Patients with sickle cell disease (SCD) suffer from anemia and painful vaso-occlusive crisis (VOC) and sometimes need blood transfusions. Delayed hemolytic transfusion reaction (DHTR) is a rare life-threatening complication observed in SCD and mimics VOC. We describe a female SCD patient undergoing three surgical procedures during which DHTR developed following the first two. Prior to a planned tonsillectomy, she received transfusion and three days after surgery developed severe hemolysis as well as pain and respiratory symptoms. On suspicion of VOC, she received additional transfusions and became hemodynamically unstable, and her hemolytic anemia worsened. Gradually, she recovered and could be discharged after two weeks; DHTR was not suspected. Sixteen months later, an arthroplasty was performed due to avascular necrosis, and again she was transfused preoperatively. Similar to the initial surgery, she developed symptoms and signs of VOC after three days, but this time, DHTR was suspected and further transfusions were withheld. Although immunosuppressive medication did not alleviate the condition, she improved on combined treatment with darbepoietin, rituximab, and eculizumab. Six months later, a second arthroplasty was performed uneventfully after prophylaxis with rituximab and without transfusion. DHTR should be considered in the presence of severe, unexplained hemolysis following a recent transfusion, and additional transfusions in this setting should be given only on vital indication. Sickle cell disease (SCD) is an inherited hemoglobinopathy prevalent in equatorial regions such as sub-Saharan Africa. This is partly because carriers (heterozygotes) of the mutated sickle cell gene are protected against fatal complications of severe malaria . The disIn case of severe anemia, blood transfusions may be indicated in SCD. Repeated transfusions often result in the formation of anti-red blood cell (RBC) alloantibodies, and alloimmunization occurs more frequently in SCD patients than in other heavily transfused patient groups . The preOur knowledge about DHTR stems from case reports and case series usually involving only one DHTR event per patient. We here present an unusual case of repeated DHTRs without documented alloimmunization in a SCD patient treated at our unit on three different occasions where we suspect DHTR as the major underlying mechanism of the severe complications observed during the first two admissions. Consequently, a different approach to the third admission led to an uneventful clinical course.A 28-year-old Nigerian female with homozygous SCD became a regular outpatient at our department in 2013 after an uneventful (without any blood transfusion) pregnancy and birth in the USA. As a child, she had received several transfusions, apparently with no complications. She had the following blood type profile: 0; D+; C-; E-; c+; e+; K-; S-; in addition to anti-C and anti-E antibodies. She had never used any medication except folic acid.9/l. She became hemodynamically unstable, and vasopressor support and two more units of RBC were given before she was intubated. She was extubated the following day, and over the next few days, her condition stabilized; the hemolytic parameters were markedly reduced by day +9, and she was discharged on day +13 with subsequent visits as an outpatient. All bacterial cultures from this admission were negative. Notably, during this first episode, DHTR was never suspected.In May 2017, she underwent an elective tonsillectomy at our hospital . Except for routine pre-transfusion screening, neither extended screening for new alloantibodies nor a direct antiglobulin test was performed. On day −1, she received a scheduled transfusion of two cross-matched units of RBC with hemoglobin (Hb) increasing from a baseline value of 7 to 9.0 g/dl. After an uncomplicated procedure, she was transferred to a local hospital with an uneventful clinical course until day +4 when she developed generalized skeletal pain and fever, but no chest or respiratory symptoms. As 9/l. We suspected DHTR, and further transfusions were avoided. Intravenous immunoglobulin therapy (30 g daily) was initiated for 4  days. On day +6, Hb plummeted to 3.5  g/dl, and LDH and total bilirubin peaked at 1470 U/l and 5.3 mg/dl, respectively, and intravenous methylprednisolone (500  mg per day for 5 days) was administered. Screening for new alloantibodies and a direct antiglobulin test were both negative. Her HbA-fraction declined from 33.5% at day 0 to 12.3% on day +6. Using the nomogram proposed by Mekontso Dessap et al. [Due to avascular necrosis of her right hip, the patient underwent an arthroplasty in September 2018. On day −3 prior to surgery, she received a planned transfusion with three cross-matched units of RBC, raising her Hb to 11.0 g/dl. Her pre-transfusion screening revealed no new alloantibodies. After an uncomplicated orthopedic procedure on day 0, she was transferred to our department. On day +3, she developed skeletal pain but no symptoms related to the newly operated hip. On suspicion of VOC, treatment was initiated with fluids, analgesics, and antibiotics. As illustrated in Due to progressive avascular necrosis of the contralateral hip, an arthroplasty was performed in March 2019. Screening for new alloantibodies and a direct antiglobulin test were both negative. As a preoperative strategy to optimize erythropoiesis, she received darbepoietin for two months as well as hydroxyurea, but no prophylactic transfusions. She maintained a preoperative Hb of about 8  g/dl, and she was also given one prophylactic dose of rituximab (1000  mg i.v.) 2.5 weeks before surgery. Both the surgery and the postoperative stay in hospital were uneventful and with no signs of hemolysis . She wasWe here present a SCD patient who received preoperative blood transfusions during two consecutive hospital admissions which were most likely complicated by DHTR. Although DHTR was not suspected during the first admission, we were alert to this complication during the second admission. Hence, we were able to provide her with adequate prophylactic treatment and avoided DHTR during her third admission.9/l) was observed in 40% of the patients. Progression to acute chest syndrome or even multi-organ failure was not uncommon in the most severe cases with an overall mortality of 6% [Retrospectively, we believe the symptoms she presented during her postoperative hospital admission in 2017 represented the start of DHTR. A main challenge in identifying DHTR in SCD patients is that the symptoms often mimic VOC. In a large French observational study of about 3,000 SCD patients, the most common presenting symptoms and signs were hemoglobinuria 94%), pain (89%), fever 64%), and severe anemia (44%) [%, pain (4%, and sHyperhemolysis refers to the destruction of allogenic and autologous erythrocytes, potentially leading to severe and fatal hemolysis ,7. In liNo new alloantibodies were detected in the acute setting during the second episode, and a direct antiglobulin test was negative. However, no repeated screenings were done between the first two episodes or after the second episode. Neither did we perform extended antibody screening for low-frequency antigens. Notably, hyperhemolysis may occur without newly formed alloantibodies and has been described in up to 1/3 of SCD patients , 8. The There is little evidence-based data for the therapeutic approach to patients with suspected DHTR, and current treatment modalities are mainly based on expert recommendations . ImmunogDHTR is a rare life-threatening complication to transfusions in SCD patients. Our patient illustrates the dilemma that DHTR can be misdiagnosed as VOC, a complication of SCD that is much more common than DHTR. Hence, a DHTR diagnosis should always be considered in the presence of an unexplained increase in hemolysis following a recent transfusion, and further transfusions should be avoided unless life-threatening anemia is present.


