Understanding antibiotic concentration-time profiles in the central nervous system (CNS) is crucial to treat severe life-threatening CNS infections, such as nosocomial ventriculitis or meningitis. Yet CNS distribution is likely to be altered in patients with brain damage and infection/inflammation. Our objective was to develop a physiologically based pharmacokinetic (PBPK) model to predict brain concentration-time profiles of antibiotics and to simulate the impact of pathophysiological changes on CNS profiles. A minimal PBPK model consisting of three physiological brain compartments was developed from metronidazole concentrations previously measured in plasma, brain extracellular fluid (ECF) and cerebrospinal fluid (CSF) of eight brain-injured patients. Volumes and blood flows were fixed to their physiological value obtained from the literature. Diffusion clearances characterizing transport across the blood–brain barrier and blood–CSF barrier were estimated from system- and drug-specific parameters and were confirmed from a Caco-2 model. The model described well unbound metronidazole pharmacokinetic profiles in plasma, ECF and CSF. Simulations showed that with metronidazole, an antibiotic with extensive CNS distribution simply governed by passive diffusion, pathophysiological alterations of membrane permeability, brain ECF volume or cerebral blood flow would have no effect on ECF or CSF pharmacokinetic profiles. This work will serve as a starting point for the development of a new PBPK model to describe the CNS distribution of antibiotics with more limited permeability for which pathophysiological conditions are expected to have a greater effect. The central nervous system (CNS) is protected by physiological barriers controlling xenobiotic penetration. To act at the CNS level, drugs must have properties allowing them to cross the blood–brain barrier (BBB). Thus, compounds with limited CNS distribution are discarded during the development process. In vitro models of the BBB  have beeΔt were close to 1 for the three compartments  was quite similar to the in vitro-scaled PSECF value (PSin vitro) but somewhat higher than the value estimated by the model (7.1-fold)  between PSECF and PSCSF was predicted by the model. All other parameters of the model were almost identical (The PS.1-fold) . Similardentical . This redentical .Metronidazole was selected as an appropriate compound to develop a minimal PBPK model, not only because data were available both in human brain ECF and CSF, which, as far as we know, is unique, but also because we previously showed that its distribution within brain ECF  and CSF ECF) was predicted from drug-specific parameters (MW and log P) and the BBB surface area. By comparison, the value given by Simcyp and used in the PBPK model (6.4 L/h) was similar to the value calculated using previously reported equations from the literature (6.3 L/h) [ECF value (8.0 L/h) was quite close to the Simcyp prediction, indicating a good correlation between these two approaches. Moreover, the determination of an in vitro efflux ratio lower than 2   but sligr than 2 A confirmr than 2 . Althoug model 6. L/h was K models . To valiobtained , a ratioobtained .ECF/CSF, VECF and Qbrain with values that can be found in patients suffering neurovascular diseases such as TBI and SAH. First, an a priori 2- to 5-fold increase in PSECF/CSF corresponding to the increase in the ratio between CSF and plasma albumin, reflecting a pathological increase in permeability of endothelial cells, was evaluated [ECF/CSF values were initially high, the increased permeability under pathological conditions had no impact on the metronidazole profiles in ECF and CSF  [ECF was allowed to vary within a narrow range of values from 5%, corresponding approximately to the threshold value (4.3%) associated with an increase in intracranial pressure [ECF may have important consequences from a pathophysiological point of view, it had no impact on metronidazole concentrations in ECF  . VECF wapressure , to 40%.s in ECF b.brain corresponding to mild hypoperfusion or oligemia , penumbra  and critical ischemia  [brain on the metronidazole profiles in ECF and CSF, which is consistent with the fact that the brain distribution is permeability-limited due to the BBB and BCSFB with their tight junctions, and not perfusion-limited  , higher and earlier peak concentrations should be observed in ECF and CSF when PSed by 5) . Howeverprofiles .sink), which was not the case for patients equipped with microdialysis. Yet the fraction of metrodinazole dose recovered in the collection bag ranged from 0.1 to 0.4%, which has, therefore, a negligible impact on total clearance and brain PK profiles . In this situation, it was assumed that CSF reabsorption was totally blocked, and thus, Qsink was set at 0 L/h, resulting in a potentially greater impact of the loss of the drug via the EVD on CSF PK profiles for drugs with low permeability  or an EVD into their lateral brain ventricles (n = 4). The demographic characteristics are detailed in This study was performed in the neurointensive care unit at the University Hospital of Poitiers (France) and was approved by the local ethics committee . The study design has been previously described in detail ,30. BrieThe metronidazole pharmacokinetic study was conducted at a steady state after at least 2 days of treatment. Brain dialysates and CSF samples were collected, respectively, during microdialysis monitoring or via the EVD over 8 h at 0.5 h intervals during the first 2–4 h and at 1 h intervals for the remainder of the experiment. Serial blood samples (between 7 and 13 per patient) were collected simultaneously over the dosing interval in all patients. Plasma unbound concentrations were determined by ultrafiltration, and all samples were assayed by high-performance liquid performance (HPLC) with UV detection (310 nm) as previously described .n = 1000) and by calculating fold errors between predicted and observed areas under the plasma, brain dialysate and CSF unbound concentration-time curves between two consecutive dosing at steady-state (AUCΔt). Predicted AUCΔt were determined based on predicted individual PK profiles for each tissue, while observed AUCΔt were calculated using the linear trapezoidal rule .The concentrations in plasma and CNS from all patients were modelled simultaneously using the non-linear mixed effect modelling approach in NONMEM 7.4 . All model estimations were conducted with the first-order conditional estimation method with interaction (FOCE INTER). Model evaluation was based on the visual inspection of diagnostic plots (goodness of fit (GOF) and individual plots) , compariA minimal PBPK modelling approach was used to describe the concentration-time profiles of metronidazole in plasma and CNS. In each patient, two individual unbound fraction values (fu) of metronidazole were calculated as the ratio of metronidazole concentrations in ultrafiltrates to corresponding total plasma concentrations. The mean value was used to convert total concentrations into unbound concentrations. The blood to plasma (B/P) ratio of metronidazole was estimated using the software tools PK-Sim  based on fu, log P and haematocrit, as defined by Rodgers and Rowland  Table 4Table 4 aB) were modelled as: B is blood volume, CO is cardiac output blood flow, Ctissue is the metronidazole concentrations in (non-CNS) tissue compartment, fd is the fraction of CO going to the tissue compartment, and Kp is the unbound tissue to blood partition coefficient. Cbrain,vasc is the metronidazole concentrations in brain vasculature, Qbrain is the brain blood flow, and CL is the total blood clearance.Metronidazole concentrations in the blood compartment (Ctissue) were modelled as:tissue, the volume of the tissue compartment, was determined by VB + Vtissue + Vbrain,vasc + VECF + VCSF = TBW due to physiological constraints, with Vbrain,vasc representing the volume of the brain vasculature compartment, VECF the ECF brain tissue volume, VCSF representing the cranial CSF volume and TBW representing the total body weight  and non-CNS tissue compartment (fd) was ≤1 , with fd,brain fixed to the physiological value calculated from Metronidazole concentrations in non-CNS tissue compartment  were modelled as:ECF and CCSF are ECF and CSF concentrations, respectively, PSECF and PSCSF are passive permeability-surface area products for the BBB and BCSFB, respectively, and Qsink is the CSF absorption rate (sink flow). Metronidazole is not thought to be actively transported; therefore, only passive diffusion across the brain barriers was considered. Passive diffusion clearance at the BBB (PSECF) was determined from a combination of system-  and drug-specific parameters (log P and molecular weight), using the prediction option incorporated in Simcyp simulator . PSECF was fixed to 6.4 L/h and PSCSF was assumed to be half of PSECF (considering SABCSFB = 50% SABBB) [Metronidazole concentrations in the brain vasculature compartment (C% SABBB) .ECF) were modelled as:bulk is the bulk flow from brain ECF to CSF. The bulk flow of ECF is a physiological convection process and was assumed to be the major mechanism of drug transfer between ECF and CSF.Metronidazole concentrations in the ECF compartment , collected as fractions during various time intervals, were described by the integral over each collection interval, represented by t1 and t2 [Brain dialysate concentrations (C1 and t2 :(5)CECF,ECF,dialysate corresponds to the dialysate concentrations corrected by the in vivo probe recovery determined for each patient using a retrodialysis-by-drug method, as previously described [In the model, Cescribed .CSF) were modelled as:EVD is the EVD flow determined experimentally during a certain time interval  were modelled for each collection interval as:EVD is the volume of EVD samples determined experimentally during a certain time interval  was also carried out.The impact of the EVD on CSF concentrations was investigated by varying Q patient . A simulECF) was fixed in the PBPK model to the value predicted by Simcyp from system- and drug-specific parameters. On the other hand, PSECF was estimated from an in vitro Caco-2 model for comparison purposes. The final protocol is detailed in the app,A-B) and basolateral-to-apical  directions. An efflux ratio was calculated as the ratio of Papp,B-A to Papp,A-B. For a ratio lower than 2, indicating passive diffusion across biological membranes [app was calculated as the mean of Papp,A-B and Papp,B-A and was scaled to PSECF,in vitro by multiplying by the physiological SABBB and the brain weight (BW)  :(9)PSECFCSF) was assumed to be half of PSECF. To validate this hypothesis, PSECF and PSCSF were estimated by the model.Permeability across the BCSFB (PSECF/CSF, cerebral blood flow or brain ECF volume would have no effect on ECF or CSF PK profiles. This work will serve as a starting point for the development of a new PBPK model to describe the CNS distribution of cefotaxime as a representative of antibiotics with more limited CNS distribution due to lower permeability and affinity for efflux pumps, which are frequently used for the treatment of CNS infections. Metronidazole was selected as a representative antibiotic with relatively rapid and extensive CNS penetration due to its sufficiently high membrane permeability and absence of affinity for efflux transport systems present at the BBB or BCSFB level to develop a PBPK model from plasma and brain ECF as well as CSF concentrations previously measured in neurocare ICU patients. This model shows that with this type of antibiotic, physio-pathological alterations of membrane permeability PS


