamoeba PCR DNA yield is possibly due to the high adsorption affinity of hBN nanoparticles to purine (Guanine—G) due to the higher thermal conductivity achieved in the PCR mixture due to the addition of hBN. Although further research is needed to demonstrate these findings in clinical application, we propose that the interfacial layers, Brownian motion, and percolation network contribute to the enhanced thermal conductivity effect. Acanthamoeba is a free-living protozoan widely found in the environment. Despite being ubiquitous, they can survive as parasites to proliferate within human or animal tissues leading to two major life-threatening infectious diseases known as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK) that often arise as mild headaches . PC. PCAcanttellanii . PCR ass−8 wt % hBN). To determine optimum hBN concentration, nano-PCR involved PCR protocol parameters, as described previously [Acanthamoeba, nano-PCR used hBN optimum concentration to test the effects of PCR parameters . Hence, we reduced temperatures in denaturation, annealing, and extension over a range of 5 °C each, while keeping the step and cycle time constant as previous [Acanthamoeba detection. After PCR and nano-PCR, 5 µL of equal PCR products were electrophoresed along with a reference DNA ladder of 1 kB  on 1% agarose gels with 1× TAE buffer and stained with ethidium bromide. Gel electrophoresis of PCR products ran at 100 V for 45 min. PCR products were visualized under UV illumination [For nano-PCR, hBN nanoparticles were added into PCR reactions to enhance the amplification of T4 genotype DNA using various hBN concentrations . Error bars were included using the function “standard error” in the Excel spreadsheet.−1. This indicates good quality of hBN used in this study involving the vibration mode of its intralayer E2g. The FESEM image of hBN shown in Convective heat transfer is a function of the thermal conductivity of the fluid. The thermal conductivity measurements on nanofluids have shown that the conductivity is inversely proportional to the size of the nanoparticle. Other possible reasons for enhanced thermal conductivity include Brownian motion of particles, molecular level layering at the liquid-particle interface, the formation of percolation network, and nanoparticle clustering . Figure Acanthamoeba T4 genotype DNA were observed for PCR bands in various cell numbers of 1000, 5000, 10,000, 20,000, and 100,000, which obtained the expected 950 bp amplicon  [Although the precise mechanism of action between hBN nanoparticle and PCR reagents requires further study, enhancement of anine—G) , which ianine—G) . Polar hanine—G) . The entAcanthamoeba detection and can be useful for clinical applications or water companies in rapid detection of Acanthamoeba. An optimal concentration of hBN, 0.4–1 × 10−8 wt % increases the DNA yield. The fidelity of PCR with hBN is a function of its concentration. The addition of hBN significantly helps in reducing the reaction step time, number of cycles, and temperature without compromising the yield with respect to positive control. Enhanced thermal conductivity induced adsorption and subsequent desorption of DNA over hBN is proposed to be the underlying mechanism of the enhancements observed in this study. However, it requires further investigation, particularly from the better PCR yield quantification point of view.In conclusion, hexagonal boron nitride (hBN) nanotechnology-based PCR improves PCR DNA yield in


Chlorella sp. or Nannochloropsis sp. in plant-based diets on antioxidant mechanisms of European sea bass (Dicentrarchus labrax) juveniles. For this purpose, three isoproteic (50%) and isolipidic (19%) diets were formulated: a practical diet, containing 15% fish meal (FM) and plant ingredients as the protein source and a mixture of fish oil and vegetable oils (40: 60) as lipid source (control diet); and two diets identical to the control but with the FM replaced by Nannochloropsis sp. or Chlorella sp. . The diets were offered to quadruplicate groups of 25 fish  for 11 weeks and then enzymatic and non-enzymatic antioxidant mechanisms and lipid oxidative biomarkers were assessed in the liver and intestine of these fish. Results showed that the antioxidant response was tissue-dependent, with the liver exhibiting lower glutathione peroxidase and glucose-6-phosphate dehydrogenase (only in Chlo group) activities, and intestine lower superoxide dismutase activity with the diets including microalgae compared to control diet. An increase of oxidized glutathione content was also observed in the intestine of fish fed the microalgae diets. Catalase and glutathione reductase activities, oxidative stress index, and total and reduced glutathione, were unaffected by dietary treatments in both tissues. Overall, the lipid peroxidation status was not compromised by the replacement of FM by microalgae.This study aimed to evaluate the effects of including microalgae  Finding sustainable ingredients alternative to the finite world fisheries products remains a major issue for the sustained growth of aquaculture . Plant fn−3 long-chain polyunsaturated fatty acids (LC-PUFA), are particularly vulnerable to ROS attack, which can lead to lipid peroxidation (LPO) –derived directly as by-products of aerobic metabolism, fatty acids peroxidation, or promoted by external factors –exceeds that of ROS removal, thus inducing tissue oxidative damage . Fish, bon (LPO) –9. To pron (LPO) .Tetraselmis sp., Nanofrustulum sp., and Tisochrysis sp.) proved adequate to partially replace FM in diets for carnivorous fish species without negative effects on growth performance  and lipid (10 to 20% DM) levels, as well as proper amino acid and fatty acid composition , the inclusion of up to 5% of Nannochloropsis sp. in diets, replacing 7.8% of FM, enhanced the fish antioxidant capacity, by decreasing serum LPO and increasing serum and liver SOD and GPX activities , replacement of 20% of dietary FM with defatted Desmodesmus sp. or Nannochloropsis oceania biomass did not affect serum SOD and CAT activities  dietary inclusion of 10–15% defatted Chlorella vulgaris did not affect plasmatic GPX and SOD activities but increased CAT activity  and focused on serum or liver antioxidant –21. Up tChlorella sp. or Nannochloropsis sp. in practical diets for the European sea bass (Dicentrarchus labrax), an economically important carnivorous fish species of Mediterranean aquaculture.Thus, the present study aimed to evaluate the effects on the intestine and liver oxidative system of including the microalgae Chlorella and Nannochloropsis were the microalgae chosen for this study as they are amongst the most used in aquaculture feeds as they have high protein content . It was directed by certified scientists , and all procedures were conducted according to the Federation of Laboratory Animal Science Association (FELASA) recommendations and the EU Directive (2010/63/EU) on the protection of animals for scientific purposes.Chlorella sp. (diet Chlo) or Nannochloropsis sp. (diet Nanno). Dietary protein content was adjusted by manipulating wheat meal and faba beans levels.Three experimental diets were formulated to contain 50% protein and 19% lipids. Also, diets were