The fertility of females with advanced age declines with aging. Therefore, for medical and social reasons, it is important to establish mechanisms to protect and improve the fertility of such populations. With widespread use of traditional Chinese medicine (TCM) in in vitro fertilization (IVF), studies have evaluated their impact on improving the fertility of females with advanced age. In this study, we performed proteomic analysis of follicular fluid to reveal mechanisms of the Erzhi Tiangui (EZTG) granule  in improving the outcomes of IVF in infertile women with advanced age.  This was a randomized, double-blind, and placebo-controlled trial in which 100 patients with advanced age were divided into the EZTG group and the placebo group by the random number table plus envelope method. Both groups were subjected to controlled ovarian stimulation with a GnRH antagonist regimen. Differences between the two groups were evaluated, including the TCM syndrome score after treatment, gonadotrophin (Gn) days and Gn doses, the number of retrieved oocytes, 2 pronucleus (PN) fertilization, 2PN cleavage, and high-quality embryos. Differentially expressed proteins were identified using the LC-MS/MS method, and their functions were determined through bioinformatics analyses. p=0.011). Eleven differentially expressed proteins were identified between the two groups. Four proteins were highly expressed, whereas seven were suppressed in the control group, compared to the EZTG group. The overall trend suggested that the apoptotic effect in the follicular fluid of the EZTG group was downregulated.  The number of high-quality embryos in the placebo group was significantly lower than that in the EZTG group (2.88 ± 1.85 vs. 4.13 ± 2.83, ChiCTR1900025139. Treatment with the EZTG granule can improve embryo quality in IVF of advanced age females with both kidney Qi and Yin deficiency syndromes. The mechanism is attributed to downregulation of apoptotic-effector protein expressions in the follicular fluid. This trial is registered with  The threshold for female reproductive aging is ≥35 years –3. Due tKidney-tonifying TCM plays a universal role in delaying human life aging and prolonging the human reproductive period –12. By eStudies involving TCM are based on two mechanisms: “holistic concept” and “syndrome differentiation and treatment.” The decline in female fertility involves multisystem, multiway, and multilink changes of the body, including proteomics. Proteomics, as a component of system biology, reflects the holistic concept of TCM and shows the advantages and roles of TCM in promoting fertility in women with advanced age. Therefore, we performed proteomic analysis of the follicular fluid to establish the mechanisms of Erzhi Tiangui (EZTG) granule  in improving IVF outcomes in infertile women with advanced age. Our findings provide a scientific, effective, and systematic theory and elucidate on the method of TCM for protecting the fertility of such populations.This study is registered in the Chinese Clinical Trial Registry (ChiCTR1900025139). The 100 infertile patients were recruited from the Reproductive and Genetic Center of Integrated Traditional and Western Medicine, the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China (PRC) from June 2015 to December 2016. Eligible participants were infertile patients undergoing IVF/intracytoplasmic sperm injection (ICSI) procedures without contraindications for adverse IVF/ICSI treatment outcomes.n = 50) was orally administered with Erzhi Tiangui, while the control group (n = 50) was administered with a placebo.Participant enrollment was performed by staff not involved in the randomization process. A computer-based random number generator was used for grouping, according to the random number allocation sequence. Women with advanced age were allocated into two groups based on computer-assisted block randomization. Sequence generation and assignment of participants to the experimental and control groups were made by a study staff member who was not involved in intervention delivery, data collection, or data analysis. Both medications (Erzhi Tiangui formula and placebo) were prepared, so that they were identical in shape, taste, and smell. The experimental group (Primary symptoms are as follows: (i) light menstrual color and thin texture, (ii) fatigue, (iii) lumbosacral soreness, (iv) light red tongue with dental marks and a thin white or less coating, and (v) both chi pulses exhibit a deep thready pulse or a deep thready and rapid pulse.Secondary syndromes are as follows: (i) dizziness and tinnitus, (ii) dry mouth and throat, (iii) dry vagina, and (iv) lower leg ache or talalgia.The kidney Qi and Yin deficiency syndrome was diagnosed only by the presence of all primary syndromes with one or two secondary syndromes.2, endometriosis, polycystic ovarian syndrome, severe malformation of reproductive organs, major operation history, and had used hormonal drugs within 3 months before the study were excluded. A couple with karyotyping abnormalities was also excluded.Married women aged between 35 and 44 years old, receiving autologous oocytes and who had kidney Qi and Yin deficiency syndromes were enrolled in the study. Patients diagnosed with premature ovarian insufficiency and who had body mass index (BMI) of ≥30 kg/mLigustrum lucidum (Nv Zhen Zi), 15 g of Lotus japonicus (Han Lian Cao), 15 g of the fruit of Chinese wolfberry (Gou Qi Zi), 15 g of Cuscuta chinensis (Tu Si Zi), 15 g of Radix Rehmanniae Preparata (Shu Di Huang), 12 g of Angelica sinensis (Dang Gui), 12 g of Paeonia lactiflora (Bai Shao), 12 g of Ligusticum wallichii (Chuan Xiong), 12 g of Rhizoma cyperi (Xiang Fu), and 9 g of Radix Glycyrrhizae Preparata (Zhi Gan Cao). The placebo granule, which was mainly composed of dextrin, was made in a similar color and shape to EZTG. Placebo granules were packaged as 3 g/bag, with the same package of the EZTG, batch number 01-FZ032-03-1. The EZTG or placebo was orally administered after being dissolved in water, 3 g each time, 2 times a day.The Drug Manufacturing Unit of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine produced the EZTG granule. The EZTG was packaged as 3 g/bag, batch number 01-FZ032-03. The daily dose is equivalent to 15 g of After the 2nd to 3rd day of menstruation and 150–300 units of exogenous gonadotropin controlled ovarian stimulation, the EZTG group was administered with Erzhi Tiangui granules, while placebo granules were administered to the placebo group for 11–14 days. After vaginal ultrasound confirmation of the follicle diameter of between 18 and 20 mm, participants were administered with a single dose of 10000 units of HCG (human chorionic gonadotropin) as a “trigger.” Then, 36 h after HCG injection, oocyte retrieval and extraction of ovarian granulosa cells were conducted under transvaginal ultrasound.The follicular fluid was obtained after oocyte retrieval, and the presence of a cumulus complex was confirmed by inverted microscopy. After centrifugation for 10 min at 3000 rpm to separate red blood cells, leucocytes, and follicle cells, the supernatant was recovered in Eppendorf (EP) tubes, labeled and refrigerated at −80°C for further examination.Briefly, XX of protein was supplemented with 50 mM ammonium bicarbonate to YY, and DTT was added to a final concentration of 10 mM for 60 min at 37°C. Iodoacetamide was added to the solution to a final concentration of 50 mM for 30 min at room temperatures in the dark. The solution was transferred to the ultrafiltration tube and centrifuged for 10 min at 14,000 rpm. Pellets were rinsed twice using 50 mM ammonium bicarbonate (containing 0.8% SDC). Trypsin was added after which samples were enzymatically hydrolyzed under incubation at 37°C for 12–16 h. Then, the enzymatic hydrolysates were rinsed using 50 mM ammonium bicarbonate and merged, followed by TFA acidification shaking, centrifugation to remove SDC, and desalting with a C18 desalting column. Desalted samples were lyophilized in a freeze-dryer and redissolved in 0.1% FA for mass spectrometry detection.μm, packed in-house; Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) at a flow rate of 8 μL/min. Peptide separation was performed on the ReproSil C18 reverse-phase column  using a linear gradient from 0 to 80% B  acetonitrile, 0.1% formic acid) in 70 min and at a constant flow rate of 200 nL/min using a splitter. Column eluent was directly sprayed into the ESI source of the mass spectrometer. Mass spectra were acquired in a continuum mode and peptide fragmentation performed in a data-dependent mode.Desalting of 100 mg lyophilized TMT-labeled peptide pools was performed on a 100 mg C18 solid-phase extraction column Sep-Pak  and further fractionated using either reverse-phase chromatography combined with elution at a high pH, isoelectric focusing on an Agilent 3100 OFFGEL fractionator  or HILIC chromatography. Each time, 18–24 fractions were collected and analyzed using a nanoflow LC-MS/MS. Nanoflow LC-MS/MS was performed on an 1100 series capillary LC system (Agilent) coupled with an LTQ-Orbitrap mass spectrometer (Thermo Scientific) operating in the positive mode and equipped with a nanoarray source. Peptide mixtures were trapped in a ReproSil C18 reverse-phase column . Protein data were retrieved from the UniProt database , with the maximum deviation of parent ion molecular weight not exceeding 10 ppm and the maximum deviation of daughter ion molecular weight not exceeding 0.02 Da. Peptide false discovery rate (FDR) was set to <1%. For each group, the experiment was repeated thrice using the MaxQuant software , city, country). We used “uniprot_Proteomes-Human.fasta” to search the original files. After configuring database files, 12 samples of original files were imported into the analysis software. Corresponding label-free quantification parameters were set and imported into the database for retrieval. The retrieved results were screened using the strict criteria (peptide FDR ≤1% and protein FDR ≤1%). For proteins to be used for quantification, they were to have ≥2 characteristic polypeptides.P < 0.05 was considered statistically significant. Quantitative data for each group were expressed as mean ± SD. The data were not statistically described using the median and quartile deviation. Comparison of means between two groups was performed using Student's t-test. Proportions were compared using the chi-square test.Statistical analysis was performed using the SPSS 19.0 statistical software . p=0.011)  .The LFQ values from MaxQuant analysis were used to characterize protein abundance. The LFQ value of the total protein for each sample was corrected. Differential proteins between EZTG and control groups were screened using a protein abundance ratio of >1.67 and <0.6. A small number of proteins that were relatively specific in the follicular fluid and played a localized role was reduced to >1.1 and <0.9. After comparisons, 11 differentially expressed proteins between the placebo and EZTG groups were identified. Four proteins were highly expressed, whereas seven were suppressed in the placebo group .The differentially expressed proteins were found to be involved in physiological processes, such as lipid metabolism, immunity, cell differentiation, proliferation, and apoptosis. Some of these proteins may be involved in several different biological function processes .Patients in the EZTG group and the placebo group had no adverse reactions during the medication period.Acceleration of apoptotic effects in elderly women results in a decline in egg and embryo quality. Abnormal growth and development of follicles and accelerated apoptosis of granulosa cells lead to follicular atresia and are the fundamental causes of the decline in ovarian reserve functions and fertility in elderly women .Many of the differential proteins found in this study are involved in apoptotic and proliferative processes. They include matrix metalloproteinases (MMPs), which are crucial in apoptosis, and metalloproteinase inhibitor-1 (TIMP-1), which binds MMPs to suppress their biological activities. In addition, TIMP-1 directly binds cell surface receptors to promote the proliferation of fibroblasts, epithelial cells, smooth muscle cells, and lymphocytes , 16. AntInsulin-like growth factor binding protein-3 (IGFBP-3) and IGFBP-6 compete with IGF receptors to bind IGF, thereby blocking IGF receptor formation and inhibiting IGF activity. Moreover, IGFBP-3 has an independent role in promoting apoptosis . The insβ Ip) is important in cell adhesion, migration, proliferation, and differentiation [Transforming growth factor beta-inducible protein ig-h3 , which enhances the essence of kidney deficiency in elderly women. The classic saying “Yang transforms Qi and Yin forms.” Yin shaping refers to the process by which Yin and Qi dominate the static state, and the invisible substance is formed into a tangible substance in the movement of “calming.” The “shaping” process involves follicle's self-development, maturation, and ovulation and increase in follicle volume, follicular fluid, and proliferation of granulosa cells. They are the functional aspects of kidney Yin. The process of follicular development and shaping is inseparable from cell proliferation. Therefore, the function of kidney Yin “shaping” predominates the whole process of follicular development.Lian, using a kidney invigoration and Yin nourishment technique, formulated the EZTG prescription , 29. TheIn this study, identification of TCM syndromes was carefully performed by experienced clinicians. Due to individual differences among patients, symptomatic severity was different. The overall dose of TCM granules cannot be adjusted. Therefore, the inability of the total dose of EZTG granules to adjust according to symptomatic severity is a limitation of this study. Moreover, we did not determine any side effects associated with the EZTG granules. In conclusion, the EZTG granule had a positive effect on advanced age women with both kidney Qi and Yin deficiency syndromes.The EZTG prescription intervention can lead to differential expression of some apoptosis-related proteins in the granulosa cell follicular fluid. These proteins should be quantitatively analyzed to establish the specific mechanism involved in replenishing vital essence to tonify kidneys during ovarian granulosa cell apoptosis and their role in assisted reproduction.