Logistic regression analyses confirmed that plasma sVCAM-1 level  is an independent predictor of CSF, and the receiver operating characteristic curve analysis showed that plasma sVCAM-1 levels had statistical significance in predicting CSF . When the sVCAM-1 level was higher than 111.57 ng/mL, the sensitivity for predicting CSF was 87% and the specificity was 73%. Conclusions: Plasma sVCAM-1 level can be used to predict CSF and was associated with the clinical symptoms of patients. It may serve as a potential biomarker for CSF in the future.Background: Coronary slow flow (CSF) is an angiographic entity characterized by delayed coronary opacification with no evident obstructive lesion in the epicardial coronary artery. Several studies have shown that the occurrence and development of CSF may be closely related to inflammation. Soluble vascular cell adhesion molecule-1 (sVCAM-1) is a biomarker related to inflammation. The aim of this study was to evaluate the correlation between plasma soluble VCAM-1 level and CSF occurrence and thus the predictive value of VCAM-1 for CSF. Methods: Forty-six CSF patients and thirty control subjects were enrolled. Corrected thrombolysis in myocardial infarction frame count (cTFC) was used to diagnose CSF. Functional status and quality of life were determined by the Seattle Angina Questionnaire (SAQ). Echocardiography was used to evaluate the systolic and diastolic function of the left ventricle (LV) and right ventricle (RV). The plasma levels of sVCAM-1, IL-6, and TNF-α were quantified by enzyme-linked immunosorbent assay. Results: Compared with the control group, the physical limitation score by the SAQ, the LV global longitudinal strain (GLS), mitral E, and mitral E/A decreased in patients with CSF, while the plasma IL-6 and TNF-α levels increased. The plasma sVCAM-1 level in the CSF group was significantly higher than that in the control group (186.03 ± 83.21 vs. 82.43 ± 42.12 ng/mL,  Coronary slow flow (CSF) is an angiographic entity characterized by non-obstructive coronary artery lesions but delayed filling at the end of a coronary artery . We haveHowever, owing to the unclear etiology and pathogenesis of CSF, it has no uniform treatment method ,6. It isVascular cell adhesion molecule-1 (VCAM-1) is an important cell adhesion molecule that is mainly expressed in cytokine-activated vascular endothelial cells. It plays an important role in the adhesion and migration of leukocytes from circulation to the surrounding tissues and participates in the occurrence and development of inflammatory reactions . In receWith this background, the present study was undertaken to find out (a) the correlation between the plasma soluble VCAM-1 level and occurrence of CSF and (b) the predictive value of VCAM-1 for CSF.We conducted a case–control study with patients that received coronary angiography because of typical angina, coronary risk factors, or abnormal electrocardiography changes from the Department of Cardiology of the First Affiliated Hospital of China Medical University from March 2018 to September 2020, and patients with coronary angiography showing all three epicardial coronary arteries at ≤40% (coronary stenosis not exceeding 40%) were included. Corrected thrombolysis in myocardial infarction frame count (cTFC) was used to diagnose CSF, where subjects with at least one main epicardial coronary artery with cTFC > 27 frames were assigned to the CSF group and those with all three epicardial coronary arteries with cTFC ≤ 27 frames were assigned to the control group . The excThis study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of China Medical University. Written informed consents were obtained from all participants.The radial artery approach was chosen for coronary angiography, the standard Judkins technique was used for multi-position angiography, and nicorandil, nitroglycerin, and calcium channel blockers were not used during the operation. Coronary blood flow velocity was evaluated by quantitatively measuring the TFC of the three main epicardial coronary arteries, namely the left anterior descending coronary artery (LAD), left circumflex artery (LCX), and right coronary artery (RCA). The acquisition speed of coronary angiography images in this study was 30 frames/s.TFC calculation: The last frame number minus the first frame number is taken as the exact number for coronary arteries. The first frame is taken as that showing the contrast agent beginning to fill the coronary artery lumen; the last frame is taken as that showing the contrast agent reaching the distal mark of each coronary artery . The distal end is defined as the distal branch of the LAD, the distal branch of the longest segment of the LCX, and the first branch of the posterolateral artery of the RCA. Due to the long LAD, the number of frames obtained is divided by 1.7 to obtain the corrected TFC for the LAD (cLAD). The sum of TFC values for the RCA, LCX, and cLAD obtained is divided by 3 to calculate the mean cTFC for each subject. TFC analyses are performed by two independent cardiologists, and any disagreements are resolved by a third cardiologist ,19.The SAQ was performed 24 h or less before coronary angiography. The subjects were required to complete the SAQ within 5 min. The questionnaire has five dimensions and 19 questions, including the degree of physical limitation (question 1), angina stability (question 2), angina frequency (questions 3–4), treatment satisfaction (questions 5–8), and quality of life (questions 9–11). Each question is scored item-by-item, and each score is converted into standard points according to the following formula: standard points = /(highest score in this aspect − lowest score in this aspect) × 100. The score range is 0–100, with higher scores indicating fewer symptoms, better functional status, and better quality of life .Echocardiography was performed within 72 h of coronary angiography using a Vivid E9 ultrasound system  equipped with an M5S phased array probe. An EchoPAC ultrasound workstation  was used for offline analysis. Image acquisition and data measurements were according to the recommendations from the American Society of Echocardiography .To assess left ventricular (LV) systolic function, LV ejection fraction (LVEF) and global longitudinal strain (GLS) were measured. To assess LV diastolic function, left atrial volume index, mitral E, mitral A, mitral e’, tricuspid regurgitation velocity, calculated mitral E/A, and mitral average E/e’ were measured. To assess right ventricular (RV) systolic function, RV fractional area change, tricuspid annulus plane systolic excursion (TAPSE), and tricuspid annulus systolic velocity were measured. To assess RV diastolic function, tricuspid E, tricuspid A, and tricuspid e’ were measured.g and 4 °C to obtain the plasma. Enzyme-linked immunosorbent assays (ELISAs) were performed to determine plasma sVCAM-1, IL-6, and TNF-α levels . All analyses were performed in triplicate.The levels of the plasma inflammatory factors sVCAM-1, IL-6, and TNF-α were measured within 24 h before coronary angiography. Blood samples were obtained from the anterior cubital vein after the patient had fasted for 12 h. The blood sample was immediately centrifuged for 15 min at 3000× t-test was used to compare the differences between continuous variables. Categorical variables are expressed in frequency and percentage. In addition, the chi-square test was used to compare the differences between categorical variables. The Shapiro–Wilk test was used to evaluate whether a variable was normally distributed. Pearson correlation analysis was used to evaluate the correlation between continuous variable parameters. Spearman correlation analysis was used to evaluate the correlation between the data parameters of each grade. Linear regression analysis and binary logistic regression analysis were performed to obtain independent variables related to coronary slow flow. Parameters that were statistically different in the two groups were included in univariate linear regression analyses. Parameters with significance in the univariate linear regression analysis were included in the multivariate linear regression analysis. Logistic regression analysis results are expressed by odds ratio (OR) and 95% confidence interval (CI). Receiver operating characteristic (ROC) curves were used to evaluate the distinguishing ability of the selected variables for predicting CSF and to obtain the cut-off values of independent influencing factors.Continuous data are expressed as mean ± standard deviation, and independent sample p < 0.05 (two-tailed) was considered statistically significant.Statistical analysis was performed using SPSS 23.0 software package . For all parameters, The demographic, medical history, laboratory value, medication, and angiographic details of the study population are shown in p = 0.008), while there are no significant differences in angina stability, angina frequency, treatment satisfaction, and quality of life between the two groups. These findings indicate that the functional status of CSF patients is decreased . In addition, the mitral E and mitral E/A for the CSF group are lower than those of the control group . These findings indicate that left ventricular systolic and diastolic function in CSF patients are reduced  and are positively correlated with mean cTFC .The plasma sVCAM-1 level for the CSF group is significantly higher than that for the control , systolic blood pressure, smoking history, fasting blood glucose, triglycerides, and total cholesterol were not associated with mean cTFC. Then, we included the parameters that were statistically different between the CSF group and the control group in the univariate linear regression analysis, including physical limitation, mitral E, mitral E/A, TAPSE, LV GLS, IL-6, TNF-α, and sVCAM-1. By univariate analysis, sVCAM-1, IL-6, TNF-α, and LV GLS are associated with mean cTFC. Upon adjusting for baseline covariates, including age, sex, body mass index (BMI), systolic blood pressure, smoking history, fasting blood glucose, triglycerides, and total cholesterol, multivariate linear regression analysis still shows that the association between plasma sVCAM-1 level with mean cTFC is significant .p = 0.001) is still an independent predictor of CSF after adjusting for age, sex, BMI, mitral E, and other variables having a p < 0.10 by univariate analysis, including physical limitation, mitral E, mitral E/A, LV GLS, TAPSE, IL-6, and TNF-α  Plasma sVCAM-1 in the CSF patient group is significantly increased. It is positively correlated with mean cTFC and negatively correlated with the SAQ physical limitation score. (2) Plasma sVCAM-1 level is an independent predictor of CSF. When it is greater than 111.57 ng/mL, the sensitivity for predicting CSF is 87% and the specificity is 73%.In recent years, an increasing number of studies have focused on patients with coronary angiography showing no obstructive coronary artery disease (CAD) nor structural heart disease that nevertheless suffer from angina-like chest pain. Such conditions may be related to coronary microvascular dysfunction . CSF patThe etiology and pathogenesis of CSF are still unclear, but there is an increasing body of evidence indicating that it is a microvascular disease ,23. InflIn assessing the relationship between certain inflammatory biomarkers, oxidative stress parameters, and CSF, Neda et al. confirmed that the pathogenesis of CSF may be closely related to inflammation . Other sThe adhesion of leukocytes to vascular endothelial cells is one of the most important features of the inflammatory process . VCAM-1 In many human heart diseases, factors such as reactive oxygen species and hemodynamics can enhance the expression of VCAM-1 in the heart, and cell infiltration and inflammation in the heart tissue can lead to continuous heart remodeling, fibrosis, and dysfunction . sVCAM-1Our study has demonstrated that plasma sVCAM-1 levels in CSF patients are significantly elevated, indicating that inflammation plays an important role in the development of CSF. This may be because VCAM-1 on the surface of vascular endothelial cells in CSF patients bound to ligands activates endothelial cells, increases the adhesion of leukocytes and vascular endothelial cells, and enhances inflammatory response. In addition, the level of sVCAM-1 was also positively correlated with mean cTFC, indicating that slower coronary blood flow velocity appeared to be associated with the inflammatory response.Turhan et al.  also fouThe SAQ is a widely accepted means by which to assess the quality of life of CAD patients . The phyThe main limitation of this research was the small sample size because of the low prevalence and the strict exclusion criteria. Therefore, we designed the case–control study to further control the confounding factors. In addition, a large sample and a prospective and multi-center study are required for further confirmation. Furthermore, because VCAM-1 is synthesized and released by many cell types, such as endothelial cells, macrophages, smooth muscle, and dendritic cells, the determination of the level of plasma sVCAM-1 does not provide any direct information on its origin. Although the findings revealed that VCAM-1 can detach from the cell surface to form sVCAM-1 in the blood, this may also be a limitation in the present study.This study revealed that plasma sVCAM-1 is significantly increased in CSF patients. Moreover, sVCAM-1 is an independent predictor of CSF and is negatively correlated with the physical limitation score obtained by SAQ. These indicate that plasma sVCAM-1 may serve as a predictive biomarker for CSF.