formulated to fulfill the species aminoacids requirements and to be isoaminoacidic. A control diet was formulated to include circa 20% protein from FM and 80% from PF, and a fish oil/vegetable oil mix (40: 60) as lipid source. This diet was formulated to be a practical diet as current commercial diets for European sea bass already includes only 15% FM. To test the feasibility of total replacement (15%) of dietary FM by microalgae, two other diets were formulated similar to the control but without FM, which was replaced by The lyophilized microalgae were provided by Buggypower, Lda.  and were produced in a marine environment and in a completely autotrophic closed production system called photobioreactors. Biomass was collected, centrifuged, freeze-dried, vacuum stored in plastic bags, and refrigerated until used. Microalgae composition is detailed in Dicentrarchus labrax) juveniles with average initial body weight (IBW) of 24 ± 1 g were obtained from a commercial hatchery  and kept in quarantine for 3 weeks. During this period fish were fed a commercial diet .The trial was carried out at the experimental facilities of the Marine Zoology Station, Porto University. European sea bass . The photoperiod regime adopted was 12:12 h light: dark, provided by artificial illumination.At the end of the trial, three fish from each tank were randomly collected 3 h after the last meal as described in Couto et al. , and eutN × 6.25) by the Kjeldahl method after acid digestion using a Kjeltec digestion and distillation units ; gross energy by direct combustion in an adiabatic bomb calorimeter ; and lipid content by petroleum diethyl ether extraction . Fatty acid methyl esters were prepared by transmethylation of total lipids extract by adding sodium methylate reagent and sequentially esterification with boron trifluoride in methanol according to Bondia-Pons et al.  were diluted to 1:9 and 1:6, respectively, and homogenized at pH 7.8 in ice-cold 100 mM Tris–HCl buffer containing 0.1 mM EDTA and 0.1% (v/v) Triton X-100. Homogenates were centrifuged at 30,000 × g for 30 min at 4°C and the resultant supernatants were separated in aliquots and stored at −80°C for further enzyme assays. All procedures were performed on ice and all enzyme activities were measured at 37°C in a Multiskan GO microplate reader .Liver and intestine samples  activity was measured at 550 nm by the ferricytochrome C method using xanthine/xanthine oxidase as a source of superoxide radicals ; catalasFor SOD, one unit of enzyme activity was defined as the amount of enzyme necessary to produce 50% inhibition of the ferricytochrome C reduction rate. All other enzyme activities were expressed as units (CAT) or milliunits  per milligram of hepatic soluble protein (specific activity). One unit of enzyme activity was defined as the amount of enzyme required to transform 1 μmol of substrate per minute under the assay conditions.Malondialdehyde (MDA) concentration was used as a marker of LPO level in the liver and intestine, following the methodology described by Buege and Aust . An aliqLiver and intestine portions were homogenized , in an ice-cold solution containing 1.3% 5-sulfosalicylic acid (w/v) and 10 mM HCl, and the whole procedures were undertaken on ice to avoid glutathione oxidation. Homogenates were centrifuged at 14,000 × g for 10 min at 4°C and the resulting supernatants stored at −80°C. Total glutathione (tGSH) and oxidized glutathione (GSSG) were measured according to Pérez-Jiménez et al. . Standarp ≤ 0.05) were determined by the Tukey's multiple range test. Statistical analyses were performed using SPSS for Windows version 25 software package.Data are presented as mean and SEM. All data were checked for normality and homogeneity of variances and normalized when appropriate. Data were analyzed by one-way ANOVA. Significant differences among means (n−6 polyunsaturated fatty acid (PUFA) content and an n−6/n−3 ratio than the control  as well as in voluntary feed intake  and feed efficiency .The antioxidant enzyme activities, LPO value, glutathione content, and OSI were significantly different between the liver and the intestine. Moreover, there were significant interactions between target tissues and diets and therefore data are presented separately for each tissue.Liver antioxidant enzyme activities are presented in Intestine antioxidant enzyme activities are presented in LPO was shown to be a sensitive biomarker of increased oxidative stress as it is a major result of oxidative damage induced by different types of stressors, including nutritional factors .Nannochloropsis sp. replacing 15.5% of FM led to a reduction in plasma and liver LPO contents. The authors speculated that such protective effects of dietary microalgae against LPO in serum and liver were related to the presence of various classes of natural antioxidants  in these ingredients  as a reducing agent, leading to the production of its oxidized form (GSSG) which is then regenerated to GSH by GR. In this process, NADPH is oxidized. NADPH is produced in the pentose phosphate pathway, being G6PDH the rate-limiting enzyme in the process  fed practical diets supplemented with 1.5% of Nannochloropsis gaditana  sufficed to prevent increased oxidative damage. This may probably represent a less energy-demanding process. Thus, the lack of induction of the antioxidant enzyme mechanisms may be regarded as potentially beneficial for fish fed microalgae diets.Our results also showed that the antioxidant defense strategies were tissue-dependent, possibly due to the different susceptibility to oxidative stress of each organ. The intestine showed an overall higher peroxidation potential (higher LPO content) than the liver, possibly due to its high cell turnover and continuous exposition to potentially toxic or allergenic compounds from the diets (such as ANF) that are prone to ROS . At the On the other hand, while the intestine is a major consumer of GSH, the liver is thought to be the main organ for GSH synthesis and export , 40. MorChlorella sp. or Nannochloropsis sp. modulate the liver and intestine antioxidant enzyme response without detrimental effects on overall lipid oxidative damage. These results are encouraging for understanding the potential role of microalgae in modulating fish redox state in the context of using microalgae as alternative protein sources in sea bass diets. But, future work should consider a deep characterization of bioactive compounds in microalgae biomass in order to identify the biological activity of these compounds and ascertain the mechanisms involved in the microalgae-modulator role in fish antioxidant mechanisms.Overall, the results of the present study indicate that dietary replacement of FM by The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by ORBEA  of CIIMAR and the Portuguese National Authority for the Animal Health (DGAV).CC and FC carried out the main experimental work and wrote the draft of the manuscript under the direction of the project designer and leaders AC and AO-T. PI supplied microalgae biomass. All authors contributed to the article and approved the manuscript.PI was employed by the company Buggypower Lda. The remaining 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.


