Klebsiella pneumoniae causes severe disseminated infections, typically with hepatic and central nervous system involvement including endophthalmitis. Case Presentation. We report a fatal case of an undocumented Chinese migrant in her 60s who presented to the emergency department with abdominal pain, lethargy, and headache over the preceding two weeks. She had a new diagnosis of diabetes mellitus on admission. Computed tomography scan of the thorax, abdomen, and pelvis showed bilateral pneumonia with liver abscesses. The patient was treated with empirical broad-spectrum antibiotics before K. pneumoniae was isolated from cerebrospinal fluid and blood cultures. The isolate was further characterised as a ST23 (ST: sequence type), serotype K1 hypervirulent strain using Nanopore sequencing. Despite admission to the intensive care unit, the patient died within 48 hrs of admission.  Hypervirulent K. pneumoniae, even in countries where it occurs infrequently. Novel, rapid, sequencing technologies can support diagnosis in unusual presentations. This case demonstrates the need for increased awareness of hypervirulent  Klebsiella pneumoniae (cKp) is an opportunistic pathogen that typically causes urinary tract infections and is a leading cause of nosocomial pneumonia. cKp infections are most common amongst those at the extremes of age or in those who are immunosuppressed. In contrast, hypervirulent K. pneumoniae (hvKp) causes severe disseminated infections in otherwise healthy individuals [Classical ividuals . hvKp isividuals –6. AlthoThe decreasing cost and size of portable sequencing devices are of increasing interest in atypical clinical presentations of infectious diseases to rapidly genotype pathogens. Having this information available routinely in clinical microbiology laboratories could aid diagnosis and ensure rapid treatment.K. pneumoniae grown on blood and cerebrospinal fluid culture.We report the case of a woman with hepatic abscesses, bilateral pneumonia, and meningitis with The infection was unfortunately fatal, and subsequent sequencing using a portable nanopore-based device further characterised the isolate as a hypervirulent strain.A female patient in her 60s presented to a hospital emergency department with a 24-hour history of vomiting. She had a two-week history of right upper quadrant abdominal pain and severe frontal headache. She did not report diarrhoea or weight loss and had no history of neck stiffness, photophobia, rash, or limb weakness. She had been generally well since arriving in the United Kingdom (UK) from China 18 months ago. She had no relevant medical history of chronic disease and she was not formally employed. No other family members had been unwell recently, and she reported no foreign travel since arriving in the UK.9/L , and severe thrombocytopaenia (platelets 17 × 109/L). Liver function tests were deranged with increased bilirubin 21 μmol/L, alanine aminotransferase 169 IU/L, and alkaline phosphatase 337 IU/L. Coagulation was abnormal with a prothrombin time of 10.4 seconds and a fibrinogen level >9.3 g/L. Other biochemical tests were unremarkable, with normal sodium (140 mmol/L), potassium (3.1 mmol/L), and creatinine (63 μmol/L) and a mildly raised urea (9.3 mmol/L). The C-reactive protein concentration was very high at 250.6 mg/L. A urine dipstick was negative for leucocytes, nitrites, and blood, but showed high (2+) glucose and very high (3+) ketones.Blood tests on admission showed a mild leucocytosis, with a white blood cell (WBC) count of 10.6 × 10The patient was treated as presumed biliary sepsis with intravenous amoxicillin and clavulanic acid . She was admitted to the acute medical unit and blood was sent for microbial culture. An ultrasound of the abdomen showed multiple solid, mixed echogenicity lesions in both lobes of the liver, the largest of which measured 41 × 34 mm, a normal gallbladder, and nondilated common bile duct. A computed tomography (CT) scan of the brain was reported as normal.3 8 mmol/L), hyperglycaemia (glucose 29.1 mmol/L), and marked ketosis (ketones 7.0 mmol/L). She was intubated for airway protection and started on an intravenous insulin and fluid resuscitation regime for diabetic ketoacidosis. She was given empirical treatment for presumed central nervous system infection with cefotaxime 2 g IV four times daily and aciclovir 10 mg/kg IV three times daily. A repeat CT scan of the brain was reported as normal, and she was admitted to the intensive care unit (ICU). A CT scan of the chest, abdomen, and pelvis showed bilateral pneumonia, appearances suggestive of an abscess within the left lobe of the liver, and moderate intra-abdominal free fluid . Venous blood gas showed metabolic acidosis , respectively. The CSF glucose was low at 1.0 mmol/L, and the red blood cell count was 640 cells/mm3. The sample was positive for xanthochromia, but no organisms were seen on Gram stain or acid-fast stain. Blood and CSF cultures subsequently grew K. pneumoniae, which was reported as sensitive to cefotaxime and gentamicin. The CSF was PCR negative for M. tuberculosis, herpes simplex DNA, Zika virus RNA, and enterovirus RNA. A blood HIV test was negative. The HbA1c level was 100 mmol/mol, suggestive of persistently high blood glucose levels in the preceding months, consistent with a new diagnosis of diabetes mellitus. Following admission to the ICU, the patient was found to have fixed and dilated pupils and, following brainstem testing, a decision was made to withdraw care. The patient died 18 hours later.The CSF was turbid and yellow , with a ® instrument . DNA was extracted and sequenced on a MinION (Oxford Nanopore Technologies); details are shown in Supplemental Material.The blood culture isolate was cultured onto Columbia Blood Agar  and incubated overnight at 37°C. A single colony was picked and subcultured overnight. The antimicrobial susceptibility of the isolate was determined using the N350 card on the VITEK2Klebsiella genomes. All read sets were trimmed, filtered, and assembled using Flye v2.7 [Basecalling was performed using Guppy v3.4.4 with three approaches: a fast option with default settings, a high-accuracy option with a methylation-aware model, and a high-accuracy option with a custom model trained on lye v2.7  and MedaKlebsiella-specific genotyping including multilocus sequence typing (MLST), AMR, and virulence determinants were performed using Pathogenwatch (https://pathogen.watch), Kaptive (https://github.com/katholt/Kaptive), and Kleborate (https://github.com/katholt/Kleborate) [pneumoniae (Supplementary Species confirmation and eborate) . ReferenK. pneumoniae. It was resistant to ampicillin but was susceptible to all other antimicrobials tested. Sequencing via MinION yielded  ∼2 million high-quality reads totalling ∼6 Gbp, giving ∼1150-fold coverage of the genome. Average read length was 3.2 Kbp, maximum read length 70.6 Kbp, and N50 6.0 Kbp. All three assemblies resolved two circular molecules: a 5.4 Mbp chromosome and a 228 Kbp plasmid.The blood culture isolate was hypermucoid  and idenK. pneumoniae. Distance-based phylogenetic analysis showed the novel isolate clustered with the ST23 (sequence type) K1 hypervirulent clinical isolates SGH10 [K. pneumoniae-trained custom model was identified as a single-locus variant of ST23, with virulence-associated features typical of CG23-I [K. pneumoniae virulence plasmid (KpVP-1) typical of ST23 [The genome sequence confirmed the species identification as es SGH10  and NTUHes SGH10  and was es SGH10 . Neverthf CG23-I : capsula of ST23 .K. pneumoniae liver abscess [We present the case of a patient with disseminated hvKp infection. hvKp causes disseminated disease and needs to be rapidly identified in clinical cases to prevent the devastating consequences of overwhelming infection. Rapid sequencing available via nanopore technology such as the MinION device enables prompt identification of pathogenic strains and allows clinicians to appropriately investigate and treat patients. The patient in this case had risk factors for hvKp, including Chinese origin, but had been residing in the UK for 18 months and had been apparently well prior to presentation. Infections caused by hvKp strains were initially reported from South-East Asia in the 1980s but have spread worldwide . Diabete abscess ; althougK. pneumoniae of capsular serotype K1 and sequence type ST23, which accounts for the majority of K. pneumoniae liver abscess cases and is widespread in South-East Asia. ST23 strains with the K1 capsule type are especially prevalent in China [We used a rapid sequencing platform to analyse the bacterial isolate and identified it as in China  and are in China . The genin China  and NTUHin China . The isoin China .K. pneumoniae [This work shows how the portable MinION device can be used to rapidly clarify the aetiology of unusual case presentations, in this case confirming that the infection was caused by a previously recognised hvKp strain with known virulence factors, and to rule out a novel emerging hypervirulent strain. However, there remain technical issues and limitations; the high-accuracy basecalling required for accurate genotyping takes substantial computing resources and clock time (≥24 hours), and best results were achieved with a model that had been previously custom-trained on eumoniae . Once a In summary, this case study demonstrates the benefits of a rapid sequencing method to identify and characterise an unusual, highly pathogenic hvKp strain. Sequencing is becoming easier to perform and less expensive, and bioinformatics analysis tools are simpler to use, heralding the possibility of using this technology in routine clinical microbiology laboratories to rapidly characterise clinically important isolates.