The PI3Kδ inhibitor idelalisib is approved for the treatment of relapsed or refractory (R/R) follicular lymphoma (FL) , 2. Howehttp://www.R-project.org). Survival curves were estimated by the Kaplan–Meier method. Cumulative incidence taking into account competing risks were estimated for NRM and RI.The aim of this European Society for Blood and Marrow Transplantation (EBMT) registry study (study code LWP 2013-N-03) was to assess the safety and efficacy of alloSCT after prior exposure to idelalisib in patients with FL. Patients aged ≥ 18 years who underwent a first alloSCT for FL after exposure to idelalisib at any time before transplant between 2015 and 2018 and who were registered with the EBMT were eligible for inclusion. Any donor type and any conditioning regimen were allowed. Baseline patient, disease, and transplant data were collected from MED-A forms. Centers were requested to provide additional treatment and follow-up information (MED-B and C forms). The primary endpoint was nonrelapse mortality (NRM) at 6 and 12 months post alloSCT. Secondary endpoints were overall survival (OS) and PFS as well as incidence of relapse (RI), engraftment, and acute or chronic graft-versus-host disease (GvHD). Informed consent for transplantation and data collection was obtained locally according to the regulations applicable at the time of transplantation. All transplant centers have been required to obtain written informed consent prior to data registration with the EBMT following the 1964 Helsinki declaration and its later amendments. Statistical analysis was performed using the survival and cmprsk packages in R 3.3.2 . Overall, 80% of all patients were in PR or better at alloSCT, including 20% CRs. In patients who had received idelalisib for bridging to alloSCT, 82% had responded to the idelalisib-containing regimen with 15% of the patients in CR. The median follow-up was 12 months. Patient characteristics details are shown in Table n = 6), infection (n = 5), relapse (n = 2), GI toxicity (n = 1), and secondary malignancy (n = 1). Six- and twelve-month incidences of NRM were 12 and 24%  grade 2–4 was observed in 45% and grade 3–4 in 24% of patients. Overall, 35% of evaluable patients-at-risk developed chronic GvHD (cGvHD), and 10% were classified as extensive disease. Eleven NRM events were reported, four within the first month, two at 6 months, and one each at 3, 7, 10, 16, and 20 months post alloSCT. Causes of death included GvHD  [While long-term results of CAR trials are still awaited, alloSCT remains the only potentially curative treatment option in advanced stage FL, even though late relapses can occur , 9–11. AIn summary, the outcome of patients who received idelalisib before alloSCT was comparable with previous reports of alloSCT in FL patients without idelalisib pretreatment. Idelalisib seems to be an effective and safe drug for bridging patients with FL to alloSCT, especially in the chemotherapy-refractory setting, with a high percentage of patients with responding disease at the time of transplant. However, further studies are required to confirm that idelalisib bridging does not increase the risk of aGvHD.


Stereotactic radiosurgery (SRS) and hypofractionated stereotactic radiotherapy (SRT) are promising treatment options for patients with multiple brain metastases in the current era of personalized medicine. Recent international guidelines propose SRS also in patients with more than three brain metastases with low-volume disease. Optimal treatment quality with sparing of healthy brain tissue is essential to avoid SRS/SRT complications such as brain necrosis. The aim of this study was to compare linac (linear accelerator)-based SRS/SRT plan quality of automated planning, intensity modulated radiotherapy (IMRT), volumetric modulated arc radiotherapy (VMAT) and manually planned dynamic conformal arc (DCA) plans as well as single- and multiple-isocenter techniques. We found that automated planning with DCA or IMRT can make linac-based SRS/SRT plan quality with single isocenter comparable with a manually planned DCA plan with a separate isocenter for each metastasis.12Gy and V5Gy of uninvolved brain, number of monitor units (MUs), irradiation time and pass rate were compared. The GI was significantly higher for VMAT than for separate-isocenter, IMRT, and all automatically generated plans. The number of MUs was lowest for VMAT, followed by automatically generated DCA and IMRT plans and highest for manual DCA plans. Irradiation time was the shortest for automatically planned DCA plans. Automatically generated linac-based single-isocenter plans for multiple BM reduce the number of MUs and irradiation time with at least comparable GI and V5Gy relative to the reference separate-isocenter DCA plans.The purpose was to compare linac-based stereotactic radiosurgery and hypofractionated radiotherapy plan quality of automated planning, intensity modulated radiotherapy (IMRT) and manual dynamic conformal arc (DCA) plans as well as single- and multiple-isocenter techniques for multiple brain metastases (BM). For twelve patients with four to ten BM, seven non-coplanar linac-based plans were created: a manually planned DCA plan with a separate isocenter for each metastasis, a single-isocenter dynamic IMRT plan, an automatically generated single-isocenter volumetric modulated arc radiotherapy (VMAT) plan, four automatically generated single-isocenter DCA plans with three or five couch angles, with high or low sparing of normal tissue. Paddick conformity index, gradient index (GI), mean dose, total V Whole brain radiotherapy (WBRT) has been the standard treatment of multiple brain metastases (BM) for many years. WBRT has side effects such as fatigue, hair loss, and neurocognitive damage with only a low local tumor control probability. Stereotactic radiosurgery (SRS) has overcome several of these limitations . Accordi3 in volume and <3 cm in longest diameter; total cumulative volume ≤15 cm3). The authors concluded that SRS without WBRT as the initial treatment for patients with five to ten BM is non-inferior to SRS alone in those with two to four brain metastases, in terms of overall survival [In 2014, Yamamoto et al.  publisheIn the radiotherapy literature, it is known that hypofractionated stereotactic radiotherapy (SRT) may decrease late side effects, compared to single-fraction treatment (SRS) . To achiThere are some studies that have compared SRS platforms for treating multiple BM. Ma et al. reported that the dose delivered to normal brain is dependent on the radiosurgery platform: Gamma Knife (superior), linear accelerator (linac) or CyberKnife . SRS plaTraditionally, linac-based SRS techniques have utilized separate-isocenter plans for the treatment of patients with multiple BM, whereby each lesion is associated with one isocenter placed in the geometric center of the lesion. Different planning techniques to irradiate multiple BM with a single isocenter can be used, such as dynamic conformal arcs (DCA), intensity modulated radiotherapy (IMRT) or VMAT. A few studies have been published that reported the irradiation of multiple BM with a single isocenter ,17,18,193, whereas the previous published studies according to Yamamoto et al. [The purpose of this study was to investigate whether linac-based SRS/SRT plan quality of single-isocenter plans, either automatically generated (DCA and VMAT) or using dynamic (sliding window) IMRT, could be comparable or even superior to the plan quality of manually planned DCA plans with separate isocenters for each metastasis as a reference. In our study, seven out of twelve patients had a total cumulative volume >15 cmClinicalTrials.gov Identifier: NCT02353000) [Patients with brain metastases <4 cm in diameter were referred for SRS/SRT if they had a Karnofsky performance score >60 and a prognosis of more than six months. Twelve patients with four to ten brain metastases (median = 6) with a total of 77 lesions were selected for this study. Nine of these patients were treated at the Haaglanden Medical Center (HMC). Three patients  were anonymized benchmark patients within a multicenter randomized phase III trial (2353000) . Most of2, with a thermoplastic mask  in combination with the Precise Bite mouth piece . For the three benchmark patients, a three-point thermoplastic mask  was used. In addition, all patients had an MRI of the brain with the following sequences: T1-weighted imaging without and with gadolinium (voxel size 1.1 × 1.1 × 1.3 mm3), T2-weighted imaging, diffusion-weighted imaging, and MR perfusion imaging, using a SE-EPI sequence. The time between the CT and MRI scans was less than one week.For each HMC patient, the acquisition of the planning CT was conducted on a CT scan , slice thickness 1.5 mm and a pixel size of 0.7 × 0.7 mm3, 1 × 18 Gy for a PTV of 10–20 cm3, and for a PTV > 20 cm3 three fractions of 8.5 Gy [The gross target volumes (GTVs) were delineated by an experienced radiation oncologist as the area of contrast enhancement on a T1-weighted MRI. This sequence was also used for auto-segmentation of organs at risk (OARs)  and edited afterwards by the radiation oncologist according to the atlas for neuro-oncology . The GTVOAR constraints were based on dose limits for these organs in quantitative analyses of normal tissue in the clinic papers (QUANTEC papers) ,26. No pFor each patient, seven non-coplanar linac-based SRS/SRT plans were created by an experienced planner and reviewed by two experienced planners: a manually planned DCA plan with separate isocenters and 2–5 couch angles each with an arc of approximately 100° for each metastasis , an inversely planned dynamic IMRT plan with three couch angles and ten beams , an Auto-Planning VMAT plan with dual arcs at three