Foveal and parafoveal retinal thicknesses were significantly related to parafoveal DCP density (p = 0.007 and p < 0.001). Foveal and parafoveal retinal thicknesses, foveal SCP and parafoveal DPC densities were significantly reduced in CD patients compared to controls . PERG and mfERG amplitudes were significantly reduced in CD patients compared to controls (p < 0.01). Conclusions: CD eyes showed reduced retinal thickness significantly related to reduced vessel density, possibly caused by a decreased metabolic demand. In addition, vessel density significantly correlated with loss of function. Background: This study investigates the relationship between retinal vascularization and macular function in patients with cone dystrophies (CDs). Methods: Twenty CD patients (40 eyes) and 20 healthy controls (20 eyes) were enrolled in this prospective case-control study. Patients underwent full ophthalmological examination, microperimetry, full-field, pattern and multifocal electroretinogram  and optical coherence tomography angiography (OCTA). Main outcome measures were as follows: foveal and parafoveal inner and outer retinal thickness; microperimetry sensitivity in the central 4° and 8°, ERG b wave amplitudes and peak times, PERG P50 and N95 amplitudes and latencies, and mfERG N1 to P1 amplitudes; and superficial capillary plexus (SCP), deep capillary plexus (DCP) and choriocapillary (CC) plexus vessel densities, divided into foveal and parafoveal region. Results: Retinal thickness, SCP and DCP densities were significantly related to PERG. A significant relationship was found between foveal and parafoveal retinal thicknesses and foveal SCP density ( Cone dystrophies (CDs) are a group of rare inherited pathologies that are characterised by cone dysfunction and loss. No explicit classification of CDs is available because some authors use this term to describe only progressive forms, while others use it to describe both stationary and progressive forms ,2. AccorFull-field electroretinogram (ERG) is considered the gold standard for diagnosis of CDs, revealing marked depression of cone responses with normal rod function or possible late rod involvement in CDs . RetinalOCT angiography (OCTA) is a new dyeless retinal imaging technique that assesses retinal and choroidal microvasculature. It has already been used to investigate vasculature alterations in the superficial capillary plexus (SCP), deep capillary plexus (DCP) and choriocapillaris (CC) in several retinal vascular diseases and hereditary retinal photoreceptor degeneration diseases, such as retinitis pigmentosa ,9,10. The aim of our study was to investigate the status of retinal and CC microvasculature and the possible relationship between retinal vascularization and function using OCTA, ERG, multifocal ERG (mfERG) and pattern ERG (PERG) in a cohort of patients with progressive forms of CDs.Twenty patients with diagnoses of CD confirmed by genetic analysis, clinical features at fundus observation, electrophysiological evidence, FAF, and spectral-domain OCT (SD–OCT), followed by the Electrophysiology Service of the Ophthalmology Clinic, Department of Medicine and Science of Ageing, University “G. d’Annunzio” Chieti- Pescara, and by the Dystrophy Unit, Ophthalmology Department, Ospedale San Raffaele, Milan, were enrolled in this retrospective case series. Characterization of allelic variants was investigated by means of a targeted next-generation sequencing (NGS) panel.Clinical diagnostic criteria for CD diagnosis were as follows: possible evidence of Mendelian inheritance; visual symptoms such as decreasing central visual acuity, photophobia, dyschromatopsia and nystagmus; reduced photopic responses at ERG flash; bull’s-eye maculopathy or macular retinal pigment epithelium (RPE) alterations at fundus examination; “bull’s-eye” ring of hyperautofluorescence or a geographic zone of hypoautofluorescence at FAF, central thinning with central or paracentral EZ attenuation or loss at OCT.Exclusion criteria were as follows: (1) best-corrected visual acuity (BCVA) lower than 1.0 logarithmic minimum angle of resolution (logMAR); (2) any retinal dystrophy or retinal disease other than CDs; (3) any optic neuropathy, including glaucoma, or any condition increasing the risk of secondary glaucoma; (4) intraocular pressure (IOP) greater than 21 mmHg; (5) medium lens opacities (according to Lens Opacities Classification System).The study was approved by the Institutional Review Board of both ophthalmologic centres and adhered to the tenets of the Declaration of Helsinki, with written informed consent signed for all patients willing to participate in the study (2019/CD01).Patients underwent full ophthalmologic examination, including BCVA evaluation, using Early Treatment Diabetic Retinopathy Study (ETDRS) charts, Goldmann applanation tonometry, slit-lamp biomicroscopy and indirect fundus ophthalmoscopy. In addition, all patients underwent full-field ERG, mfERG and PERG with the Retimax , microperimetry (MP) by means of an MP-1 Microperimeter , SD OCT  and OCTA .Color vision assessment was performed using the Ishihara pseudoisochromatic plates and the Farnsworth Munsell 100-hue test. ERG, PERG and mfERG were recorded for each patient according to the International Society for Clinical Electrophysiology of Vision (ISCEV) protocols ,12,13. TP50 and N95 amplitudes and latencies on PERGs were analysed for each patient. Furthermore, we analysed the ratio between N95 and P50 amplitudes (N95/P50). In the mfERG, the ocular fundus was segmented by an array of 61 hexagons testing a central retinal area of 25°, and