In CSF samples from early induction chemotherapy, VEGF-A concentration were uniformly elevated in the CNS-positive group compared to those patients without unambiguous meningeal infiltration . Expression of miR-181a, a VEGFA-regulating microRNA which showed increased level in CNS leukemia in our previous experiments, was then paralleled with VEGF-A concentration. A slight correlation between the levels of miR-181a and VEGF-A indicators in CSF and BM samples was revealed . After validating in international cohorts, the joint quantification of miR-181a and VEGF-A might provide a novel tool to precisely diagnose CNS involvement and adjust CNS-directed therapy in pALL.Central nervous system (CNS) involvement is a leading cause of therapy-refractory pediatric acute lymphoblastic leukemia (pALL), which is aggravated by underdiagnosing CNS disease with the currently used cell-based approach of cerebrospinal fluid (CSF) diagnostics. Our study focused on developing novel subcellular CNS leukemia indicators in the CSF and the bone marrow (BM) of patients with pALL. Serial liquid biopsy samples ( Pediatric acute lymphoblastic leukemia (ALL) is still the leading cause of cancer-related long-term disability influencing the quality of life not only in childhood but also in adulthood . In ALL,Previous studies suggest that subcellular biomarkers in the CSF may help to identify undiagnosed CNS leukemia cases. Earlier, more than 30 humoral factors and a set of molecular markers have been tested in the CSF, although, most of these candidate markers were proved not to be eligible for diagnostics in real clinical cohorts . RecentlTo establish a clinical diagnostics study on CNS leukemia with state-of-the-art candidate subcellular biomarkers, we analyzed serial liquid biopsies from the CNS and bone marrow (BM) niches in children with ALL by measuring VEGF-A and ITGA6 concentrations and correlating these data with miR-181a expressions.g for 15 min two times, while cell- and debris-free CSF samples were gained after centrifugation at 300 g for 10 min. Processed BM PFP and CSF samples were then stored at −80°C. In our study, we examined CNS-positive  ALL (n = 9) and matched CNS-negative  patients (n = 9) based on sex, age at the diagnosis and immunophenotype. In addition, patients (n = 6) diagnosed with spinal muscular atrophy (SMA) were selected as CSF reference samples of non-cancerous origin. Further information about the patient cohort is available in The study is based on patients (aged ≤ 18 years) diagnosed with ALL in two Hungarian pediatric hematology centers . We collected CSF and BM of patients at diagnosis and at day 15 of the induction cycle of the ALL IC-BFM 2009 trial protocol, between October 2015 and August 2019. Consecutive lumbar punctures are the standard of care for evaluating leukemic CNS involvement depending on cytomorphology of the CSF. In the treating centers mentioned above, CSF samples were analyzed by cell counting chamber and cytospin preparate to assess the initial CNS status of the patient  and ITGA6  in accordance with the manufacturer’s guidelines. Due to the fact that we expected low protein concentrations, we transferred 75 µl of the samples to each well instead of the recommended 50 µl to assure a significant absorbance when performing the VEGF-A assay. The ITGA6 measurement was performed according to the manufacturer’s instructions, with a volume of 100 µl per sample. Both ELISA kits contained standard samples which were serially diluted to generate a calibration curve. ELISA reactions were processed on pre-coated plates and spectrophotometry was applied to read the final optical density values.In our previous study, we aimed to detect and quantify miR-181a levels in BM PFP and CSF samples of the same patient cohort . After sAll statistical analyses were performed using R version 4.0.3 . In order to determine the concentration of the analyte within a sample, we used the non-linear 4-Parameter Logistic (4 PL) curve model. We fit the data obtained for the standard samples to the 4 PL curve by Levenberg-Marquardt algorithm (drc package). Concentration estimates were calculated as the natural logarithm of VEGF-A or ITGA6 absolute levels for each sample. Linear regression models were used to assess the statistical significance between each subgroup, where gender, age at diagnosis and ALL immunophenotype were considered as cofactors. The correlations between the expression level of miR-181a and the estimate of concentration was compared using Pearson’s method and Student’s t-test. Alpha level of 0.05 was used as criterion for statistical significance after false discovery rate correction, where it was relevant.KMT2A-rearranged, P2RY8-CRLF2 translocated, CDKN2A deleted and ETV6 deleted).ELISA measurements were performed on 33 CSF and 32 BM PFP samples collected from 18 children with ALL and on CSF reference samples from six SMA patients. The baseline characteristics for this cohort is described in + and CNS− patients, two commercial ELISA tests were used. Due to the paucity of cell-derived material in CSF compared to other body fluids, we questioned whether these proteins can be detected in CSF supernatant. VEGF-A was measurable in all CSF samples in the CNS+ as well as the CNS− group, as seen in see− patients compared to the CNS+ group. As opposed to these results, ITGA6 was not detectable in our CSF or BM samples using this method.To evaluate the VEGF-A and ITGA6 content of cell-free CSF and platelet-free BM plasma samples from both CNS+ ALL group compared to CNS− patients when analyzing CSF samples from day 0 and day 15 of the induction chemotherapy . This means a mildly more than 1.5-fold difference in concentrations between the two subgroups , as seen in + cohort, we were not able to demonstrate a change in VEGF-A level between d0 (rich in blasts) and d15 (paucity of blast cells) CSF samples. However, change in BM VEGF-A levels from d0 to d15 was not proportional to flow cytometry-based BM residual disease dynamics  were uniformly elevated in the CNSdynamics , so the + and CNS− ALL patients, we addressed whether VEGF-A levels correlates with a previously proposed marker of CNS leukemia, miR-181a. Relative miR-181a expressions in the same patient cohort were determined by delta-delta cycle threshold (ddCt) algorithm as we previously published [n = 46, Pearson’s r = 0.36, p = 0.015), as displayed in After the identification of VEGF-A concentration gap between CSF samples of CNSublished . As of i+ and CNS− ALL defined based on cytomorphologic examinations. The CSF level of VEGF-A was significantly elevated in patients with meningeal disease compared to those without CNS involvement. The role of VEGF-A was previously investigated in preclinical models of CNS leukemia. Münch et al showed that patient derived ALL cells transplanted onto mice invaded the CNS niche by transmigrating through microvascular endothelial cells facilitated by VEGF-A signaling [VEGFA upregulation in the nutrient-poor CNS microenvironment [VEGFA expression in these cells [− samples were not analyzed parallelly. In a study with pediatric and adult acute leukemia patients, levels of soluble VEGF-A receptors and their ratio to VEGF-A concentration in CSF and serum were proposed as a prognostic marker of leukemia and a contributor of CNS involvement evolution [As the identification and follow-up of pediatric ALL manifestation in the CNS compartment present difficulties in current clinical practice, sensitive and easy-to-measure novel markers are needed to develop. Our study proposes a subcellular approach of CNS leukemia diagnostics. We measured VEGF-A and ITGA6 concentrations in CSF samples of children with CNSignaling . Similarironment . Howeverse cells , but CNSvolution . NeverthOur VEGF-A concentration results in BM PFP samples showed no difference between day 0 and day 15 of the treatment , however+ and CNS− groups [The positive correlation between VEGF-A level and regulatory RNA miR-181a expression in BM PFP and CSF samples of childhood ALL cases was also observed. MiR-181a seemed to be a sensitive subcellular marker of CNS disease in our previous study . There i− groups . If we sITGA6 was not measurable in cell-free ALL samples by the ELISA method we used. ITGA6-expressing blasts are actively engaged in adhesion and migration on the abluminal side of vessels to the CNS as multiple studies suggest . The abs− group. Yet, this study provides the rationale for future studies of subcellular elements, soluble proteins and miRs, in the diagnostics of CNS leukemia.In summary, our data represents the first clinical observation of VEGF-A based CNS status evaluation and the identification of VEGF-A and miR-181a co-expression in ALL. However, several limitations should be considered when interpreting our results. The rarity of CNS involvement in childhood leukemia resulted in low patient numbers. Another bias is the selection of patients without CNS disease, which relied on insensitive conventional diagnostics and carries the risk of involving patients with occult CNS involvement in the CNS


It was found that ultrasonic vibration perpendicular to the middle surface of the cavity is beneficial in forming transverse microstructure, but is not conducive to generating longitudinal microstructure. Increase in injection pressure can improve molding qualities for both the longitudinal micro groove and the transverse micro groove. Increase in injection speed is not conducive to forming the longitudinal micro groove but benefits formation of the transverse micro groove. When ultrasonic vibration is applied at the injection and packing stages, molding quality of the longitudinal micro groove becomes worse, while that of the transverse micro groove becomes better.It is challenging to fabricate micro features on an injection-molded polymer product. Ultrasonic vibration induced into micro-injection molding is helpful for flow of polymer melt. In this paper, a micro-injection mold integrated with ultrasonic vibration was designed and fabricated, and micro embossment was machined on the surface of the ultrasonic vibrator. Poly(methacrylic acid methyl ester) (PMMA) was used for injection molding experiments, with four ultrasonic power levels , three injection speed levels (60, 80, and 100 cm With rapid development of micro electro-mechanical system (MEMS) technology, polymer products with microstructural characteristics have been widely used in many fields, such as optics, clinical medicine and micro machinery. For injection molding of polymer products that have microstructures, molding quality largely depends on design of the microstructural cavity and on injection process parameters including mold temperature, polymer melt temperature, injection speed, injection pressure, and packing pressure. During micro-injection molding, fluidity of polymer melt at cavity microstructure is poor, which leads to low precision of microstructural reproduction. Increasing temperature can reduce viscosity of polymer melt, thus improving its flow capacity. When cavity temperature is raised close to the glass transition temperature of polymer, microstructural replication precision can be improved significantly ,2. HowevPower and amplitude of the ultrasonic vibrator are limited by the internal space of the injection mold. In order to maximize the effect of ultrasonic vibration on formation of the microstructure, we machined the micro embossment on the surface of the ultrasonic vibrator with electric discharge machining (EDM) to directly use the ultrasonic vibrator as the microstructural cavity insert. For the first time, this work unveiled the relationships between replication of products and orientation of microstructures during ultrasonic vibration-assisted injection molding. The ultrasonic vibration field can promote unwinding of polymer chains. Moreover, cavitation and thermal effects of the ultrasonic wave can transform mechanical vibration energy into the internal energy of polymer melt so that polymer melt near the vibrating cavity cools slowly. These two aspects result in low viscosity and good flowing ability of polymer melt near the vibrating cavity.