Material and Methods. 503 children under 12 years old (8.7 ± 1.3) were included with anthropometric evaluation ). Levels of opiorphin (OPI), free cortisol, alpha-amylase (sAA), and secreted immunoglobulin (sIgA) were determined by quantitative assays (ELISA) in unstimulated saliva. Unpaired t-test, Welch test, and Mann–Whitney U test were applied for appropriate group comparisons, and the correlation between variables was analyzed with Spearman's rank coefficient. Results were considered significant at p < 0.05.  The use of easily accessible biomarkers for assessing young patients' health is weighty. This cohort study is aimed at measuring stress/immune biomarkers in the saliva of healthy school-age children and comparing subgroups according to age, sex, and stress perception. p = 0.031). sAA was negatively correlated to low mood self-declaration in children in the last two weeks .  sIgA and sAA exhibited significant differences depending on age and sex: IgA (ng/mL): 86 ± 68.6 vs. 104.9 ± 72.1 for (6-7 y.o.) and (8-11 y.o.), respectively, and 108.1 ± 80.1 vs. 94.6 ± 62.2 for male and females, respectively; sAA (U/mL): 78.9 ± 54.4 vs. 100.5 ± 81.2 for (6-7 y.o.) and (8-11 y.o.). No difference related to age or sex between groups was observed for cortisol and OPI. However, OPI levels were higher and correlated to prior stress exposure in children (0.31 ± 0.4 vs. 0.26 ± 0.5 ng/mL,  sIgA and sAA can be used as sex- and age-related biomarkers in children 6-12 y.o., which is not the case for free cortisol and opiorphin. However, OPI reflected previous exposure to stress, suggesting its use for evaluating stress-related changes in children. Psychological stress impairs homeostasis in many aspects, including immune dysregulation  with sigSaliva is a promising medium that has been used to evaluate both acute and chronic stresses . SeveralCortisol has been often used to assess the activity of the hypothalamic-pituitary-adrenal (HPA) stress axis system and received extensive attention in research on stress physiology . Life siSalivary amylase (sAA) was also recently suggested as an indicator of sympathetic nervous system (SNS) activation but significantly different from cortisol. Typical sAA concentration reaches a peak in the late afternoon or evening . In acutIgA is a secreted glycoprotein, part of the adaptive immune system, that acts synergistically with other inherited mucosal defense factors, such as alpha-amylase, lactoferrin, and lysozyme . Similarμ- and δ-opioid receptors in the central nervous system (CNS), therefore displaying analgesic and antidepressant properties [Opiorphin (OPI) is a polypeptide recently discovered in human saliva, increasing the bioavailability of enkephalins acting on operties –25. Salioperties , which aoperties –29. OPI operties , 28, 30 operties .These four biomarkers may be easily measured in saliva, but in variable amounts depending on age and sex, as well as in response to psychological stress . The maiThe study was approved by the Ethics Committee, Poznan University of Medical Sciences (resolution no. 542/14 from 6 December 2014). Parents or legal guardians gave written informed consent for the participation of their children in the study. Prior to the examination, the purpose of the study was explained to the children. They were also asked to express their opinions on the study.Subjects were healthy schoolchildren attending an urban district school in the years 2019-2020 . Ten primary public schools were randomly selected in the aforementioned area and invited to participate in the study. Six schools agreed. During meetings with the parents, professionals explained the details of the study and its noninvasive nature. Then, parents registered their children for final participation. After a general interview, children were screened for inclusion/exclusion criteria. The final sample was then divided into two subgroups according to age: 6-7 (group 1) and 8-11 (group 2) years old (y.o.). The rationale for age categories was based on Poland's educational system and hormone-related changes in children at those ages. As puberty might affect opiorphin production, we excluded children over 12 y.o. since sex steroid impregnation usually does not occur before this age . The schInclusion criteria were as follows : childreExclusion criteria were divided into four categories: (1) school attendance: child absent from school more than 4 weeks in the recent period; (2) general health: permanent somatic diseases, mental or neurodevelopmental disorders, hereditary disorders (first-degree relatives), pharmacotherapy, endocrine therapy, and dietary supplements; (3) oral health: urgent or nonroutine dental treatment, ongoing orthodontic treatment/wear of an orthodontic appliance, and any general or oral treatment prone to alter salivary flow and composition; (4) communication: inability to answer questions and lack of cooperation during physical measurements or salivary sampling.MINI-KID, a proprietary structured questionnaire completed by a specialist in child and adolescent psychiatry –36, was Life or health of the child was seriously endangered (yes/no)Child witnessed an event in which the life or health of another person was at risk or someone died (yes/no)Child experienced physical violence  or psychological violence  (yes/no)Child has witnessed physical or mental violence against another person (yes/no)Child has experienced the death of someone close to him (yes/no)There were severe problems in the family  (yes/no)Child was separated from one of the parents for many days (yes/no)Serious problems with grades at school (yes/no)Another very stressful event (yes/no)“…Tick the events that took place in the child's life in the last 6 months”:Evaluation of all the salivary biomarkers was performed according to subgroups SE1 and SE0.2 (m2) with cut-offs adopted by the International Obesity Task Force (IOTF) to estimate normal or abnormal weight according to age.Body height, weight, waist, and hip circumferences were evaluated. Body height in a standing position was measured with the SECA 216 wall-mounted stadiometer with an accuracy of 0.1 cm. Body mass was recorded in lightweight clothing on a digital scale with an accuracy of 0.1 kg. Waist circumference was determined halfway between the lower edge of the rib arch and the upper iliac crest using metric tape . Hip cirSpecial attention was paid to standardize collection. All participants were referred to the medical school offices between 9 : 00 and 10 : 00 a.m. for examination and saliva sampling, performed by the same qualified dentist (E.P.).Unstimulated whole saliva was collected according to a previously described methodology , 41. Recr2 = 0.998, p < 0.001) [The concentration of free cortisol in the saliva was quantitatively determined by ELISA using the ELISA Kit DES6611 for the in vitro diagnostic (IVD) . The standard curve ranged from 0 to 30 ng/ml, the intra- and interassay variability coefficients were assessed to be below 6%, and the standard curve was statistically significant (< 0.001) , 43. Corr2 = 0.982, p < 0.001) [The sAA concentrations were quantitatively determined by ELISA using the ELISA Kit DEEQ6231 for the in vitro diagnostic (IVD) . The standard curve ranged from 0 to 500 U/ml, the intra- and interassay variability coefficients were assessed to be below 5%, and the standard curve was statistically significant (< 0.001) , 45. sAAμg/ml, the intra- and interassay variability coefficients were assessed to be below 5%, and the standard curve was statistically significant  [μg/mL.Salivary IgA concentrations were quantitatively determined by the ELISA method using the ELISA Kit DEXK276 for the in vitro diagnostic (IVD) . The standard curve ranged from 0 to 400 < 0.001) . Levels r2 = 0.985, p < 0.001). OPI levels are expressed in ng/mL.The quantification of OPI in saliva was performed using a commercial enzyme immunoassay  according to the manufacturer's instructions. The measuring range of the kit was 0.156–10 ng/ml and sensitivity 0.094 ng/ml. The intraplatelet coefficient of variation was <8% with an interplatelet variation coefficient of <10%, and the standard curve was statistically significant  for a wavelength of 450 nm ± 10 nm. A four-parameter algorithm (4 parameters logistic) was used to assess concentration in the tested samples. All tests were performed by an investigator blinded to clinical data and the status of the samples (group affiliation).in vitro diagnostic tests certified by CE IVD certificates. Measurements were selected only at the quantification level, and when two technical replicates were obtained, it was detected with an acceptable difference of <10%.Cortisol, sAA, and IgA were tested with validated We used all the obtained nominal measurements for the calculations, treating them as individual natural variability, as we did not observe extreme deviations in results. We have not adopted any arbitrary cut-off thresholds to distinguish between the correct and the abnormal levels, mainly due to the lack of diagnostic standards for the examined children age group.U test. Spearman's rank correlation was used to detect the relationship between variables. In order to rule out interfering variables that may be biasing the correlation results, we used covariance analysis ) [p value of <0.05 was considered statistically significant.Statistical analyses were conducted in Statistica v13.3 . Normality of distribution was assessed with the Shapiro-Wilk test, and equality of variances was checked using Levene's test. Due to the lack of normality for most of the variables, more restrictive nonparametric tests were used. Comparison of two unpaired groups was performed using the Mann–Whitney ANCOVA))  to checkp > 0.05, Mann–Whitney U test), respectively. Not surprisingly, the anthropological measurements of body weight, height, BMI, and waist size were significantly different according to age and gender . Children from both groups were Caucasians of Polish origin attending a public school for primary education. There were no differences between confessed religions (mainly catholic), domestic-owned animals (mainly dogs), or living places. The MINI-KID questionnaire excluded children at risk of mental disturbances, and the TESI questionnaire included children who experienced stressful events. More than half reported a stressful experience in the past and feeling low mood during the past two weeks . The main characteristics of the sample are summarized in The final sample consisted of 503 children , 6-11 yep < 0.038 and p < 0.026, respectively). A significant difference (p < 0.017) was observed for sAA, found in higher concentration in group 2 aged 8-11 (p < 0.017), especially for boys (p < 0.049). No differences in the levels of cortisol and OPI were detected depending on sex  and age  or any anthropometric parameter. However, OPI was detected in statistically significant higher concentration in children having experienced distressing events   for this condition.Salivary measurements are shown in 031) see . The resp < 0.05) for all children (p < 0.05), was evidenced. OPI showed a significant correlation between age and height in group 1, together with sAA in the whole group of boys (p < 0.05). OPI was the sole marker positively correlated and sAA negatively correlated with children declaring prior distressing experiences and low mood (p < 0.05) (p > 0.05) (Correlations are shown in children . A simil < 0.05) . There w > 0.05) .p < 0.001) as cortisol on body mass index (p < 0.046).Four covariance models (ANCOVA) shown in  were buiThe main results of this study are significant differences in sIgA and sAA among children depending on age and gender. Correlations between salivary immune biomarkers sIgA, sAA, age, sex, and parameters of developmental status were evidenced. Opiorphin was detected in statistically higher concentrations in children reporting previous experiences of stressful events.