average responses for the implicit times and amplitudes of N1 (first negative component) and P1 (first positive component) of the first-order kernel were calculated for five regional ring groups (R1 to R5). Amplitude was measured from the baseline to the trough (N1) or the peak (P1) of the deflection. N1 to P1 amplitudes (from the first negative to the first positive peak) on mfERGs in the five recorded rings were considered for analysis. Patients were instructed to look at a fixation point incorporated into the stimulus dome for ERG or to the centre of a cross on a video display for PERGs and mfERGs to ensure accurate execution of the exam. Correct fixation was monitored with the help of an infrared CCD camera positioned in the ganzfeld stimulator during ERG or by direct observation of the patient fixating on the video display during PERG or mfERG acquisition.2) in 1-dB steps, and the duration of each stimulus was 200 ms. To assess central macular retinal sensitivity, differential light threshold values were compared by calculating the mean of the central 4° and 8° of the macular area, which was averaged automatically by the MP-1 software programme for the mean sensitivity in a polygon. To assess fixation, fundus movements were tracked during the examination, and where the patient gazed on the fixation target and the fixation pattern were assessed. To evaluate fixation location, the fixation target centred on the fovea was defined. Fixation location was expressed as a percentage of fixation points located within the 2° and 4° central areas. Eyes with >50% of the preferred fixation points located within the central area were classified as having predominantly central fixation. Eyes with >25% but <50% of the preferred fixation points located within the central area were classified as having poor central fixation. Eyes with <25% of the preferred fixation points located within the central area were classified as having predominantly eccentric fixation.Microperimetry was performed by means of the MP-1 microperimeter. All patients were dilated with tropicamide 1% eye drops, and after pre-test training, 5 minutes were allotted for adaptation to the dark. This test is routinely carried out with an automated eye-tracking system, which provides real-time compensation for eye movements and allows improved presentation of a stimulus at a predefined retinal location. During the test, the patient was encouraged to fixate on a red cross target, 2° in diameter, on a white monochromatic background at 4 asb. Then, retinal sensitivity was tested using a customised radial grid centred on the fovea with 77 Goldman III stimuli covering the central 20°. The stimulus intensity ranged from 0 dB to 20 dB .Vascular retinal layers were visualised and segmented as previously described in the SCP, DCP and CC .The projection-resolved algorithm was used to remove projection artifacts from the inner vascular plexus in the deep vascular plexus. This algorithm retains flow signals from blood vessels while suppressing projected flow signals in deeper layers. Images were reviewed by two investigators (LT and MBP) for segmentation accuracy; if segmentation errors were observed, then they were corrected using the segmentation and propagation tool from AngioVue . Final images were reviewed again to confirm segmentation placement in all B-Scans.Objective quantification of vessel density was carried out for each eye using SSADA software . A quantitative analysis was performed on the OCTA en-face images for each eye using AngioVue software as previously described [Vessel densities of the SCP, DCP and CC were evaluated in the foveal and parafoveal areas. Vessel density was defined as the percentage of the area occupied by vessels in a circular region of interest (ROI) of 3 mm in diameter positioned on the centre of the foveal avascular zone and including the foveal area (1 mm of diameter) and the parafoveal area, which constitute the remaining part inside the ROI.Two independent observers subjectively examined vessel anomalies inside the 3 × 3 mm ROI on OCTA and evaluated vessel caliber , the vessel course  and density  of the superficial and deep retinal capillaries.Foveal macular thickness (MT), parafoveal MT, parafoveal MT in the superior and inferior hemi-macular areas (S-Hemi MT and I-Hemi MT) from the ILM to the retinal pigment epithelium (ILM-RPE) (whole retina), parafoveal MT in the S-Hemi and I-Hemi areas from the ILM to the IPL  and parafoveal MT in the S-Hemi and I-Hemi areas from the IPL to the RPE (outer retina) were automatically calculated by software on OCTA 3 × 3 mm volume scans. A circular ROI centred on the Foveal Avascular Zone (FAZ) with a diameter of 3.0 mm was used for retinal thickness analysis: the central foveal area (1 mm in diameter) and the parafoveal area constituted the remaining part inside the ROI .The acquisition protocol for SD–OCT  included a horizontal and vertical B-scans centered on the fovea with enhanced depth imaging (EDI) mode in all patients.Subfoveal choroidal thickness (SFCT), measured vertically from the outer border of the RPE to the inner border of the sclera, was measured using the inbuilt manual caliper on EDI–OCT scans.The main outcome measures were foveal and parafoveal retinal thicknesses in the inner (from the ILM to the IPL), outer (from the IPL to the RPE) and whole (from the ILM to the RPE) retina; SCP and DCP densities and CC density, which were tested in the whole retina and the foveal and parafoveal areas; FAZ area; dark-adapted 0.01 ERG, dark-adapted 3.0 ERG and light-adapted 3.0 ERG b wave amplitudes and latencies