Φ 2.0 mm were arranged at the four corners of the cavity. The ultrasonic vibrator was placed at the center of the cavity. In order to enhance uniformity of polymer melt filling, a fan-shaped gate was used. As shown in The upper part of the ultrasonic vibrator assembled in the cavity insert was a cylinder with a diameter of 16 mm so that micro embossment on the surface of the ultrasonic vibrator could be rotated with respect to the cavity insert. When length direction of the microstructure was parallel to the polymer-melt filling direction in the cavity, it was called longitudinal microstructure, as shown in 3, an elastic modulus of 3.33 GPa, tensile strength of 66.00 MPa, and flexural strength of 120.00 MPa. Because of its good mechanical properties and excellent transparency, PMMA has been widely used to fabricate optical products for diverse applications [The polymer used for injection molding was PMMA  c. The seications ,18,19,20The structure of the ultrasonic-assisted injection mold is shown in The ultrasonic vibrator was made of titanium alloy for the sake of strength. The micro embossment on the surface of the ultrasonic vibrator was machined by EDM. Considering the ability to resist arc ablation, the material of the tool electrode was copper–tungsten alloy with 75% tungsten content ,22. The 3/s). Ultrasonic power had four levels . Constant processing parameters included a PMMA melt temperature of 240 °C, mold temperature of 60 °C, packing pressure of 20 MPa, packing time of 5s, and ultrasonic time (starting from injecting moment) of 4 s. Ultrasonic frequency was 21 kHz. All quantitative values in the text and figures were reported as means ± standard deviation (SD), with n = 5 samples per group.The PMMA was dried before injection molding due to high moisture absorption. Drying temperature was 85 °C and drying time was 4 h. Processing parameters of injection molding experiments are illustrated in In micro-injection molding, high mold temperature (approaching the glass transition temperature of polymer) is more likely to be adopted for the sake of improving flow capacity of polymer melt ,24,25,26Every time processing parameters were changed in the experiments and the first five plastic parts were discarded. Then, the injection molding machine was considered to work stably, and the next five plastic parts were taken as experimental samples.The measuring position was selected at the midpoint of the length of the micro groove. Polymer products obtained by different molding parameters were sliced. Then, cross-section photos of the micro grooves were taken by a universal tool microscope. It was found that the rounded corner at the opening of the molded micro groove was larger than the rounded corner at the root of the micro embossment of the mold cavity because the root of the cavity micro embossment was difficult to fill completely. That is to say, the opening width of the molded micro groove was larger than the root width of the micro embossment of the cavity. In addition, opening width of the molded micro groove varied greatly with processing parameters, while depth and bottom width of the molded micro groove were more stable and rarely affected by processing parameters. Therefore, influence of processing parameters on opening width of the micro groove is mainly discussed. 3/s. Comparison of the average values of each group of sample data is shown in When injection pressure was 60 MPa, changes of longitudinal micro groove opening width with injection speed and ultrasonic power were illustrated in It can be seen that with rise in injection speed, opening width of the longitudinal micro groove increased, which meant falling replicability and worse molding quality. This phenomenon can be explained by the fact that when injection pressure is low, high injection speed only urges polymer melt to rapidly spread forward in the direction of inertial motion, and the “detail” of filling the longitudinal micro embossment of the cavity is omitted. When polymer melt reached the end of the flow path and completed filling the macro area of the cavity, cavity pressure peaked but was insufficient to drive polymer melt into the space around the longitudinal embossment of the cavity because polymer melt viscosity increased sharply as temperature dropped.3/s in the absence of ultrasound. This shows that ultrasonic vibration has a negative effect on formation quality of the longitudinal micro groove. Because orientation of macromolecules usually coincided with flow direction of polymer melt, macromolecules were oriented along length direction of the longitudinal micro groove, i.e., perpendicular to the cross-section of the longitudinal micro groove. Thus, for the longitudinal micro groove, ultrasonic vibration perpendicular to the middle surface of the cavity had no effect on the orientation state of macromolecules. Not only that, but polymer chains that were arranged along the length direction of the longitudinal micro groove were pushed away from the micro embossment of the cavity by ultrasonic vibration perpendicular to the middle surface of the cavity, resulting in increased opening width of the molded longitudinal micro groove. There was one exception: when injection speed was 100 cm3/s. In that case, because high-speed filling without ultrasound led to excessive opening width of the longitudinal micro groove, there was an obvious reduction of micro groove opening width at 300 W ultrasonic power. This indicates that ultrasonic vibration of low power may affect the stacking state of polymer chains that are oriented along the length direction of the longitudinal micro groove to a certain extent and make macromolecules move closer to the root of the cavity micro embossment.In addition, with rising ultrasonic power, opening width of the longitudinal micro groove increased in general. In When injection pressure was 90 MPa, changes of longitudinal micro groove opening width with injection speed and ultrasonic power were illustrated in 3/s in the absence of ultrasound. The minimum value (182 μm) of sample data of longitudinal micro groove opening width was as obtained in 3/s, and ultrasonic power of 0 W. Moreover, ultrasonic vibration still enlarged the opening width of the longitudinal micro groove, but enlargement was not as dramatic as when injection pressure was lower (60 MPa). In 3/s), opening width of the transverse micro groove was nearly 90 μm larger than that of the longitudinal micro groove, and increasing injection speed could reduce opening width of the transverse micro groove. This phenomenon can be explained by the fact that no two sides of the transverse micro groove were formed at the same time.When injection pressure was 60 MPa, changes in transverse micro groove opening width with injection speed and ultrasonic power were shown in As shown in It can also be seen in 3/s almost coincided with each other when ultrasonic power was 900 W. This indicates that the promoting effect of ultrasonic vibration surpasses the promoting effect of high speed on micro transverse groove formation.Moreover, ultrasonic vibration makes the effect of injection speed on opening width of the transverse micro groove become weak. As shown in 3/s and ultrasonic power was 600 W. The minimum value (183 μm) of sample data of transverse micro groove opening width was as obtained in 3/s, and ultrasonic power of 600 W.When injection pressure was 90 MPa, changes in transverse micro groove opening width with injection speed and ultrasonic power were shown in The ultrasonic-assisted micro-injection mold designed in this paper took the surface of the ultrasonic vibrator as a component of the microstructural cavity. A micro embossment with a cross-section size of 117 μm high and 145 μm wide at the top and 175 μm wide at the root was machined on the surface of the ultrasonic vibrator. Improvement measures for formation quality of the longitudinal micro groove parallel to the filling direction and the transverse micro groove perpendicular to the filling direction were discussed. A comprehensive comparison of different injection molding experiments showed that ultrasonic vibration perpendicular to the middle surface of the cavity is beneficial to formation of the transverse micro groove, but, however, unfavorable to formation of the longitudinal micro groove. If ultrasonic vibration is used to improve molding quality of micro grooves, the gate should be arranged to make the filling direction of polymer melt not parallel to the length direction of the micro groove; that is to say, the micro groove of the polymer product should avoid the position of the longitudinal micro groove.Without raising mold temperature close to the glass transition temperature of polymer, formation quality of the longitudinal micro groove was better than that of the transverse micro groove when ultrasonic vibration was not applied. The way to improve molding quality of the longitudinal micro groove is to increase injection pressure. The ways to improve molding quality of the transverse micro groove are (1) to increase injection speed when injection pressure is low, (2) to introduce ultrasonic vibration when injection pressure is low, and (3) to increase injection pressure.3/s, injection pressure of 90 MPa, and mold temperature of 60 °C. The transverse micro groove obtained a minimum opening width of 183 μm under conditions including ultrasonic power of 600 W, injection speed of 100 cm3/s, injection pressure of 90 MPa, and mold temperature of 60 °C.The longitudinal micro groove obtained a minimum opening width of 182 μm under conditions including ultrasonic power of 0 W, injection speed of 60 cm