α-amylase release during stress [In the present study, concentrations of sIgA and sAA were correlated to age and anthropometric parameters. The concentration of sAA showed significant developmental differences in particular for boys, especially for those of the older age group. Other studies focusing on human development have shown that salivary components and salivary gland function may alter with age . The secg stress , 50, andg stress –53.In our study, the increase in sAA levels with age and physical developmental changes suggests a possible application of saliva biochemical analyses to monitor young children's health. Saliva has been increasingly suggested as a good, easily obtainable sample to assess the health status. In this study, the inverse correlation between mental stress among children in group SE1 and sAA was relatively weak. The covariance models (ANCOVA) testing the effects of interaction between clinical factors and salivary parameters did not indicate sAA as a significant one.Similarly to sAA, sIgA levels in the present study were different depending on age. This finding is consistent with previous findings reported by other researchers among children and adult subjects –58. HoweOPI was the only salivary biomarker elevated in children who self-reported previous exposure to stress. Statistical analysis (ANCOVA test) showed that OPI levels were not dependent on age, sex, and body weight , thus reinforcing the statistical link between OPI levels and past stressful events. Although declarative in nature, to our knowledge, no study to date has compared opiorphin to healthy children with and without self-declared past distressing events. OPI has been described as a potent endogenous antinociceptive  and streTherefore, further research specifically exploring the role of stress needs to be continued among such young individuals.μg/dl and 148.98 nmol/l, which is within normal range [No evidence of specific reactive secretion differences was found between children for cortisol levels. Moreover, cortisol levels were unchanged among those who declared previous stressful events, suggesting a well-functioning HPA system. Cortisol levels, measured at the same time point as sIgA and sAA, did not show sex-related differences. However, the covariance models (ANCOVA) showed a dependence between BMI and salivary cortisol. This interrelation may suggest a dependent release from separate systems and individual diurnal settings , 69. In al range .In the present study, much attention was paid to collect a well-controlled cohort matched according to age and sex, with similar demographic features, representative of the general population. The group aged 8-11 was the largest, while the group aged 6-7 was lower in all chosen schools. A possible explanation for such unequal distribution is the overlap between school education and kindergarten for the youngest children. The study excluded children attending kindergartens and focused on those attending schools. Schools share a similar education system regarding rhythms, activities, and programs, which is not the case for kindergartens.We also used a standardized instrument for evaluating the psychological stress among such young children with professional analysis performed by the child and adolescent specialists. However, the exposure to stress was only declarative, as evaluated by the TESI questionnaire. Neither further investigations of the nature of the stress nor assessment of the severity of stress was undertaken. We may also assume that the level of perceived stress for the same stressor may differ from person to person , 73. FurAnother possible bias of the study was the acute psychological stress that may occur during physical measurement and salivary sampling in the school area, despite all precautions taken. We also limited our explorations to a quartet of salivary biomarkers when a larger set of biomarkers would have provided additional valuable information, especially inflammatory biomarkers as well as oral health indicators.Results of the present study among healthy children indicate that sIgA and sAA can be used as possible sex- and age-related biomarkers in children 6-12 y.o., which is not the case for free cortisol and OPI. The importance of sIgA and sAA findings may support monitoring the age-sex differences, especially in the prehormonal period of life.However, OPI levels reflected previous exposure to stress, suggesting its evaluation of stress-related changes in prepubertal children. Interactions between BMI and salivary cortisol release suggest the dependence of such secretion with abnormal body weight in obese children.


Indications and cutoff value of deformities to determine surgical procedures for flexed knee gait are not clear. The aim was to determine the influence of none or mild, and moderate preoperative knee flexion contracture on the improvement of gait after orthopedic surgery in children with bilateral cerebral palsy (CP).Inclusion criteria; bilateral CP, Gross Motor Function Classification System level I-III, and pre- and post operative-gait analysis. The 132 individuals identified were categorized into 2 groups based on the severity of knee flexion contracture , and then matched according to the exact same soft tissue and/or bony orthopedic surgical procedures performed. The indication for surgery was to prevent progressive development of knee flexion contracture and stance phase flexed knee gait. Pre- and postoperative physical examination and gait analysis data were analyzed retrospectively.Sixty (30 + 30) children, with mean age 10.6 years in each group, were included. The average follow-up time was 17 months.p = 0.004, and in group 2 from 60 (13) to 69 (15), p = 0.001. Knee flexion in stance improved in group 1 from 21.4 (16.1) to 12.1 (16.0) degrees, p = 0.002, and in group 2 from 32.2 (14.2) to 17.0 (15.9), p = 0.001. Step length improved in both groups, p = 0.017 and p = 0.008, respectively.Gait Deviation Index (GDI) improved in group 1 from mean 66 (SD 19) to 74 (15), p = 0.018 and standing function, Gross Motor Function Measure (GMFM-D), p = 0.001. Knee flexion contracture decreased in group 1 from mean 4.6 (5.3) to 2.1 (8.3) degrees, p = 0.071 and in group 2 from 17.2 (4.9) to 9.6 (9.3), p = 0.001.Only in group 2 significant improvement was noted in walking speed, There was no statistical difference between groups in pre-post improvement of GDI or other variables, except GMFM-D.Relative mild to moderate preoperative knee flexion contracture does not influence the short-term improvement of gait after orthopedic surgery in children with bilateral CP. Walking with excessive knee flexion in stance phase, i.e., crouch gait, is a common gait pattern in children with bilateral cerebral palsy (CP) , 2. StudDuring growth, especially in adolescence, knee flexion contracture may develop with increased knee flexion in gait , 11. FleSurgical treatment for flexed knee gait with more or less knee flexion contracture is performed at multiple levels , 13–17. The exact indications and severity of deformities for surgical treatment addressing flexed knee gait, with or without knee flexion contracture are still debated . SeveralHence the significance of knee flexion contracture and how it may influence the outcome of different orthopedic surgical procedures to improve and prevent the progression of flexed knee gait, is not clear and needs to be further studied.The aim of this study was to determine the influence of preoperative, none or mild knee flexion contracture with moderate knee flexion contracture, in matched surgical groups, on the improvement of gait in ambulatory children with bilateral CP.From the Epic medical records database , 132 children with bilateral CP who underwent orthopedic surgery, with both pre and postoperative three-dimensional gait analysis between 2004 and 2015 were identified retrospectively. All gait analysis assessments were performed at the same gait laboratory. The inclusion criteria were: diagnosed as bilateral CP; GMFCS level I to III at the preoperative visit; preoperative gait analysis performed within 12 months before surgery; and postoperative gait analysis between 10 and 30 months after surgery. The children were categorized into two groups based on the preoperative severity of knee flexion contracture. Group 1 comprised those with none or less than 11° of knee flexion contracture; group 2 comprised  those with knee flexion contracture greater than or equal to 11°. In addition, the two groups were matched according to orthopedic surgical procedures performed. When the exact same set of procedures was noted in one child from group 1, with another child in group 2, they were considered matched, which resulted in 30 + 30 children. The surgical procedures performed are listed in Table Three-dimensional gait analysis was performed with an eight-camera motion analysis system  to obtain kinematic gait data. The markers were attached to specific anatomical landmarks in accordance with a modified Helen-Hayes model , 25. The. The popliteal angle was measured supine with the hip flexed to 90 degrees, and the knee was extended until the pelvis starts to rotate and at this position, the goniometer measurement was assessed. The hamstring muscle spasticity is measured by the modified Ashworth Scale, a 6-point rating scale for the level of muscle resistance to passive movement ) and left side (10.4° [SD 8.6]) on preoperative physical examination. Results from the right side were used as the representative data of each individual except GMFM-D, which evaluates the bilateral standing function.No difference was found in knee flexion contracture .Graphical exploration with frequency histograms and standard plots was performed to evaluate normal distribution of collected data. Since the data were normally distributed, parametric tests were used. Pearson’s correlation coefficient to study the association of different variables with knee flexion contracture and paired-sample t-tests and independent-sample t-tests to compare groups. A n = 30 in each group) were included in the final analysis. Break down of detailed surgical procedures (Table p = 0.020).Based on the severity of knee contracture  and matching with surgical procedures performed, 60 individuals , but not in group 1 . Knee flexion contracture decreased in group 1 (p = 0.071), but not as significantly as in group 2 (p = 0.001)  in whom the same set of orthopedic surgical procedures was performed but had different severity of pre-operative knee flexion contracture, we found that the severity of knee flexion contracture did not directly influence the degree of improvement of postoperative gait pattern. The same improvement was found in gait kinematics, i.e., GDI, and step length despite the severity of knee flexion contracture. In addition, popliteal angle improved, and hamstring muscle spasticity deceased in both groups. Not surprisingly, individuals with more severe knee flexion contracture (group 2) had lower gross motor function categorized by GMFCS when compared with those with less severe knee flexion contracture (group 1) as previous studies showed [By comparing pre and postoperative data retrospectively between matched groups  children. In addition, the follow-up time was short (< 3 years). A longer follow-up time should be investigated in future studies to understand the long-term outcome predictively of the preoperative knee flexion contracture. Moreover, details of each child’s previous interventions were not provided since it is nearly impossible to obtain with high accuracy. However, the children were all assessed and treated at the same medical center according to the same algorithm of one senior orthopedic surgeon. The exact post-operative care, time of casting, degree and time of weight bearing, and the amount and physiotherapy program performed is not provided. Furthermore, the groups could not be perfectly matched according to GMFCS level and we had not included other variables that could influenced the composition of the two groups and the result such as, pre- and post-operative training, availability of orthosis and assistive devices. Possible cognitive, behavioral disabilities and co-morbidities i.e. seizures, could also influence and make the comparisons of groups difficult. Nevertheless, the groups were relatively comparable regarding age, height, weight and follow-up time, and fairly good regarding gender and using assistive devices Table .In children with bilateral CP who had relatively mild to moderate preoperative knee flexion contracture, the severity of knee flexion contracture does not directly influence the short-term improvement of gait pattern after soft tissue and/or bony orthopedic surgery.Our results indicate that although physical examination of passive-range-of motion is an important and easily accessible variable, the severity of knee flexion contracture does not necessarily determine the indication for specific orthopedic surgical procedures. A comprehensive multi-level assessment in the surgical treatment plan is most likely warranted.