and light-adapted 30 Hz flicker ERG; PERG P50 and N95 amplitudes and latencies; the ratio between N95 amplitude and P50 amplitude (N95/P50); mfERG N1 to P1 amplitude in the five recorded rings; retinal sensitivity on microperimetry at 4°, 8° and 20°; and fixation location within the 2° and 4° central areas. Correlation between morphological and functional parameters were evaluated in patients with CD.n = 20, an effect size of 0.4 and an alpha error rate of 0.05.Power analysis was performed for testing the regression between retinal vascularization and function parameters in patients with CDs on the basis of previously observed data or published results. The power of the study was 80% considering The quantitative variables were summarised as the mean and standard deviation (SD) or the median and interquartile range (IQR) according to their distributions. Qualitative variables were summarised as frequencies and percentages. Shapiro–Wilk’s test was performed to evaluate departures from normal distribution for each variable. U test was performed to evaluate significant differences in functional and morphological parameters between the CD patients and controls.The Mann–Whitney ® SPSS Statistics v 20.0 software .The effects of vessel density and retinal/choroidal thickness on functional parameters were analysed using different linear mixed models using the eye as the unit of analysis and considering the effect of the paired eye as a random effect. A linear mixed model was used to regress morphological and functional parameters on the fixed-effect factors assuming an unstructured covariance matrix. False discovery rate correction (FDR) was applied to control the family-wise type I error rate, and a q-value less than 0.05 was considered statistically significant. The statistical analysis was performed using IBMTwenty patients  with diagnoses of CD and 20 controls  were enrolled in this retrospective case series.p = 0.874). Thirteen out of 20 patients had a confirmed genetic analysis, whilst seven patients refused the examination for personal reasons (The mean age of patients with CD was 32.25 ± 14.13 years (range 13–62 years), while the mean age of controls was 32.85 ± 13.61 years (range 17–63 years) ( reasons .p < 0.001).The mean best-corrected visual acuity (BCVA) values were 0.7 ± 0.3 logMAR in the CDs group and 0.0 ± 0.1 logMAR in the control group (p < 0.01)  .p < 0.001)  .Dark-adapted 0.01 ERG was not significantly different compared to the control, group, dark-adapted 3.0 ERG and light-adapted 3.0 ERG b wave amplitudes and latencies were significantly reduced, and light-adapted 30 Hz flicker ERG amplitude was also significantly reduced; on the contrary, there was not a significant difference of 30 Hz flicker ERG latency between cases and controls . Three pRetinal sensitivity on MP at 4° and 8° was significantly reduced in patients with CDs compared to that in the normal subjects . FixatioRetinal microcirculation was visualised in detail in all cases. In all patients (40 eyes) in the CDs group, vessels were rarefied to various extents in the foveal and parafoveal areas in both SCP and DCP. The vessel calibre and course did not show any alterations. An increased foveal avascular area due to interruption of the perifoveal anastomotic arcades was evident in the SCP and DCP .p = 0.010), and DCP density was mainly reduced in the parafoveal area (p = 0.008), compared to the control group. The CC density did not show significant differences between the two groups ; this reduction was observed in the whole, inner and outer retina (p < 0.001). Reductions in retinal thickness were observed in both the foveal and parafoveal areas in CD patients compared to that in the controls (p = 0.101).The SFCT was 251.0 µm (203.0–269.2) in CDs and 272.0 µm (226.0–332.0) in controls (PERG (N95) was significantly related to parafoveal retinal thickness (whole retina and inner retina) and to parafoveal SCP and DCP densities .Parafoveal retinal thickness and parafoveal SCP and DCP densities showed a trend toward a significant prediction of mfERG (N1P1A ring 5) .p < 0.001 and b = 2.33; p = 0.018, respectively) as well as between whole-retina thickness in the parafoveal area and parafoveal SCP density   . p = 0.002)  as well as between inner retinal thickness in the parafoveal area and parafoveal DCP density  (p = 0.621).A significant relationship was also found between inner retinal thickness in the parafoveal area and parafoveal SCP density  . A signi= 0.005) . There wIn this study we investigated the retinal and CC microvasculature status in patients with CDs using OCTA and evaluated the correlation between anatomical and functional data by means of electrophysiology testing. Our group of eyes with CDs was characterised by a reduced retinal vessel density, which was related to reduced retinal thickness. Moreover, we demonstrated a direct correlation between reduced vessel density and loss of function, as documented by PERG and mfERG impairment. Retinal and choroidal vessel alterations have been observed in rod–cone dystrophy using OCTA, characterised by SCP, DCP and CC density reduction in the macular area and in the mid-peripheral retina when assessed using widefield OCTA. This evidence confirmed previous clinical findings of vessel changes in retinitis pigmentosa characterised by vessel attenuation ,15,16,17Clinical evidence of vessel alterations in selective or prevalent cone loss, such as CDs or cone–rod dystrophy (CRD), is very limited in the literature. Vessel attenuation has been reported in patients with CRD on fundus examination . In our series, we