Conclusions: This suggests that comorbidities have an additive impact on the cognitive profile of children with ADHD. Design fluency may be a sensitive measure for capturing the subtle cognitive deficits that are likely to be involved in these disorders.Background: Attention deficit/hyperactivity disorder (ADHD) is often associated with frontal executive impairment in children. Oppositional defiant disorder (ODD) and anxiety disorders (AD) frequently accompany ADHD, but the impact of these comorbid disorders on cognition remains elusive. The five-point test (FPT), a design fluency task, has been shown to be sensitive to neurological damage, specifically to frontal lobe lesions in patients with brain injuries. The purpose of this study was to compare the performances of neurotypical children with that of children with ADHD, ADHD-ODD, and ADHD-AD on the FPT in order to examine whether these groups could be distinguished from one another based on their cognitive profile. Methods: A total of 111 children aged 8 to 11 years old participated in the study. Six measures from the FPT were used to characterize their performance. Results: Statistically significant differences between groups were observed for five of the six FPT measures. Essentially, children with ADHD-ODD made more repeated designs than the three other groups (control  Attention deficit/hyperactivity disorder (ADHD) is characterized by persistent inattention and hyperactivity/impulsivity and is one of the most prevalent neurodevelopmental disorders . The disDesign fluency was initially developed by Jones-Gotman and Milner  as the nDecreased frontal/prefrontal activity has been reported in individuals with ADHD e.g., ,23,24) a,24 a23,2To our knowledge, only four studies have tested children with ADHD using design fluency. In the first one, Loge, Staton, and Beatty  comparedDespite the potential interest of design fluency in ADHD, there are limited and rather incomplete and inconsistent data. Part of the discrepancy may stem from the lack of consideration for the frequent comorbidities associated with ADHD in childhood. Between 30% and 66% of children with ADHD also present with comorbid ODD ,38,39 anIn children, ODD is believed to be associated with abnormalities of the dorsolateral prefrontal cortex and the cerebellum ,43,44,45Similarly, several studies support the involvement of the prefrontal cortex in anxiety e.g., ,62). Pos. Pos62])No study to date has used design fluency to compare the performance of children with ADHD alone and comorbid ADHD. In this study, we used six measures of the FPT for characterizing ADHD with or without comorbidity. Considering the study by Lee et al.  and the ® were excluded from the study since atomoxetine cannot be eliminated from the body fast enough.A total of 111 children aged 8 to 11 years  participated in the study. The control group (without ADHD) consisted of 34 children  from regular schools. The clinical groups consisted of 77 children  divided into three groups see : ADHD  a developmental and behavioral assessment with questionnaires completed by parents and teachers, (b) a cognitive assessment (of attention and executive functions), and (c) a diagnostic evaluation of mental disorders based on the Diagnostic and statistical manual of mental disorders, fourth edition (DSM-IV)  and condThe parent and teacher versions of the ARS-IV  were useThe parent (CBCL/6-18) and teacher (TRF) versions of the ASEBA  were useThis questionnaire was developed by professionals working at the specialized ADHD clinic in Montreal and was used to screen for the presence of mental retardation or neurological problems that would have led to the exclusion of a participant.The FPT  requiresTwo examples of correct designs were shown. The child could then ask any question before starting. Six scores were used: (a) total number of designs (TD) to assess design production, i.e., generativity; (b) total number of repeated designs (RD) to assess self-regulation, i.e., cognitive flexibility and inhibition ; (c) number of correct designs (CD), i.e., TD minus RD, a relative measure of design production; (d) ratio number of RD on TD (RT), to appreciate the importance of repetition relative to design production; (e) ratio number of RD on CD (RC); and (f) total number of strategies (TS) to assess planning. Numerical strategies consisted of adding or subtracting a line from the previous design to create a novel one, and spatial strategies consisted of rotating or mirror flipping the previous design. Construct validity of the FPT is good (0.4 to 0.7). Test-retest reliability is acceptable for CD (0.51) and good for RT 0.78), but remains unknown for the other measures. Design fluency improves with age and is independent of gender and intelligence quotient (IQ) ,74,75,76, but remPreliminary analyses were realized. First, one-way analysis of variance (ANOVA) or chi-squared test were performed to determine potential differences between groups in terms of age, gender, or medication. Second, correlation analyses  were carried out between age, gender, medication intake, and FTP scores. Considering the descriptive aim of theses analyses, a significance threshold of 0.05 was considered.A series of covariance analyses (ANCOVA) were conducted to determine a statistically significant difference between control, ADHD, ADHD-ODD, and ADHD-AD groups on each score of the FPT , with age as a covariable (given the paucity of FPT age-weighted scores for the age range of our sample). ANCOVA is considered robust to violations of normality and heterogeneity of variance . When a Considering the exploratory aim of the current study, we kept the significance threshold of 0.05. A Bonferroni correction could be used for a more conservative approach. The correction implies a significance threshold of 0.008 (0.05/6). Interpretations are made using the conventional 0.05 threshold, but readers can refer to 0.008 for more conservative results. Analyses were conducted using IBM SPSS (version 23).F = 2.025, p = 0.115, η² = 0. 075  = 1.49, p = 0.475, but did differ between the control and the clinical groups, X2 = 8.11, p = 0.044. The percentage of participants that took medication did not differ between clinical groups, X2 = 0.20, p = 0.903. The three groups with ADHD differed significantly from the control group in terms of symptoms of inattention and hyperactivity , positive correlations with design production scores in the low (TD: p = 0.002) and moderate (CD: p < 0.001) range, and a moderate correlation with the strategy score (TS: p < 0.001). Gender  only showed a low correlation with the repetition scores . Medication intake in the clinical groups  was not associated to any measure. Given that age was the only variable significantly correlated with the majority of FPT scores, subsequent analyses examining group differences included age as a covariate.p = 0.001; RC: p = 0.007) to moderate , and high (CD: p < 0.001) range. The relative production score CD did not correlate with the absolute repetition score RD, but showed a low negative correlation with repetition ratio measures , as well as a high correlation with the strategy score (TS: p < 0.001). Finally, the repetition score RD was highly associated with repetition ratio scores , which were very highly associated with each other (p < 0.001). The strategy score TS was not associated with any of the repetition scores.Correlations examining the relationship between the FPT scores indicated that the absolute production score TD was positively associated with all FPT scores, from the low  = 3.939, p = 0.010, η² = 0.100; CD, F = 2.857, p = 0.041, η² = 0. 075; RD, F = 6.277, p = 0.001, η² = 0.151; RT, F = 5.303, p = 0.002, η² = 0. 131, and RC, F = 4.546, p = 0.005, η² = 0. 114. No group effect was observed on TS, F = 0.714, p = 0.546, η² = 0.020.Results show a statistically significant effect of group on TD, p = 0.009), and a significant difference on CD between ADHD-AD and ADHD (p = 0.030). The ADHD-AD group did not significantly differ on TD from the control (p = 0.733) and the ADHD (p = 0.097) groups, nor on CD from the control (p = 0.132) and ADHD-ODD (p = 0.280) groups. No other comparison on TD and CD were significant.With regards to the design production measures (TD and CD), multiple pairwise comparisons of adjusted means for age revealed a significant difference on TD between ADHD-AD and ADHD-ODD (p < 0.001), ADHD (p = 0.008), and ADHD-AD (p = 0.008) groups, on RT between ADHD-ODD and the control (p = 0.001) and the ADHD (p = 0.009) groups, and on RC between ADHD-ODD and the control (p = 0.005) and the ADHD (p = 0.014) groups. The ADHD-ODD group did not significantly differ from ADHD-AD on RT (p = 0.235) and RC (p = 0.490). No other comparisons on RD, RT, and RC were significant.Concerning the repetition measures , pairwise comparisons revealed a significant difference on RD between ADHD-ODD and the control  and that the ADHD groups would produce more repeated designs in comparison to the control group.The results confirm our first hypothesis. The comorbid groups offered poorer performance than the ADHD-alone and control groups. Children with ADHD-ODD made more repeated designs and showed a greater ratio of repeated designs when compared to children with ADHD, ADHD-AD, and non-ADHD children. This suggests lower self-regulation during design production in children with ADHD-ODD, resulting in poor inhibition of already produced responses. Previous studies support a perseveration tendency in children with behavioral disorders ,82. The Furthermore, confirming our first hypothesis, children with ADHD-AD produced fewer total designs and correct designs than the ADHD and ADHD-ODD groups, and showed a tendency to produce fewer correct designs than the control group. This suggests that AD impedes generativity in children with ADHD. To our knowledge, no study has compared the design fluency performance of children or adults with ADHD and ADHD-AD. However, studies on individuals with anxiety disorders suggest that the diminution of generativity in the FPT could be due, in part, to frontal and prefrontal cortices dysfunctions. Adults with obsessive-compulsive disorder (OCD) produced fewer correct designs than a control group on the FPT , consistAll in all, these results suggest that the dysfunctions associated with ADHD, ODD, and AD are additive. Although these disorders have a common neurobiological basis which may lead to similar cognitive performance, each disorder appears to be associated with specific circuits that are likely to overlap, but also to have a distinct impact on performance, as the results of the present study suggest.With regards to our second hypothesis, the results do not support the prediction that the ADHD-alone group would underachieve relative to the control group. Both groups provided similar performances on all measures of the FPT. This is in line with previous studies that observed equivalent generativity in children with and without ADHD ,36. The There might be other explanations for the lack of a difference between our control and ADHD-alone groups, which could also account for the cognitive heterogeneity observed in ADHD. First, some authors suggested the possibility that executive dysfunction in ADHD may be partially due to an impairment of automatic processing e.g., ,94,95). ,95. 94,9Finally, the results of the correlational analyses are interesting from a clinical point of view. They are congruent with previous studies showing an effect of age, but not of gender, on productivity in design fluency ,74,75,76Although a rigorous, standardized, and comprehensive diagnostic procedure was used, we did not control for the severity of symptoms related to ADHD, ODD, and AD, and the ADHD-AD group may have included different anxiety disorders that may have masked their effect on the performance. Future research should compare groups of children with different anxiety disorders while controlling for the severity of anxiety (and other disorders) symptoms. Furthermore, the small sample size may explain the absence of a significant difference in generativity between the ADHD-AD and the control groups. Finally, adding process measures to global performance scores may help better characterize the cognitive profiles of children with ADHD with and without comorbidities, and identify differences between groups (see ).In this study, children with comorbid ADHD performed less well than children with or without ADHD on the FPT, a measure of executive function that recruits and requires the coordination of multiple cognitive processes. Compared to other groups, children with ADHD-ODD made more repeated designs, which may reflect poorer self-regulation, while children with ADHD-AD produced fewer designs, showing lower generativity that may reflect an excess of inhibition. The performance of comorbid ADHD groups on the FPT may be associated with neurobiological dysfunctions that are specific to ODD and AD. Neural networks connecting the amygdala to the frontal and prefrontal regions are suspected. A deficit in cognitive coordination may also contribute to the poorer performance of the ADHD-ODD and ADHD-AD groups. In conclusion, the results of the present study suggest that the cognitive impairments associated with ADHD, ODD, and AD are additive. Although further research is needed to confirm this, it still remains that the presence of comorbidities is associated with greater functional impacts that are not always corroborated by the child’s performance in the context of neuropsychological studies or clinical assessment. Considering the high frequency of these comorbidities, it appears important to develop tools that are more sensitive to the cognitive deficits that may characterize children with ADHD, with or without comorbid disorders. This would help to understand the impact of comorbid disorders on everyday functioning and guide specific interventions. The present study suggests that design fluency could be helpful in this regard.