This study aimed to assess the diagnostic accuracy of serum LDH to pleural ADA ratio for malignant pleural effusion (MPE) through an original study and meta-analysis.We retrospectively collected data from 145 patients with MPE and 117 cases of benign pleural effusions (BPE). The diagnostic performance of CR and a typical biomarker of MPE, carcinoembryonic antigen (CEA), were analysed using the receiver operating characteristic (ROC) curves and the area under the curve (AUC) as a measure of accuracy. The overall diagnostic accuracy of CR was summarised by a standard diagnostic meta-analysis.Significantly higher CR and pleural CEA values were observed in the MPE patients than in the BPE patients. At a cut-off value of 14.97, CR showed high sensitivity (0.91), low specificity (0.67), and high AUC (0.85). The combination of CEA and CR increased the AUC to 0.98. The meta-analysis included seven studies involving 2,078 patients. The pooled values for sensitivity, specificity, positive/negative likelihood ratio, and diagnostic odds ratio of CR were 0.96, 0.88, 7.70, 0.05, and 169, respectively. The AUC of the summary ROC of CR was 0.98.CR has a high diagnostic accuracy for predicting MPE, especially when used in combination with pleural CEA. It is aLactate dehydrogenase (LDH) is a ubiquitous enzyme that occurs in high concentrations in the liver, kidney, myocardium, skeletal muscles, and red blood cells . It playAdenosine deaminase (ADA) is a known biomarker of benign tuberculous pleural effusion ,17. It i2.2.1.In this retrospective study, we considered 261consecutive patients suffering from pleural effusion who were admitted to our hospital for further investigation between February 2015 and October 2015. We included all patients who had received a confirmed diagnosis of benign pleural effusion (BPE) or MPE and who had completed routine laboratory tests associated with pleural effusion, as well as tests of serum LDH, pleural ADA, and pleural CEA. Patients with inconclusive final diagnosis and incomplete data were excluded from this study. On admission, all patients signed the informed consent for their anonymized clinical data to be analysed and published for scientific research purposes. This study was approved by the Ethics Committee of our hospital and was conducted based on the principles outlined in the Declaration of Helsinki.2.2.In this study, MPE was diagnosed based on the presence of malignant cells in pleural effusion or pleural biopsy specimens . In pati2.3.p < .05 were considered statistically significant.We collected data on age, sex, and routine biochemical analysis results on admission, includingcolourof pleural fluid; levels of pleural glucose, proteins, ADA, and CEA; and serum LDH. Patient demographic data and disease characteristics were summarised and expressed as mean ± standard deviation (mean ± SD). Intergroup differences were assessed for significance using the Mann-Whitney U test. We analysed the receiver operating characteristic (ROC) curves and the area under the ROC curves (AUC) to assess the overall diagnostic performance of CR and pleural CEA. The AUCs of different indexes were compared using the nonparametric approach . Further2.4.We systematically examined the PubMed, EMBASE, Web of Science, Chinese National Knowledge Infrastructure, Wanfang, and Weipu databases to identify studies on MPE published before June 2020. The following search terms were used in each database: “malignant pleural effusion”, “malignant pleural fluid”, “lactate dehydrogenase”, “LDH”, “adenosine deaminase”, “ADA”, “cancer ratio”, “sensitivity”, “specificity”, and “accuracy”. We included original clinical research articles that reported true positive (TP), false positive (FP), false negative (FN), and true negative (TN) data with respect to the use of CR for MPE diagnosis. Only articles published in English and Chinese were considered. Studies were excluded from this meta-analysis for the following reasons: (1) there were on data of sensitivity and specificity; (2) they were not based on human subjects; (3) Conference proceedings and studies published only as abstracts.All records were imported into Endnote for further review. Two reviewers (YZ and XL) independently evaluated the studies initially based on titles and abstracts and subsequently based on full text. After removing inappropriate and duplicate studies, the reviewers assessed the quality of the included studies using a revised version of QUADAS-2 .2: an I2 > 50% indicated significant heterogeneity. The diagnostic performance of CR and CEA was assessed based on the summary ROC (SROC) curve. Potential publication bias was evaluated by using Deeks’ test [p < .05 indicated statistical significance.The indices of test accuracy, sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR), along with 95% confidence intervals (CIs),were pooled from each study using a bivariate model. Heterogeneity among all eligible studies was evaluated based on Iks’ test . Meta-an3.3.1.Supplementary Figure 1. We found that a large proportion of patients with MPE suffered from lung cancer (n = 121), metastatic cancer (n = 13), hematological malignancies (n = 8), and pleural mesothelioma (n = 3). A large proportion of the BPE patients suffered from tuberculous pleural effusion (n = 68), followed by parapneumonic effusion (n = 44), hepatic pleural effusion (n = 2), pleural effusion due to acute pancreatitis (n = 2), and chylothorax (n = 1).We collected data on the clinicopathological characteristics of a total of 145 patients diagnosed with MPE and 117 patients diagnosed with BPE . The sel3.2.p < .001). Conversely, pleural ADA levels were significantly higher in patients with BPE than those with MPE . Therefore, CR levels were significantly higher in MPE patients (32.96 ± 25.31) than in BPE patients .Patients with MPE had significantly higher levels of serum LDH than those with BPE . With the maximisation value of the Youden index, the best cut-off was 15, along with a sensitivity value of 0.91 and a specificity value of 0.67. On the other hand, CEA had an AUC of 0.91 at a cut-off value of 2.57 ng/ml, along with a sensitivity value of 0.81 and a specificity value of 0.94. A combination of the two markers increased the sensitivity (0.93), specificity (0.92), and AUC values (0.98), which was significantly higher than CR alone (p < .05).ROC curves were created to summarise and compare the diagnostic performance of CR and pleural CEA as markers for MPE . At a cu3.4.We performed a meta-analysis of 7 studies (including the present one) involving 860 patients with MPE and 1,218 patients with BPE . These sQuality assessment of eligible studies is shown in To evaluate the diagnostic accuracy of CR with respect to MPE, we calculated pooled values of sensitivity . However, we could not perform a meta-regression analysis to investigate the source of heterogeneity due to the limited number of included studies. We found no significant evidence of publication bias based on the Deek’s funnel plot  and specificity (I2 = 96.83%)  and specificity (0.88). The SROC curve, which assesses overall test performance and depicts the trade-off between sensitivity and specificity, gave an AUC of 0.98, indicating a high overall accuracy. The DOR value combines sensitivity and specificity data into a single number ranging from 0 to infinity; higher DOR values indicate better discriminatory test performance . The meaThere are several advantages with respect to the clinical utility of CR for the diagnosis of MPE. First, nearly all patients with undiagnosed pleural effusion undergo routine blood and pleural biochemical tests. Therefore, it is easy to obtain data on LDH and ADA levels, without incurring any additional costs. Second, this data can help guide treatment plans: patients with high CR values must be treated with caution, and further diagnostic examinations such as repeated cytologic test, invasive procedures such as medical thoracoscopy and pleural biopsy should be considered. While, the best cut-off value of CR has not been established, a presepcified threshold value is needed since a data-driven threshold may overstate an index test .CEA has been widely used in the diagnosis of MPE. A meta-analysis based on 45 studies showed low pooled sensitivity (0.54) and high pooled specificity (0.94) for CEA when diagnosing MPE, the low sensitivity of CEA limited its role in screening MPE . The preThe results of our study must be interpreted with caution in the light of several limitations. Due to the retrospective nature of our study, we were unable to obtain all the relevant data required for our analysis from the hospital’s medical records, and we didn’t calculate the sample size and also can’t calculate the diagnostic performance of CEA ratio for MPE. Additionally, most of the included studies used patients with tuberculous pleural effusion and parapneumonic effusion as controls. Other aetiologies of pleural effusion, such as heart failure, chemical pleurisy, or connective tissue disease were not included. In order to extend our results, future work must include clinical data from a larger cohort of patients with many different types of pleural effusion. The limited number of studies included in our meta-analysis was also another source of bias, especially since we could not evaluate covariates as possible sources of the observed heterogeneity .5.In summary, our study and meta-analysis demonstrate that CR is an effective diagnostic marker for MPE, especially in combination with CEA. Further research is required to validate these findings.