couch angles , two automatically generated DCA plans with three or five couch angles , and two automatically generated DCA plans with five couch angles with high and low sparing of normal tissue (NT) of the brain . Manually planned DCA plans with a separate isocenter for each metastasis were used as a reference.The last six plans used a single isocenter  with 6 MV photon energy with flattening filter (dose rate = 500 monitor units (MUs)/min) equipped with an integrated Agility multi-leaf collimator , consisting of 160 interdigitating leaves with a width of 5 mm, was modelled for all treatment planning purposes. Monte Carlo and Collapsed Cone algorithms were used for dose calculation with a dose grid of 1 mm in iPlan RT Dose/MBM Elements and Pinnacle, respectively.In Pinnacle, a three dual VMAT arcs with a collimator angle of 45° arrangement spanning of 360° at couch angle of 0° and 160° at couch angle of 60° and 300° was chosen. In automated single-isocenter DCA, the default number of couch angles was five . In addition to these five angles, we also used three couch angles  in order to make a comparison with the other three-couch angle techniques and to study the influence of five instead of three couch angles on the plan quality. The algorithm of MBM Elements v.1.6 starts by considering a maximum of five couch angles and two independent arcs of 160° per couch angle, but only relevant arcs remain . In v.2.Separate-isocenter DCA (separate), IMRT, VMAT, MBM Elements v.1.6 with three couch angles (SIDCA_1), MBM Elements v.1.6 with five couch angles (SIDCA_2), MBM Elements v.2.0 with high NT (SIDCA_3), and low NT (SIDCA_4) plans were compared for 12 patients.max, dose to 0.03 cc, was calculated. For separate-isocenter DCA plans, a summation plan in iPlan was used.The plans were evaluated by visual inspection and by analyses of dose volume histograms (DVHs) from each TPS for the PTVs and OARs. For the critical structures  D12Gy and V5Gy of the uninvolved brain, i.e., brain volume—GTVs  , the Pad quality ), were s quality ,32.p-value ≤ 0.05 was considered statistically significant.To assess the difference in plan quality, analysis of variance (ANOVA) for repeated measures was performed for dosimetric parameters between various planning techniques. Paired T-tests were performed to compare the techniques pairwise. For statistical analysis, IBM SPSS version 22  was used. A two-sided The mean total CI was the highest (0.73 ± 0.1) for dynamic IMRT and automated SIDCA_4 (MBM Elements v.2.0 with low NT) see . Accordip = 0.003), IMRT  and all automated SIDCA plans (p = 0.012 or lower). In comparison to the separate-isocenter plans, the GI of the IMRT (p = 0.008) and SIDCA_1 plans with three angles (p = 0.013) were significantly higher. Moreover, the GI of the IMRT plans was significantly higher than the SIDCA_3 with high  and SIDCA_4 with low  sparing of the uninvolved brain. The GI of SIDCA_2 plans with five angles and the SIDCA_3 with high and SIDCA_4 with low sparing of the uninvolved brain did not differ significantly from the separate-isocenter plans (paired T-test). For ten patients irradiated in a single fraction, the GI was comparable with the results for 12 patients.The GI was significantly higher (worse) for VMAT plans (7.1 ± 3.0) compared to separate-isocenter DCA , between VMAT and automated SIDCA_2 plans with five couch angles (p = 0.044).The mean dose to the uninvolved brain for the SIDCA_2 with five angles (3.0 ± 2.0 Gy) was comparable to the manual separate-isocenter plans (3.1 ± 1.7 Gy). Based on the ANOVA, the mean dose of the uninvolved brain was not significantly different between different plans, neither for ten patients irradiated in a single fraction. However, based on paired T-tests, a statistically significant difference was found between the separate-isocenter DCA and VMAT plans , were comparable with the manual DCA plans (54 ± 58 cm3). According to the ANOVA results, the total V12Gy did not differ significantly across all planning techniques for either all patients or the ten single-fraction patients.The total V5Gy, the automatically generated SIDCA plans were comparable with the manual separate-isocenter plans. According to the ANOVA, the total V5Gy of the uninvolved brain was not significantly different between different plans, neither for ten patients irradiated in a single fraction. However, according to paired T-tests, a statistically significant difference was found between the separate-isocenter DCA and VMAT plans (p = 0.045), between VMAT and SIDCA_2 (p = 0.041) as well as SIDCA_3 with high NT sparing (p = 0.032).For the total Vp = 0.003) and other plans (p = 0.001 or lower). The number of MUs was the smallest for VMAT plans, followed by automatically generated SIDCA and IMRT plans and much lower than for the separate-isocenter DCA plans. According to the ANOVA, the total number of MUs is not significantly different between all SIDCA plans, neither for ten patients irradiated in a single fraction.The number of MUs was the highest for the separate-isocenter plans and significantly higher than for IMRT plans (p = 0.035). According to the ANOVA, the irradiation time was not significantly different between the VMAT and all automated SIDCA plans (p = 0.12). The irradiation times of IMRT plans were significantly longer than those of the last five plans. Similar results were found for ten patients irradiated in a single fraction.Irradiation time was longest for manually planned separate-isocenter DCA plans and significantly longer than for the IMRT plans (p = 0.000), IMRT , SIDCA_1, SIDCA_2, and SIDCA_4 plans (p = 0.000). Also, according to the ANOVA, the pass rate was significantly different between the VMAT and all other plans (p = 0.000).The pass rate was significantly lower for VMAT plans (87.7 ± 4.8) compared to separate-isocenter DCA  and for SIDCA_1, SIDCA_2, and SIDCA_4 plans than for separate-isocenter DCA plans (p = 0.015–0.029), but this value is still very low. For all patients and all plans, the maximum doses to OARs were within our limits and thus clinically acceptable, but varied between treatment techniques ; in the V12Gy, no statistically significant differences were found. Moreover, automatically generated linac-based SRS/SRT plans result in a lower number of MUs and are more time-efficient compared to manual separate-isocenter DCA plans. The pass rate in Mobius was significantly lower for VMAT plans compared to separate-isocenter DCA, IMRT, and SIDCA plans (except SIDCA_3).Recent international guidelines proposed SRS also in patients with more than three BM with low-volume disease. Optimal treatment quality with sparing of healthy brain tissue is essential to avoid SRS/SRT complications such as brain necrosis. We found that automated planning with DCA or IMRT can make linac-based SRS/SRT plan quality with single isocenter comparable with a manually planned DCA plan with a separate isocenter for each metastasis. The Paddick GI was significantly worse for VMAT plans, because island blocking (open leaves between metastasis) is not automatically suppressed in Pinnacle (see an example of BEV in 5Gy) than VMAT plans with comparable CI in our study. Gevaert et al. [Previously published studies ,15,17,19t et al.  conclude12Gy, but required a significantly lower number of MUs when compared to RapidArc plans [According to Narayanasamy et al., multiple BM planning software produced equivalent conformity, dose falloff, and brain Vrc plans . We foun3 and two patents were treated with three fractions, whereas the previously published studies according to Yamamoto et al. [3). Hofmaier et al. [®, Elekta, Stockholm, Sweden) for smaller metastasis (≤11.9 cm3). They found that MBM Elements can often generate treatment plans with steeper dose gradients, superior healthy brain sparing and fewer MUs as compared to VMAT plans and recommend to perform such study with larger and more irregularly shaped lesions. This is exactly what we did in our study and found that automated SIDCA results in plans with significantly better GI and shorter irradiation time, not only for smaller metastases but also for large metastases. Also, we studied the V12Gy/ and V5Gy/ of the uninvolved brain, and the gradient index as a function of the total PTV  and gradient index is seen in 12Gy, shows a tendency to increase more than if there would be a linear relation with the PTV, as shown in Moreover, in our study, seven out of twelve patients had a total PTV > 15 cmo et al. , we did o et al.  studied r et al.  also com PTV see . The smaOne would expect a better OARs sparing with separate-isocenter DCA plans because of a possibility to avoid the critical structures. According to ANOVA in our study, the maximum dose to brain stem, chiasm, optic nerves, and eyes were not statistically significantly different between the seven planning techniques. This result was not published before.Our study has some limitations. A small number of patients was analyzed. Additionally, it is probably possible to get better results with VMAT than SIDCA using another treatment planning system.5Gy) compared to VMAT and IMRT. According to ANOVA, the maximum dose to brain stem, chiasm, optic nerves, and eyes were not statistically significantly different between the seven planning techniques.In conclusion, automatically generated linac-based single-isocenter SRS/SRT plans for multiple BM reduce the number of MUs and irradiation time with a comparable GI relative to the separate-isocenter DCA plans, which were our reference. SIDCA with 5 couch angles results in the best plan quality (lower GI and V