Posterior lamellar keratoplasty and especially Descemet membrane endothelial keratoplasty (DMEK) are gaining interest worldwide. Little is known about the influence of donor factors on DMEK outcome. Here we provide an overview of the existing peer-reviewed literature on this topic and present the design of the upcoming cooperation study COMEDOS (Cologne-Mecklenburg-Vorpommern DMEK Donor Study).A literature search of PubMed and MEDLINE was conducted to retrieve articles published between September 2013 and May 2021. Seventeen peer-reviewed articles were selected. Design and concept of the prospective COMEDOS are outlined.Main interest parameters were the donor diabetes mellitus status, age, and lens status. There is a large heterogeneity regarding the sample size, study design, and investigated parameters. There seems to be a consensus that younger donors are associated with tighter rolls, a more difficult preparation, and unfolding setting. Diabetic donors seem to increase the risk of tissue tearing due to adherences and result more frequently in preparation failure. The COMEDOS aims not only to analyze the diabetes status of the donor, but also to correlate all donor systemic comorbidities and their ophthalmologic history to the DMEK clinical outcome. Furthermore, a correlation of Descemet membrane lamella preparation and surgery outcome is planned.Currently, there is a lack of knowledge regarding the effect and impact of donor tissue characteristics on DMEK outcome and complications. An in-depth investigation is planned by the upcoming COMEDOS to close this knowledge gap. The field of corneal transplantation has been revolutionized during the recent years by introducing Descemet membrane endothelial keratoplasty (DMEK). It has become the procedure of choice for the treatment of corneal endothelial diseases such as Fuchs endothelial dystrophy in many countries –4. This Furthermore, rolling characteristics are very different in different donor ages. Besides properties of the recipients’ eye, the tendency of DM to form a roll is the most important factor in determining how atraumatic the unfolding of the graft can be performed inside the anterior chamber. Schaub et al. reported that donor age can play a role in the graft preparation and the unfolding behavior of the graft lamella during surgery; donor tissue from elderly patients (above 70 years of age) tends to roll less than tissue obtained from younger donors (below 50 years of age) –10. AlsoOverall, there is a lack of knowledge about the long-term effect and impact of donor tissue characteristics on DMEK outcome and complications , 11. ThiNevertheless, studies with large sample sizes with meaningful follow-up and solid long-term assessments of donor characteristics on clinical outcome are still lacking in the field of DMEK. This knowledge gap has an important impact on clinical work as it could influence the prediction of surgery outcome. By considering preexisting donor conditions and correlating these with graft characteristics, the outcome of the DMEK surgeries could be positively influenced and complications could be prevented. In times of donor shortage, preventing tissue discarding because of graft preparation complications is also a key issue.In this context, the objectives of this article are (i) to review the currently existing peer-reviewed literature on this topic and (ii) to present the objectives and design of the upcoming COMEDOS which aims to correlate donor characteristics and DMEK outcome.A search of electronic databases was conducted to retrieve articles published between September 2013 and May 2021 in PubMED and MEDLINE. Search term combination used were as follows: “DMEK” or “Descemet Membrane” AND “donor” OR “graft” OR “preparation.” Only publications which matched the search terms in the same context were included. Publications in English or German were included; other languages were excluded. All types of research  were included. A literature management software  was used to manage the records.Only studies on humans were selected. Only studies reporting on the influence of donor tissue characteristics on DMEK were selected.A spreadsheet software  was used for standardized data extraction. Data recorded per publication included as follows: country and region of data collection, number of cases by indications, year of publication, study setting , study design , and diagnostic base .Cologne DMEK database was established in 2015 and includes all data related to DMEK surgeries performed in the Department of Ophthalmology, University Hospital of Cologne. Since 2011, more than 4000 DMEK surgeries have been performed at the Department of Ophthalmology in Cologne. Our DMEK database includes records of more than 3000 patients with a minimum follow-up of 1 year. The clinical information contained is demographics, medical history, preoperative status of the eye, details of surgery, details of donor tissue, and postoperative outcomes . There are no exclusion criteria. The prospective Cologne DMEK database is filled retrospectively through the existing data collection systems. All medical data is handled confidentially according to the “Good Epidemiological Practice (GEP)” guideline and with ethics committee approval by the Ethics Commission of Cologne University’s Faculty of Medicine (14–373). Pseudonym using consecutive numbering is being used without patient abbreviation or birthdate. Only designated investigators can access the REDCap data collection program. The development of the database provides a broad clinical data base and an easy access for future clinical studies in this field on reporting the long-term outcomes and safety of this procedure [The rocedure –25.A total of 353 articles matched the search terms. After filtering and screening for relevance by title and abstract, 17 articles were found suitable to include in the final review  and the Society for Transplantation Medicine Mecklenburg-Vorpommern (GTM-V) (https://www.gtm-v.de/), which will dispose of a large donor history database as well as merge and correlate information of donor characteristics to clinical DMEK outcome.COMEDOS is a DFG funded (SCHR 1666/2–1) collaborative study between the largest German DMEK database and the Multi-Tissue Bank Mecklenburg-Western Pomerania (GBM-V) , culture time, culture medium, death-to-preservation time, death-to-use time etc.) will be included and correlated with the tissue preparation characteristics of the donor graft  and the impact on the clinical outcome of the DMEK-surgery.One aim is to analyze the impact of different donor characteristics  on the clinical outcome following DMEK surgery .Also, we plan to evaluate the role of donor characteristics on the graft behavior during tissue preparation and surgery  on the clinical outcome. To achieve this, we plan to collect stripping data in relation to the general condition of the donor tissue from over 2000 donors  and also to perform histological examinations from donor Descemet membrane remnants or discarded tissue.www.gbm-v.de).All donor collection parameters and data regarding the donor graft  are standardized and meticulously documented by the certified Multi-Tissue Bank Mecklenburg-Western Pomerania . All three surgeons were involved in the documentation of a standardized questionnaire at the end of every surgery. The questionnaire includes graded information about the graft preparation characteristics .The statistical analyses will be performed in collaboration with the Institute for Medical Statistics and Computational Biology (IMSB), University of Cologne.The study has been registered in the “German Clinical Trials Register (Deutsches Register Klinischer Studien) — DRKS00028034.”The current review provides an overview of the literature which analyzed the influence of donor factors like age, diabetes, and lens status on the clinical outcome of DMEK. The study methods and cohort sizes of the existing literature are heterogenous and have several limitations: variation of sample sizes, period of follow-up, and graft preparation variability (shortly before surgery by the surgeon and eye bank preparation ahead) are some. Also, the diabetic status and complication degree of the patient were not defined in the cited studies. As previously reported by Luke et al., the overall quality of data is low and further research on this field is warranted .However, there seems to be a consensus that the preparation of the DM graft is influenced by age and comorbidities of the donor. These factors reflect themselves in the rolling behavior of the graft intraoperatively. Donor age seems to be an important factor regarding the rolling behavior of the graft . Some suAnother factor to keep in mind in relation to older donors is the lens status as the prevalence of pseudophakia increases with age. This factor can influence the endothelial cell density (ECD). Lapp et al. reported that pseudophakic eyes are comparable to those with grafts from phakic eyes . This coDuring graft preparation, the separation of the DM from the underlying stroma represents a delicate step. Diabetic donor corneas seem to be more prone to DM splitting and are linked to difficulties during graft preparation , 40, 41.Currently there are no guidelines of strong evidence on how the DMEK surgeon should choose the donor tissue. This is in contrast to several previous studies in the field of PK and DSAEK.As postulated by large cohort studies in PK and DSAEK, several donor factors like donor age, gender, and diabetes seem to play a role on the recipient’s clinical outcome , 22. OneSuch large studies are necessary for guiding surgeons on the suitability of donor tissue and impact on outcome. However, there is still a lack of knowledge and large studies are missing in the field of DMEK. In our opinion, further research is required to reinforce these data with strong statistical measures and correlation of the effect of other donor characteristics on DMEK graft preparation. Future research should include an evaluation of the impact of pre-existing diseases and previous surgeries of the donor on the clinical outcome and complications. A correlation of a standardized evaluation of the intraoperative graft preparation features on outcome is needed. In addition, the intraoperative graft behavior should be followed-up for a minimum of 2 or 3 years to assess the clinical outcome of DMEK surgery.In this review, we demonstrated that there is a huge gap of knowledge regarding the impact of donor tissue characteristics on DMEK outcome and complications . The upc


Pythium insidiosum, has been increasingly reported in humans and animals worldwide. Antifungal drugs usually fail to control the pathogen. The surgical removal of an infected organ is the treatment of choice. Many affected patients die due to advanced infection. A timely and accurate diagnosis could lead to a better prognosis in pythiosis patients and save their lives. Although a standard culture method is available in microbiological laboratories, it is time-consuming, laborious, and insensitive for P. insidiosum identification. Immunological assays have been developed to improve the diagnosis of pythiosis. However, immunological methods are commercially unavailable and primarily detect anti-P. insidiosum antibodies, which constitute indirect evidence of pythiosis, making it challenging to differentiate a past from a recent infection. Moreover, such immunological tests cannot diagnose patients with a local infection, such as in the eye. Nucleic acid-based tests (NATs) are efficient for the direct and rapid detection of P. insidiosum DNA in trace-amount or culture-negative specimens. The reagents and equipment required for NATs are usually available in molecular diagnostic laboratories. Herein, we provide a systematic review to comprehensively present the principal and clinical usages, advantages, and limitations of such NATs in the detection of P. insidiosum. Various NATs have been established to detect P. insidiosum, which can be classified into amplification-based  and non-amplification-based  techniques. This concise review on NATs constitutes an up-to-date reference with which healthcare professionals can learn about and decide upon which detection method is suitable for their respective laboratory environments.Pythiosis, a life-threatening infectious condition caused by  Pythium insidiosum [P. insidiosum infection. The surgical removal of the infected organ  is the treatment of choice for pythiosis. Many pythiosis patients die due to advanced infection or inadequate treatment [Pythiosis is a life-threatening infectious disease of humans and animals caused by the pathogenic oomycete sidiosum . The infsidiosum ,2. Cutansidiosum ,2. Medicreatment . A delayreatment ,4. P. insidiosum from infected tissue. Such a laborious and time-consuming method requires experienced personnel to perform the procedure and interpret the result [P. insidiosum and the prompt treatment of affected patients. Several alternative detection methods, relying on sensitive and specific immunological and molecular technologies, have been implemented to overcome the