F3:4 and F3:5 lines were grown in Blacksburg and Orange, Virginia in three years, respectively, and were measured for KTI content using a quantitative HPLC method. We identified three SNP markers tightly linked to the major QTL associated to low KTI in the mapping population. Based on these SNPs, we developed and validated the KASP assays in a set of 93 diverse germplasm accessions. The marker Gm08_44814503 has 86% selection efficiency for the accessions with low KTI and could be used in marker assisted breeding to facilitate the incorporation of low KTI content in soybean seeds.Trypsin inhibitors (TI), a common anti-nutritional factor in soybean, prevent animals’ protein digestibility reducing animal growth performance. No commercial soybean cultivars with low or null concentration of TI are available. The availability of a high throughput genotyping assay will be beneficial to incorporate the low TI trait into elite breeding lines. The aim of this study is to develop and validate a breeder friendly Kompetitive Allele Specific PCR (KASP) assay linked to low Kunitz trypsin inhibitor (KTI) in soybean seeds. A total of 200 F Glycine max L. Merr.) is widely recognized as the richest and least expensive source of vegetable protein for livestock, poultry and aquaculture production, which is also used as a source of metabolizable energy . However, soybean seeds have several anti-nutritional factors (ANFs) that affect animal nutrient digestion and absorption, reducing animal growth performance in meat production [Soybean (Trypsin inhibitor (TI), one of the ANFs in soybeans, is the major proteinase inhibitor accounting for about 6% of the total protein in soybean seed . TI restkti) [Two accessions were originally identified from the USDA’s Germplasm collection: PI 157,440 (‘Kin-du’) and PI 196168, completely lacking the Kunitz trypsin inhibitor protein  and bothkti) . Despitekti) ,13 due tkti) . Additiokti) ,13,15. Tkti)  after ‘Kktiti, the recessive form of KTi gene [The use of molecular markers for marker-assisted selection (MAS) have been proved to speed up the breeding process and efficiently introgress recessive alleles into crops ,19,20. UKTi gene . HoweverKTi gene ,27. SNPsKTi gene . Moreove2 population of SP6A-206 x PI 542,044 (low KTI). However, Patil et al. did not report what phenotyping system they used to discriminate the low KTI lines from the high ones, making not clear if the KASP clustering correspond to true low and high KTI phenotypes. Moreover, we tested the reported SNP genotyping assay in the Glenn (high KTI) x PI 547,656 (low KTI) population with no successful results, perhaps because PI 542,044 and PI 547,656 do not have common low KTI genes. In 2018, Rosso et al. developed an HPLC method as a reliable method to quantify KTI in soybean seeds. showing that this quantification method was strongly correlated with two popular activity assays for measuring total TI activity. Additionally, the HPLC method overcomes the flaws of activity assays because it can detect low levels of KTI on nonfunctional mutated soybean lines, which is impossible for activity assays, and can measure only KTI, not the mixture of KTI and BBTI as activity assays do. Therefore, the objectives of this study were: (i) to identify SNP markers associated with the QTL that confers low KTI in soybean seeds; and (ii) to develop and validate a breeder -friendly KASP SNP genotyping assay linked to low KTI content derived from ‘Kin-du’ as the KTI free donor, that could be used in marker-assisted selection to incorporate the low KTI trait into elite breeding lines.In a recent article, Patil et al. (2017) reported a development of a SNP genotyping assay for detection of low KTI in soybean in an F3:4 (data not shown) and F3:5 lines from the mapping population and the 93 PIs selected for the SNP validation. Analysis of variance and least squares means Student’s t-test showed there was a significant (p = 0.007) variation in KTI concentration between the two parents, Glenn and PI 547,656  and also between years   . Frequen 0.0001) .H2) of KTI was estimated to be 54% and was calculated using variance components for KTI of the mapping population across two years (2016 and 2017) and two locations .The broad-sense heritability (qKTI08. The additive effect (Add) of qKTI08 was −1.88 mg/g in 2016 and −3.99 mg/g in 2017, indicating that the allele at this QTL inherited from the low KTI parent (PI 547656) reduced KTI content  were selected for establishing the genetic linkage map. The linkage map generated by SMA from the mapping population consisted of 20 chromosomes, which spanned 1278.69 cM and were defined by 1272 SNP markers. The constructed map had a coverage ranging from 0.097 to 0.312 cM with an average coverage of 0.215 cM . One QTL content . When ane affect .The SNPs Gm08_44814503 and Gm08_45270892 were located at 85.22 and 88.74 cM on chromosome 8 (LG A2), respectively, in the most recently developed high density linkage map for soybeans . Based oqKTI08 region on chromosome 8 that were significantly associated  with low Kunitz trypsin inhibitor content across years and locations were identified using single marker analysis (SMA) on IciMapping. qKTI08   in soybean seeds has a negative impact on the feed and food industry since it affects animal nutrient digestion and absorption, reducing animal growth performance in meat production . The bes14 mg/g) . We estitability . A modertent 0.5–.14 mg/g tability ,32, howeqKTI08) associated with KTI was detected in PI 547,656 on chromosome 8 with a confidence interval from Gm08: 44,814,503 to Gm08:45270892  associated with low concentration of KTI previously reported. In this study, one QTL  with low KTI content across years and locations , which were polymorphic between parents, were used to verify true hybrids. A total of 200 individuals of the F2 were advanced to F3 in 2015. The parents and F2:3 lines were separately planted in single, 3.05 m long rows with 0.76 m row spacing and about 30 seeds per meter arranged in a randomized complete block design in Blacksburg, VA, USA in 2015. Two parents and a population of F3:4 lines were grown in Blacksburg and Orange, VA, USA in 2016, and two parents and a population of F3:5 lines were grown in the same two locations in 2017 using the same experimental design. Parental soybean lines were chosen based on the amount of KTI in the seeds. Glenn, developed and released by Virginia Tech in 2009, was chosen as a normal KTI concentration parent. PI 547,656 (cultivar name: L81-4871) was chosen as a low KTI parent. PI 547656, derived from a cross ‘Clark 63′ x PI 157,440 (‘Kin-du’), was developed by the USDA-ARS and the Illinois Agricultural Experimental Station in 1986 [3:4 and F3:5 mapping population were phenotyped for KTI content using a quantitative HPLC method [3:4 phenotypic data are not shown. Briefly, 10 mg of finely grounded soybean seed powder was mixed with 1.5 mL of 0.1 M sodium acetate buffer, pH 4.5. Samples were vortexed and shaken for 1 h at room temperature. The samples were centrifuged at 12,000 rpm for 15 min. One-mL of the supernatants were filtered through a syringe with an IC Millex-LG 13 mm mounted 0.2 µm low protein binding hydrophilic millipore (PTFE) membrane filter . The Kunitz trypsin inhibitor in solution were separated on an HPLC Agilent 1260 Infinity series , equipped with a guard column (4.6 × 5 mm2) packed with POROS® R2 10 µm Self Pack® Media and Poros R2/H perfusion analytical column . The mobile phase A consisted of 0.01% (v/v) trifluoroacetic acid in Milli-Q water and mobile phase B was 0.085% (v/v) trifluoroacetic acid in acetonitrile. The injection volume was 10 µL and the detection wavelength was 220 nm. The final KTI concentration of each sample was calculated using the initial weight of each sample to calculate the KTI concentration on a dry weight basis with results reported as milligrams per gram (mg/g).The two parents and 200 progenies of the FC method . F3:4 ph3:5 lines of the mapping population and each PI from the validation panel were collected and stored at −80 °C until DNA extraction. Leaves were freeze dried using a FreeZone 6 Liter Console Freeze Dry System (−56 °C and 0.220 mbar) and about 200 mg of leaf tissue were placed in a 2.5 mL tube. Tissue was ground to fine powder in liquid N2 using glass stirring rods. Total genomic DNA was isolated from leaf tissue (0.20 g) by a modified protocol from the CTAB method of [A bulk sample of young leaves from three individuals of each Fethod of .3:5 lines were sent to USDA–ARS Soybean Genomics and Improvement Laboratory  and genotyped using the Illumina BARCSoySNP6K_v2 Beadchip containing 6000 SNPs. Single-nucleotide polymorphism genotyping was conducted according to Song et al. 2013 [Fifty nanograms of genomic DNA for the parental lines and the population of Fal. 2013  on the Ip < 0.0001 was used as a threshold for significant markers. In the composite interval mapping (CIM) and simple interval mapping (SIM), the empirical significance threshold was determined by 1000-time permutation with a walk speed of 1 cM and significance level of 0.05. MapChart [Linkage maps were constructed by JoinMap v4  using a MapChart  was usedG. max genome Glyma.Wm82.a1  following Zhu et al. [The SNPs tightly linked to major QTL identified in the mapping population  were conu et al. . Brieflyu et al. . Thermocu et al. .The 6749 MGs IV and V soybean accessions collected by the National Genetic Resources Program (USDA Soybean Germplasm Collection) were arbitrarily grouped in 500 clusters based on their genetic distance which were calculated using 42,509 SNPs that were included in the SoySNP50K . One accp = 0.05. Histograms of KTI concertation were also performed by JMP. Analysis of variance was used to determine phenotypic differences between parents, Glenn and PI 547656. Variance-component heritability estimates were calculated by analyses of variance using R software. Broad-sense heritability of seed coat deficiency was estimated using the equation: H2 is heritability, s2g is genotypic variance, s2ge/e is genotype x environment interaction variance, s2 is error variance, r is the number of replications, and e is the number of environments [Concentration of KTI, collected in 2016 and 2017, were analyzed using JMP statistical version Pro 14.2.0 (SAS Institute). KTI concentration was analyzed by analysis of variance and the non-parametric method Kruskal–Wallis test for the 2016 and 2017 data at two locations, Blacksburg and Orange, VA, USA. Student’s t test was used to determine if mean of KTI concentration of parents and lines differed significantly at ronments .