Few data is available on the risk/benefit balance of native kidney biopsy (KB) in very elderly patients.Multicenter retrospective cohort study in the Aix-Marseille area: the results of KB and medical charts of all patients over 85 years biopsied between January 2010 and December 2018 were reviewed.104 patients were included. Median age was 87 years. Indications for KB were: acute kidney injury (AKI) in 69.2% of patients, nephrotic syndrome (NS) with AKI in 13.5%, NS without AKI in 12.5%, and proteinuria in 4.8%. Median serum creatinine was 262 μmol/L, 21% of patients required dialysis at the time of KB. Significant bleeding occurred in 7 (6.7%) patients, requiring blood cell transfusion in 4 (3.8%), and radiological embolization in 1 (1%). The most frequent pathological diagnoses were: non-diabetic glomerular diseases , hypertensive nephropathy (27.9%), acute interstitial nephritis (16.3%), renal involvement of hematological malignancy (8.7%), and acute tubular necrosis (6.7%). After KB, 51 (49%) patients received a specific treatment: corticosteroids (41.3%), cyclophosphamide (6.7%), rituximab (6.7%), bortezomib (3.8%), other chemotherapies (3.8%). Median overall survival was 31 months.KB can reveal a diagnosis with therapeutic impact even in very elderly patients. Severe bleeding was not frequent in this cohort, but KB may have not been performed in more vulnerable patients. As life expectancy increases, the proportion of elderly patients with renal diseases raises , 2: the Kidney biopsy (KB) is an invasive diagnostic procedure aimed at identifying kidney diseases and guiding treatment to prevent progression to chronic kidney disease and end-stage kidney disease (ESKD). Nowadays, there is consensus of opinion that an age over 60–65 years is not a contraindication to kidney biopsy (KB)  to perfoTo evaluate the risk/benefit balance of KB in very elderly patients, we analyzed the indications, adverse events and therapeutic impact of KB in a cohort of consecutive patients over 85 years of age.In this retrospective multicenter cohort study, we analyzed the medical charts of all patients aged ≥85 years who underwent a native kidney biopsy (KB) in 9 nephrology centers from the Aix-Marseille area between 2010 and 2018. The same pathologists (LD and JT), from the Laboratory of Pathology of the University Hospitals of Marseille (AP-HM), are in charge of all KB in this area (550 native KB each year), which allowed the screening of all cases.All the procedures followed were in accordance with the Helsinki Declaration of 1975, as revised in 2000.The French authority on data protection “Commission Nationale Informatique et Libertés” (CNIL) authorized data collection for this study (authorization number: 2211854).age, gender, history of hypertension, diabetes, progressive cancer or cardiovascular diseasephysical examination: blood pressure, edema, body mass index, extra-renal symptomslaboratory values: serum creatinine, estimated glomerular filtration rate (eGFR) using the MDRD formula, serum albumin, urine protein/creatinine ratio, presence of hematuriamain indication for KBpathological diagnosisKB-related adverse eventstherapeutic intervention after KBkidney function evolution and date of dialysis initiation if applicabledate of the last follow-up and date of death if applicable.For each case, clinical information was collected from the medical chart with the help of the referring nephrologist. Recorded data included:The following immunological tests were collected when available: antinuclear antibodies, anti-double-stranded DNA antibodies, anti-neutrophilic cytoplasmic antibody, anti-phospholipase A2 receptor (PLA2R) antibodies, rheumatoid factor, cryoglobulinemia, complement C3 and C4, and serum and urine electrophoresis. Serological tests for hepatitis B, hepatitis C, syphilis and HIV were also collected.Indications for KB were classified as: acute kidney injury (AKI), nephrotic syndrome (NS), coexisting AKI and NS, proteinuria without AKI, and chronic kidney disease (CKD).≥3.5 g/day with serum albumin < 3 g/dL. Significant proteinuria was defined as a urinary protein/creatinine ratio ≥ 0.5 g/g.AKI was defined, according to the KDIGO guidelines, as an increase in serum creatinine of at least 26.5 μmol/L within 48 h, or as an increase in serum creatinine to × 1.5 time baseline . NS was All KB were analyzed by the same renal pathologists (LD and JT), by light microscopy and immunofluorescence. For light microscopy, araldite-embedded sections were stained with Masson’s trichrome and Jones silver impregnation (2 and 0.2 μm sections respectively). For Immunofluorescence, 4 μm frozen sections were incubated with anti-Immunoglobulins, C3, and C1q antibodies .p-value< 0.20) were fed into the multivariable models. All statistical tests were two-sided and the threshold for statistical significance was p < 0.05. Analyses were performed using IBM SPSS Statistics 20.0 .Descriptive statistics are presented as medians and interquartile ranges for continuous variables and as absolute values and percentages for categorical variables. The study endpoints were overall survival (OS) and renal survival, computed from the date of the biopsy to the date of the event  or censored at the date of the last follow-up in event-free patients. Median survival rates were estimated using the Kaplan-Meier method. Hazard ratio were estimated using Cox regression model. All factors associated with outcomes in the univariate analyses  patients and are detailed in Table Pathological results are displayed in Table Overall, a disease likely to respond to a specific treatment was identified in 61 patients : 56.9% (41/72) of patients with AKI, 71.4% (10/14) of patients with NS and AKI, 69.2% (9/13) of patients with NS without AKI, and 20% (1/5) of patients with proteinuria.n = 1), myeloma cast nephropathy (n = 2) or monoclonal Ig light chain-associated Fanconi disease (n = 1).A specific therapy was administered to 51 patients  following KB Table , among wFollow-up data were available for 96 patients (8 were lost to follow-up), their outcomes are displayed in Fig. 2 were independent predictors of impaired renal survival.Overall survival is shown in Fig. We report here a large cohort of very elderly patients who underwent a native KB and show that the adverse events were not frequent and that the result of KB led to a specific therapeutic strategy in half of patients. In patients for whom no specific treatment was indicated, KB may have prevented the initiation of a potentially harmful treatment (such as corticosteroids). This work shows that for some patients, even if they are very old, the KB remains a useful procedure with therapeutic implications, especially in case of AKI and/or NS.In the present cohort, the bleeding risk of KB was significant but not prohibitive (3% requiring transfusion), without KB-related death. The same level of complication was identified in other series of elderly patients . Even ifAlthough patients were not selected in this retrospective study, an indication bias is likely in these very elderly subjects, as clinicians may have refrained from performing a KB in the frailest patients or in patients with mild kidney involvement. This may explain why a large majority of patients from this cohort displayed AKI. The question of the futility of performing an invasive procedure such as KB in very old patients, whose life expectancy is probably limited, may arise. Yet, although their median age was 87 years, only 16.7% (16/96) of patients died within 6 months after KB. For comparison purposes, the 1-year survival rate for incident patients on dialysis is 68% in France .The diagnostic benefit of KB in the very elderly has been reported before , 19. Mou≥80 in Japan [AKI is associated with increased morbidity and mortality, especially in elderly subjects: the in-hospital mortality of elderly patients with AKI ranges from 15 to 40% –24. Pre-in Japan  69% of pAcute interstitial nephritis (AIN) was the second most frequent diagnosis after hypertensive nephropathy in this cohort. Most AIN were immuno-allergic, related to the prescription of proton pump inhibitors, vitamin K antagonist or antibiotics, which are often prescribed in the elderly. In this condition, KB allows the withdrawal of the drug, and sometimes the prescription of corticosteroids.Pauci-immune crescentic GN was the third most frequent diagnosis in this cohort. A high frequency of crescentic GN in elderly patients was also reported in several studies from different countries , and the detection of light chain amyloid deposits in a patient with NS can lead to the initiation of a chemotherapy which was not indicated on haematological parameters only. This was the case for 5 patients with hematological diseases from the present cohort.There are some limitations to this study, in addition to its retrospective design. First, no data was available on the evaluation of frailty, autonomy and quality of life of the elderly patients included here. Second, no data was gathered on the clinical characteristics and outcomes of elderly patients with AKI and/or NS who were not proposed a KB. This hampers the evaluation of the “right indications for KB” and of the benefit of KB in the global population of elderly patients.Yet, the focus of this work on very elderly patients (≥85 years), and the comprehensive analysis of KB indications, results, and therapeutic impact, together with the information on patients’ outcomes, brings new information to the field.In conclusion, this study provides detailed information on the benefits and risks of KB in a population of very elderly patients in France. The reasonable bleeding risk and the high proportion of pathological diagnoses with a therapeutic impact, especially in patients with AKI and/or NS, pleads for the interest of this invasive procedure in some very elderly patients, irrespective of their age.