microbiological culture method’s limitations in identifying P. insidiosum [P. insidiosum infection. Moreover, an immunological test cannot detect anti-P insidiosum antibodies in patients with a local infection, such as in the eye, which is an immunological privilege site that lacks certain types of host immune response  [P. insidiosum-specific antigen. The unavailability and inaccessibility of an immunological test prevent its usage in clinical laboratories. The conventional method for diagnosing pythiosis is a microbiological culture to isolate e result ,5. Moreoe result ,6,7. Thesidiosum . Althougduction) ,10. No iConsidering another diagnostic modality, the detection of the DNA or RNA sequence specific to a target pathogen by nucleic acid-based tests (NATs) is an effective procedure for reliable microbial identification ,11. OnceP. insidiosum. Since published details on these P. insidiosum-detecting NATs are scattered, we performed a systematic review to collect and summarize the current information on the principles and application of such assays, including the recently developed next-generation sequencing (NGS) technology. Moreover, the advantages and limitations of each assay are provided, making it convenient for healthcare professionals to learn and decide which detection method is suitable for use in their respective laboratory environments. It should be noted that the specific genotypes  of P. insidiosum have recently been proposed as a new species called P. periculosum [P. insidiosum in this study, as do all the collected publications regarding NATs.During the past decades, various NATs have been established for detecting iculosum . HoweverP. insidiosum-detecting NATs deposited in 2 public databases, PubMed  and Google Scholar , following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [Pythium insidiosum OR Pythium sp. OR pythiosis” AND “molecular OR analysis OR diagnosis OR detection OR identification OR differentiation OR evaluation OR sequencing OR genome sequence”. Regarding inclusion criteria, all studies that described the detection and identification of P. insidiosum by NATs were gathered in the collection. Regarding exclusion criteria, irrelevant original articles that reported non-nucleic-acid-based diagnostic approaches  and studies without available full text were removed from this review. The reference list of each recruited article was manually checked to find any other studies describing a P. insidiosum-detecting NAT not identified from PubMed and Google Scholar searches.We conducted a systematic review to collect the studies on idelines . The keyP. insidiosum. The other researcher (T.K.) double-checked the relevance of the selected studies. The full texts of the recruited articles were read in detail and further evaluated against the inclusion and exclusion criteria to obtain the final set of publications used in this study. Information about the principles and applications of the NATs for P. insidiosum was extracted and summarized for the presentation and visualization of the data. The test principles of NATs for P. insidiosum were divided into non-amplification and amplification techniques, as defined by Mothershed and Whitney [Duplicate publications from the literature search were removed. One researcher (T.S.) screened the titles and abstracts to collect only the reports on NAT for  Whitney . MoreoveP. insidiosum. Manual checks of the reference lists in the collected articles identified two additional articles reporting P. insidiosum-detecting NATs. Adhering to the inclusion and exclusion criteria  using the defined keywords see the . Reviewi see the , a totalThe general principle of NATs relies on detecting organism-specific DNA or RNA sequences. This diagnostic modality is suitable and efficient for clinical microbial detection due to its high specificity, sensitivity, and short turnaround time ,15. It cP. insidiosum. These techniques can be classified into two groups, namely, (i) non-amplification and (ii) nucleic acid amplification, as shown in P. insidiosum-detecting NATs are summarized below. Moreover, the primer sequences referred to in each NAT are listed in Many NAT techniques have been established to facilitate the detection of P. insidiosum [Nucleic acid hybridization can identify specific DNA or RNA without target sequence amplification . This mesidiosum . Dot-blot hybridization is a nucleic acid-based detection technique for high throughput or large-scale sample testing. It relies on the direct and semi-quantitative detection of a specific nucleotide sequence dotted on a membrane without prior electrophoresis-based separation and gel-to-membrane transference of the target sequence . TechnicP. insidiosum [HinfI site-specific deoxyribonuclease digestion of the intergenic spacer I (IGS-1), located between the LSU and 5S rRNA sequences of P. insidiosum. The hybridized target sequence of the pathogen can be detected by a digoxigenin (DIG)-11-dUTP-labeled probe that generates a chemiluminescent signal on X-ray film. By evaluating 104 different Pythium species and some other pathogenic microorganisms, the detection probe specifically hybridized target DNA samples extracted from all 22 genetically diverse isolates of P. insidiosum without cross-hybridization with the other organisms tested. However, Schurko et al. only tested the performance of this detection technique against the P. insidiosum DNA extracted from pure cultures [P. insidiosum in the clinical setting. Nevertheless, apart from its multistep procedure and relatively high cost, dot-blot hybridization using the P. insidiosum-specific probe requires trained personnel to perform the assay, which could limit its routine uses in clinical laboratories.Schurko et al. were the first to develop the dot-blot hybridization technique for the detection of sidiosum , as depicultures . Future P. insidiosum DNA in a clinical specimen or pure culture. Such techniques include sequence homology analysis [PCR was first invented in 1985 by Mullis and colleagues . This DNanalysis , conventanalysis ,47, nestanalysis ,32,40,41analysis , and reaanalysis ,48, as d(1)Sequence homology analysisP. insidiosum [https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 6 November 2022) [The PCR amplification of a target gene followed by the direct sequencing and homological search of an obtained DNA sequence against a public database  is a widsidiosum . Such aner 2022) . A commoer 2022) . P. insidiosum in a sample isolated from a pythiosis patient. Badenoch et al. used the universal fungal primers NS1 and NS2 [P. insidiosum reference strain CBS777.84, thereby successfully identifying an unknown etiologic pathogen. Sequence homology analysis is an effective strategy that can be performed in a general molecular laboratory in order to identify and confirm the presence of  and NS2  to ampli and NS2 . A BLAST and NS2 . The obtP. insidiosum infection in a horse by sequencing the amplified 18S rRNA sequence using the universal fungal primers NS1 and NS8 [P. insidiosum isolated from a human case [P. insidiosum infection in a Brazilian horse by amplifying and analyzing a 900 bp rDNA sequence using the primers ITS4 and ITS5 [P. insidiosum sequences deposited in GenBank. The pathogen sequence was amplified and further confirmed using additional FFPE tissue blocks and primers targeting ITS1 and cox1 sequences [P. insidiosum DNA could be detected in 13 samples [Afterward, many investigators employed this sequence homology analysis strategy, using different sets of primers, to reach definitive diagnoses of pythiosis. For example, Reis et al.  confirme and NS8 . Vanittaman case . Rivierrman case . Mendozaman case . Bosco eman case  detectedand ITS5 . Salipanand ITS5  used theand ITS5 . Using tand ITS5 , they amequences . Using tequences , Sharma  samples . Konradt samples  confirme samples . P. insidiosum in the environment. For example, Supabandhu et al. [P. insidiosum in 59 organisms isolated from rice paddy fields, irrigation channels, and water reservoirs by sequencing the ITS regions using the primers ITS1/ITS4 [P. insidiosum. However, as a downside, this technique involves multiple steps and is time-consuming, especially with respect to the sequencing process, which may take weeks to obtain a sequence for homology analysis, resulting in a delayed diagnosis. Besides its clinical diagnostic applications, sequence homology analysis has been used to survey the presence of u et al.  confirmeTS1/ITS4  and ITSpTS1/ITS4 . As ment(2)Conventional PCRP. insidiosum DNA and reach a definitive diagnosis of pythiosis. A famous target sequence is rDNA [P. insidiosum-specific primers, can detect the pathogen in a biological sample without requiring DNA sequencing of the PCR product. The assay interpretation relies on the presence of a PCR product at the expected size on an electrophoresis gel. Vanittanakom et al. designed two specific primers (ITSpy1 and ITSpy2) that can detect P. insidiosum by conventional PCR [P. insidiosum rDNA-specific primers ITSpy1 and ITSpy2 failed to amplify some isolates of P. insidiosum [exo1). Based on the DNA samples extracted from P. insidiosum isolates and various fungal species, the conventional PCR technique using the primers Dx3 and Dx4 showed 100% detection sensitivity and specificity [The conventional diagnostic PCR assay requires organism-specific primers, template DNA, PCR reagents, thermal cycler, and gel electrophoresis equipment to amplify and detect  is rDNA . Within onal PCR . Later, sidiosum . They incificity .(3)Nested PCRP. insidiosum [P. insidiosum but not from other control organisms, indicating the high detection sensitivity and specificity of the assay. However, Vanittanakom et al. showed that the inner primers PI-1 and PI-2 failed to identify one Thai P. insidiosum strain [P. insidiosum in various clinical specimens, such as frozen and ethanol-fixed infected animal tissue [P. insidiosum in the tissue samples from horses  and rabbits  with pythiosis. Alternative primer sets, such as ITSpy1 and ITSpy2 [A nested PCR assay is a modified version of the conventional PCR method. Technically, this amplification assay is designed to increase the detection sensitivity and specificity of a normal PCR reaction . It conssidiosum . In the m strain , proceedm strain  can idenl tissue . Moreovel tissue  demonstrd ITSpy2 , ITS1 and ITSpy2 , and ITSd ITSpy2 , could rP. insidiosum habitats in the environment, the nested PCR assay, using the outer primers ITS1 and ITS4 and the inner primers PI-1 and ITS2R, was able to identify the organism in question in irrigation water and soil samples in northern Thailand [P. insidiosum in FFPE-biopsied tissues from dogs. They amplified a 623 bp amplicon of the ITS2 non-coding region using the pan-fungal primers ITS3 and ITS4 [P. insidiosum-specific amplicon [P. insidiosum, with the annealing temperatures of 68 °C for the outer primers  and 58 °C for the inner primers . Moreover, the single-tube, nested PCR method has been used to detect P. insidiosum infection in patients with suspected fungal keratitis [P. insidiosum—even in a trace amount of specimen.As part of a survey of the Thailand . More reThailand  developeand ITS4 . The newamplicon . As per amplicon . Althougamplicon . For exaamplicon , in whiceratitis ,41. The (4)Multiplex PCR (m-PCR)m-PCR is a variant PCR technique that utilizes more than one primer pair to simultaneously amplify multiple target sequences in a single reaction ,11. ThisP. insidiosum strains. These investigators employed several genotype-associated single nucleotide polymorphisms (SNPs) presented within the rDNA sequence to design a common forward  and