Maximal strength-speed exercise is a powerful stimulus to acutely increase concentrations of circulating steroid hormones and homocysteine [Hcy]. There is some evidence that antioxidant beverages rich in polyphenols can attenuate [Hcy] levels and modulate endocrine responses in favor of an anabolic environment. Polyphenols-rich pomegranate (POM) have been reported to possess one of the highest antioxidant capacities compared to other purported nutraceuticals and other food stuffs. Studies focused on proving the beneficial effect of POM consumption during maximal strength exercises have only measured physical performance, muscle damage, oxidative stress and inflammatory responses, while POM effects on [Hcy] and hormonal adaptations are lacking. The aim of the present study was to investigate the effect of consuming natural polyphenol-rich pomegranate juice (POMj) on the acute and delayed [Hcy] and steroidal hormonal responses to a weightlifting exercises session.Nine elite weightlifters (21.0 ± 1 years) performed two Olympic-weightlifting sessions after ingesting either the placebo (PLA) or POMj supplements. Venous blood samples were collected at rest and 3 min and 48 h after each session.p < 0.01) and testosterone/cortisol [T/C] ratio increased immediately following the training session in both PLA and POMj conditions (p = 0.003 for PLA and p = 0.02 for POM). During the 48 h recovery period, all tested parameters were shown to recover to baseline values in both conditions with significant increases in [C] and decreases in [T/C] (p < 0.01 for PLA and p < 0.05 for POMj) from 3 min to 48 h post-exercises. Compared to PLA, a lower level of plasma testosterone [T] was registered 3 min post exercise using POMj supplementation (p = 0.012) and a significant decrease  in plasma [Hcy] was registered during the 48 h recovery period only using POMj. A moderate correlation was observed between [Hcy] and [T] responses .Compared to baseline values, circulating cortisol [C] decreased (In conclusion, supplementation with POMj has the potential to attenuate the acute plasma [T] response, but did not effect 48 h recovery kinetics of [Hcy] following weightlifting exercise. Further studies investigating androgen levels in both plasma and muscular tissue are needed to resolve the functional consequences of the observed acute POMj effect on plasma [T].NCT02697903. Registered 03 March 2016.Clinical Trials.gov, ID:  Punica granatum is an ancient fruit originating from the Middle East  and hormonal responses during intensive strength exercise. Therefore, the aim of the present study was to investigate the influence of natural polyphenol-rich pomegranate juice (POMj) supplementation on acute and delayed [Hcy] and steroidal hormonal responses following an intensive weightlifting training session. We hypothesized that the repeated consumption of POMj during the 48 h proceeding the training session mixed with an acute consumption 1 h prior exercises would reduce the acute and delayed responses of [Hcy] and modulate the responses of steroidal hormones in favor of an anabolic environment.The sample size was calculated a priori, using procedures suggested by Beck  and the Nine elite male weightlifters  volunteered to participate in this study. The participants were recruited on the basis of: (i) they trained at least five sessions/week, (ii) they had at least 3 years of experience in Olympic weightlifting, (iii) they did not have any injury, (iv) they did not use any antioxidant or anti-inflammatory drugs, and (v) they did not consume foods rich in antioxidants or polyphenols  during https://clinicaltrials.gov/ct2/show/NCT02697903).The study was conducted according to the Declaration of Helsinki and the study’s protocols and procedures were fully approved (identification code: 8/16) by the local ethics committee of “Habib Bourguiba hospital, Sfax, Tunisia” before the commencement of the assessments. Additionally, all ongoing and related trials for this intervention were registered with Clinical Trials.gov ) was assessed for each participant in each Olympic movement. 1-RM was determined in three trials interspersed by 5 min recovery after an ascending warm-up from 40 to 80% of the athlete’s estimated maximum , 20. TheParticipants consumed 250 mL of the PLA or POMj supplementations three times daily over the 48 h that proceeded the two training sessions with 8 h intervals between each 250 mL serving of the supplement. Moreover, participants consumed an additional opaque and unmarked 500 mL can of PLA or POMj 60 min before commencing the training sessions , 26 to fGiven that randomly assigning the supplements would have resulted in some participants consuming the POMj supplement before the PLA supplement, and since the beneficial effects of POMj could persist for up to 3 weeks after consumption , we elecBeverages were prepared by an agri-food engineer. The natural POMj was prepared from a fresh pomegranate fruit 48 h before the beginning of the experimentation and was frozen and stored at − 4 °C. No additional chemical products were added to the natural POMj. PLA juice consisted of a pomegranate-flavored drink containing mineral water, natural identical flavor (pomegranate), stabilizers (Arabic-gum) with caloric sweeteners  added to match the POMj energy content and avoid any confounding effect of different caloric content of the beverages . To the Concerning the POMj processing, a previous study showed that the polyphenol content of POMj may vary between 900 and 2300 mg/L , 55 whenOne can of POMj  contained 2.56 g of total polyphenols, 1.08 g of orthodiphenols, 292.6 mg of flavonoids and 46.75 mg of flavonols, 64 g of total carbohydrates , and 1046 kJ of energy. The PLA drink  did not contain antioxidants, vitamins or polyphenols, but comprised 60 g total carbohydrates , and 983 kJ of energy . The absorbance was measured at λ = 765 nm, using a spectrophotometer . The concentration of o-diphenolic compounds in the methanolic extract was determined by the method of Dridi-Gargouri et al. , [T] and [C] were determined using Architect Ci 4100 d’ABOTT. The intra-assay and inter-assay coefficients of variation were < 4% for all assays performed. The ratio testosterone/cortisol [T/C] was derived from the respective concentrations. Additionally, to account for any change in plasma volume shifts post exercise, hematological parameters  were assessed within 3 h in a multichannel automated blood cell analyzer .2) by using between-groups sum of squares and the total sums of squares for all ES. The magnitude of η2 was interpreted following the Cohen’s guidelines as follows: small: 0.01, medium = 0.06 and large: 0.14. For each studied main effect in ANOVA analysis, ES was calculated as partial eta-squared (ηp2) and interpreted for the ANOVA analysis to interpret determine the magnitude of the change score and was assessed using the following criteria: < 0.2 = trivial, 0.2–0.6 = small, 0.6–1.2 = moderate, 1.2–2.0 = large, 2.0–4.0 = very large, and > 4.0 = extremely large [p < 0.05 and data are presented as mean ± SD unless otherwise stated.All statistical analyses were performed using STATISTICA 10.0 Software. Normality of the data distribution was confirmed using the Shapiro-Wilks-W-test. To analyze the effect of POMj supplementation on the [Hcy] and steroidal responses during training sessions , a two-way [supplement (PLA and POMj) × time (pre and 3 min post training session)] ANOVA with repeated measures was employed. To analyze the effect of POMj supplementation on the recovery kinetics of the selected parameters, a one-way ANOVA was utilized. When significant main effects were observed, Tukey’s honest-significance-difference (HSD) post-hoc tests were conducted. The effect size (ES) for each studied interaction in ANOVA analysis was calculated using the eta squared  in response to the weightlifting exercises in both PLA and POMj conditions Table . Additio, [T] and [C]), the T/C ratio and the [Hcy] before and after the weightlifting sessions  are presented in Fig.  = 7.48, p = 0.026 η2 = 0.05) with a significant decrease pre-post training session only registered during the POMj condition , resulting in lower post-session values of [T] during the POMj condition compared to the PLA condition (p = 0.012).Mean and SD values for the steroidal hormones  = 99.78, p = 0.000 ηp2 = 0.93) and [T/C]  = 63.64, p = 0.000 ηp2 = 0.89) parameters with [C] level significantly decreased from pre- to post- training session in both PLA  and POMj  conditions; while [T/C] level significantly increase immediately after the training session  for POMj. However, no significant difference between both conditions was registered for [C] and [T/C] ratio 3 min post training session (p > 0.05). Similarly, no significant effect of supplementation condition or of training session were observed for the [Hcy] during the weightlifting training session (p > 0.05).Significant main effect of the training session was observed for both [C] (Fp = 0.04 and Δ rate of decrease = − 14 ± 12.9%). However, both PLA and POMj condition showed a significant increase in plasma [C]  and a significant decrease in [T/C] ratio .Table p > 0.05). Similarly, for all tested parameters, no significant differences were observed between the plasma levels registered at pre- and 48 h post- exercise sessions indicating that in both conditions, 48 h recovery period was sufficient to recover the baseline values of [Hcy], [T] and [C] (p > 0.05).For all tested parameters, no significant difference was shown between PLA and POMj in plasma levels 48 h post training session (r = − 0.50 and p = 0.002). Concerning the inter-relation between the steroidal hormones measures, a significant low correlation was found between [T/C] ratio and [T] (r = 0.04 and p = 0.02), while a significant moderate negative correlation was found between [T/C] ratio and [C] (r = − 0.05 and p = 0.003) with no significant correlation was found between [T] and [C] (p > 0.05).Table r = − 0.5) to [T] responses.The aim of the present study was to investigate the effect of a specific supplementation protocol  with a natural POMj on the acute and delayed [Hcy] and steroidal hormone responses following a weightlifting training session. In response to the training session, circulating [C] decreased and [T/C] increased in both PLA and POMj conditions, while [T] decreased only following POMj supplementation (− 9.8%). During the 48 h recovery period, all tested parameters were shown to recover to baseline values in both conditions with significant increases in [C] and decreases in [T/C] from 3 min to 48 h post-exercises. The slight increase in [Hcy] immediately following exercise, returned to baseline during the 48 h recovery period in the POMj condition, with the acute and delayed responses of [Hcy] shown to be negatively correlated  and/or increases in [C] can lead to an imbalance between anabolism and catabolism in favor of the latter  and [C] before and after strength exercises, but the existing findings are inconsistent. Indeed, while [T] increased significantly from pre to post training session in the studies of Weiss et al.   [The discrepancies between these findings could be due to factors related to the training session such as relative intensity to the maximum load (% of 1 repetition maximum [1RM]) , number to 126%) , 70. It to 126%) . In accoRegarding the effect of POM supplementation on the tested parameters, the present results demonstrated that [T] decreased significantly from pre- to post- exercise only during the POMj condition with no significant difference between PLA and POMj in the [C] and [T/C] responses. These findings are not consistent with those of Al-Dujaili and Smail . who repThe exact mechanisms explaining the post-exercise decrease in plasma [T] following POMj intake still not well established . HoweverThe delayed response of the tested parameters is in agreement with studies conducted by Häkkinen et al. , Jensen The present results also showed a significant negative relationship between [Hcy] and circulating [T]. This association is in line with previous findings suggesting [T] as independent predictor and regulator of plasma [Hcy] concentration in both health and diseases individuals , 91. HcyTaken together, these preliminary findings suggest a possible beneficial effect of dietary intake of natural polyphenol-rich POMj (250 ml POMj × 3 times daily) on acute plasma [T] response following high-intensity strength exercise in an athletic population. However, given that the potential applications of this work  are most relevant for an athletic population, further large-scale research in both athletic and non-athletic populations is needed to corroborate these preliminary observations and to elucidate the potential underlying mechanisms and translational potential of our novel observations in the current study.This study is the first to test the efficacy of POMj supplementation on [Hcy] and circulating steroidal hormones. Supplementation with POMj has the potential to attenuate the acute plasma [T] response, suggested to predict and regulate plasma [Hcy], but with no potential effect on the recovery kinetics of the tested parameters as all parameters returned to baseline values 48 h post exercise in both the PLA and POMj conditions. Additional large, randomized trials considering a longer intervention period with different doses of POM intake and investigating large androgens and inflammatory cytokines in both plasma and muscle tissue are warranted to resolve the functional consequences of the observed acute POMj effect. Since resistance training is now recommended to enhance health outcomes in the general population , further