HTS was correlated with plant height, outer/rosette leaf length, and petiole length. A high-density genetic map was constructed. Quantitative trait locus (QTL) analysis resulted in the identification of 22 QTLs for leafy head-related traits, which included five HTS QTLs. Bulked segregant analysis (BSA) was used to confirm HTS QTLs and identify candidate genes based on informative single-nucleotide polymorphisms. Interestingly, the HTS QTLs colocalized with QTLs for plant height, outer/rosette leaf, and petiole length, consistent with the observed phenotypic correlations. Combined QTL analysis and BSA laid a foundation for molecular marker-assisted breeding of Chinese cabbage HTS and directions for further research on the genetic regulation of this trait.The agricultural and consumer quality of Chinese cabbage is determined by its shape. The shape is defined by the folding of the heading leaves, which defines the head top shape (HTS). The overlapping HTS, in which the heading leaves curve inward and overlap at the top, is the shape preferred by consumers. To understand the genetic regulation of HTS, we generated a large segregating F Brassica oleracea), Chinese cabbage (Brassica rapa), and lettuce (Lactuca sativa). The leafy head is composed of curving leaves, and these leaves form various head shapes depending on the curvature of the distal end, hereafter referred to as the tops of leaves: leaves with outward curving tops, leaves with inward curving tops that do not overlap, and inward curving leaves with their tops overlapping or forming a spiral1. Leaf morphology is determined by leaf cell division and cell elongation rates along the main axes of the leaf4. The heading leaves curve inward to form a leafy head at the heading stage. The upward curvature of Chinese cabbage heading leaves is facilitated by increased growth of abaxial cells5. As a storage organ, the leafy head is an important source of mineral nutrients, crude fiber, and vitamins. Head traits include head weight (HWe), compactness, size, and shape. The Chinese cabbage overlapping head type, in which the heading leaves curve inward and overlap at the top, is the shape that current consumers prefer6. However, the molecular mechanism of leafy head formation and regulation of head shape is not fully understood.Leafy heads are an important trait of leafy vegetable crops, such as cabbage : round heads with flat rosette leaves, cylindrical heads with rosette leaves with wavy margins, oblong heads with shrinking rosette leaves, and cone-like heads with incurved rosette leaves. In this paper, they also identified that allelic variation in the TCP4 gene in the miR319a recognition sequence modulated head shape by differentially arresting cell division in different leaves. Sun et al.9 also showed the correlation between rosette leaf traits, such as leaf length (LL), width, and midvein length, with leafy head traits in a diverse set of 152 Chinese cabbages. Although the phenotypic correlation between rosette leaves and leafy heads has been described, it is unclear how rosette leaves influence leafy head growth and how this is regulated at the molecular level.The vegetative growth of Chinese cabbage is divided into four stages: seedling, rosette, folding, and heading stages. The leaves in these four stages are different in shape, size, and physiological function13. Seq-BSA revealed genomic regions enriched with candidate genes related to plant hormones associated with Chinese cabbage heading type and leafy head formation15. In addition, several studies have described gene expression profiles during Chinese cabbage development and constructed rosette and inner leaf gene expression networks19. Although these studies provide insight into the general pathways involved in the formation of leafy heads in Chinese cabbage, they do not explain the genetic control of the relation between rosette leaf growth and leafy head formation.Genetic information from quantitative trait loci (QTLs), sequencing‐based bulked segregant analysis (Seq‐BSA), and RNA-sequencing approaches provides clues for understanding Chinese cabbage leaf and leafy head traits. QTLs have been identified for Chinese cabbage heading traits, such as head diameter, head height, and HWe, which are all important components of head yield and quality2 population of B. rapa derived from a cross between heading Chinese cabbage and nonheading pak choi and analyzed the association between rosette/heading leaves and several heading traits, including the leafy head top shape (HTS). In addition, a combined approach of BSA of pools with contrasting head traits and QTL analysis was used to study rosette leaf growth and the HTS trait at the genetic level. Several colocalizing genomic regions for rosette leaf traits and HTS were identified. Further investigation of these regions resulted in candidate genes for the HTS trait in Chinese cabbage.In this study, we phenotyped an F2 plants. This resulted in 228 plants in class 4 (inward curving leaves that overlap at the top of the leafy head), 389 plants in class 3 (inward curving leaves that do not overlap at the top of the leafy head), 591 plants in class 2 (with outward curving outer heading leaves), and 99 plants in class 1 (that did not form a leafy head). Based on these four classes of the HTS trait, it was hypothesized that the qualitative trait HTS was controlled by two major genes with recessive epistasis (2MG-RecessiveI) based on SEgregation Analysis (SEA) software analysis (Table + (60 plants), 2− (63 plants), 2 (521 plants), 2+ (7 plants), 3− (47 plants), 3 (233 plants), 3+ (109 plants), 4− (28 plants), and 4 (200 plants). The scores of the parental genotypes were PC-101 = “1”, CC-48 = “2”, and F1 = “3”, Based on these ten phenotypic classes, it was hypothesized that the qualitative trait HTS is controlled by four major genes with additive and epistatic effects (4MG-AI) based on SEA software analysis , the outer/rosette leaf dimensions (O) leaf width (LW), LL, leaf blade length (LaL), leaf midvein length (LvL), LA, LPL, leaf petiole width (LPW), and leaf petiole area (LPA), PH, and PWe values were lower than those of both groups of heading plants (both OC: outward curving heading plants and O: overlapping heading plants) Fig. . These d2-104 genetic map consisted of ten linkage groups and was constructed using 3194 bin markers  and composite IM (CIM) methods  for HTS were detected on A04, and three QTLs  were detected on A05. HTSQ1A4 and HTSQ7A5 had the highest logarithm of the odds (LOD) scores (4.833 and 4.895) and explained ~6.1% and 4% of the variation, respectively. For leaf-related traits, three QTLs for OLL  and two QTLs for OLPL (OLPLQ1 and 2) were detected on A05. Three QTLs for HLPL  were detected on A09, and two QTLs for head leaf vein length (HLvLQ1 and 2) were detected on A06. For leafy head traits, a total of four QTLs  for the PH traits were detected on A04, with LOD values ranging from 3.879 to 4.622, and one QTL for the HWe (HWeQ1A8) trait was detected on A08. By the CIM analysis method, two QTLs for HTS, namely, CIM_HTSQA4 and CIM_HTSQA5, were detected on A04 and A05, which explained ~16.9% and 17.6%, respectively. Together, these findings accounted for 34.5% of all the phenotypic variation in the HTS trait. For the OLL, OLPL, and PH traits, single QTLs were detected on A05 (CIM_OLLQA5 and CIM_OLPLQA5) and A04 (CIM_PHQA4). The genes in these QTL regions are shown in Table For all mapped QTLs of the Chinese cabbage leaf and leafy head traits, overlapping genomic regions were identified between HTS and PH and the outer/rosette leaf traits OLL and OLPL Fig. . HTS trap < 0.05).We generated three independent plant DNA pools, each representing different head-type shapes. In the bulks, the plants were all barcoded, and SNP markers with different allele frequencies between bulks with different phenotypes were selected (Bra001163 (embryo defective 2016: EMB2016) in the development category and Bra016948 (glutamate synthase 2: GLU2) in the N-metabolism category, were identified in all three comparisons  were in the protein category, four genes  were in the cell and cell wall category, and two genes (Bra020751 and Bra039445) were in the RNA category. Among them, Bra017274 , Bra029597 , and Bra039421  included SNPs causing amino acid changes (Table Bra014031 (A08:4344956..4347618) and cell wall degradation gene Bra014180 (A08:2743942..2745200) with SNPs causing amino acid changes were among these 16 genes in HWeQ1A8.As expected, some of the candidate genes with significant allele frequency differences in the “N vs. O,” “N vs. OC,” and “O vs. OC” comparisons colocalized with QTL regions on A04, A05, A06, A08, and A09 Fig. . For thees Table . Seventyes Table , and 59%6. HTS refers to the shape of the top of the leafy head, which is affected by the shape, overlap, and curvature of the outer heading leaves. Currently, with the improvement in people’s living standards, Chinese consumers, especially from Northern China, prefer leafy heads of the overlap head-type shape. For this reason, it is an important agronomic trait for breeders to screen, and it is interesting for researchers to study because it involves variation in leaf curvature. Recently, several papers have been published about QTL mapping of the Chinese cabbage leafy head. Most of these studies analyzed leafy head formation (head or nonhead), the heading degree , and the heading shape index (head width diameter/length diameter)15. Only one paper investigated HTS, based on a cross between two heading Chinese cabbages14. Due to the different parental genotypes used in their mapping study, a different candidate region on A06 was detected. This study provides new insights into the HTS phenotype by combining phenotypic observations and genetic analyses using a large F2 progeny from a cross between Chinese cabbage and pak choi.The leafy head of Chinese cabbage is an important agronomic trait associated with both yield and quality. Compared to heading Chinese cabbage, nonheading plants (such as pak choi) are generally smaller and produce less biomass . For heading Chinese cabbage, the head shapes include round, oblong, cylindrical, and cone-like head shapes2 population was segregated into various HTS types, and we tried to fit