three reverse  primers capable of detecting and, at the same time, classifying the organism into three distinct phylogenetic genotypes. The primers ITS1, R1, R2, and R3 together generate several amplicons with defined sizes , in which the amplicon number and sizes define the P. insidiosum genotype, such as clade I (two amplicons of 490 and 660 bp), clade II (an amplicon of 660 bp), and clade III (an amplicon of 800 bp), as described in P. insidiosum isolates and 22 control fungi. The SNP-based m-PCR assay has been used to identify and genotype P. insidiosum isolated from affected animals (primarily horses and dogs) in South America [P. insidiosum in corneal specimens from patients with suspected ocular pythiosis. The d-PCR assay generated 580 bp and 233 bp amplicons from the P. insidiosum’s target sequences. Based on the samples analyzed by the investigators, the d-PCR assay showed 100.0% detection specificity and 92.8–100.0% detection sensitivity. Rujirawat et al.  develope America  and Egyp America , as well America . Recentl America  describe America ) sequenc(5)Real-time or quantitative PCR (qPCR)qPCR is an amplification technique for explicitly identifying and quantifying a nucleic acid material of interest in a biological sample . qPCR ofP. insidiosum, and has aided in the early diagnosis of infectious diseases. Keeratijarut et al. [P. insidiosum by targeting the exo1 gene, as depicted in P. insidiosum isolates and 58 various control fungi. The analytic sensitivity (limit of detection) of qPCR is 1 × 10−4 ng gDNA template, as opposed to 1 ng for the conventional PCR.Due to its extremely high detection sensitivity and specificity and relatively short turnaround time, qPCR has been widely used to detect many pathogens, including t et al.  developeP. insidiosum cox2 gene, giving rise to an assay, namely, qPCR-HRM. HRM can discriminate sequence variations based on sequence length, nucleic acid composition, GC content, and strand complementarity. Their assay rapidly detected and genotyped P. insidiosum and assessed the presence of non-specific amplicons and primer dimers. Based on the SNPs presented within the resulting 91 bp amplicons, qPCR-HRM can differentiate 15 P. insidiosum isolates from 28 of the related organisms tested. Moreover, HRM curve patterns can classify P. insidiosum into three assigned clades . The limit of detection of qPCR-HRM is 1 pg of a genomic DNA template. The post-PCR dissociation (melting) curve is another qPCR characteristic that can be used to confirm whether the fluorescence signal is generated from the target sequence and not from non-specific PCR products. Worasilchai et al.  incorporP. insidiosum  and DNA quantity assessment .Overall, qPCR has become a more popular amplification technique because it improves analytical sensitivity and reduces turnaround time. All processes of qPCR occur in a single, closed tube, thereby minimizing carry-over contamination and, thus, the acquirement of a false positive result. Apart from diagnostic applications, qPCR can be used in an epidemiological study of P. insidiosum.Although PCR-based amplification techniques have been widely used for various molecular analyses, they require dedicated thermocycler instrumentation with precise temperature control, limiting their usage in a resource-limited setting . Over th(1)Helicase-dependent amplification (HDA)P. insidiosum using thermophilic HDA (tHDA) was first described by Worasilchai et al. [cox2 gene in a 2 hr amplification reaction at 65 °C. The resulting 91 bp amplicon was subjected to digestion with the restriction enzyme CviKI-1 to obtain 42 bp and 49 bp fragments—specific characteristics of P. insidiosum. tHDA has a detection limit of 100 fg  of the genomic DNA template. HDA is an in vitro isothermal nucleic acid amplification method first developed by Vincent et al. . It has i et al. . Their a(2)Loop-mediated isothermal amplification (LAMP)Bst DNA polymerase large fragment. The LAMP reaction occurs under isothermal conditions set between 60 and 65 °C for a specific duration . Like the other isothermal amplification techniques, LAMP does not require a thermocycler for DNA denaturation, annealing, or extension, as is required for standard PCR-based assays [LAMP is a unique isothermal amplification technology developed by Notomi et al. . This med assays . The assd assays . LAMP prd assays .P. insidiosum. Their LAMP products can be detected by observing SYBR Green illumination via the naked eye or by observing a ladder pattern of the amplicons on an electrophoresis gel. Compared with m-PCR [−4 ng of genomic DNA template). Based on clinical sample evaluation, LAMP showed 88% sensitivity and 100% specificity. LAMP is another molecular assay that could be used to facilitate the early detection of P. insidiosum in resource-limited areas. LAMP is the most widely applied isothermal amplification technology for the rapid and straightforward identification of pathogenic microorganisms . Htun etth m-PCR , LAMP exP. insidiosum strains to establish a genome database of this pathogen [P. insidiosum in a severely ill patient with a suspected infectious condition, for which the standard microbial identification procedures failed to identify the etiological microorganism [P. insidiosum. The mNGS is a novel, rapid, and robust method for microbial detection, especially in urgent clinical situations where the causative agent is unknown. However, this technology comes with a high cost and is not available in many laboratories, limiting its use, at the moment, as a routine microbial detection assay.The emergence and application of NGS technology for obtaining microbial whole-genome sequences (WGSs) have markedly changed the approach to improving the clinical management of patients with infectious diseases . As an apathogen ,76,77,78organism . They emP. insidiosum  that amplify a size-specific amplicon, bypassing the need for DNA sequencing. Nested PCR exhibits extremely high sensitivity, allowing it to detect a trace amount of the pathogen DNA in a clinical specimen. Recognized limitations of this assay include a longer turnaround time and a higher risk of carry-over contamination. Multiplex PCR can simultaneously amplify multiple target sequences, leading to the ability to detect and genotype a pathogen specifically. However, it tends to have a relatively lower detection sensitivity. Real-time PCR has high detection sensitivity and specificity, demonstrates the ability to check and quantify the target sequence in real time, and provides a short turnaround time. As a downside, like most PCR-based techniques, real-time PCR requires P. insidiosum-specific primers, and it is susceptible to an inhibitory substance in a test sample, not to mention the need for a more sophisticated and expensive machine. LAMP and had can detect P. insidiosum DNA without requiring a thermal machine to generate a test result. Nevertheless, their higher chance of carry-over contamination (in the case of LAMP) and non-specific products, expensive reagents, and susceptibility to an inhibitor in a test sample (in the case of HDA) limit the use of these isothermal assays. Recently, NGS has been reported as a novel and robust method for detecting P. insidiosum. It does not require a postulation for what is the most-likely pathogen, which is helpful for healthcare workers unfamiliar with P. insidiosum. The requirements for expensive equipment and reagents, skillful personnel, and extensive bioinformatic analysis prevent the clinical use of this technique.Various NATs have been established for detecting sidiosum . Each teP. insidiosum using sequence homology analysis in 2001, other NAT techniques have been implemented for the molecular diagnosis of pythiosis. However, the technical and clinical details on the use of NATs for detecting P. insidiosum are scattered, making it difficult for healthcare workers to learn and remain updated with respect to these techniques. Here, we performed a systematic review to collect and summarize the current information on the principles and applications of P. insidiosum-detecting NATs. Over the past two decades, many NAT techniques for the diagnosis of pythiosis have been reported in the literature and can be classified into non-amplification-based and amplification-based methods (P. insidiosum have been depicted in an easy-to-understand diagram (P. insidiosum in a sample with high detection sensitivity and specificity. However, the diagnostic advantages and limitations of each NAT technique compared with one another (P. insidiosum. It constitutes an up-to-date reference with which healthcare professionals can learn about the P. insidiosum-detecting NATs and thereby choose a suitable detection method according to their respective laboratory settings.Since the first report of NAT for the detection of  methods . The non diagram . As show another  should b


Groove—defined as the pleasurable urge to move to a rhythm—depends on a fine-tuned interplay between predictability arising from repetitive rhythmic patterns, and surprise arising from rhythmic deviations, for example in the form of syncopation. The perfect balance between predictability and surprise is commonly found in rhythmic patterns with a moderate level of rhythmic complexity and represents the sweet spot of the groove experience. In contrast, rhythms with low or high complexity are usually associated with a weaker experience of groove because they are too boring to be engaging or too complex to be interpreted, respectively. Consequently, the relationship between rhythmic complexity and groove experience can be described by an inverted U-shaped function. We interpret this inverted U shape in light of the theory of predictive processing and provide perspectives on how rhythmic complexity and groove can help us to understand the underlying neural mechanisms linking temporal predictions, movement, and reward. A better understanding of these mechanisms can guide future approaches to improve treatments for patients with motor impairments, such as Parkinson’s disease, and to investigate prosocial aspects of interpersonal interactions that feature music, such as dancing. Finally, we present some open questions and ideas for future research. In the field of musicology, the term groove was coined in the context of African-American musical genres, such as R&B, jazz, soul, disco, funk, and hip-hop, where it can refer to esthetic qualities of the music, specific rhythmic patterns, or the musicians’ way of effortlessly synchronizing and interacting with each other . In contHere, we focus on the pleasurable, movement-inducing aspect of groove. In this approach, groove is genre-independent, in that every rhythmic pattern and performance evoking the pleasurable urge to move possesses the quality of groove. Although soul, disco, funk and related genres may be more likely to induce groove, the pleasurable urge to move can also be experienced while listening to rock, jazz, electronic dance music, and many other genres.We argue that groove, when defined as the pleasurable urge to move to a rhythm, depends on a fine-tuned interplay between predictability and surprise. The predictability arises from repetitive rhythmic patterns, and the surprise arises from slight deviations from these patterns, for example in the form of syncopation. This tension represents the sweet spot of the groove experience, and is commonly found in rhythmic patterns that are simple enough for us to interpret and predict, but complex enough to keep us challenged and engaged . We willThe neural mechanisms that are engaged from the moment we start to listen to music to the moment we are