Attention deficit hyperactivity disorder (ADHD) is one of the most common mental disorders diagnosed in children below the age of 12 years. It is characterized by hyperactivity, inattention, and impulsive behavior. ADHD affects the social, academic, and psychological aspects of children and adolescents. Children with ADHD struggle with school tasks and performance. They have lower grades than their peers and have difficulties interacting with their friends.Oppositional defiant disorder (ODD) is a mental disorder characterized by disruptive behavior, a pattern of angry and irritable mood, argumentative, and vindictive behavior. Children with ODD struggle with forming friendships and have problems at school. Conduct disorder (CD) is divided into the childhood onset and the adolescent onset types. The childhood onset is associated with poor outcomes in adulthood, an increase in criminal behavior, violence, and progression to antisocial behavior. Children with CD are at increased risk for substance use disorders (SUD) and antisocial personality disorder. The current literature review is aiming to provide an overview of the psychosocial impact of comorbid ODD and CD in children with ADHD. The results of this study review showed the comorbidity of ODD and CD is very strong. ODD is a strong predictor of CD in boys. The presence of comorbid ODD and ADHD in children is a significant predictor of adolescent onset CD. The comorbidity of ADHD with ODD and CD worsens symptom severity and is associated with high psychosocial dysfunction. Children with ADHD and comorbid ODD and CD have difficulties with school, friends, and trouble with the police.  Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder diagnosed in children below the age of 12 years. It is characterized by difficulty paying attention, excessive activity, disruptive behavior, and impulsivity. The prevalence of ADHD among children and adolescents is 7.2% . ChildreCD is a mental and behavioral disorder in children. Children with CD suffer from disruptive behavior and have problems with following rules. Children with this disorder have repetitive and persistent patterns of aggression, destruction, and deceitfulness. CD is twice more common in boys compared to girls. The prevalence of CD in the United States is 6% to 16% in males and 2% to 9% in females. Symptoms appear earlier in boys at a median age of 11 years compared to girls with a median age of 15 years. Children with CD have educational difficulties; they often struggle with psychosocial functioning and isolation. Other psychiatric comorbidities are common with CD, such as depression, ADHD, substance abuse, and ODD [ODD is a disruptive behavioral disorder in children; it consists of a pattern of angry and irritable mood, argumentative, and vindictive behavior . The lifCD and ODD frequently coexist together. ODD is believed to be a risk factor to CD . ODD co-The objective of this literature review is to explore the impact of comorbid disorders like CD and ODD on children and adolescents diagnosed with ADHD. We are looking to increase our understanding of how children with these comorbid disorders function socially, psychologically, and academically comparable to children with ADHD only. Our study review aims to find out why these disorders commonly occur together and what common risk factors that might coexist. We have reviewed past research studies that discussed ADHD, CD, and ODD as separate disorders and as comorbid disorders.Methods and resultsData were searched on PubMed using regular keywords: "Attention deficit hyperactivity disorder (ADHD)", "Conduct disorder (CD)", "Oppositional defiant disorder (ODD)". Table ResultsThe total number of keywords search results was 39,520. The following is a breakdown of the keywords searched and the volume of results: Attention deficit hyperactivity disorder (ADHD) keyword search results was 37,126· Conduct disorder (CD) keyword search results was 1,244· Oppositional defiant disorder (ODD) keyword search results was 1,150Only research articles related to human studies that were published since 2002 in the English language were included in this study. All types of research articles were included except for books and documents. After the manual screening of each article, the relevant research studies for this literature review were selected. A total of 31 articles were selected for this study to determine the impact of comorbid CD and ODD in children with ADHD on psychosocial functioning and behavioral adjustment.DiscussionBased on the study results, ADHD is associated with impairment in psychosocial functioning. Children with comorbid CD, ODD, and ADHD have significant learning and intellectual disabilities. Children with these disorders struggle psychologically, socially, and academically.Attention Deficit Hyperactivity Disorder ADHD is divided into three subtypes: predominantly inattentive, predominantly hyperactive-impulsive, or combined. Children with ADHD struggle to perform academically in school. They typically have lower scores and grades compared to their peers and have higher rates of school dropouts. The poor academic performance is linked to a lack of attention rather than hyperactivity. Girls are more likely to struggle with attention problems compared to boys who struggle with hyperactivity and impulsivity. About 20% to 25% of children with ADHD have learning disabilities . There iChildren and adolescents with ADHD struggle socially and are often isolated from their peers. They have high conflict levels within their families. This behavior is possibly due to the difficulty in emotion regulation . ChildreConduct Disorder CD is divided into childhood onset and adolescent onset types. The onset of CD in childhood is associated with poor outcomes in adulthood, such as an increase in criminal behavior, violence, and progression to antisocial behavior. Children with CD are also at an increased risk for substance use disorders (SUD) and personality disorders such as antisocial personality disorder . NeuroimOppositional Defiant DisorderAccording to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V), ODD consists of three dimensions: irritability and anger, vindictiveness, and argumentative behavior. Children with ODD struggle with forming friendships and have problems in school . ComorbiComorbidity of CD and ODDCD and ODD are disruptive behavior disorders that commonly coexist with 96% in the clinical samples and 60% in the general population. ODD is a strong predictor of CD in boys but not in girls. Some studies found CD is associated with behavioral problems, while ODD is more commonly linked to emotional regulation issues. However, more recent evidence has indicated that ODD includes both emotional and behavioral problems. ODD was considered a mild form of CD that forms at an early developmental stage. The Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) mentioned that all features of ODD are essentially present in CD, and diagnosis of CD essentially includes ODD .Whether CD and ODD are completely separate disorders or expressions of one underlying disorder is a cause of scientific debates. Some argued that ODD is an exaggeration of common features like the rebellious behavior that happens normally in adolescents; therefore, it should be considered as a temperament dimension, not a separate categorical disorder. The repetitive pattern of behavior in which there is a violation of the basic rights of others is an important aspect that characterizes CD. Although both CD and ODD are more common in boys compared to girls, CD is significantly more common in boys than ODD. A longitudinal study  showed ODD develops at a median age of 2.5 years compared to CD that develops at a median age of 5.5 years. CD is associated with a high risk of SUD such as alcohol use disorder (AUD) and tobacco. The comorbidity of the two disorders increases the risk of SUD. Children with comorbid CD and ODD are at high risk to develop antisocial personality disorder, anxiety, and depression . Comorbidity of ADHD, CD, and ODDADHD, CD, and ODD were under one heading in the DSM-IV: “Attention Deficit and Disruptive Disorders". In DSM-V, ADHD is considered a neurodevelopment disorder. CD and ODD are classified as disruptive, impulse-control, and conduct disorders.ADHD and ODD: About 47% of children with ADHD have ODD . Those cADHD and CD: About 30% to 50% of children with ADHD have comorbid CD. Some studies showed the presence of ADHD predicts antisocial behavior and is an indicator of the severity of the symptoms developed in CD. Children with ADHD and comorbid CD have higher rates of criminal behavior as adults, SUD, and school dropouts . While AThe presence of comorbid ODD and ADHD in children is a significant predictor of adolescent onset CD type, especially in females . ADHD, OA cross-sectional study n=1,029) found out that children with ADHD and intellectual disability are at significant risk to develop ODD and show more symptoms of comorbid CD . ADHD is9 found oThe comorbidity of ADHD with ODD and CD worsens symptom severity and is associated with high psychosocial dysfunction Figure . Figure Study LimitationOur study is based on reviewing research studies in the last 18 years, so possibly we missed other important contributions from studies before this. We did not perform a systematic review in our study and no quality assessment of the selected research studies was done.ADHD is a common neurodevelopmental disorder in children. Comorbid disorders, such as CD, ODD, anxiety disorders, and learning disabilities, are frequently associated with ADHD. ODD and CD are disruptive disorders that usually coexist together. SUD and antisocial personality disorder are comorbid disorders with ODD and CD. Children and adolescents with ADHD, CD, and ODD are at an increased risk to develop other problems like anxiety disorders, SUD, and mood disorders. Those children struggle to function among their peers, family, and school. Learning and intellectual disabilities are also common. ADHD, CD, and ODD share common risk factors, genetics, and symptoms. Children presented with ADHD should be carefully screened for the presence of other comorbid disorders like CD and ODD. Properly analyzing and addressing the common psychosocial risk factors is a crucial step in the prevention of these comorbid disorders. Creating educational programs and support groups to ease the burden of the comorbid disorders on the families of the affected children is necessary. Creating effective networking between families, teachers, and health care providers are essential. Further research studies are required to address the genetic and neurobiological aspects of these comorbid disorders.