a genetic model to the segregation data of the leafy head shape based on the HTS phenotypes of the 1307 F2 plants. However, regardless of whether we scored the HTS into four or ten classes, we could not clearly select the most likely genetic model. This was likely caused by the fact that the scored phenotype is a combination of traits that are all under the control of different genetic loci20. To reveal genetic regulation, we should identify and score the subtraits that make up the final head shape-type trait. These subtraits include the curvature of the petiole, angle of the midvein to the soil, head size and weight, LL, and curvature of the distal end of the leaf blade.HTS was judged based on the heading leaf top shape at the heading stage. The FArabidopsis, outer (old) rosette leaf senescence proceeds during plant growth to recycle nutrients that become available for the actively growing parts22. However, in Chinese cabbage, rosette leaves remain active until the leafy heads mature, likely performing photosynthesis in the fast-growing leafy head18. Both this study and previous studies showed a correlation between the size and shape of rosette leaves and leafy head shape9. HTS is clearly associated with outer rosette LL and leaf petiole length (LPL) (this study). These results indicate that the outer leaves not only provide the products for leafy head growth but also affect the shape of the head.We phenotyped many leaf traits, for both the outer rosette leaves and the outer leafy head leaves, at the heading stage. Interestingly, we showed that the traits related to the shape and size of the outer rosette leaves were correlated with HTS. Surprisingly, these same traits of the heading leaf itself were not correlated with HTS. In addition, when we compared the leaf traits of the overlapping “O” HTS group with those of the outward curving “OC” HTS group, significant differences were detected between their outer/rosette leaf and petiole traits (LL and LPL), while the shapes of their heading leaves did not differ. Normally, in 9. In addition to environmental variation, in these studies, the timing of phenotypic data collection differed: we measured the rosette leaf traits at the heading stage in this study, but phenotyped the growing rosette leaves at rosette stages ) in a previous QTL study. In a study by Sun et al.9, QTLs for rosette LPL also varied over time (DAS), and generally, LOD scores were lower when measured at later stages. The phenotyping of the leafy heads was performed at the same developmental stage in both studies, the heading stage. In both studies, QTLs for the leafy head trait “weight” (HWe) were detected at the top of A08  have been identified on A05 and contribute to phenotypic variation for these pod-related traits24. In rice, colocalization between PH- and flowering time-related QTLs was identified, and the major gene Ghd7, which has pleiotropic effects on PH and heading date, was identified25. Here, we detected two QTLs for the HTS trait. Colocalization of HTS with outer/rosette LL and rosette LPL (OLL/OLPL) was identified on A05, and that of HTS with PH was identified on A04. These results were consistent with the phenotypic results: there were strong correlations among outer/rosette LL, LPL, and HTS traits, and significant differences existed in the OLPL, OLvL, and HLvL traits between “O” and “OC” HTS plants. This phenomenon was not unexpected and indicated pleiotropic effects of single genes or tight linkage. We identified an HTS QTL on A05. Interestingly, in an independent study on an F2 population derived from a cross between two Chinese cabbages with different HTSs, BSA of the “O” and “OC” types identified significant SNPs on A05 (21700337..24709181) overlapping with the QTL region for HTS identified in this study , at the seedling, rosette, and heading stages Table . All can26. During these phases, the arrangement of cells is indicative of changes in leaf shape4. Our BSA results showed many candidate genes in the cell wall and cell cycle category. In particular, Bra039421 was mapped to the HTS QTL_CIM on A05. The SNP (A/C) located in exon 1 (540 bp) of Bra039421 resulted in an amino acid change from “Gln” to “His.” According to classic cell theory, changes in cellular behavior are responsible for mutant morphology27. Thus, the genes in these QTLs may have functions that play roles in HTS formation in Chinese cabbage. Interestingly, genes common between the “N vs. OC” and “N vs. O” comparisons were mainly localized on A08 in the HWe QTL. Heading Chinese cabbage plants were consistently heavier than nonheading plants. We identified several candidate genes for this heading/nonheading comparison (N vs. OC/O), which were related to total PWe. Bra014180 in the cell wall degradation functional category encodes USPL1 (AT1G49320 in Arabidopsis) targeted to protein storage vacuoles, which is particularly interesting because it is closely related to seed development, protein storage vacuoles, and lipid vesicle morphology and behaves like a storage protein28. In addition, Bra014180 with an SNP (A/G) on exon 2 (444 bp) caused an amino acid change (Ile/Met) and displayed higher transcript abundance in pak choi than in two Chinese cabbage varieties at early heading stages, with fold change >1.5 and adjusted p value ≤ 0.01.Leaf growth is strongly associated with leafy head formation in Chinese cabbage and includes leaf primordium initiation, leaf polarity formation, cell division, cell expansion, and cell differentiation phases2 population from a cross between a nonheading pak choi and a heading Chinese cabbage and identified correlations between HTS and other rosette leaf traits. These correlations between rosette leaf traits and head-type shape were confirmed by the colocalized QTLs for these traits. This finding is a good foundation for further research on the genetic regulation of the HTS trait in Chinese cabbage and provides tools for molecular marker-assisted selection of leafy head shape in Chinese cabbage. In addition, by combining QTL and BSA results, we identified a few genes with interesting annotations as candidate genes for quantitative trait loci for leafy head formation. These results will help us understand the genetic control of head shape traits in cabbage.In conclusion, we phenotyped both leaf and leafy head traits of an F2 population was derived from a cross between a heading Chinese cabbage  and a nonheading pak choi   plus their parental lines were sown in seeding soil in a greenhouse in September 2017, and 2 weeks later, the plants were transplanted to an open field at Hebei Agriculture University  and grown under short-day conditions until December 2017.An Fna) Fig. . The F2 2 plants (n = 1307)  by visual observation at the heading stage for the F07) Fig. .2 plants that represented the different HTS classes  software, including the mean, minimum, maximum, standard deviation, variance, and t test, using p < 0.05. The correlation coefficients between traits were analyzed using an R package (“corrplot” package: http://www.R-project.org) with the Pearson’s correlation coefficient.The inheritance model analysis for a plant quantitative trait (HTS) was analyzed by SEA software2 progeny plants (n = 104) by the CTAB method. The genotyping-by-sequencing (GBS) method was used to generate ApeKI-associated DNA fragments for sequencing on the Illumina HiSeq2000 platform at the Gene Denovo Company, China, and SNP genotyping and evaluation were then performed30. The “Chiifu” genome V1.5 was used as the reference sequence for the alignment of sequenced reads by the BWA software31. A total of 258,443 genetic variations , with indels representing 9% of the total, were identified, and 71.8% of these SNPs resided in intergenic regions. Among the intergenic SNPs discovered, 11.3% were within 5 kb upstream, and 10.5% were within 5 kb downstream, of an open-reading frame. In addition, 14.9% of the SNPs were observed in exonic regions and 13.2% in introns. For the SNPs located in the coding region (exon), 56.9% were synonymous (silent) mutations, and 40.4% resulted in a change in amino acids (nonsynonymous substitutions) or stop codons. For the SNPs located in introns, 162 SNPs were observed at intron splicing sites that potentially alter the function of these genes.Genomic DNA from individual plants was extracted from young leaves of parents and their F2 population. After filtering, a set of 16,570 SNPs were discovered through GBS, of which 8998 were “aa × bb”, 1165 were “hk × hk”, 2612 were “lm × ll”, and 3795 were “nn × np” segregation types32. As the F2 population was derived from a cross between homozygous parents (DH lines), 8898 “aa × bb” parental markers that formed 5973 bins were used as input data for “JoinMap” to construct the genetic linkage map. After creating population nodes, “ML mapping” was used to assign the markers into linkage groups, and “Kosambi’s” mapping function was used to construct genetic maps.The 234,421 SNPs were filtered for those that showed polymorphisms between parent lines (CC-48 and PC-101) and had allele frequencies >60% in the F2 population. SNPs in the F2 population were only used if the parental source of the alleles could be unambiguously assigned. The F2 plants (n = 104) were genotyped for a set of 8998 markers covering the ten linkage groups. A subset of 3194 specific SNP markers was used to construct the F2 genetic linkage map in this study. Genetic mapping was performed using an R package34. Based on the genetic map, QTL regions for the phenotyped traits were identified by IM analysis with the R/qtl package35. The LOD critical values (Table p < 0.05)36.A total of 8998 polymorphic homozygous SNPs in both parents (aa × bb) were used to generate a genetic map of the Fes Table  for acce2 plants into three bulks  corresponding to the different leafy head shapes. These three bulks were genotyped with the 8998 selected SNP markers. Markers showing allelic polymorphisms between different phenotype bulks for leafy head shape were selected based on allele frequency. We selected subsets of polymorphic SNPs by comparing different bulks .In addition, a BSA approach was used to identify genomic regions linked to the leaf blade top curvature trait. Four leafy head phenotypes were distinguished: no leafy head, with leaves that do not form a leafy head and are fully curved outward (N: 17 plants). Three groups of 30 plants with different heading types were selected (outward curve (OC): 30; intermediate phenotype: 30; and overlapping (O): 30). For BSA, we divided 77 FFigure S5Table S1Table S2Table S3Table S4Table S5Table S6Table S7Figure S1Figure S2Figure S3Figure S4