Spinal cord ischemia/reperfusion (SCI/R) injury is a devastating complication usually occurring after thoracoabdominal aortic surgery. However, it remains unsatisfactory for its intervention by using pharmacological strategies. Oxidative stress is a main pharmacological process involved in SCI/R, which will elicit downstream programmed cell death such as the novel defined necroptosis. Astragalin is a bioactive natural flavonoid with a wide spectrum of pharmacological activities. Herein, we firstly evaluated the effect of astragalin to oxidative stress as well as the possible downstream necroptosis after SCI/R in mice. Our results demonstrated that astragalin improves the ethological score and histopathological deterioration of SCI/R mice. Astragalin mitigates oxidative stress and ameliorates inflammation after SCI/R. Astragalin blocks necroptosis induced by SCI/R. That is, the amelioration of astragalin to the motoneuron injury and histopathological changes. Indicators of oxidative stress, inflammation, and necroptosis after SCI/R were significantly blocked. Summarily, we firstly illustrated the protection of astragalin against SCI/R through its blockage to the necroptosis at downstream of oxidative stress. Ischemia reperfusion injury is a secondary pathological condition caused by restoration of blood perfusion to the primary ischemic tissues . In clinα (TNF-α) [In pathology, excessive reactive oxygen species elicited by ischemia and reperfusion overwhelms the redox balance of cells thus leading to oxidative stress. It has been recognized that oxidative stress as a pivotal pathological mediator for ischemia reperfusion and prevention to oxidative stress can effectively protect against ischemia reperfusion injury –7. Cell  (TNF-α) , 9. In t (TNF-α) , 10–13.Recently, a novel modality of programmed cell death has been discovered, namely, necroptosis, which is similar with passive necrosis in morphology but is regulated by genes like apoptosis . Studiesβ, and collagen-induced inflammation as well as allergic inflammation [Astragalin is a bioactive natural flavonoid extracted from a number of plants . Extensiammation –28. Meanammation –31; in mammation . Based on = 6 in each group). Animal experiments were approved by the Ethics Committee of Experimental Research, Harbin Medical University, and performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Procedures of SCI/R were operated according to the previous studies [Adult male C57BL/6 mice weight about 25 g were randomly divided into four groups: the sham group, the SCI/R group, the SCI/R with astragalin administration group, and the SCI/R with necrostatin-1 treatment group  at room temperature for 24 h. Then, the tissues were embedded in paraffin and cut transversely into serial sections with a thickness of 6 μm. Standard hematoxylin and eosin (HE) staining of tissue slides was performed followed by the manufacturer's suggestion of HE commercial kit .After 72 h of SCI/R, mice were anesthetized by isoflurane before decollation to flense the spinal cord tissues. The T6 to L5 segments of the spinal cord tissues were fixed in paraformaldehyde , which catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, is one of the most important antioxidant enzymes. Glutathione  is the most abundant low-molecular-weight thiol, and GSH/glutathione disulfide is the major redox couple in animal cells. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acid peroxidation in the cells. An increase in free radicals causes overproduction of MDA. Malondialdehyde level is commonly known as a marker of oxidative stress. After 72 h reperfusion, blood was harvested from the eyeballs of mice before separation to gain serum. The serum SOD activity, GSH concentration, and MDA content were measured in accordance with the manufacturers' direction for commercial SOD, GSH and MDA kits .α is predominantly produced by macrophages. The two markers are associated with apoptosis. The spinal cord tissues harvested after 72 h reperfusion were homogenized in cold PBS using a manual homogenizer; then, the spinal cord homogenated lysis was gained after cryogenic ultracentrifugation to subsequently detect the IL-6 and TNF-α. Commercial enzyme linked immunosorbent assay kits of IL-6 and TNF-α were purchased from R&D Systems, Inc. . Measurement protocol of IL-6 and TNF-α was according to the manufacturers' instructions.IL-6 is a pleiotropic proinflammatory cytokine that is mainly secreted by monocytes, and TNF-Spinal cord tissue protein was extracted using RIPA lysis  added with phosphatase and protease inhibitor cocktail . Protein quantification was determined by the Bradford method. Tissue protein extracts were denatured in boiled water bath and then loaded into the lane of sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, the protein was separated and then transferred onto nitrocellulose membranes . The membranes loaded with target proteins were blocked in 5% nonfat milk for 2 h and incubated in primary antibody buffer at 4°C overnight. After primary antibody incubation, the membranes were incubated in HRP-conjugated secondary antibody buffer for 2 h at room temperature. Final protein bands were visualized with the Immobilon Western Chemiluminescent HRP Substrate .Astragalin was purchased from Chengdu Pulis Biotech Co., Ltd. (480-10-4). Necrostatin-1 was from Sigma . Antibodies to HMGB1 were purchased from Cell Signaling Technology . Antibody to RIP1 was purchased from Proteintech . Antibody to RIP3 was purchased from Santa Cruz Biotechnology . Antibody to MLKL was purchased from Signalway Antibody . Secondary antibodies conjugated with HRP were from Jackson Laboratories .P < 0.05 was defined as significantly statistical difference.The density of protein blots assayed by western blotting was semiquantified by the ImageJ software . Softwares of Microsoft Excel  and GraphPad Prism v.5.01  were used to analyze the experimental results. Data are expressed as mean ± SEM. Variance comparison among multiple groups was determined using one-way ANOVA with Bonferroni post hoc analysis. ∗P < 0.001). However, astragalin treatment could increase the Basso mouse score from 12 h to 72 h after SCI/R compared to the SCI/R group, which was similar to the necrostatin-1 group, indicating an improvement to the motor function (∗P < 0.05). Similarly, results from HE staining in In order to evaluate the pharmacological effect and cellular mechanism of astragalin to SCI/R, according to the preexperiments, mice were treated with astragalin and necrostatin-1 after the operation of descending thoracic aorta and left subclavian artery occlusion, and the procedure of astragalin and necrostatin-1 administration is showed in P < 0.001). However, when compared to the SCI/R group, after astragalin administration, the SOD activity and GSH level were remarkably salvaged and increased (∗∗P < 0.01), and MDA was significantly reduced (∗P < 0.05). The results in the necrostatin-1group was parallel to the astragalin group  but was significantly reduced by astragalin and necrostatin-1 administration (∗P < 0.05). At the same time, compared to the Sham group, the level of IL-6 was also apparently increased in the SCI/R group (#P < 0.001), and astragalin as well as necrostatin-1 decreased the IL-6 after SCI/R (∗P < 0.05). These data indicated alleviation of astragalin to the inflammation after SCI/R.Since inflammation is a pivotal phenomenon triggered by cell necrosis after ischemia reperfusion injury, we further evaluated the changes of proinflammatory factors TNF-P < 0.001). Nevertheless, the administration of astragalin could effectively block these aberrant upregulation of these proteins , which was parallel with the RIP1 inhibitor necrostatin-1, demonstrating that necroptosis was truly induced in SCI/R and could be inhibited by astragalin administration.Necroptosis has been recognized as a novel pharmacological target after ischemia reperfusion injury . In ordeOur present results show that the dyskinesia and histopathological deterioration after SCI/R are apparently alleviated by the natural flavonoid astragalin, and we have also verified amelioration of astragalin administration to oxidative stress and inflammation, which are similar to the effects of RIP1 inhibitor necrostatin-1. Besides, it has been illustrated the obvious necroptosis after SCI/R and the inhibition of astragalin to necroptosis. Therefore, we conclude that astragalin is a protective agent to SCI/R, and the potential cellular mechanism is based on its alleviation to oxidative stress-induced necroptosis.Restoration of blood flow after ischemia triggers reactive oxygen species redundancy; thus, the cellular antioxidant defenses are overwhelmed, and cells are stuck in a state of oxidative stress, which has been seen as a key pathological process induced by ischemia reperfusion injury . SOD andα from inflammatory cells manifests inflammatory cascades, and reduce of these factors in tissues or serum after SCI/R reflects a potential anti-inflammatory effect [α and IL-6 in spinal cord tissue homogenate after SCI/R, astragalin treatment generated apparent anti-inflammatory effect by reduce to these two classical inflammatory factors (Figures Activation of native innate immune cells and infiltrating leukocytes after SCI/R gives rise to inflammation, which is another pivotal pathological factor of SCI/R injury . Releasey effect , 43. Sim Figures , in lineThe recently illustrated necroptosis is a serious cellular endpoint after ischemia reperfusion injury , 46. OxiIn summary, our work firstly proved the protection of the natural flavonoid astragalin against SCI/R. and the cellular mechanisms of its downregulation to oxidative stress stimulated necroptosis , thus pr


The impact of thrombocytopenia on infection in patients with ST-elevation myocardial infarction (STEMI) remains poorly understood.To evaluate the association between thrombocytopenia and infection in patients with STEMI.Patients diagnosed with STEMI were identified from January 2010 to June 2016. The primary endpoint was in-hospital infection, and major adverse clinical events (MACE) and all-cause death were considered as secondary endpoints.p < 0.001). Prevalence of in-hospital MACE (30.1% (56/186) vs. 16.4% (199/1215), p < 0.001) and all-cause death (8.1% (15/186) vs. 3.8% (46/1215), p = 0.008) revealed an increasing trend. Multivariate analysis indicated that thrombocytopenia was independently associated with increased in-hospital infection  and MACE , but not all-cause death . After a median follow-up of 2.85 years, thrombocytopenia was not associated with all-cause death at multivariable analysis .A total of 1401 STEMI patients were enrolled and divided into two groups according to the presence (n = 186) or absence (n = 1215) of thrombocytopenia. The prevalence of in-hospital infection was significantly higher in the thrombocytopenic group (30.6% (57/186) vs. 16.2% (197/1215), Thrombocytopenia is significantly correlated with in-hospital infection and MACE, and might be used as a prognostic tool in patients with STEMI.The online version contains supplementary material available at 10.1186/s12872-021-02210-3. Infection is an uncommon  but potentially devastating complication in patients with ST-elevation myocardial infarction (STEMI). Infection in these patients is usually associated with a significantly increased risk of mortality and morbidity, prolongs hospitalization, and increases healthcare costs –3. Given9/L [Thrombocytopenia is a common laboratory abnormality in patients presenting with acute myocardial infarction (AMI) and always defined as a platelet count < 150 × 109/L . Baselin9/L . However9/L –9.Helicobacter pylori (H. pylori) infection was on the rise as a cause of immune thrombocytopenia (ITP) [9/L) was a potential