Background: Hyponatraemia is associated with increased morbidity, increased mortality and is frequently hospital-acquired due to inappropriate administration of hypotonic fluids. Despite several attempts to minimise the risk, knowledge is lacking as to whether inappropriate prescribing practice continues to be a concern. Methods: A cross-sectional survey was performed in Danish emergency department physicians in spring 2019. Prescribing practices were assessed by means of four clinical scenarios commonly encountered in the emergency department. Thirteen multiple-choice questions were used to measure knowledge. Results: 201 physicians responded corresponding to 55.4% of the total population of physicians working at emergency departments in Denmark. About a quarter reported that they would use hypotonic fluids in patients with increased intracranial pressure and 29.4% would use hypotonic maintenance fluids in children, both of which are against guideline recommendations. Also, 29.4% selected the correct fluid, a 3% hypertonic saline solution, for a patient with hyponatraemia and severe neurological symptoms, which is a medical emergency. Most physicians were unaware of the impact of hypotonic fluids on plasma sodium in acutely ill patients. Conclusion: Inappropriate prescribing practices and limited knowledge of a large number of physicians calls for further interventions to minimise the risk of hospital-acquired hyponatraemia. Hyponatraemia, defined as a plasma sodium (P-Na) < 135 mmol/L, is the most common electrolyte disorder affecting 15% to 30% of hospitalised patients, and is associated with increased mortality, morbidity, and length of hospital stay . WorldwiIn healthy individuals, the body maintains P-Na within the normal range 135 to 145 mmol/L) by controlling water intake (thirst/nausea) and excretion [5 mmol/L Fortunately, hospital-acquired hyponatraemia is largely preventable by appropriate use of intravenous fluids as first suggested by Moritz and Ayus in 2003 for maintenance fluids in children . Since tSeveral attempts have been made by medical associations and national regulatory authorities to reduce the risk of hospital-acquired hyponatraemia caused by intravenous fluids ,13,14,15The primary aim of this study was to explore intravenous fluid prescribing practices among physicians working at EDs in Denmark, in order to determine whether further interventions are needed to reduce the risk of hospital-acquired hyponatraemia.We conducted a cross-sectional survey amongst physicians working at EDs throughout Denmark using a self-administered questionnaire  patient with hypovolaemia.A child in need of maintenance intravenous fluids without hypovolaemia and hyponatraemia.A hypovolaemic and hyponatraemic (P-Na = 110 mmol/L) patient without severe symptoms of hyponatraemia.A hyponatraemic (P-Na = 118 mmol/L) patient with severe symptoms of hyponatraemia.Four clinical scenarios  describing different situations encountered in the ED where IV fluids are administered were used to measure prescribing practices of intravenous fluids ,21. The After each scenario, participants were asked to select the first-line treatment of choice between eight commonly used intravenous fluids with different electrolytes and/or carbohydrates content and tonicity see . In all The primary outcome measures were prescribing practice measured as correct answers to the scenario questions and selection of hypotonic fluids as this is associated with hospital-acquired hyponatraemia.Renal water excretion in the acutely ill patient.Intravenous fluids impact on P-Na in the acutely ill patient.Hyperglycaemia and P-Na.Severe symptoms of hyponatraemia.Patients at high risk of severe symptoms.Prevention and treatment of over-correction of hyponatraemia.After the four scenarios, participants were asked to answer 13 factual knowledge questions within the following topics:http://www.limesurvey.org) for the electronic version of the questionnaire.We used LimeSurvey version 2.67.2  of correct responses. For the scenarios, also selection of hypotonic fluids was summarized (counts and percentages).Simple logistic regression was used to explore predictors of prescribing practice. The aim of this analysis was to decide whether we should target the interventions at certain EDs or towards certain groups of physicians. Based on responses to the four scenarios, we defined a priori, critical prescribing practice as comprising of none or one correct response, and excellent prescribing practice equivalent to three or four correct responses. Excellent prescribing practice indicates no need for additional risk minimisation measures, and a critical prescribing practice indicates a need for an intensified and differentiated intervention. The following predictors were assessed: age, number of weekly treated patients with intravenous fluids, position, years of practice, region, size of the hospital, complexity of services they provide, and type of patients they serve.All data handing and analysis was performed using SPSS .Fifteen (71%) out of 21 hospitals, and 23 (61%) out of the 38 invited EDs participated in the study. Four (11%) EDs refused, and 11 (29%) did not respond to the invitation.The primary analysis is based on the 201 respondents who responded to all scenarios, representing 55.4% (201/363) of the total population of physicians working at EDs in Denmark based on the estimated source population from 2014  answered all four questions correctly, 5.0% (10/201) three, 59.2% (119/201) two, 27.9% (56/201) one, and 6.5% (13/201) answered none correctly. In terms of excellent and critical prescribing practices, this corresponds to 6.5% and 34.4%, respectively. The fluids selected for each scenario are detailed in In scenario 2 and 3, hypotonic fluids included the strongly hypotonic fluids see . In scenIn the first scenario, describing normo-natraemic and hypovolaemic women with potentially increased intracranial pressure (meningitis), 71.6% (144/201) choose the correct intravenous fluid  to restore normal hydration status. Hypotonic solutions including the moderate hypotonic solution, Ringer’s acetate, were incorrectly selected by 24.9% (50/201). Due to the low sodium content, hypotonic fluids are inappropriate to restore the circulatory volume. Therefore, isotonic saline solution and Ringer’s acetate/lactate are the fluids of choice for most patients with hypovolaemia . HoweverThe second scenario presented a 5-year-old boy in need of maintenance intravenous fluid. The correct option  was selected by 10.4% (21/201). The most commonly selected fluid (83 out of 201 corresponding to 41.3%), though incorrect, was the isotonic saline solution. Hypotonic intravenous fluids were incorrectly selected by 29.4% (59/201).The third scenario was a 75-year-old hypovolaemic and hyponatraemic (P-Na = 110 mmol/L) woman in treatment with thiazide diuretics without severe symptoms of hyponatraemia. The correct fluid  was selected by 67.7% (136/201), and 10.4% (21/201) selected hypotonic intravenous fluids incorrectly.The fourth scenario was a 28-year-old hyponatraemic (P-Na = 118 mmol/L) woman with severe symptoms of hyponatraemia  ,27,28,29Percentage of correct responses to the knowledge questions are listed in The predictive value of characteristics of respondents and EDs on critical prescribing practice is presented in The most striking finding of this study was the choice of hypotonic fluids in high-risk patients; a quarter of respondents selected a hypotonic fluid for a patient with potentially increased intracranial pressure and more than a quarter selected a hypotonic maintenance fluid for a child. Moreover, if a patient develops hyponatraemia during hospitalisation due to intravenous fluid treatment, it would probably not be linked to the administration of intravenous fluids, since most physicians were unaware of the impact of hypotonic fluids on P-Na in acutely ill patients. Finally, even if the respondents were able to link the treatment of intravenous fluids to hyponatraemia, less than one-third knew how to treat a patient with severe symptoms of hyponatraemia, which is a medical emergency.Children and patients with potentially increased intracranial pressure are patient-populations at particular risk of hyponatraemic encephalopathy upon hypotonic intravenous fluid treatment. Consequently, the European product information of physiologically hypotonic intravenous fluids was updated in 2017/2018 to include, amongst others, a warning of hospital-acquired hyponatraemia in these patients ,27,30, aNumerous studies of maintenance intravenous fluids in children have shown that isotonic fluids are effective in preventing hospital-acquired hyponatraemia . FurtherOf note in scenario 2, describing a child in need of maintenance fluids, physicians working at a paediatric ED performed better than physicians working at adult EDs when comparing those correctly answered (isotonic NaCl with 5% glucose); 16% versus 4%, respectively. However, when looking at selection of hypotonic fluids, physicians working at a paediatric ED performed more poorly; 40% selected a hypotonic fluid as opposed to 17% by physicians working at adult EDs.Our results are consistent with both a recent study conducted in Spain which reveals that 29% of all paediatricians there continue to use hypotonic fluids as maintenance intravenous fluid ; as wellIdentifying hyponatraemia from mismanagement of intravenous fluid therapy and establishing a causal association between the two can be difficult. Our study may shed light on this challenge. In the knowledge questions for example, participants were asked about intravenous fluids’ impact on P-Na in patients with reduced renal water excretion. Since only 24.0% to 46.2% was able to classify the selected and commonly used intravenous fluids correctly, our findings suggest a lack of knowledge about tonicity and its physiological effects, which may preclude recognition of the association between hypotonic fluids and hyponatraemia.In the clinic, linking hypotonic intravenous fluid treatment and hyponatraemia is further challenged. Firstly, differing personnel are involved in taking care of patients; one person might set up the intravenous fluid, but another may later inspect the patient and assess the treatment. Secondly, the presenting symptoms  and, to some extent the severe symptoms , are nonspecific and easily overlooked, which could delay a diagnosis. The length of time separating the administration and a diagnosis complicates and hinders the recognition of hyponatraemia and its association to intravenous fluid treatment. Confused labelling, whereby many strong hypotonic fluids  are labelled as ‘isotonic’ though become physiologically hypotonic once