tapping our foot in time with the beat or start dancing, rely on the human brain’s ability to integrate external stimuli with internal representations, expectations, or predictions . This coWithin the context of groove, PC has predominately been deployed to interpret the inverted U-shaped relationship between groove ratings and the degree of rhythmic complexity . RhythmiPanels A and B of According to PC, there are two ways to minimize prediction errors and thereby reduce uncertainty . One wayvia covert or overt motor processes. Recent work has demonstrated a direct link between music-evoked reward and moderate levels of prediction error and uncertainty in the context of melodic and harmonic expectations , a set of subcortical nuclei involved in motivation and motor control, along with dopaminergic transmission within the BG, have been implicated in all of the processes discussed above, including beat and meter-based perceptual and motor timing , music-eve loops .One way to test the role of the BG and dopamine in groove is to compare Parkinson’s disease (PD) patients with healthy controls. PD results from dopaminergic dysfunctions in the BG caused by neuronal degeneration of the substantia nigra pars compacta (SNc). Such dopaminergic depletion disrupts disinhibitory mechanisms between the BG and the motor thalamus, altering the fine-tuning between initiation and suppression of the activity in the motor loop, and giving rise to the characteristic motor symptomatology. In terms of groove, a recent study showed that the inverted U-shaped relationship between rhythmic complexity and pleasurable desire to move is shifted from moderately complex rhythms in healthy individuals, toward less complex rhythms in PD patients, who seem to prefer little incongruence between the internal predictive model and the stimulus . NotablyResearch into auditory-motor activity and the neural correlates of rhythm perception  has led By comparing the experience of groove in PD patients and healthy controls, we contribute to our understanding of the underlying neural mechanisms linking temporal predictions, movement, and reward. In turn, a better understanding of these mechanisms can guide future approaches to better treat motor deficits, and improve the quality of PD patients’ personal and social life.participatory discrepancies and hypothesizes that they are necessary to make music involving and socially meaningful (When people come together to listen to or make music, the level of rhythmic complexity that hits an individual’s sweet spot for an optimal groove experience depends on their biology and cultural background. In his interviews with musicians, Charles Keil noted that “each person has a unique feel for time and that bringing different or discrepant personalities together generates different kinds of groove” , p.8. Keaningful , 1995. Taningful .Music and dance are so intertwined that some cultures do not distinguish between them , and groFrom the perspective of predictive processing, beat and meter are mental models that can be shared by all dancers and musicians in a group. If the models are alike, i.e., ‘distributed’ across dancers, they facilitate synchronous movements, which in turn can promote shared affective experiences . In addiBoth dancing and performing music in a group involve moving in time together. However, dance and music improve sensorimotor integration in both shared and unique ways . For exaIn dance, music making, and music listening, groove research can help us to better understand the interactions between temporal processing, movement, social behavior, and pleasure in both the general population and individuals with motor impairments. Elucidating the neural mechanisms underlying groove—especially the role of the basal ganglia—will contribute to our understanding of the integration of motor driven predictive timing and reward processes more generally.One open question is how the experience of groove differs when comparing individual versus collective situations of music listening or dancing. It is also unclear how the type of rhythm-related movements affects groove experiences. Future research could, for example, compare whole body engagement in dance with specific movements of particular body parts, such as finger tapping or playing an instrument. Another future direction of groove research could be to investigate activities outside the field of music. Senn and colleagues define the verb to groove as “playing music together in an effortless and rhythmically well-coordinated manner” , p.46. TThe original contributions presented in this study are included in the article, further inquiries can be directed to the corresponding author.JS: conceptualization and writing – original draft, review and editing. TEM, VP-N, and OVFE: writing – original draft, review and editing. PV: writing – review and editing. All authors contributed to the article and approved the submitted version.


Dichelostemma congestum; in Butomus umbellatus (Alismataceae), in Tacca chantrieri (Dioscoreaceae), and in umbellate structure in Fritillaria imperialis (Liliaceae). We compare these with racemes found in three members of the subfamily Scilliioideae (Asparagaceae). We find there are three convergent developmental programs that generate umbellate inflorescences in the monocots, bostryx-derived, cincinnus-derived and raceme-derived. Additionally, among the bostryx-derived umbellate inflorescence, there are three instances of parallel evolution found in the Amaryllidaceae, in two members of Brodiaeoideae (Asparagaceae), and Butomus umbellatus, all of which share the same generative developmental program. We discuss the morphological modifications necessary to generate such complex and condensed structures and use these insights to describe a new variant of metatopy, termed horizontal concaulesence. We contextualize our findings within the broader literature of monocot inflorescence development, with a focus on synthesizing descriptive developmental morphological studies.Inflorescence structure is very diverse and homoplasious, yet the developmental basis of their homoplasy is poorly understood. To gain an understanding of the degree of homology that these diverse structures share, we characterize the developmental morphology and anatomy of various umbellate inflorescences across the monocots and analyzed them in an evolutionary context. To characterize branching order, we characterized the developmental morphology of multiple inflorescences with epi-illumination, and vascular anatomy with Laser Ablation Tomography, a novel high-throughput method to reconstruct three-dimensional vasculature. We used these approaches to analyze the umbellate inflorescences in five instances of presumed homoplasy: in three members of the Amaryllidaceae; in three members of the Asparagaceae, including a putatively derived raceme in  The mechanism by which developmental variation underlies novel and adaptive morphologies is a key research program in evolutionary and developmental (Evo-Devo) biology . Under tsensu M. H. Wake, is a macroevolutionary manifestation of evolutionary forces independently acting upon a homologous generative developmental program . The developmental morphology of a wide number of taxa in the family Amaryllidaceae has been investigated, and the morphological consensus is that the inflorescence comprises an indeterminate primary axis with cymose lateral branches , however the developmental morphology of this endangered species has not yet been investigated , at the time used to affiliate Tacca with Amaryllidoideae  (Limnocharis flava (Alismataceae) and Butomus umbellatus (Butomaceae)   and in tomaceae) . While d observe .via morphological parallelism or convergent evolution. To test this question, we characterize the developmental morphology of a condensed inflorescence evolved independently in five lineages  , as raceme developmental morphology is consistent through ought monocot    and Butobellatus  (Butomac monocot . To comphy (LAT)  of ButomPlant were either purchased, collected from the wild or sourced from New York Botanical Gardens . All pur2O) with vacuum infiltration and stored for at least 24 h. Samples were washed once in 50% EtOH and then taken through a graded EtOH dehydration series  for 1 h at each step. Samples were stained for 1–4 h in a 1% w/v Nigrosin solution . Allium hollandicum inflorescence is interpreted as a thyrse, as previously described for other ornamental Alliums  that in turn gives rise to a secondary lateral branch that terminates into a flower (sensu Wilder). Secondary branches are displaced from the primary stem axis appearing horizontal adjacent to the primary axis, as opposed to branching from the axis  while that of Fritillaria persica can be considered a sciadium (ly fused . In contsciadium .Only unopened inflorescences that had emerged from the stem were available. The inflorescence was interpreted as comprising two cincinni (aka scorpioid cymes), surrounded by four inflorescence bracts, each branching centrifugally along the transverse plane . FlowersOrnithogalum nutans  The bostryx- (aka helicoid cyme) derived umbellate inflorescence, (2) The cincinnus- (aka scorpioid cyme) derived umbellate inflorescence, and (3) the racemose-derived umbellate inflorescence which is considered a true umbel .Dichelostemma congestum, (b) the inflorescence of Amaryllidaceae, including Allium triquetrum, studied here, and (c) the inflorescence of Butomus umbellatus. They share a generative program of inflorescence development involving multiple bostryces (aka helicoid cymes) that lack vertical internodal elongation of the primary axis (Butomus umbellatus) or not , and how many bostryces are present in the inflorescence . This parallel evolution likely applies to other umbellate taxa that fall in these groups, specifically, Limnocharis flava  which has been described as a cincinnus but diagrammatically resembles a bostryx  Brodiaeoideae, excluding ary axis . Within  bostryx  and vari bostryx . Even thTacca chantrieri is a convergent case of umbellate inflorescence originating a different type of thyrse those with cincinni. A cincinnus and a bostryx, both cymes, differ in their three-dimensional floral arrangements  , which is expressed in the order in which flowers open, has been noted in taxonomic studies of Dichelostemma  . DSD is ft (DSD) , howeverlostemma . Order olostemma . Umbellalostemma . In DichMusa, the lateral branching system  is interpreted to be a cincinni based on the order of initiation of the individual flower primordia , Metanarthecium  and Japonolirion   , 2013, a, is speed and scalability. Serial sections of 1um increments can consistently be obtain within a few minutes. This consistency and large amount of data facilitates computational 3D reconstruction as there is no need to interpolate between section. Although, computational 3D reconstruction, can be done with serial section obtain by traditional sectioning . The maiStreptopus amplexifolius, Symphytum officinale, Tricyrtis hybrida)  , but canhybrida)  and the hybrida) . In thesDichelostemma venustumTriteleia laxa   . The typidaceae) . InteresTacca, with one exception, the inflorescence of Allium hollandicum  can be found below: JM-G designed, executed experiments (excluding laser ablation tomography), and wrote the manuscript. TA, IR, and AH contributed to morphological preparation and imaging. CS performed laser ablation tomography. WM provided coolers and growth conditions for plants. All authors edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with one of the author DS.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.