risk stratification tool for severe Clostridium difficile infection (CDI) [Recent studies have reported that patients with coronavirus disease 2019 (COVID-19) exhibited severe thrombocytopenia , and Helia (ITP) , while aia (ITP) , which iia (ITP) , and modon (CDI) .Given the potential value of thrombocytopenia in the prediction of infection, and in order to improve risk stratification for these patients, we sought to investigate the association between thrombocytopenia and infection in patients with STEMI.Patients diagnosed with STEMI were enrolled from January 2010 to June 2016 in Guangdong Provincial People’s Hospital. STEMI was diagnosed if patients presented within 12 h from the onset of symptoms including typical chest pain lasting for ≥ 30 min, not responsive to nitrates, with ST-segment elevation of ≥ 0.2 mV in at least two contiguous leads, or left bundle-branch block . The exc9/L [Thrombocytopenia was defined as a platelet count < 150 × 109/L . The pri9/L , and wast-tests. Continuous variables with a non-normal distribution were expressed as median and interquartile ranges, and analyzed using Wilcoxon rank sum tests. Categorical variables (which are presented as percentages) were compared using the chi-squared test or Fisher’s exact test (as appropriate). In-hospital outcomes and follow-up outcomes were calculated using a logistic regression model or Cox regression model in multivariable analysis. As for sample size consideration, we applied a rule of thumb, namely, that the number of events per variable should be 10 or greater. We expected about no more than 11 factors in the development model; thus, at least 110 infections were needed. According to the incidence of infections (10%) in the previous study [p < 0.05 was considered significant. Data analyses were carried out using SAS v9.4 .Continuous variables with a normal distribution were presented as mean ± SD, and compared using two-sample us study , the fin9/L). The clinical characteristics of the study population at baseline are shown in Table A total of 2283 patients with STEMI at our hospital were identified between January 2010 and June 2016; of these, 882 patients were excluded according to the exclusion criteria. Finally, 1401 patients  diagnosed with STEMI undergoing PCI were enrolled and analyzed in this study , with no significant differences in various types of infection (p = 0.804), as well as in-hospital MACE (30.1% (56/186) vs. 16.4% (199/1215), p < 0.001) than non-thrombocytopenia cases during hospitalization. The thrombocytopenia group also had a higher proportion of in-hospital death (8.1% (15/186) vs. 3.8% (46/1215), p = 0.008) and longer hospital stay  than the non-thrombocytopenia group (Table p < 0.001), but not severe thrombocytopenia defined as PLT < 50  due to the small sample size possibly  . After adjustment for potential confounding factors , multivariate analysis showed that thrombocytopenia remained a significant predictor of infection risk in such patients  (Table p = 0.002) but not all-cause death  . However, upon fully adjusted multivariate Cox regression analysis, this significant association disappeared  , 1.52; 95%CI 1.04–2.20; The present study investigated, for the first time, the relationship between thrombocytopenia and infection in patients with STEMI undergoing PCI. We found that thrombocytopenia was associated with a significantly increased risk of in-hospital infection, in-hospital MACE but not all-cause death. These findings suggested that serial monitoring of platelet counts was essential to identify patients with STEMI at high risk of adverse outcomes.Platelets are anucleate blood cells that play a crucial role in the maintenance of hemostasis . ThromboIn our analyses, we demonstrated that the prevalence of thrombocytopenia in STEMI patients was 13.3%, while previous studies have reported the incidence of thrombocytopenia in patients with ACS varied from 1.6 to 13% , 26. TheWith regard to the predictive value of thrombocytopenia for infection and the underlying mechanisms of the association between them, some factors might be notable. First, patients with thrombocytopenia receive many transfusions of red blood cells and platelets in necessity, both of these interventions are proinflammatory , 30. SecIn addition to thrombocytopenia, our study firstly found that the levels of serum albumin and hemoglobin was independently associated with the risk of infection. Leonardi and colleagues reported that declining hemoglobin content, with or without overt bleeding, was independently associated with heightened risk of 1-year mortality among patients with ACS managed invasively . Plakht Our study highlights the notion that thrombocytopenia is infrequent in patients diagnosed with STEMI, but is associated with a markedly increased prevalence of infection and in-hospital MACE, and should not be underestimated. Early identification of thrombocytopenia is essential for appropriate clinical care of STEMI patients. Hence, dynamic monitoring of platelet count might be encouraged for cardiologists among high-risk patients to avoid complications.This study also had several main limitations. First, the causes of thrombocytopenia were not elucidated clearly, and the diagnosis of thrombocytopenia according to one complete blood count (CBC) test might be not sufficient while minor variation might occur and platelet agglutination might result in technical CBC diagnosis of thrombocytopenia. Second, our study was carried out at a single center with a relatively small study cohort. Third, we evaluated a cohort of Chinese patients, so caution is needed when trying to apply our conclusions to another population. Fourth, we failed to collect the information about blood product transfusion, which might be a potential confounding factor. Finally, because of a lack of studies investigating the outcome of infection in thrombocytopenia patients, the potential molecular mechanisms could not be explored.In summary, the present study indicated that thrombocytopenia during hospitalization was independently associated with an increased prevalence of infection and in-hospital MACE in patients with STEMI. Thrombocytopenia could be used as a simple prognostic tool for these patients.Additional file 1: Table S1. Association of platelet count on infection after multivariable adjustment.


Protein post-translational modification (PTM) is a key issue to investigate the mechanism of protein’s function. With the rapid development of proteomics technology, a large amount of protein sequence data has been generated, which highlights the importance of the in-depth study and analysis of PTMs in proteins.We proposed a new multi-classification machine learning pipeline MultiLyGAN to identity seven types of lysine modified sites. Using eight different sequential and five structural construction methods, 1497 valid features were remained after the filtering by Pearson correlation coefficient. To solve the data imbalance problem, Conditional Generative Adversarial Network (CGAN) and Conditional Wasserstein Generative Adversarial Network (CWGAN), two influential deep generative methods were leveraged and compared to generate new samples for the types with fewer samples. Finally, random forest algorithm was utilized to predict seven categories.https://github.com/Lab-Xu/MultiLyGAN.In the tenfold cross-validation, accuracy (Acc) and Matthews correlation coefficient (MCC) were 0.8589 and 0.8376, respectively. In the independent test, Acc and MCC were 0.8549 and 0.8330, respectively. The results indicated that CWGAN better solved the existing data imbalance and stabilized the training error. Alternatively, an accumulated feature importance analysis reported that CKSAAP, PWM and structural features were the three most important feature-encoding schemes. MultiLyGAN can be found at The CWGAN greatly improved the predictive performance in all experiments. Features derived from CKSAAP, PWM and structure schemes are the most informative and had the greatest contribution to the prediction of PTM.The online version contains supplementary material available at 10.1186/s12859-021-04101-y. As a common occurrence in the body, protein-translational modification (PTM) plays an important role in regulating various physiological processes and functions. PTM refers to the process of covalent modification of individual amino acid residues on a protein after the mRNA has been translated into a protein . HoweverAlternatively, protein lysine modifications (PLMs), prevalent PTM types, which occur at active ε-amino groups of specific lysine residues in proteins and are critical for orchestrating various biological processes. So far, a series of computational prediction tools have been developed. These predictors firstly employed feature construction methods including sequences and physicochemical properties. Then, machine learning algorithms were adopted to train models. The published predictors about seven types of lysine modified sites are as follows: (1) Acetylation: NetAcet , PAIL 33, BRABSBThe data imbalance issue was characterized by prediction bias across widely divergent categories, therefore minimizing the bias is essential for downstream exploration in the prediction of PTMs. Here, we aim to harness deep generative methodology to solve the issue. In 2014, Goodfellow et al. first proposed the Generative Adversarial Nets (GAN) . GAN achTo further study the underlying mechanisms and the relationship of features and some specific modifications, Random Forest was utilized as a classifier and explain feature importance. The whole pipeline MultiLyGAN is shown in Fig. C) and independent test error rate (EI) can be measured to evaluate statistical model performance , Confusion Entropy (CEN), Matthews Correlation Coefficient (MCC) and Cross-validation error rate  were easily predicted to be S7 (Ubiquitination); samples within S2 (Glycation) were prone to classified into S7 and S1; some samples labelled as S3  were wrongly predicted as the S1, S7 and S5 (Succinylation); samples labelled as S4 (Methylation) were specifically mis-predicted as S1; samples labelled as S5 were easily incorrectly predicted as S1; samples in S6 (SUMO) were mispredicted to S1 and S7; and S7 is easily mispredicted as S1. Therefore, acetylated sequences harbor the largest similarity with other modifications, indicating its function may be interconnected with other types. Sumoylation (S6) and ubiquitination (S7) were easily confused, further demonstrating the sequence or functional correlations between the two processes.Compared with CGAN in Table 1, S2, S4, and S6, PCC + CWGAN + RF performed significantly better than PCC + CGAN + RF. No statistically better performance in S3, S5, and S7 was shown compared PCC + CWGAN + RF with PCC + CGAN + RF.Table I was 0.1451 after CWGAN in the independent cohort. Table 1 and S3, PCC + CWGAN + RF harbored more precise prediction than PCC + CGAN + RF. The CWGAN and CGAN showed no discriminative difference in the remaining types.Profiling the independent dataset which is orthogonal to training set, the results in Table We investigated one case study on distinguishing two confusing modifications easily to be misclassified, which was illustrated in our paper. For example, Ubiquitination and Acetylation Fig.  were repIn addition to the improvement of predictive performance, we evaluated CGAN and CWGAN on loss variations in neural network training. This paper adopted the average distance to evaluate simulation data after GAN training. Firstly, the mean of all real data was calculated. Secondly, the Euclidean distance was calculated between the mean value of the simulated data and the real data for each category (modification) as the distance Fig. a. CGAN's1) was depicted in Fig. We calculated the loss