the glucose is metabolized, challenges the issue further.Our findings are in accordance with the NICE guideline that states that there is a general lack of knowledge amongst health professionals concerning the composition of intravenous fluids and that mismanagement is rarely reported .Although isotonic maintenance fluids reduce the risk of hospital-acquired hyponatraemia, it will not eliminate the problem completely as hospital-acquired hyponatraemia is common in children receiving isotonic fluids as well . In addiThe results of this study should be presented in perspective to its strengths and limitations. The strength of our study is that we were able to approach the complete source population. In addition, the scenarios were based on real-life cases and developed in close collaboration with a clinician, and expert within the field of intravenous fluid treatment.There were several limitations to this study. First, we cannot conclude whether inappropriate prescribing practice contributed to adverse patient outcomes. However, it was not considered feasible to measure actual patient outcomes in our study due to lack of intravenous fluid administration recording and the underreporting of complications to intravenous fluid therapy. A scenario-based survey was therefore considered a more valid method to measure prescribing practice ,21.Secondly, with regard to prescribing practice and knowledge, we do not know how those who agreed to participate differ from those who refused or did not respond to the invitation. Together with a response rate on 55.4%, this clearly limits the generalizability of our study. However, it is worth noticing that the response rate represents 55.4% of all physicians working at an ED in Denmark, and there was a balanced distribution with regards to region, type of hospital, and experience, with a slight overweight of experienced physicians as consultants accounted for 44% of our study compared to 35% in the population . TherefoLastly, there is uncertainty and controversy about whether scenarios reflect actual clinical practice. The Hawthorne effect, a tendency to perform better when observed, seems inevitable . In addiOur findings support the recommendations by NICE and several other studies regarding the need for guidance on intravenous fluid therapy ,43,44.It is important to recognise that intravenous fluids are medicines and intravenous fluid prescribing should be held in the same regard to that of any medicines in order to avoid serious complications from intravenous fluid treatment. Thus, regular monitoring is needed so that responses to intravenous fluid treatment can be evaluated and altered or stopped as appropriate. When monitoring P-Na, it is important to use the same technique as differing techniques can yield different results .The association between hyponatraemia and hypotonic intravenous fluids.Intravenous fluids’ impact on P-Na in patients with reduced water excretion.Treatment of patients with severe symptoms of hyponatraemia.This study indicates that inappropriate prescribing practices may be caused by insufficient knowledge, which can help with tailoring the needed intervention. Based on our findings, the following areas may require additional attention when setting up an educational program for prescribers:In our regression analysis, we observed no significant differences in prescribing practices between senior and junior doctors and the other selected characteristics, hence there is no reason to differentiate or target the intervention at certain EDs or towards certain groups of physicians.In conclusion, the current prescribing practices of a large numbers of physicians working at EDs in Denmark calls for further interventions to reduce the risk of hospital-acquired hyponatraemia.


A large amount of the current literature has focused on the characteristic symptoms of attention deficit hyperactivity disorder (ADHD) in children and adolescents. In contrast, less attention has been devoted to ADHD clinical subtypes in adult patients. We evaluated 164 consecutive adult ADHD (A-ADHD) outpatients using DSM-5 criteria and many specific rating scales and questionnaires. A principal component factor analysis was performed on clinical and symptomatological variables to describe potential clinical variants. We sought to determine different A-ADHD variants focusing on demographic and clinical features. A four-factor solution was identified, and patients were clustered, according to their z-score, in 4 subgroups. The first was marked out by Emotional Dysregulation (ED), the second by Substance Use (SU), the third by Core-ADHD Symptoms (Co-ADHD) and the fourth by Positive Emotionality (PE). Predominantly ED patients showed worse overall function, early treatment with antidepressants and a greater presence of borderline personality disorder than predominantly Co-ADHD patients. Predominantly SU patients reported high rates of bipolar disorder and severe general psychopathology. The PE factor was related to hyperthymic temperament and hypomania and showed a higher level of functioning. Females with A-ADHD showed a lower risk of being included in SU, and A-ADHD patients with co-occurring delayed sleep phase had less risk of being included in the SU factor than the prevailing Co-ADHD group. Our empirically based description of four clinical A-ADHD variants shows several aspects beyond the definition given by the DSM-5 diagnostic criteria. Neurodevelopmental disorders include a broad group of disabilities involving some forms of disruption of brain development, and group together a very heterogeneous range of neuropsychiatric conditions where each condition may differ substantially from the others in clinical presentation and etiology . AttentiThe evolutionary trajectory of the disorder into adulthood is variable, and about half of children and young adolescents with ADHD show an attenuation of hyperactivity when they grow up . UsuallyIn epidemiological surveys, the prevalence of A-ADHD is estimated to be around 2.5–4%  and the In the DSM-5, ADHD is described according to a two-dimensional model comprising inattentive, hyperactive-impulsive or combined subtypes. This model underestimates several clinical aspects of fundamental importance to the clinical management of adults with the disorder.However, given the proposals made in scientific research, the current categorical approach to nosography may be intended to be overcome by a more appropriate etiopathogenetic classification system of mental diseases consistent with the existence of definite neurobiological processes that support a variety of psychiatric conditions that share the same pathophysiology. These pathophysiological traits may be explained by the clinic as transdiagnostic psychological/psychiatric defects and vulnerabilities that play the role of “endophenotypes”. In particular, The Research Domain Criteria (RDoC) project ,13 develAttractively, the RDoC provides a dimensional viewpoint on psychiatry, ranging from normal to pathological, including diagnostic subthreshold psychological/psychiatric settings, psychological/psychiatric precursors and predisposing factors. Prime patterns of these neurobiologically based systems considered in the RDoC transdiagnostic approach to the pathogenesis of addiction, and in mood and impulse control dysfunctions, are reward-processing systems and inhibitory systems.Different variants of A-ADHD have been described, with different courses ,15. HoweThe term “emotional dysregulation” is used to identify a series of affective and behavioral phenomena found in many psychiatric disorders ; it is oThe aim of this study is to explore the presence of different clinical patterns in a population of A-ADHD subjects so as to more accurately describe the heterogeneity of the disorder in adulthood.This is a multicentric, cross-sectional, observational study. The sample included 164 consecutive outpatients admitted, with the same protocol, to Psychiatric Unit 2 at the University of Pisa or to the Department of Neurology and Psychiatry of the Sapienza University in Rome. All patients were diagnosed with A-ADHD according to DSM-5 criteria. No exclusion criteria were applied, except for the patient’s age (≤18 years old). Demographic data, psychiatric history, past psychiatric treatment, past ADHD specific treatment and self-reported drug use were recorded at study entry. Resident and senior psychiatrists of the ADHD research groups evaluated the recruited patients by holding a clinical interview and specific diagnostic tests. The study was conducted between 2016 and 2019, in accordance with the WMA Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects. All patients enrolled in the study filled in and signed an informed consent document. Both the consent form and the experimental procedures were approved by the ethics committee of the University of Pisa, in accordance with internationally accepted criteria for ethical research .The diagnosis of ADHD was made according to DSM-5 criteria and confirmed by using the Italian version of the semi-structured, clinician-administered Diagnostic Interview for ADHD in adults (DIVA 2.0). When possible, a caregiver/family member of the patient participated in the interview to provide retrospective and collateral information. Each interview comprised three parts: nine criteria for attention deficit, nine criteria for hyperactivity-impulsivity and a third part evaluating the age of onset and the level of impairment. The presence/absence of each item was investigated both in childhood/adolescence and adulthood. If collateral information provided by the caregiver was not available, the diagnosis was based exclusively on the patient’s recollection.Conner’s Adult ADHD Rating Scales—Observer: Screening Version (CAARS-O:SV): this is a 30-item questionnaire for the caregiver/family member. It evaluates the presence of symptoms and behaviors associated with ADHD in adults, and rates symptoms on a Likert scale, ranging from 0 “never” to 3 “very often”. CAARS-O:SV is a tool designed for the DSM-V criteria, although there is no updated version yet. Nevertheless, it is appropriate to assess the severity of A-ADHD regardless of the version of the DSM. The Screening Version provides four subscale scores: Inattentive Symptoms, Hyperactive-Impulsive Symptoms, Total ADHD Symptoms and ADHD Index. Higher scores indicate clinically significant levels of ADHD symptoms .Adult ADHD Self-Report Scale (ASRS): this 18-question scale was used to assess the frequency of self-reported adult ADHD symptoms derived from the DSM-IV criteria. Answers are expressed on a Likert scale, ranging from 0 “never” to 4 “very often”, and a higher total score indicates significant levels of