Disease-related malnutrition is associated with adverse outcomes such as increased rates of morbidity and mortality, prolonged hospital stay, and extra costs of health care. This study was conducted to assess nutritional status among patients and to determine the risk factors for malnutrition in Iran university f.Persian Nutritional Survey In Hospitals (PNSI) was a cross-sectional study that conducted in 20 university hospitals across Iran. All the patients with age range of 18 to 65 years, who were admitted or discharged, were assessed by subjective global assessment (SGA).2, respectively. Malnutrition (SGA-B & C) was identified in 23.92% of the patients, 26.23 and 21% of whom were among the admitted and discharged patients, respectively. The highest prevalence of malnutrition was in burns (77.70%) and heart surgery (57.84%) patients. Multivariate analysis presented male gender , malignant disease , length of hospital stay , and polypharmacy  as independent risk factors for malnutrition. Malnutrition was not associated with age (P = 0.10).In total, 2109 patients were evaluated for malnutrition. Mean values of age and body mass index were 44.68 ± 14.65 years and 25.44 ± 6.25 kg/mThis study provides an overall and comprehensive illustration of hospital malnutrition in Iran university hospitals, finding that one out of four patients were malnourished; thus, appropriate consideration and measures should be taken to this issue.The online version contains supplementary material available at 10.1186/s12937-021-00741-1. Disease-related malnutrition is associated with adverse outcomes such as increased rates of morbidity and mortality, prolonged hospital stay, impaired wound healing, high readmission rate, and increased costs for health care –4.Malnutrition development is associated with insufficient dietary intake or malabsorption, increased nutritional needs, complications of the underlying disease, or a combination of these factors . There iStudies performed in the United States indicate that approximately 32.7% of hospital patients are either malnourished or at nutritional risk , they al2 [There are different reports of malnutrition prevalence in Iran, Hosseini reported 5.7% of patients on admission and 11% of discharged patients had malnutrition based on body mass index (BMI) ≤18.5 kg/m2 . Forty-t2 . Another2 . A recen2 .Proper nutritional status can play a notably important role in lowering the incidence of the malnutrition-disease defective cycle. Despite the importance of disease-related malnutrition and the great economic costs of this condition imposes on health systems, there is no comprehensive data on disease-related malnutrition and related risk factors in Iran hospitals. Therefore, this study aimed to assess nutritional status among patients in Iran hospitals and to determine different risk factors for malnutrition in hospitalized patients.P = 0.32, Z = 95%, d = 0.02) [Persian Nutritional Survey In Hospitals (PNSI) is a multicenter, cross-sectional study conducted in 20 public hospitals which were selected based on a random stratified-cluster method. The sample size was estimated as 2100 patients, based on the relevant formula , and dat = 0.02) . InformeIn each hospital, the investigators referred to included clinical wards and assessed newly admitted or discharged patients on a specified date consecutively. The inclusion criteria comprised of admitted or discharged patients within the age of 18 to 65 years with Iranian nationality, also we excluded duplicated patients. Patients undergoing surgery on the data collection day, outpatients, patients with trauma or eating disorders, and patients admitted to maternity, obstetric, pediatric, orthopedic, and emergency departments or intensive care units (ICUs) were excluded from the study. The study was conducted from 24th to 28th November 2015. STROBE checklist of study is available as supplementary material Patient characteristics, i.e., gender, date of birth (age), underlying disease, the main affected organ, comorbidity, the number of different prescriptions per day, and history of ICU stay and surgery were recorded. The length of hospital stay was calculated for discharged patients from the date of admission and date of the survey. Body weight was measured by standard Seca scale  in light clothes to the nearest 1 kg. Body height was assessed by Seca portable stadiometer  to the nearest 1 cm. Mid-arm circumference (MAC) was measured in mid-acromion and olecranon process interval at the non-dominant relaxed arm with a non-stretchable tape measure to the nearest 0.1 cm.The nutritional status of patients was assessed by subjective global assessment (SGA) and anthropometric measures (BMI and MAC) for all the admitted and discharged patients included in the study .SGA is a valid and reliable tool for assessing nutritional status in hospitalized patients , 20. Amo2) is most commonly used for assessing nutritional status. BMI was calculated as weight (kg) divided by squared height (m2) [Body mass index  . If BMI P-value less than 0.05 was considered statistically significant.Quantitative variables with normal distribution were expressed as mean, standard deviation, and range. Quantitative variables with non-normal distribution were reported as median and range. Stratified variables were reported as frequency and percentage. Analysis of nominal qualitative data was performed by non-parametric tests such as the Chi-squared test. Mann-Whitney test was used for ordinal qualitative variables; for quantitative variables with normal distribution, t-test was run. Odds ratios (OR) were reported with 95% confidence interval. To identify independent risk factors logistic regression was used. A n = 1055) were male and 50% (n = 1054) were female. Demographics of the study population are shown in Table 2, respectively. In 8.72% (n = 183) of the patients, BMI was less than 18.50 kg/m2.Participant flow chart is presented in Fig. n = 479 cases), which is determined by combining the SGA-B  and –C  also 76.07% (n = 1523) of patients were identified as SGA-A .The nutritional data of the patients are presented in Table n = 799) of the respondents, of whom 217 subjects were well-nourished and 36 subjects were malnourished.Weight loss in the last 3 months was reported by 37.31% (P < 0.05). The highest prevalence of malnutrition was observed in burns department (77.70%), followed by cardiac Surgery (57.84%), and hematology departments (50.00%). The lowest number of patients with malnutrition was in the ophthalmology department (2.51%) , malignant disease , number of medications  and length of hospital stay  were independent risk factors for malnutrition. Age and disease type were not associated with malnutrition (P > 0.05).Analysis of factors which may have been associated with malnutrition shows male gender  . In EuroThe results of the present study are in line with those of a national study conducted in Germany using SGA that reported 28% of patients suffered from malnutrition. A study in European hospitals showed that the prevalence of malnutrition ranged between 10 and 50% depending on the studied region . A studyThere are various reports on the prevalence of malnutrition around the world globe, which might be due to features of the studied samples, the tool used to identify malnutrition, and the type of assessed centers. Moreover, it should be noted that the mean age of patients in this study (44.68 years) was less than other studies (63.93 years) performed in Australia and (52.21 years) Latin America, while age was shown to be an independent risk factor for malnutrition. Additionally, most patients in the present study did not have malignant diseases that could affect the prevalence of malnutrition.The present study did not demonstrate any association between age and malnutrition that could be pertinent to the low mean age of the samples 44.68 years) [ years 8 and incoThe current study was the first national survey of hospital malnutrition and associated factors in Iran public hospitals which used a valid and reliable tool for nutritional assessment malnutrition, however, there were some limitations such as didn’t assess of malnutrition trend during hospitalization, because of the cross-sectional design of the study, another limitation was assessment by different investigators in hospitals. In further studies status of nutritional care and management of patients can be assessed, also effect of interventional measures in decline and management of problem could be evaluated.The data of this study provide an overall and comprehensive illustration of the nutritional status of patients and identify groups at higher risk for malnutrition in Iranian university hospitals. Control and management of this problem need a structural approach that should contain all these aspects: 1- administration of a universal assessment to screening patients for nutritional risk to identify patients with or at risk of malnutrition, 2- handle multidisciplinary supportive nutrition care to provide appropriate nutritional therapy, 3- take measures to promote continuity of care after discharge from acute care hospitals. Notable, improve knowledge and consciousness of clinician’s team to consider malnutrition as a specific disease entity that concludes significant patient and economic outcomes and requires active management.This study revealed that almost 24% of patients in public hospitals suffer from malnutrition. Male gender, malignancy, polypharmacy, and length of hospital stay were identified as independent risk factors for malnutrition. While the present study cannot determine to what extent and how malnutrition affects the outcomes, there is evidence showing effects on outcome and health costs, in this regard screening and nutrition interventions can improve the results.Additional file 1. STROBE checklist for nutritional epidemiologic studies.