of the generator (Gloss) and the loss of the discriminator (Dloss) during 50,000 iterations to compare the advantages and disadvantages of the two algorithms. The Gloss and Dloss in training CGAN and CWGAN on the acetylation modification  to conduct the same steps for comparable results. The tenfold cross-validation and independent matrices of SMOTE were lower than CWGAN, and higher than no-augmentation Table , 1 and 4RF gave the order of importance for the 1497-dimensional features. According to the importance degree, the first nine most important features were from the PWM-encoding scheme. The frequency of different amino acids appearing in different positions of the sequence fragment was significantly different, which provided important information. The cumulative importance of different encoding schemes is summarized in Fig. To validate the performance of MultiLyGAN, the comparison of our models with the MusiteDeep  was perfI of 0.1451 by independent test, which were better scores than those obtained by CGAN. Meanwhile, CWGAN performed better in each of the seven modifications than CGAN.In this work, we propose a new pipeline to predict the seven types of modified sites, where the GAN was utilized to solve the data imbalance problem. We translated the multilabel prediction problem into a multiclass prediction problem. Overall, 2340 dimensional features were constructed by combining eight different sequences and five structural information-encoding schemes. Finally, 1497 dimensional features were obtained after PCC feature extraction. Through CWGAN, the generated simulation data were closer to the real data. CWGAN yielded Acc of 0.8549, MCC of 0.8330, CEN of 0.2250, and EC) and independent test error rate (EI) . To reduce redundancy and bias, fragments with high sequence similarity (40% or more pairwise sequence identity) were removed. After deleting homology, we obtained 46 2-hydroxyisobutyrylated, 3273 acetylated, 38 butyrylated, 16 carboxylated, 29 crotonylated, 143 formylated, 402 glutarylatd, 1454 glycated, 19 hydroxylated, 1467 malonylated, 1208 methylated, 37 neddylated, 108 phosphoglycerylated, 223 propionylated, 169 pupylated, 1855 succinylated, 1302 sumoylated and 3468 ubiquitinated lysine-centered fragments. These data totally contained 18 different modification types.We collected 18 kinds of lysine modification samples from the CPLM2.0 database , involvi18 types of labels for each fragment. After data integration, there were a total of 58 schemes, encompassing 18 for one label, 28 for two labels, and 12 for three labels. The labels with less than 500 samples were deleted. The remaining samples are single-label data, including Ubiq (3253), Ace (3194), Succ (1692), Glyca (1416), Malon (1253), Sumo (1213) and Meth (1172). Since the feature construction consisted of the structural information of each amino acid, we discarded fragments with the length less than 17. Finally, we attained Ubiq (3185), Ace (3114), Succ (1645), Glyca (1399), Malon (1224), Sumo (1174) and Meth (1147). The detailed data of each type are shown in Fig. We merged the data with the same ID, site and fragments. Eighteen types of modifications contribute to theoretically 2), Succ 192, Glyca1 was set as Ace, S2 as Glyca, S3 as Malon, S4 as Meth, S5 as Succ, S6 as Sumo, and S7 as Ubiq. Thus, the total dataset S can be defined as:In alphabetical order, SL is the length of the fragment):Fourteen specific physical and chemical properties were selected to construct features. A 14-dimensional vector was obtained for every amino acid  value, and a 1200-dimensional vector was obtained.There are 400 dimensions for each margin L, thus, each sample can be encoded as L-dimensional vector:The position weight matrix was calculated by category to obtain the frequency information of the amino acids at each position. According to the above description, the total length of the sample fragment is L:A reduced-letter code 8 is selected, and each amino acid is encoded as an 8-dimensional vector by acid, basic, aromatic, amide, small hydroxyl, sulfur, aliphatic 1 and aliphatic 2. Therefore, a sample of length L is encoded as a vector of 8 × http://antares.protres.ru/fold-amyloid/ to predict the amyloidogenic region of the sample and finally obtain the L-dimensional vector:Using L-dimensional vector:Under BE (Binary Encoding), each amino acid is encoded into a 20-dimensional binary vector, resulting in a 20 × L-1 = 17–1 = 16, ω = 0.05, physicochemical properties = . Each peptide fragment ultimately obtained a (20 + L-1)-dimensional vector:Select λ = L-1 = 17–1 = 16, ω = 0.05, physicochemical properties = . Each protein fragment ultimately obtained the characteristics of a (20 + 2(L-1))-dimensional vector:Select λ = 10-helix (G), α-helix (H), L-dimensional vector for each protein fragment. Combining sequence and structural features, each peptide was translated into a 2359-dimensional vector  model, which integrates CGAN and Wasserstein’s distance. The objective of GAN is to learn best parameters for generator so as to minimize the JS divergence between the real distribution For the generator, the input includes the prior noise distribution z and the categorical label y embedded as a seven-dimensional vector by one-hot encoding method. There are several main improvements in the network. CWGAN deleted the sigmoid function of the last layer of D. The loss function for G and D no longer used logarithmic transformation. Instead, it used the clip function to update the function and replace Adam with the RMSProp optimization method. Different from common WGAN that outputs the divergence of generative samples in comparison to realistic samples, the discriminator in CWGAN further adds the estimation of whether generative samples are matched to the conditional information. Therefore, CWGAN can generate samples with a specific category.Using the sample amount of the seventh type of data as a reference, the CWGAN simulation was performed on the other six types, and the simulation data were consistent with the seventh type of data generated. The parameters tuning of CWGAN and the finial parameters of CWGAN are shown in Table 8 and Table 9 of Additional file: Random forest (RF) is a prevalent bagging approach of machine learning. The parameters of RF are shown in Table 10 of Additional file: Two-classification and Multiclassification system indicators are in Additional file: Additional file 1. S1: Sequence preprocessing. The supplementary material introduces amino acid window sliding technology and feature construction that convert amino acid sequences into numerical vectors.Additional file 2. S2: CWGAN generative model.Additional file 3. S3: Supplementary tables.Additional file 4. S4: Classification system indicators.


AtFeSOD gene in comparison to its wild type (WT). Compared to their WT, the TG plants showed less Cd accumulation and improved growth, physiology, antioxidant and detoxification systems. These results demonstrate the role of SOD in determining Cd-tolerance.Cadmium (Cd), a readily absorbed and translocated toxic heavy metal, inhibits plant growth, interrupts metabolic homeostasis and induces oxidative damage. Responses towards Cd-stress differ among plant cultivars, and the complex integrated relationships between Cd accumulation, detoxification mechanisms and antioxidant defenses still need to be unraveled. To this end, 12 Egyptian maize cultivars were grown under Cd-stress to test their Cd-stress tolerance. Out of these cultivars, tolerant (TWC360 and TWC321), moderately sensitive (TWC324) and sensitive (SC128) cultivars were selected, and we determined their response to Cd in terms of biomass, Cd accumulation and antioxidant defense system. The reduction in biomass was highly obvious in sensitive cultivars, while TWC360 and TWC321 showed high Cd-tolerance. The cultivar TWC321 showed lower Cd uptake concurrently with an enhanced antioxidant defense system. Interestingly, TWC360 accumulated more Cd in the shoot, accompanied with increased Cd detoxification and sequestration. A principal component analysis revealed a clear separation between the sensitive and tolerant cultivars with significance of the antioxidant defenses, including superoxide dismutase (SOD). To confirm the involvement of SOD in Cd-tolerance, we studied the effect of Cd-stress on a transgenic maize line (TG) constitutively overexpressing  Cadmium (Cd) is recognized as an important pollutant due to its high toxicity, even at low concentrations, and high solubility in water ,2. It isTo avoid injuries induced by Cd toxicity, plants have developed several defense mechanisms . These m2•−), hydrogen peroxide (H2O2) and hydroxyl radical (•OH) /2: where DWM: dry weight at mild stress, DWS: dry weight at severe stress and DWC: dry weight of control. Cultivars scored the highest and lowest shoot and root TI were selected as tolerant and sensitive to be evaluated for their growth and physiological response under cadmium stress. Four cultivars  were selected according to their lowest and highest TI, respectively. The selected cultivars were subjected to Cd-stress in an experiment that was carried out during May–June 2019 in the same conditions as mentioned above with temperature range of 35 °C for maximum and 22 °C for minimum. Fifty days after sowing, morphological criteria were estimated. Shoot and root samples were dried in an oven at 70 °C and kept for estimation of Cd content. Fresh ones were used in the rest of the biochemical analyses.In this experiment, 12 maize cultivars, as commonly cultivated in Egypt, were tested in pot experiment to select those are the most tolerant and the most sensitive cultivars to Cd-stress. Seeds of the investigated cultivars were obtained from the Agriculture Research Center, Giza, Egypt. The experiment was carried out during May–June 2018 in Beni-Suef University, in an open field condition and under natural light in a randomized complete block design. The temperature ranged between 36 °C as maximum and 27 °C as minimum. Plastic pots (14 cm diameter and 15 cm height) were filled with one kg sand: clay soil (3:1) for each pot. Cadmium was applied as different concentrations . The transgenic line was induced by overexpressing the FeSOD gene from Arabidopsis  under the control of the cauliflower mosaic virus 35S promoter, the backcross used were of Pa91 × H99 to the H99 parent. Seeds of wild and transgenic lines were provided by Frank Van Breusegem [4·8/3H2O; 0.0, 46.5 and 372.1 mg Cd Kg−1 dry soil). Pots were covered with plastic wrap, irrigated daily with tap water and maintained at the original soil water content. Seedlings were grown for 24 days after sowing in the growth chamber under controlled conditions . Growth parameters (fresh and dry weights) were estimated and fresh shoots were kept at −80 °C for biochemical analyses.To test the effect of enhancing the antioxidants on Cd-tolerance, in association with Cd accumulation, we used a Cadmium content in both shoot and root was estimated using the protocol of Cottenie, et al. , while tplant/Cdsoil, where Cdplant is Cd concentration in harvested plant material (mg kg−1) and Cdsoil is Cd concentration in soil (mg kg−1).Bioconcentration factor (BCF) = Cdshoot/Cdroot, where Cdshoot is Cd concentration in plant shoot (mg kg−1) and Cdroot is Cd concentration in root (mg kg−1).Translocation factor (TF) = Cd−1 protein min−1. Phytochelatins content was measured according to de Knecht, et al. [−1 FW.Glutathione-S-transferase (GST) activity was assayed as described in Habig, et al. . The act, et al.  by extra2 concentration in the leaf chamber were kept at 25 ± 0.5 °C and 400 μmol mol−1, respectively. All parameters were estimated inside the growth room at noon [gs), this was determined using a Leaf Porometer  [0, Fm) were assayed for 30 min and photochemical efficiency of PSII was calculated as Fv/Fm, where Fm  = Fm − F0. Photosynthetic pigments were extracted and determined according to Markwell, et al. [−1 FW.The light saturated photosynthetic rate was determined with a portable photosynthesis system . The temperature and CO at noon . ConcernWA, USA) . Fully e, et al. . The con, et al.  and expr2O2) as well as lipid peroxidation and protein oxidation were assayed. Hydrogen peroxide (H2O2) was extracted in fresh tissue using 0.1% TCA and estimated by the reaction with 1M potassium iodide (KI). The absorbance was read at 390 nm [−1 FW. Malondialdhyde (MDA), a product of lipid peroxidation and a thiobarbituric acid reactive substance (TBARS) was estimated according to Jambunathan [−1 FW. Protein oxidation was measured through carbonyl quantification [−1 protein.To reveal the oxidative stress induced by the uptake and accumulation of Cd, the content of hydrogen peroxide  is the amount of enzyme needed to induce 50% inhibition in the rate of NBT photoreduction. The method of Elavarthi and Martin [2O2. The enzyme activity was expressed in terms of mM H2O2 mg−1 protein min−1. The method of Kumar and Khan [2O2 into pulpurogallin at 430 nm. The activity of the enzyme was expressed as μ mole pulpurogallin mg−1 protein min−1.Ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) were assayed in a semi- high through put set up [2, and 1 mM ascorbic acid (ASC). The activities of APX, MDHAR, DHAR, and GR were assayed in a microplate following the method of Murshed, et al. [21 cm21 extinction coefficient. GR activity was estimated following the change in absorbance at 340 nm and calculated from the 6.22 mM21 cm21 extinction coefficient. The activity of glutathione peroxidase (GPX) was determined as described by Drotar, et al. [21 cm21 extinction coefficient. Enzymes activities were expressed as μ mol mg−1 protein min−1.By using a pre-cooled mortar and pestle, a fresh maize sample (0.2 g) was pulverized in liquid nitrogen and extracted with 1.2 mL of 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA.Na reagent  and bovi reagent . The actd Martin  was follt set up . Enzyme , et al. . The act, et al. , in a co2O was mixed in equal volume with the extract. The absorbance was read at 600 nm using a microplate reader  and FRAP was calculated using trolox standard curve. Flavonoid extraction was performed by homogenizing fifty mg of plant tissues in 0.5 mL ethanol (80% v/v) and supernatants were collected after centrifugation. Flavonoids concentration was assayed following the modified AlCl3 colorimetric method and expressed as mg quercetin g−1 FW [v/v). Then, reduced ascorbate (ASC) and glutathione (GSH) contents were analyzed using HPLC as described in Potters, et al. [−1 FW. Tocopherols were extracted in fresh samples using hexane. After centrifugation, the extract was dried using  and then resuspended in hexane once more. Tocopherol separation and quantifications were performed using HPLC analysis. Dimethyl tocol (DMT) was used as internal standard (5 ppm). Data were analyzed through Shimadzu ClassVP6.14 software, and tocopherols concentration was expressed as mg g−1 FW.Total antioxidant capacity was measured following the Benzie and Strain  method fn g−1 FW . About 0, et al. . HPLC (Sp < 0.05) among means at stress levels within the individual cultivar were compared by Duncan’s multiple range test. Data were statistically analyzed by using SPSS . Multi Experimental Viewer  was applied to generate the principal component analysis (PCA). Differences between WT and TG within each Cd-stress level were analyzed using Student’s t-test at p < 0.05.Each experiment was repeated twice, and since data were in the same trend, results of the first were shown. Two-way ANOVA was applied to study the effect of the two factors, Cd-stress (Cd) and cultivars (C), and their interaction (Cd × C). For the experiments with the transgenic line, two-way ANOVA was applied for the two factors; Cd-stress (Cd), and SOD and their interaction (SOD × Cd). Significant differences (2·2.5H2O Kg−1 soil), representing control, mild and severe Cd-stress, respectively. The cultivars were screened according to the morphological parameters  were regrown in the following season (May 2019) to confirm the results of the screening experiment and to study the mechanisms underlying their differing tolerance. The cultivars were investigated for their morphological and physiological responses as well as Cd uptake and accumulation under Cd-stress. The four cultivars showed a differential growth of their shoots and roots in response to Cd-stress A–D that To elucidate whether the variation in tolerance of the investigated cultivars is related to their uptake, translocation and accumulation of cadmium, we estimated cadmium content in shoot and root, in addition to the bioconcentration factor (BCF) and translocation factor (TF). The results showed differences among the four investigated maize cultivars concerning Cd uptake (BCF), Cd translocation from root to shoot (TF) and Cd content in both root and shoot. While SC128 accumulated the highest cadmium content in the shoot, the highest content in the root was found in TWC360 at severe Cd-stress . MoreoveTo study if the cultivar TWC360 manages Cd detoxification better than sensitive cultivars, we studied the enzyme GST and its substrate GSH to clarify their role in the detoxification of Cd in the investigated cultivars A–D. The 2O2 and lipid peroxidation in the investigated cultivars in response to Cd-induced oxidative stress and membrane damage. H2O2 content significantly increased in shoots as well as roots of SC128 and TWC324, whereas TWC360 and TWC321 were not affected , consistent with our biochemical results. While, the treated tolerant cultivars (T-TWC321 and T-TWC360) and the moderately sensitive one (T-TWC324) were located on the lower side of PCA2, according to their managing detoxification and antioxidant defense (POX and SOD) under Cd stress. Similar to shoot, the separation along PCA1 in case of root (represented 52% of variances) was according to Cd-exposure and separated the responses of control and treated cultivars (2O2 and MDA), while the tolerant cultivars showed a positive correlation with the detoxification mechanisms (GST and GSH) and the antioxidant enzymes, including SOD. Interestingly, in both principal component analyses for shoot and root, the stressed tolerant cultivar (T-TWC321) located beneath its non-stressed respective one (C-TWC321) due to its less sensitivity and Cd content as well as its high maintenance of biomass under Cd-stress. Overall, PCA analysis indicated that SOD and APX (as antioxidant defenses) and the stimulation of GST in addition to the accumulation of GSH (as detoxification mechanisms) are probably the defense strategies that the tolerant cultivars use to cope with Cd-stress.For more clarification, we performed two independent principal component analyses for each of the shoot and root A,B to vaultivars B. Cd-tre2•− to H2O2 and O2 [FeSOD maize transgenic line under Cd-stress to investigate its growth, physiology and chemical responses. We compared the performance of shoots of the transgenic line AtFeSOD (TG) and the wild type (WT) under Cd-stress. At normal conditions, there was no significant difference between WT and TG in all the investigated parameters, except a higher SOD activity (Superoxide dismutase (SOD) is considered the first line of defense responsible for scavenging ROS by dismutating O2 and O2 ,59. Our activity . Under iactivity A,B.gs) decreased in both WT and TG, but to a lesser extent in TG  and the activity of H2O2 degrading enzymes CAT, POX, APX [The variability in biomass of the four cultivars probably was linked to their potential to uptake and accumulate Cd in the shoot/root. The four cultivars showed differential Cd uptake and accumulation, where cultivars with less Cd accumulation in their shoot, accompanied with stability of biomass, modulated the antioxidant defense mechanisms more effectively to cope with Cd-stress. These results indicate that the tolerance of cultivar TWC360 may be associated with the restricted Cd translocation to the shoot, since tolerant genotypes were reported to have a lower metal content in their shoots and a lower shoot: root Cd ratio than sensitive ones . The hig and •OH ,70,71, a and •OH ,73 as on and •OH . In cont and •OH . The sev and •OH . FurtherPOX, APX . The rooArabidopsis gene for FeSOD [AtFeSOD (TG) showed enhanced tolerance and improvement of the plant growth and development under Cd stress. That response might be implemented through the activation of antioxidant system resulting in high ROS scavenging and less oxidative damage (lower MDA and protein oxidation) as well as enhanced photosynthesis (higher Chl a + b and Fv/Fm).In addition to its potential role in Cd-stress mitigation according to our results, existence of a maize transgenic line overexpressing an or FeSOD ,77 allowor FeSOD ,78,79. For FeSOD ,80. SimiAtFeSOD overexpressing maize coupled with a declined Cd in shoot suggests that a relation between the balanced generation of ROS and the controlled accumulation of Cd in shoots exists. The enhanced functionality of proteins, resulting from the lower protein oxidation, and the more integrated membranes indicated by lower lipid peroxidation could confer more control on membrane permeability. Lessening Cd accumulation also could be due to the decrease in the transport of Cd to shoot. The enhanced photosynthesis probably supplied assimilates and produced more energy necessary for restricting Cd uptake and translocation. Additionally, lessened accumulation of Cd in leaves could play an indirect role in the increased content of chlorophyll. In this regard, Cd may induce a competitive inhibition of the absorption of some elements that are essential for chlorophyll biosynthesis or by the dysfunction of the enzymes associated with chlorophyll synthesis by interacting with sulfhydryl-rich regions [The TG also showed a lessening in the accumulation of Cd in its leaves. Consistent with a previous study , the enh regions ,82.2•− to H2O2. In order to prevent H2O2 from generating the highly reactive and toxic radical (•OH), the plant employs CAT, POD, APX and GPX enzymes to reduce H2O2 to H2O [AtFeSOD also enhanced the plant ability to increase the activity of other antioxidant enzymes. For example, CAT, POD and GPX activities were increased in the TG line over those of WT exposed to Cd stress. That response indicates a vital role of those enzymes in detoxification of ROS [pcs mutant decreased Cd tolerance compared to WT plants [The SOD enzyme is the most vital superoxide anion scavenger that catalyzes converting of O2 to H2O . Thus, tn of ROS ,84,85. Mn of ROS ,87,88. Cn of ROS . ApparenT plants , while mT plants . MoreoveT plants . It seemAtFeSOD overexpressing transgenic maize line highlighted the role of SOD in Cd-tolerance. The transgenic line showed improved growth and tolerance, less Cd content and enhanced photosynthesis, as well as a stimulated antioxidant defense and detoxification mechanisms. The significant role of SOD in Cd-tolerance was therefore indicated. However, further studies are needed to reveal whether that significance is associated with Cd-accumulation.Cadmium interrupts metabolic homeostasis and causes inactivation of proteins, thereby inducing excessive ROS generation. By studying the response of maize cultivars varying in tolerance to Cd-stress, we conclude that the reduction in biomass of sensitive cultivars was due to the failure of managing either antioxidant or detoxification mechanisms. The tolerant cultivar had lower Cd accumulation and showed a more efficient ROS scavenging system and detoxification mechanisms. Studying the response of an