ADHD symptoms .Brief Temperament Evaluation of Memphis, Pisa, Paris and San Diego (brief TEMPS-M): this is the short version of the 110-item questionnaire evaluating the self-reported affective and anxious temperaments. The brief TEMPS-M consists of 35 items, with a five-point Likert scale ranging from 1 “not at all” to 5 “very much”. The questionnaire assesses five types of temperament: depressive, cyclothymic, hyperthymic, irritable and anxious temperaments .Barratt Impulsiveness Scale (BIS-11): this is a self-report questionnaire designed to investigate the personality/behavioral construct of impulsiveness and is considered the gold standard measure of impulse control. The BIS-11 consists of 30 questions; answers are expressed as a range going from 1 “never/rarely” to 4 “almost always/always”. The scale is structured following a factorial analysis covering six first-order  and three second-order factors  .Difficulties in Emotion Regulation Scale (DERS): this is a 36-item self-evaluation scale of six subdomains of emotional regulation . Answers range from 1 “almost never” to 5 “almost always”, and higher total DERS scores reflect greater difficulties in regulating emotions .Reactivity, Intensity, Polarity and Stability questionnaire (RIPoSt-40): this scale was used to quantify emotional dysregulation in its different facets. It consists of 40 self-administered questions with answers given on a Likert scale going from 1 “never” to 6 “always”. Results can be divided into four subscales: affective instability, positive emotionality, negative emotionality and emotional impulsivity. A second-order scale can be identified as follows: negative emotion dysregulation, comprising affective instability, negative emotionality and emotional impulsivity .Hypomania Check List-32 (HCL-32): used to evaluate lifetime hypomanic symptoms, it consists of 32 questions with yes/no answers and has a two-factor structure (active/elated hypomania and risk-taking/irritable hypomania) [Brief Psychiatric Rating Scale (BPRS): 18-item tool commonly used by clinicians to assess current psychopathology and symptom severity .WHO Disability Assessment Schedule (WHODAS 2.0): a 36-item questionnaire used to explore self-reported functioning and disability in six major life domains: cognition, mobility, self-care, getting along, life activities, participation. Answers are given on a Likert scale ranging from 1 “no difficulty” to 5 “extremely difficult/cannot do” .The clinical evaluation was based on the following scales:Self-reported substance use was expressed by a four-level scale with increasing time of use: 0 = “never used” for non-SUD patients, 1 = “abstinent for more than one year”, implying use only in the past, 2 = “abstinent for less than one year” as recent use and 3 = “used for more than one year” as chronic use. We considered alcohol, THC and cocaine.A principal component factor analysis was performed on clinical and symptomatological variables in order to reduce the heterogeneity among our A-ADHD patients. The factors which allowed the best possible reconstruction of the data were extracted by means of principal component analysis (PCA); we selected factors with an eigenvalue greater than 1. We then rotated our factor matrix according to Varimax with Kaiser normalization in order to achieve a simple structure and interpret the factor loadings. The loadings with a magnitude greater than the conventional cut-off of 0.40 were selected and used to describe the factors. In order to make factor scores comparable, they were standardized by converting them into z scores. All the subjects were then assigned to one of the different subtypes based on the highest z-scores obtained for each factor (predominant factor). This procedure gave us the opportunity to classify patients on the basis of the highest-ranking symptomatological cluster.We also explored differences between the four different A-ADHD subtypes regarding demographic data, past psychiatric treatment and psychiatric comorbidities. For continuous variables, we used the Kruskal–Wallis test; for categorical variables, we used Pearson’s Chi-squared test with post hoc Bonferroni corrections.Third, we performed a backward multinomial logistic regression to test clinical and demographic variables as possible predictive values of A-ADHD subtypes.We used the statistical routines of IBM SPSS Statistics for Macintosh, Version 25.0 .In our sample of 164 patients with a diagnosis of A-ADHD, 112 (68.3%) were males and 52 (31.7%) females. The mean age was 28.3 (sd = 10.7) years, mean body mass index was 24.3 (sd = 5.1). Of these 164 subjects, 42 (25.6%) were unemployed, and 122 (74.4%) were employed; the mean number of years of schooling was 13.40 (sd = 3.18). Regarding marital status, most of the subjects  were single, while 24 (16.6%) were married. A total of 85 (51.8%) subjects had received previous psychiatric drug treatment and more precisely, 53 (32.3%) with antidepressants, 37 (22.6%) with antipsychotics. Only 45 (27.4%) had been diagnosed which allowed them to receive specific treatments (methylphenidate or atomoxetine) for ADHD in childhood or adolescence.Regarding psychiatric comorbidities, 134 (81.7%) of our patients had at least one comorbid psychiatric disorder: 66 (40.2%) had anxiety disorders, 66 (40.2%) had bipolar disorder, 7 (4.3%) had major depressive disorder, 72 (43.9%) had borderline personality disorder, 10 (6.1%) had obsessive compulsive disorder, 28 (17.1%) had specific learning disabilities, 13 (7.9%) had tic disorders, 8 (4.9%) had an intellectual disability, 13 (7.9%) had autism spectrum disorder, 19 (11.6%) had feeding and eating disorders and 60 (36.3%) had delayed sleep phase disorder.Factor analysis, which is reported in p = 0.006).The comparisons of demographic and clinical variables among the, predominantly, Emotional Dysregulation, Substance Use, Core-ADHD Symptoms and Positive Emotionality groups of patients are reported in In the multinomial logistic regression analysis , we studA positive association was found for the ED-predominant group with borderline personality disorder. The SU-predominant group was positively associated with bipolar disorder and negatively with female gender, professionally employed and delayed sleep phase disorder. Lastly, the PE-predominant group was negatively associated with delayed sleep phase disorder.The demographic and clinical characteristics of our patients were consistent with those reported in other A-ADHD samples ,36. The In a recent report, the DSM-5 distinction between predominantly inattentive, hyperactive/impulsive and combined subtypes  has beenThe importance of emotional dysregulation in the psychopathology of neurodevelopmental disorders has been confirmed by many empirical observations ,40. MostEmotional dysregulation is characterized  by rapidIn our first factor, the severity of emotional dysregulation (measured by DERS-TOT and RIPOST-ED) and the associated affective temperaments (TEMP-depressive and TEMP-cyclothymic) are grouped together, so suggesting relative independence of the DSM-5 core-ADHD features. This result is consistent with other reports . A signiAs for positive emotionality, in our sample, due to the presence of hyperthymic temperament, RIPOST-Positive Emotionality and HCL-32 total scores, it seems to be relatively independent of the severity of emotional instability, supporting the possible existence of an A-ADHD subtype with less difficulty in keeping emotional control ,49 and aConsistent with the view that substance use is very common in A-ADHD patients, we observed a high rate of SUD in our sample ,51. In oComparative studies on demographic and clinical characteristics shown by the various clinical subtypes showed that patients with stable employment are more common in the PE than in the SU group. This observation is consistent with research showing that subjects with positive emotionality tend to be less sensitive to negative feedback while being better adapted socially . They alWe will now compare various previous treatments that rely on psychiatric drugs: as expected, ED-predominant patients were more frequently treated with antidepressants than those with predominant PE. Emotional dysregulation is, indeed, associated with negative affect and more frequently prompts medical attention. In addition, the prolonged use of antidepressants may be associated with emotional instability ,66 and mBipolar disorder and SUD frequently coexist ,71 and tGender differences resulted when multinomial logistic regression was applied, with A-ADHD females showing a lower risk of having SUD. The same finding was reported by Sobanski et al. , which hIt is known that females have greater difficulty in regulating negative emotions in respect to males; this leads to a propensity towards comorbidities in the emotional spectrum and anxiety disorders ,77,78, wLimitations: In our sample, there was an imbalance between males and females, which may skew the generalization of our results. Additionally, the sample was relatively uneducated, lacking college graduates and also lacked marital statuses other than single, which is not generalizable to most of the adult population. However, our sample gives a good description of individuals with A-ADHD who come for a consultation at a level II psychiatric clinic. As an additional issue, most of our A-ADHD subjects had never been diagnosed with the disorder, they had never been medicated or were aware of their symptoms. Thus, they may not have had the awareness to report accurately on their ADHD symptoms. However, this fact often happens in real life; narrative inconsistency and alexithymia are typical of patients with A-ADHD. Thus, the clinical variants highlighted by us are not negatively affected by that. At the most, this fact accounts for the diagnostic difficulties encountered by psychiatrists.In this study, we have described four clinical variants of A-ADHD, distinguished as ED, SU, CAS and PE. Predominantly ED patients showed worse overall functioning, frequent early treatment with antidepressants and common association with borderline personality disorder. A-ADHD patients with predominant SU were frequently males with comorbid bipolar disorder who showed severe general psychopathology. The predominantly CAS variant showed co-occurring delayed sleep phase, and the PE variant was distinguished by hyperthymic temperament and hypomania, with a relatively high level of functioning.Our empirically based description of four clinical variants of A-ADHD shows several aspects that elaborate on details of the DSM-5 diagnostic criteria. This clinical perspective may have pertinent implications for patient identification and management in various psychiatric settings.