NF1 gene mutation analysis was performed on 27 families with NF1 in which at least one affected family member developed MPNSTs. Of the 27 NF1 germline mutations, five were large deletions spanning (or nearly spanning) the gene (18.5%), substantially more than such deletions in NF1 in general, consistent with increased risk of MPNSTs in such cases.Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor syndrome in which benign plexiform neurofibromas are at risk of transforming into malignant peripheral nerve sheath tumors (MPNSTs), a very rare soft-tissue sarcoma. The prognosis of patients with MPNSTs is poor, with most studies reporting <50% survival at five years. However, studies evaluating MPNSTs are limited and report heterogeneous results. Because no MPNST-specific evidence-based treatment guideline exists, individual institutional experiences are very informative to the field. The main objective of this study was to investigate and report MPNST prognostic clinical and genetic biomarkers from our institution's Orthopedics service experience treating 20 cases from 1992 to 2017. Most patients were treated with resection and adjuvant radiation. Extended follow-up, averaging 11.4 years (ranging 1.1 to 25.1), revealed excellent five-year survival rates: 70% for overall and 60% for metastatic disease. An S100 B immunonegative tumor phenotype was associated with a significantly worse outcome than MPNSTs with positive S100 B stain. In addition,  NF1 gene, of which over 5000 have been identified spanning the large locus. The clinical hallmark of NF1 is the neurofibroma, a benign peripheral nerve sheath Schwann-cell tumor. Patients can develop few to thousands of neurofibromas in their lifetimes. Neurofibromas are classified as “cutaneous” when they are superficial, involving nerve endings in the skin; larger and usually deeper “plexiform neurofibromas” involve peripheral nerves [Neurofibromatosis type 1 (NF1) is an incurable progressive autosomal dominant disease. Despite an NF1 incidence of 1 in 3000 people, the first effective systemic therapy with the MEK inhibitor selumetinib was only recently FDA approved, in 2020 [l nerves . Overalll nerves  and thusNF1 mutation effect on the risk of developing an MPNST. Some studies reported a higher incidence of deletions spanning the entire NF1 locus and surrounding genes in NF1 patients who develop an MPNST than the NF1 population overall [Because of its rarity, there have been few clinical trials specifically for patients with MPNSTs and information about natural history and response to therapies in the literature is sparse. Typically, MPNSTs are managed with treatment protocols for soft-tissue sarcomas, as protocols specific to MPNSTs have not been established . Early s overall , while o overall .NF1 mutation data from the MPNST population at our institution to identify prognostic factors.There are a limited number of publications reporting MPNST survival, most with small case numbers and variable outcomes, not unexpected for such a rare tumor. Thus, experience from institutions managing patients with MPNSTs is important to add to the field's knowledge . The purAs approved by the University of Florida Institutional Review Board, a retrospective medical chart review of 1992 to 2017 was performed from 1992 to 2017 to gather data on our institution's Division of Orthopedic Oncology's experience of treating patients with MPNSTs. The data were extracted from an EPIC electronic medical record service and from the Enneking/Anspach Research Center database in the Department of Orthopedics and Rehabilitation. Inclusion criteria were (1) diagnosis of MPNSTs, (2) definitive treatment performed at our institution, and (3) at least 12 months of follow-up data. Demographic and clinical information collection included gender, self-reported ethnic group, age at initial diagnosis, tumor stage at first diagnosis, time to recurrence, diagnosis of NF1, family history of NF1, therapeutic history, immunohistochemical S100 B data from a pathology service, and survival status.NF1 mutation analysis was performed on DNA samples from the IRB-approved Wallace Genetics Bank from 27 NF1 families bearing at least one MPNST occurrence. Previously existing data included some NF1 cDNA Sanger sequencing, while current testing included PCR and sequencing of 13 exons  of the 57 exons in the locus (NCBI gene sequence: NG_009018.1)  .ribed in ). In adda priori at p < 0.05. Descriptive statistics were computed for demographic and clinical variables. Kaplan–Meier survival curves were generated to assess the overall survival of the study population and compare between demographic, clinical, survival, and pathological variables.All data were analyzed using statistical software . The significance level for analyses was set A total of twenty subjects  qualified for this study's retrospective chart review. Demographic characteristics are provided in With respect to race, 12, 6, and 1 subject(s) were Caucasian, black, and Hispanic, respectively. Race was not identified in the medical record of one subject. The mean total length of follow-up was 136.9 months. The survival rate for the entire study population was approximately 70% .Two subjects had local recurrence, and five had subsequent metastasis. The mean time to local recurrence and time to metastasis were 134.9 and 113.6 months, respectively. Both subjects with local recurrence received radiation therapy. At the last follow-up for all subjects, 14 were alive and 6 were deceased. Two of the five subjects who developed subsequent metastasis were alive at the last follow-up (2018) and survived an average of 197.9 months (range 85.7 to 309.8) after diagnosis of metastasis. Four of these metastases targeted the lung, and one subject developed metastasis in the brain. The five-year survival rate of patients with metastatic disease was 60%.p=0.45). Black subjects were found to have better cumulative survival outcomes than Caucasian and Hispanic subjects; however, the difference was not significant (p=0.25). The most common primary MPNST locations in this patient population were the lower extremities (n = 11) and the upper extremities (n = 8), followed by the neck (n = 1). Six subjects had a diagnosis of NF1. Five subjects were NF1-negative, and the NF1 status for nine subjects was indeterminate. Although subjects with negative NF1 status were found to have better cumulative survival outcomes, the difference was not significant (p=0.39).The female gender was associated with better cumulative survival outcomes, and the difference was not significant (n = 2), intermediate (n = 3), and high (n = 15). Low-grade tumors were found to have better cumulative survival than intermediate and high, although the difference was not significant (p=0.63). AJCC 8th Education Stage classification of the patients was as follows: IA = 2, II = 4, IIIA = 8, IIIB = 6. Patients with stage IA and II tumors were all alive at the last follow-up. Overall comparison between stages failed to reveal a significant difference in cumulative survival (p=0.26). S100 B immunohistochemistry status was reported positive in 9 tumors, negative in 4, and could not be determined in 7. Subjects with positive S100 B status were shown to have a significantly better cumulative survival outcome (p=0.003) (Tumor-grade classifications were low (p=0.003) .n = 2) against subjects who did not receive chemotherapy (n = 18), the two subjects receiving chemotherapy had a poorer outcome, although it was nonsignificant (p=0.69). When comparing estimated overall survival in subjects who received radiation (234.1 months) against those who did not (193.9 months), subjects who received radiation had better survival, but the difference was not significant (p=0.89).Three subjects were treated with resection alone (1 subsequently developed metastasis), 15 were treated with resection and adjuvant radiation, and two subjects were treated with resection and chemotherapy. Only one of the resections involved an amputation. When comparing cumulative survival in subjects who received chemotherapy (NF1 mutations among these 27 families were distributed as follows: nonsense (9), missense (4), frameshift (5), splicing (5), deletion spanning most of the gene (1), and whole-gene deletion (4).Among banked DNAs, there were 27 NF1 cases who developed MPNSTs and/or had a positive family history of MPNSTs. As shown in The purpose of this study was to evaluate clinical prognostic factors and genomic biomarkers in patients with MPNSTs treated at our institution. As an academic tertiary-care institution, our institution is a referral center for cancer patients, including those with MPNSTs, from throughout the US as well as internationally. Most similar studies have reported less than a 50% five-year MPNST survival rate –30. Our Consistent with previous reports, we noted that subjects with S100B-positive tumors had significantly better outcomes than those with S100B-negative tumors . S100 B NF1 mutations associated with MPNSTs was higher in our population than the 4–5% rate of such deletions in NF1 patients in general [The frequency of whole-gene deletions (14.8%) in the 27 germline  general . This fr general , 34, 35. general .Although a significant difference in survival was not found based on presence of an NF1 diagnosis, patients with NF1 trended toward a poorer outcome, particularly males: one was still alive at the last follow-up at 314.8 months after metastasis, but three (including one with metastatic disease) had survival under five years . The two NF1 female subjects are both still alive (108.78 months after metastasis and 256.61 months after diagnosis). This study was underpowered to detect relationships between race, gender, and other factors with respect to survival. Similar studies with larger cohorts found individual variables associated with survival, such as tumor size , 22, 29,Our study had a number of limitations, the foremost being sample size and another being lack of NF1 status in earlier cases. Although a larger sample size might aid in identifying additional significant prognostic factors, it is important to report individual institution experiences because of the lack of evidence-based established therapeutic regimens for MPNST. Information from institutions with better outcomes may be very useful to other groups. Toward this goal, we excluded patients who had received prior MPNST treatment at outside institutions. Although this allowed evaluation of survival outcomes based solely on care at our institution, a more varied subject group might have been more representative of the overall US MPNST patient population experience.NF1 deletions are associated with an increased risk of MPNST.Given the rarity and lack of clinical trial data for MPNSTs, treatment parameters and survival data from multiple institutions are needed to move toward improved treatment and prognosis. It is critical that patients with rare tumors such as MPNSTs be referred to institutions experienced with such cancers. Our institutional experience with MPNST cases treated in the Orthopedics oncology service, with treatment favoring surgical resection and adjuvant radiation, showed an outstanding five-year survival of 70%. This work also highlighted the potential use of S100 B immunostaining in prognosis. In addition, our study added to the evidence that whole-gene germline