observed reductions in vessel density in the SCP, mainly in the foveal area, and in the DCP in the parafoveal area of CDs patients, whilst alterations of choriocapillaris vessel density were not found.In addition, a reduction in both inner retina (GCC complex) and outer retina thickness were observed in the CD patients compared to controls. The vessel density alterations, particularly those in the DCP, significantly correlated with macular thickness. The macular function was significantly related to SCP and DCP densities and to whole-retinal and inner-retinal thickness. PERGs were significantly related to parafoveal DCP density, and MfERG showed a trend toward a significant relationship with parafoveal retinal thickness and parafoveal SCP and DCP densities. It has been previously reported that in CDs, reduced retinal thickness is mainly related to thinning of the outer nuclear layer ,20.In our study, both inner retina (GCC complex) and outer retina were reduced. Several authors have investigated the relationship between GCC, RNFL and bipolar cell dysfunction and photoreceptor loss ,22,23,24Reductions in inner retinal layers due to inner retinal cell apoptosis, such as ganglion cell apoptosis after photoreceptor degeneration, have been described in inherited retinal diseases (IRDs), such as RP ,24.In addition, in prominent cone dysfunction diseases, such as cone–rod dystrophy, transsynaptic degeneration with involvement of the inner retinal layers as a consequence of outer retinal photoreceptor cell degeneration has been suggested ,26. A hyDifferently from disorganisation of the retinal inner layers (DRIL) observed in retinal pathologies such as diabetic retinal edema and retinal vein occlusion retinal edema where boundaries between the ganglion cell–inner plexiform layer complex, inner nuclear layer, and outer plexiform layer cannot be identified for a certain extent, in CDs, the inner and outer retina show reduced thickness with preserved retinal interfaces in the inner part and absence of outer retinal structures to a different extent such as ELM and EZ band, or reduced thickness of photoreceptor outer segment and RPE .Macular vessel depletion of CDs is also typical of other hereditary macular dystrophies such as Stargardt disease (STDG) ,30. PatiWe hypothesise that vessel depletion of the SCP and DCP of CD patients is a secondary event of inner retinal and outer retinal atrophy due to a reduced metabolic demand. Photoreceptors in healthy eyes have a high oxygen consumption, so progressive destruction of the outer segment of the retina in retinal dystrophies may reduce the demand for oxygen. Photoreceptor metabolism mainly depends on glycolysis and accounts for the majority of oxygen consumption in the retina, with cones containing 2-fold and 10-fold more mitochondria than rods in mice and primates, respectively, suggesting higher oxidative metabolism than rod ,32. The Choroid thinning has been described in IRDs, such as RP, rod–cone dystrophies and CDs, using SD–OCT, which is probably secondary to a loss of photoreceptor and RPE cells. Vascular endothelial growth factor, which is produced by RPE cells, has been demonstrated to participate in choroidal maintenance; therefore, it has been suggested to play a role in choroidal atrophy ,34,35. IThe normal subfoveal choroidal thickness and CC density detected in our study could be related to the stage of the disease or to the genotype of the included patients.It is known that PERG P50 is partly ganglion cell-derived, with a contribution from structures distal to the GC in the visual pathways. The PERG N95 response is related to ganglion cell activity. In this study, the correlation between PERGs and parafoveal DCP density demonstrated that macular ischemia is related to poor macular function.The alteration of PERG N95 observed in this study and the significant correlation between GCC thickness and N95 amplitude and latency confirm the occurrence of ganglion cell disfunction in CDs.The MfERG response mainly derives from both bipolar and photoreceptor cells; however, a contribution from ganglion cells has also been hypothesised . The altOur study has some limitations. One of the limits is the small sample size, although CDs are relatively rare diseases. In addition, possible segmentation failures that are characteristic of eyes with CDs could partially have reduced the reliability of vessel density measurements.In conclusion, our data showed that our group of CD eyes was characterised by a reduced vessel density , which was related to reduced retinal thickness in both the inner and outer retina. Reduced retinal thickness is related to photoreceptor apoptosis and transsynaptic degeneration with involvement of the inner retinal layers as a consequence of outer retinal photoreceptor cell degeneration. The reduced vessel density could be a consequence of retinal atrophy due to a reduced metabolic demand. In addition, a direct correlation was found between vessel density and loss of function. These findings support the concept of vessel involvement as an important feature in the pathogenesis of CDs. The role of capillary loss in macular function deficits requires further investigation. Thus, OCTA could be used as an adjunctive tool to the armamentarium of the retinal specialists to monitor the progression of vascular involvement in cone dystrophy and correlate to the severity of vascular damage to the loss of function. CDs could be characterised by different severity of vascular depletion detectable using OCTA and could be classified in different phenotypes correlating with function.


Candidatus Phytoplasma solani infection on antioxidative metabolism in leaves and roots of carrot (Daucus carota L.). Disease symptoms appeared at the end of June in the form of the chlorosis on some of the leaves, which became intensely red one week later, while the previously healthy leaves from the same branch becme chlorotic. A few days later, all leaves from the infected leaf branch were intensely red. Infected plants also had slower growth compared to the healthy ones with fewer leaf branches developed. The roots of infected plants were less developed, seared, or gummy with or without brown-colored root hair. The presence of the pathogen was detected by sequencing the 16S rRNA. National Center for Biotechnology Information (NCBI) BLAST analyses of the obtained sequence revealed 100% identity of tested strain with deposited Ca. Phytoplasma solani strains from various countries and hosts, all belonging to the “stolbur” group (16SrXII-A). Identity of 99.74% was found when the tested Serbian strain (MF503627) was compared with the reference stolbur strain STOL11 (AF248959). The oxidative damage of membranes in carrot cells was accompanied by a decrease in the content of photosynthetic pigments. Furthermore, for the determination of specific scavenging properties of the extracts, in vitro antioxidant assay was performed. In phytoplasma-infected carrot leaves, there was a greater reduction in the level of glutathione content (GSH); however; flavonoids and anthocyanidins seem to be responsible for the accompanied increased antioxidative capacity against hydroxyl radical and hydrogen peroxide.The present study examined the effects of  Phytoplasmas are plant pathogens from the class Mollicutes that inhabit phloem sieve elements of host plants . A wide Candidatus Phytoplasma genus are obligate parasites that require plant host and insect vectors for spread and survival. Unlike those biotrophic pathogens that do not require insect vectors, and which activate the JA-dependent pathway but inhibit the SA-dependent pathway [Oenothera biennis plants were obtained when infected by Candidatus Phytoplasma solani [Members of the  pathway , phytopl pathway . Based o pathway  suggestea solani . SA playa solani ,11,12,13a solani ,15.Daucus carota L.) has been considered an important vegetable crop in Serbia, showing a trend of increasing the surfaces on which it is grown over the years. The first occurrence of phytoplasmas on carrot in Serbia has been reported by Duduk et al. [In Serbia, phytoplasmas have been found in cereals, vegetables, spices, and medicinal plants ,17,18,19k et al. . In latek et al.  stated tThe aim of the study was to identify the strain of Ca. Phytoplasma solani and to test the response to the infection in diseased carrot plants. The extent of oxidative stress induced by the infection was measured by levels of reduced glutathione content (GSH) and polyphenols. Damage in phytoplasma-infected carrot plants was determined by measuring a malondialdehyde (MDA), which is a product of oxidative membrane degradation, and bleaching of photosynthetic pigments. Furthermore, in vitro assay for the determination of specific scavenging properties of leaves and roots extracts was performed to reveal the ability of tested plants to cope with oxidative burst caused by determined phytoplasma pathogen.The first disease symptoms were observed at the end of June, in the form of chlorosis on some of the leaves. One week later, chlorotic leaves became an intense red color, while the previously healthy leaves from the same branch become chlorotic. A few days later, all leaves from the infected branch were intensely red a. InfectDuring the colelection of carrot samples , climatic conditions  did not contribute to the disease incidence, although they were different in both years. During June and July, average temeratures in 2016  were lower than in 2017  and precipitations in 2016  were higher than in 2017  . One clear band on the position of 1784 bp was obtained after PCR amplification of tested Serbian strains from symptomatic carrot leaves using two universal primers pairs (P1/P7 and P1A/P7A). Band presence was not visible in any of the negative controls. National Center for Biotechnology Information (NCBI) BLAST analyses of the obtained sequences of Serbian carrot strains (1553 nt in size) revealed 100% identity with already deposited sequences of Ca. Phytoplasma solani strains from various countries and hosts , all belWe observed that carrot plants at the same developmental stage responded differently to the infection with Ca. Phytoplasma solani in two successive years, based on a larger difference in lipid peroxidation between asymptomatic and symptomatic leaves and roots measured in 2017. However, in 2016, a smaller accumulation of sugars, reduced glutathione, and total polyphenols were accompanied by intensified cell membrane peroxidation in asymptomatic leaves and roots . Almost no difference in the content of total sugars between symptomatic and asymptomatic plants was observed in 2016. However, a significant increase in total sugars was measured in symptomatic leaves in comparison to asymptomatic leaves of carrot grown in 2017. The increase in total sugars was accompanied by an almost threefold increase in GSH level in both symptomatic leaf and root. Despite the activation of antioxidative defense in plants with symptoms of infection, the degradation of membrane lipids was 10 times lower in intensity in plants collected in 2016, irrespective of the presence of symptoms. An impaired antioxidative response (GSH and antioxidative capacity) to infection with Ca. Phytoplasma solani in 2016 compared to 2017 resulted in about 50% and 20% higher oxidative stress (LP) in symptomatic leaves and roots, respectively. In both years, the activity of PAL was induced in leaves, but not in root after plant infection. A significant increase was observed only for 2017 . A slight increase in the accumulation of polyphenols was observed in 2016 but not in 2017. Proanthocyanidins and anthocyanins had the opposite changes. While proanthocyanidins decreased in symptomatic plants, anthocyanins increased. The contents of both types of photosynthetic pigments, chlorophylls, and carotenoids decreased in plants with visible symptoms of infection. Contents of chlorophylls in leaves of symptomatic plants were significantly decreased in comparison to asymptomatic plants, contrary to anthocyanins, which accumulated in infected leaves . Significant differences between tested years were observed for antioxidant response to pathogen infection when antioxidant tests  radical) were performed . If 100%Capsicum annuum L.) [Zea mays L.) [Nicotiana tabacum L.) [Pastinaca sativa L.) [Vitis vinifera L.) [Vaccinium corymbosum L.) [Vinca minor L.) [Macrosteles laevis (fam. Cicadellidae), was revealed to be one of the causal agents of carrot disease for the first time in Serbia. Mixed infection with both previously mentioned Ca. Phytoplasma solani groups (16SrI and 16SrXII) was also found in Slovenia, with specific distribution through the plant—in leaves (16SrI), between root and stem (16SrXII), and with both of them inhabiting root [Candidatus could be improved through the sequencing of some non-ribosomal DNA-fragments such as tuf, secY, or vmp1 [O. biennis plants was more vulnerable to the oxidative stress induced by Ca. Phytoplasma solani compared to the root [Stolbur Ca. Phytoplasma solani is highly distributed through different regions of Serbia since 1949, when it was discovered for the first time on pepper (nuum L.) . Ever simays L.) , tobaccoacum L.) , parsniptiva L.) , grapevifera L.) , blueberosum L.) , periwininor L.) , etc., dinor L.) . Mixed iinor L.) . This sting root . A 100%  or vmp1 ,32. The  or vmp1 , with di or vmp1 , such asthe root . Similarthe root . The actthe root ,8,35. Rethe root . Carrot cultivar Bolero F-1  was planted in commercial fields in the second half of April covering 3 ha nearby Futog, Bačka region of Vojvodina, Northern Serbia . Monitoring and visual inspection of Ca. Phytoplasma solani development were performed bi-weekly during the carrot vegetation period. In the middle of July 2016 and 2017, for all experiments in the study, samples were collected from 20 points in the surveyed field, containing 40 symptomatic plants with intensely red leaves and 20 plants with no symptoms a,b. No sMeteorological data  were monitored over the study years (2016–2017) from April to September in weather station Futog, nearest to the selected experimental field  . Averages monthly temperatures and precipitations were presented in 2, 0.2 µL of 20 mM dNTP mixture, 1 µL of each of the primer sets (10 µM), and 0.2 µL of KAPA Taq polymerase (5 U µL−1). PCR mix with 2 μL of DNAase/RNase-free water, instead of sample DNA, served as a negative control in each PCR reaction. Cycling conditions and the number of cycles for both primer pairs were set as follows: 2 min at 95 °C for initial denaturation, 34 cycles of denaturation for 60 s at 95 °C, annealing for 120 s at 50 °C, and extension for 180 s at 72 °C, followed by 10 min at 72 °C for final elongation step. PCR products were visualized on 1% agarose gel stained with ethidium bromide, and they were checked for a band presence on the predicted position in relation to a 1 kb GeneRuler DNA ladder  under UV transilluminator. PCR products obtained with P1A/P7A primers were purified with Qiagen QIAquick PCR Purification Kit and sent for sequencing to Macrogen Europe B.V. Meibergdreef 31, 1105 AZ, Amsterdam, the Netherlands. The obtained sequence of one Serbian carrot strain generated in this study was deposited into the National Center for Biotechnology Information (NCBI) GenBank database to get an accession number. The sequence of Serbian carrot strain was used for further phylogenetic analysis and comparison with other sequences of Ca. Phytoplasma spp. available in the GenBank (http://www.ncbi.nlm.nih.gov/BLAST/ (accessed on 12 November 2020)). Phylogenetic analysis was performed to check the relatedness between strains of three formally described species within Stolbur phytoplasma group 16S rXII, the ones transmitted by polyphagous planthoppers from the Cixiidae family, to determine the position of tested Serbian strain in relation to them. For this purpose, sequences of Ca. Phytoplasma solani strains obtained from Serbia and other countries, affecting diverse host plants, as well as sequences of reference Ca. Phytoplasma strains , retrieved from the NCBI database were used for phylogenetic analysis. Strains used for comparison are listed in Acholeplasma palmae strain , which is the secondary product of the oxidation of polyunsaturated fatty acids, as it is explained in method by Hodges et al. . ThiobarA modified sulfuric acid-UV method  was usedL-phenylalanine against blank solution (1h at 37 °C in water-bath). The amount of cinnamic acid produced was determined from a trans-cinnamic acid standard curve, and PAL activity was expressed as U/g fw. Phenylalanine ammonia-lyase  activity was performed according to the protocol given by Gerasimova et al. . The con2CO3. Absorbance at 765 nm was recorded after 60 min of incubation at room temperature. The total tannin content was determined by the same Folin–Ciocalteu procedure after removal of tannins by adsorption on an insoluble matrix . Calculated values were subtracted from the total phenolic contents, and total tannin contents were expressed as mg GAE/g dw. The determination of total flavonoids content was performed according to Pękal and Pyrzynska [3 reagent (0.1 g of AlCl3 and 0.4 g of CH3COONa) in reaction medium. Absorbance was recorded at 430 nm against a blank, and the amount of flavonoids was calculated as a rutin equivalent from the calibration curve of rutin standard solutions and expressed as mg rutin/g dw. Total polyphenols and tannins content was determined by Folin–Ciocalteu method  and expryrzynska  with plaProanthocyanidins were determined by a butanol-HCl assay . Prepareg). Absorbance was recorded at 440, 662, and 644 nm. Chlorophyll a and b contents were calculated from equations described in the applied method and given as mg/g dw. Total carotenoids content was determined according to a slightly modified method described by de Carvalho et al. [g). Absorbance of the petroleum ether phase was recorded at 470 nm. The total carotenoid content was calculated from β-carotene standard curve and expressed as β-carotene equivalents in mg/g dw.Chlorophyll a and b contents were determined according to method described by von Wettstein . Fresh lo et al. . Plant mAntioxidant capacity was tested by three antioxidant tests that measure the scavenging activity of 1.1-diphenyl-2-picrylhydrazyl (DPPH) free radicals (DPPH test), superoxide anion (NBT test), and hydroxyl radical (•OH). The antioxidant activity of methanol dry extracts was assessed based on the DPPH test . Anothert-test (p < 0.05). Statistical analyses were performed using STATISTICA for Windows version 13 .Obtained results were expressed as means ± standard error and were tested using Student’s Ca. Phytoplasma solani accumulated the product of oxidative degradation of membranes, MDA, to much higher extent compared to the root. In addition to polyphenols, which preferentially accumulated in symptomatic carrot plants in leaves, a significant role of GSH in the antioxidative defense of carrot leaves was indicated. However, a twice as large increase of reduced glutathione and total polyphenols (including anthocyanins) in symptomatic as in asymptomatic leaves of carrot were not efficient in the antioxidative protection against lipid peroxidation and degradation of photosynthetic pigments. Despite a much smaller accumulation of total polyphenols and GSH (10%), the oxidative effect on lipids in roots was less pronounced (18%) compared to the leaves (61%). Results implied that leaves compared to the root of the carrot were preferentially exposed to the oxidative stress induced by Ca. Phytoplasma solani, which can be explained by (1) favoring the photosynthetic electron transfer to molecular oxygen that produces superoxide anion and other reactive oxygen species (ROS), and (2) impaired antioxidative action against the superoxide anion of leaves extracts.